(IJISA-V6-N2-9)

超高效液相色谱-三重四极杆质谱联用仪同时检测水中9种亚硝胺朱翔;李伟;刘玉灿;段晋明【摘要】采用超高效液相色谱-三重四极杆质谱联用仪(UPLC-MS/MS)优化了9种亚硝胺的质谱运行参数,并通过对比不同流动相、固相萃取柱和定容溶剂对水中9种亚硝胺检测强度的影响,确定以甲醇-10 mmol/L碳酸氢铵溶液为流动相,使用椰壳活性炭萃取柱对样品进行预处理,超纯水作为定容溶剂.在最优实验条件下,9种亚硝胺的线性范围为5 ～ 150 ng/L,相关系数(r2)为0.997～0.999,检出限和定量下限分别为1.3 ～2.8 ng/L和4.0～8.5 ng/L;日内(n=5)和日间(n=6)相对标准偏差分别为3.5％～ 8.4％和2.8％～7.5％.3种不同实际水样在25 ng/L和100 ng/L 加标浓度水平下,回收率达80.4％～109.6％.使用该方法对6个不同给水处理厂和污水处理厂出水中的亚硝胺浓度进行检测,其中N-亚硝基二甲胺(NDMA)的检测浓度最高,分别为10.2 ng/L和45.6 ng/L.【期刊名称】《分析测试学报》【年(卷),期】2014(033)008【总页数】7页(P866-872)【关键词】超高效液相色谱-三重四极杆质谱联用仪;亚硝胺;固相萃取;定容溶剂【作者】朱翔;李伟;刘玉灿;段晋明【作者单位】西安建筑科技大学环境与市政工程学院,陕西西安710055;西安建筑科技大学环境与市政工程学院,陕西西安710055;西安建筑科技大学环境与市政工程学院,陕西西安710055;西安建筑科技大学环境与市政工程学院,陕西西安710055【正文语种】中文【中图分类】O657.63;O623.731亚硝胺是一类致癌、致畸、致突变的物质[1-2]，在食物、橡胶制品、香烟中均有检出[3-5]，也有研究报道在氯或氯胺消毒后的水中检出亚硝胺类物质，尽管其浓度相对较低但其毒性却高于常见的卤化消毒副产物(三卤甲烷和卤乙酸)[6-7]。

中国预防兽医学报Chinese Journal of Preventive Veterinary Medicine第42卷第12期2020年12月V ol.42No.12Dec.2020doi ：10.3969/j.issn.1008-0589.202002012用于CRISPR 文库筛选的N2a-Cas9细胞系的构建张纪文，王露露，李芳，刘杏，赵东明*，步志高*（中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室，黑龙江哈尔滨150069）摘要：为了进行CRISPR 文库筛选，本研究采用慢病毒转导的方法利用N2a 细胞构建表达Cas9蛋白的N2a-Cas9单克隆细胞系。

实验将LentiCas9-Blast 、pSPA X2和pMD 2.G 3种质粒共转染人胚胎肾细胞（HEK293T ）获取高滴度的重组慢病毒，收集重组慢病毒并感染N2a 细胞，经杀稻瘟菌素（Blasticidin ）筛选，通过有限稀释法获得含有Cas9基因的多个单克隆细胞株。

Western blot 结果显示候选细胞系高水平表达Cas9蛋白；利用慢病毒表达报告基因载体系统检测显示候选细胞系的Cas9蛋白具有很高的切割活力；利用CellTiter-Glo 试剂检测结果显示，Cas9基因在候选细胞系内高表达但不影响细胞增殖活性。

本研究利用慢病毒转导系统构建了稳定表达Cas9蛋白的N2a-Cas9单克隆细胞系，为基于CRISPR/Cas9全基因组高通量筛选技术探究神经细胞内关键宿主基因调控狂犬病病毒嗜神经性提供研究基础。

关键词：基因编辑；慢病毒；高通量筛选；鼠神经瘤母细胞中图分类号：S852.65文献标识码：A文章编号：1008-0589（2020）12-1292-04Construction of N2a-Cas9cell line for genome-wide CRISPR screeningZHANG Ji-wen,WANG Lu-lu,LI Fang,LIU Xing,ZHAO Dong-ming *,BU Zhi-gao *(State Key Laboratory of Veterinary Biotechnology,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150069,China)Abstract :In this study,mouse neuroblastoma (N2a)cells were used to construct N2a-Cas9cell line which stably expresses Cas9protein by using lentivirus transfection technology.This constrcted cells line laid the foundation for CRISPR library screening.First,three plasmids including LentiCas9-Blast,pSPA X2,and pMD 2.G were co-transfected into human renal epithelial cells (HEK293T)to obtain recombinant lentivirus with high titers.N2a cells were infected with the recombinant lentivirus,and then screened by Blasticidin.Finally,several monoclonal cell lines expressing Cas9protein were isolated by limiting dilution.The expression of Cas9expression in N2a-Cas9cell line was determined by western blot,and the high cleavage activity of Cas9was also examined by using lentivirus-based reporter vector system.By using CellTiter-Glo ®reagent,the cell proliferation activity was well detectable and not decreased in N2a-Cas9cell lines,where Cas9gene was expressed in a high level.Therefore,N2a-Cas9cell line was successfully constructed using a lentiviral transfection system,which can provide a basis for genome-wide CRISPR screening to identify crucial host factors regulating Rabies virus neuro-tropism.Key words :gene editing;lentivirus;genome-wide CRISPR screening;murine neuroblastoma收稿日期：2020-02-11基金项目：兽医生物技术国家重点实验室自主课题（SKLVBP201801）作者简介：张纪文（1993-），男，河南驻马店人，硕士研究生，主要从事兽医微生物及其分子生物学研究.*通信作者：E-mail ：****************；*********************Corresponding author张纪文，等.用于CRISPR文库筛选的N2a-Cas9细胞系的构建第12期CRISPR-Cas9（Clustered regularly interspaced short palindromic repeat sequences/CRISPR-associated protein 9）是存在于细菌或古生细菌中的一种抵御外源DNA 侵入的适应性免疫反应系统[1]。

HNIW 的合成研究有着重要意义。

利用核磁共振氢谱（H-NMR ）、质谱(MS)等方法虽然能够定性分析HBIW [10]，但不能很好的定量检测。

已有关于以四氢呋喃-水作为流动相采用液相色谱检测HBIW 的报道[11]，但是HBIW 的保留时间仅为2.83 min ，易于受到其它成分的干扰，需要对该方法进行进一步完善。

另外，长期使用四氢呋喃不仅会腐蚀仪器零部件，而且能通过呼吸道进入人体内引起头晕、呕吐等中毒症状[12-13]。

因此建立一种方便高效、安全可靠的HBIW 检测方法非常有必要。

本研究采用高效液相色谱法，以异丙醇-乙腈为流动相建立了一种准确可靠的HBIW 分析方法，为HNIW合成研究提供技术支持。

图1 HBIW的结构图Fig.1 Structure diagram of HBIW2、材料与方法2.1实验仪器与试剂Agilent1260型高效液相色谱仪（美国安捷伦科技公司），配有SPD-20A 型紫外检测器、LC-20AT 溶液输送单元、DGU-20A5R 脱气单元、CTO-20A 柱温箱、SIL-20A 自动进样器和LabSolutions LC 色谱工作站；0.22 μm 滤膜（天津市津腾实验设备有限公司）。

异丙醇（色谱纯，上海麦克林生化科技有限公司）；乙腈（色谱纯，北京迈瑞达科技有限公司）；正辛醇（分析纯，上海麦克林生化科技有限公司）；HBIW 样品由北京理工大学合成，纯度为99%，所有实验用水均为二次蒸馏水。

2.2色谱分析条件美国安捷伦公司Poroshell 120 EC-C18色谱柱（150 mm ×4.6 mm ，5 μm ）；流动相为异丙醇-乙腈（体积比为60:40）；检测波长为241 nm ；柱温箱温度为30℃；流速为0.3 mL/min ；进样量为10 μL 。

2.3溶液配制六苄基六氮杂异伍兹烷（HBIW ）的储备液：精确称取六苄基六氮杂异伍兹烷（HBIW ）10 mg ，然后用正辛醇溶解，加入到10 mL 容量瓶中并稀释至刻度线，配成1000.0 μg/mL 的储备液。

I.J. Intelligent Systems and Applications, 2014, 02, 76-87Published Online January 2014 in MECS (/)DOI: 10.5815/ijisa.2014.02.10Quality Model and Artificial Intelligence Base Fuel Ratio Management with Applications toAutomotive EngineMojdeh PiranIndustrial Electrical and Electronic Engineering SanatkadeheSabze Pasargad. CO (S.S.P. Co), Shiraz, IranE-mail: SSP.ROBOTIC@Farzin PiltanSenior Researcher at Research and Development Unit, SanatkadeheSabze Pasargad Company, (S.S.P. Co), Shiraz, IranE-mail: Piltan_f@; Mehdi AkbariIndustrial Electrical and Electronic Engineering SanatkadeheSabze Pasargad. CO (S.S.P. Co), Shiraz, IranE-mail: SSP.ROBOTIC@Mansour BazregarIndustrial Electrical and Electronic Engineering SanatkadeheSabze Pasargad. CO (S.S.P. Co), Shiraz, IranE-mail: SSP.ROBOTIC@Abstract—In this research, manage the Internal Combustion (IC) engine modeling and a multi-input-multi-output artificial intelligence baseline chattering free sliding mode methodology scheme is developed with guaranteed stability to simultaneously control fuel ratios to desired levels under various air flow disturbances by regulating the mass flow rates of engine PFI and DI injection systems. Modeling of an entire IC engine is a very important and complicated process because engines are nonlinear, multi inputs-multi outputs and time variant. One purpose of accurate modeling is to save development costs of real engines and minimizing the risks of damaging an engine when validating controller designs. Nevertheless, developing a small model, for specific controller design purposes, can be done and then validated on a larger, more complicated model. Analytical dynamic nonlinear modeling of internal combustion engine is carried out using elegant Euler-Lagrange method compromising accuracy and complexity. A baseline estimator with varying parameter gain is designed with guaranteed stability to allow implementation of the proposed state feedback sliding mode methodology into a MATLAB simulation environment, where the sliding mode strategy is implemented into a model engine control module (“software”). To estimate the dynamic model of IC engine fuzzy inference engine is applied to baseline sliding mode methodology. The fuzzy inference baseline sliding methodology performance was compared with a well-tuned baseline multi-loop PID controller through MATLAB simulations and showed improvements, where MATLAB simulations were conducted to validate the feasibility of utilizing the developed controller and state estimator for automotive engines. The proposed tracking method is designed to optimally track the desired FR by minimizing the error between the trapped in-cylinder mass and the product of the desired FR and fuel mass over a given time interval. Index Terms—IC Engine Modeling, Nonlinear Methodology To Control, Chattering Free Baseline Sliding Mode Methodology, Artificial Intelligence, Sliding Mode Methodology, Baseline Methodology, Fuzzy Inference EngineI.IntroductionInternal combustion engine:Modeling of an entire internal combustion (IC) engine is a very important and complicated process because internal combustion engines are nonlinear, multi inputs-multi outputs (MIMO) and time variant. There have been several engine controller designs over the previous years in which the main goal is to improve the efficiency and exhaust emissions of the automotive engine [1-4]. Specific applications of air to fuel (A/F) ratio control based on observer measurements in the intake manifold were developed by Benninger in 1991 [5]. Another approach was to base the observer on measurements of exhaust gases measured by the oxygen sensor and on the throttle position, which was researched by Onder [6]. These observer ideas used linear observer theory.Hedrick also used the measurements of the oxygen sensor to develop a nonlinear, sliding mode approach to control the A/F ratio [7]. All of the previous control strategies were applied to engines that used only port fuel injections, where fuel was injected in the intake manifold. Current production A/F ratio controllers use closed loop feedback and feed forward control to achieve the desired stoichio metric mixture. These controllers use measurements from the oxygen sensor to control the desired amount of fuel that should be injected over the next engine cycle and have been able to control the A/F very well [8-9].Quality Modeling of IC engine: In developing a valid engine model, the concept of the combustion process, abnormal combustion, and cylinder pressure must be understood. The combustion process is relatively simple and it begins with fuel and air being mixed together in the intake manifold and cylinder. This air-fuel mixture is trapped inside cylinder after the intake valve(s) is closed and then gets compressed [14]. When the air-fuel mixture is compressed it causes the pressure and temperature to increase inside the cylinder. Unlike normal combustion, the cylinder pressure and temperature can rise so rapidly that it can spontaneously ignite the air-fuel mixture causing high frequency cylinder pressure oscillations. These oscillations cause the metal cylinders to produce sharp noises called knock, which it caused to abnormal combustion. The pressure in the cylinder is a very important physical parameter that can be analyzed from the combustion process. After the flame is developed, the cylinder pressure steadily rises, reaches a maximum point after TDC, and finally decreases during the expansion stroke when the cylinder volume increases. Since cylinder pressure is very important to the combustion event and the engine cycle in spark ignition engines, the development of a model that produces the cylinder pressure for each crank angle degree is necessary. Result Management based on Nonlinear Controller Methodology to FR Adjust:A nonlinear robust controller design is major subject in this work. Controller is a device which can sense information from linear or nonlinear system to improve the systems performance [10-13]. The main targets in designing control systems are stability, good disturbance rejection, and small tracking error[14-20]. Several IC engines are controlled by linear methodologies (e.g., Proportional-Derivative (PD) controller, Proportional- Integral (PI) controller or Proportional- Integral-Derivative (PID) controller), but in uncertain dynamic models this technique has limitations [21-45].Result Management Based on Sliding Mode Methodology:Sliding mode methodology (SMM) is a significant nonlinear optimal control fuel ratio methodology under condition of partly uncertain dynamic parameters of system. This controller is used to control of highly nonlinear systems, because this type of optimal method is a robust and stable [46-69]. Conversely, pure sliding mode controller is used in many applications; it has two important drawbacks namely; chattering phenomenon, and nonlinear equivalent dynamic formulation in uncertain dynamic parameter [31-50]. It is possible to solve this problem by combining sliding mode controller and baseline law which this method can helps improve the system’s tracking performance by adjusting controller’s coefficient [51-55].Result management based on Artificial Intelligence:In recent years, artificial intelligence theory has been used in nonlinear systems. Neural network, fuzzy logic and neuro-fuzzy are synergically combined with nonlinear methodology and used in nonlinear, time variant and uncertain plant (e.g., IC engine). Fuzzy logic controller (FLC) is one of the most important applications of fuzzy logic theory. This controller can be used to control nonlinear, uncertain, and noisy systems. This method is free of some model techniques as in model-based controllers. As mentioned that fuzzy logic application is not only limited to the modelling of nonlinear systems [31-36] but also this method can help engineers to design a model-free controller. The main reasons to use fuzzy logic methodology are able to give approximate recommended solution for uncertain and also certain complicated systems to easy understanding and flexible. Fuzzy logic provides a method to design a model-free controller for nonlinear plant with a set of IF-THEN rules [32-69]. The applications of artificial intelligence such as neural networks and fuzzy logic in modelling and control are significantly growing especially in recent years.Objectives: based on previous research IC engine is MIMO, nonlinear and time variant system. One of the most active research areas in field of internal combustion engine (IC engine) is the identification, mathematical modeling and control of the internal combustion engine. Since plant modeling, as accurate as possible, is the fundamental part of any model based control approach, the system is modeled using analytical mathematical method. Sliding mode methodology is one of the robust and stable methods to tune the fuel ratio in IC engine. This method is tuned based on sliding surface slope, to adjust this parameter; based-line methodology is applied to sliding mode method. Artificial intelligence is used to estimate the dynamic model of IC engine. Proposed method is caused to reduce or eliminate the chattering as well as trajectory tracking fuel ratio in switching sliding mode highly stable method.This paper is organized as follows; second part focuses on the modeling dynamic formulation based on Lagrange methodology, fuzzy logic methodology and sliding mode controller to have a robus t control. Third part is focused on the methodology which can be used to reduce the error, increase the performance quality and increase the robustness and stability. Simulation result and discussion is illustrated in forth part which based on trajectory following and disturbance rejection. The last part focuses on the conclusion and compare between this method and the other ones.II.TheoremMathematical Modeling of IC Engine Using Euler Lagrange:In developing a valid engine model, the concept of the combustion process, abnormal combustion and cylinder pressure must be understood. The combustion process is relatively simple and it begins with fuel and air being mixed together in the intake manifold and cylinder. This air-fuel mixture is trapped inside cylinder after the intake valve(s) is closed and then gets compressed [6-9]. When the air-fuel mixture is compressed it causes the pressure and temperature to increase inside the cylinder. In abnormal combustion, the cylinder pressure and temperature can rise so rapidly that it can spontaneously ignite the air-fuel mixture causing high frequency cylinder pressure oscillations. These oscillations cause the metal cylinders to produce sharp noises called knock, which it caused to abnormal combustion. The pressure in the cylinder is a very important physical parameter that can be analyzed from the combustion process. Since cylinder pressure is very important to the combustion event and the engine cycle in spark ignition engines, the development of a model that produces the cylinder pressure for each crank angle degree is necessary.The dynamic equations of IC engine can be written as: *+*+[][][][][][](1)There for to calculate the fuel ratio and equivalence ratio we can write:*+*+,*+{[][][](2)*+[]}-To solve , we can write;*+Where(3)Where is the ratio of the mass of air.Matrix is a matrix:[](4) Matrix engine angular speed matrix()[](5)Where, Matrix mass of air in cylinder for combustionmatrix () is a matrix.[](6)The above target equivalence ratio calculation will becombined with fuel ratio calculation that will be usedfor controller design purpose.Sliding Mode methodology: Consider a nonlinearsingle input dynamic system is defined by [10-17]:( )(⃗)(⃗)(7)Where u is the vector of control input, ( ) is thederivation of, ̇ ̈() is the statevector, ( ) is unknown or uncertainty, and ( ) is ofknown sign function. The main goal to design thiscontroller is train to the desired state;̇ ̈(), and trucking error vector is defined by [18-30]:̃̃̃()(8)A time-varying sliding surface ( ) in the statespace is given by [31-45]:()( )̃(9)where λ is the positive constant. To further penalize tracking error, integral part can be used in sliding surface part as follows [10]:()( )(∫̃)(10) The main target in this methodology is kept the sliding surface slope () near to the zero. Therefore, one of the common strategies is to find input outside of ( )[10].()| ( )|(11) where ζ is positive constant.If S(0)>0()(12) To eliminate the derivative term, it is used an integral term from t=0 to t=∫()∫()()()(13)Where is the time that trajectories reach to the sliding surface so, suppose S ( ) defined as()()()(14)And()()()()()|()|(15)Equation (15) guarantees time to reach the sliding surface is smaller than | ( )|since the trajectories are outside of ( ).()()(16)suppose S is defined as()( )̃( ̇ ̇)()(17)The derivation of S, namely, can be calculated as the following;( ̈ ̈)( ̇ ̇)(18) suppose the second order system is defined as;̈ ̈( ̇ ̇)(19)Where is the dynamic uncertain, and also since, to have the best approximation ,̂ is defined aŝ̂ ̈( ̇ ̇)(20)A simple solution to get the sliding condition when the dynamic parameters have uncertainty is the switching control law:̂(⃗)()(21)where the switching function ( ) is defined as [11,16](){(22)and the (⃗) is the positive constant. Suppose by (11) the following equation can be written as,()[̂ ( )](̂)||(23)and if the equation (15) instead of (14) the sliding surface can be calculated as()( )(∫̃)( ̇ ̇)( ̇ ̇)()(24)in this method the approximation of is computed as [26]̂̂ ̈( ̇ ̇)()(25)Baseline methodology: The design of a baseline controller to control the fuel ratio was very straight forward. Since there was an output from the fuel ratio model, this means that there would be two inputs into the baseline controller. Similarly, the output of the controller result from the two control inputs of the port fuel injector signal and direct injector signal. In a typical PID controller, the controller corrects the error between the desired output value and the measured value. Since the equivalence ratio and fuel ratio are the two measured signals, two controllers were cascaded together to control the PFI and DI inputs. The first was a PID controller that corrected the error between the desired equivalence ratio and the measured equivalence ratio; while the second was only a proportional integral (PI) controller that corrected the fuel ratio error.()()()(26)()()()(27)∑(28)∑(29)( ∑)(30)(31)III.MethodologyBaseline Sliding Mode Controller Methodology: The design of a baseline controller to control the sliding surface slope was very straight forward. Since there was an output from the sliding surface slope model, this means that there would be two inputs into the baseline controller. Similarly, the output of the controller result from the control input of the sliding surface slope. In a typical PID controller, the controller corrects the error between the desired output value and the measured value. Since the sliding surface slope is the measured signal, two controllers were cascaded together to control the sliding surface slope. The first was a PID method that corrected the error between the desired FR and the measured FR; while the second was only a proportional integral (PI) method that corrected the sliding surface, error and integral of error.()()()(32)()∑(33) ( ()∑ )(34)( ()∑ )(()∑ )(35)Artificial intelligence (Fuzzy) Based Management Baseline Sliding Mode Methodology: this part is focused on design SISO fuzzy estimation baseline sliding mode methodology for system’s managment based on Lyapunov formulation. The firs type of fuzzy systems is given by()∑() ( )(36) Where( )()( ()())()∏()∑(∏( )) are adjustable parameters in (23) . ()( ) are given membership functions whose parameters will not change over time.The second type of fuzzy systems is given by()∑*∏(())+∑*∏(())+(37)Where are all adjustable parameters. From the universal approximation theorem, we know that we can find a fuzzy system to estimate any continuous function. For the first type of fuzzy systems, we can only adjust in (23). We define ( | )as the approximator of the real function ( ).(|) ( )(38) We define as the values for the minimum error:[|(|) ( )|](39)Where is a constraint set for . For specific | (|)()| is the minimum approximation error we can get.We used the first type of fuzzy systems (23) to estimate the nonlinear system (11) the fuzzy formulation can be write as below;( | ) ( )∑[()]∑()(40)Where are adjusted by an adaptation law. The adaptation law is designed to minimize the parameter errors of . A MIMO (multi-input multi-output) fuzzy system is designed to compensate the uncertainties of the nonlinear system. The parameters of the fuzzy system are adjusted by adaptation laws. The tracking error and the sliding surface state are defined as:(41)(42) We define the reference state as(43)̇(44)The general MIMO if-then rules are given by(45) Where are fuzzy if-then rules; ( ) and ( ) are the input and output vectors of the fuzzy system. The MIMO fuzzy system is define as() ( )(46) Where( )[](47)()( ()())()∏( )∑(∏( )) and ( ) is defined in (40). To reduce the number of fuzzy rules, we divide the fuzzy system in to three parts:()()*()()+(48)()()*()()+(49)()()*()()+(50) The control input is given by,{[][][]*+[]}-()()()( )(51)and are positive constants. The adaptation law is given by()()()(52)Where and are positive diagonal matrices.The Lyapunov function candidate is presented as∑∑∑(53)Where andwe define()()()( )(54) From (29) and (30), we get()()()()()̂()( )(55) Since ̇ ̇ and ̈ ̈ ̇ , we geṫ(()())()()(56) Then ̇()() can be written aṡ()()() ( )(57)Where ̃ ̈()()̃̃()()()()̃The derivative of iṡ∑∑∑(58)We know that ̇( ̇()() ) from (45). Then()()∑∑∑(59) We define the minimum approximation error as[(|)(|)(|)](60) We plug (49) in to (50)()() ∑∑∑[()(|) (|)(|)()()( )]∑∑∑()∑()∑()∑()∑∑∑()∑( ())∑( ())∑( ())()∑( ())∑( ())∑( ())Then becomes()∑( ||)∑( )|| ](61)Since can be as small as possible, we can find that | |||()Therefore, we can get () forand () Figure 3 shows the fuzzy estimator variable structure.IV.Results and DiscussionThis section is focused on compare between baseline methodology to FR optimization (BLO) and artificial intelligence (fuzzy) baseline Sliding Mode methodology (FBSMM).These controllers were tested by MATLAB/SIMULINK environment.FR trackingManaging:Based on equations in sliding mode methodology; this method performance is depended on the gain updating factor () and sliding surface slope coefficient (). These two coefficients are computed by gradient descent optimization in pure SMM. After this process the main important challenge is, optimizer is depending on sliding surface slope coefficients so baseline method is apply. Figure 1 shows FR tracking performance in BLO and FBSMM without torque load disturbance.Fig. 1: FBSMM and BLO for adjust FR without torque load disturbance Fig. 2: FBSMM and BLO for adjust FR with 10% torque load external disturbanceBased on Figure 1 it is observed that, BLO has oscillation in this nonlinear system, caused to instability in tuning the FR but FBSMM has steady in response. BLO’s overshoot is 1.3% but BSMM’s overshoot is 0%.Managing the disturbance rejection: Figures 2 and 3 are show the power disturbance elimination in BLO and FBSMM with torque load disturbance. The manage disturbance rejection is used to test the robustness comparisons of these two methodologies. Avariablelimited torque load with predefined of 10% and 20% the power of input signal value is applied to this system. Based on Figure 2; by comparing FR trajectory with 10% torque load disturbance of relative to the input signal amplitude in BSMM and BLO, BSMM’s overshoot about (0%) is lower than BLO’s (2.3%) . Based on Figure 2, it is observed that FBSMM’s performance is better than BLO and it also can eliminate the chattering in presence of 10% disturbance.Fig. 3: FBSMM and BLO for adjust FR with 20% torque load external disturbanceBased on Figure 3; by comparing FR trajectory with 20% disturbance of relative to the input signal amplitude in FBSMM and BLO, FBSMM’s overshoot about (0.32%)is lower than BLO’s (3.11%). Based on Figure 3 it is observed that, these two methodologies have oscillation in presence of 20% torque load uncertainty but FBSMM is more stable and more robust than BLO.V.ConclusionRefer to this research, IC engine modeling based on Lagrange and an artificial intelligence based manage baseline sliding mode methodology is proposed for tuning the FR in internal combustion engine based on nonlinear methodology. Manage the stability and convergence of the fuzzy baseline sliding mode controller based on switching function is guarantee and proved by the LYAPUNOV method. The simulation results exhibit that the fuzzy baseline sliding mode methodology tuning the FR very well in various situations. The FBSMM gives significant steady state error managing performance when compared to BLO. The main goal in this research were modeling and manage or eliminate the chattering that proposed methodology reach to this objective by adding baseline and fuzzy method to high impact robust and stable nonlinear based methodology. The second main goal in this research was reduce dependence on sliding surface slope which baseline methodology does thi s objective in this research. When applied 20% torque load disturbances in FBSMM the RMS error increased approximately 0.1% (percent of increase the BSMM RMS error=( ) that in BLO the RMS error increased approximately 8% (percent of increase the BLO RMS error=( ). AcknowledgmentThe authors would like to thank the anonymous reviewers for their careful reading of this paper. This work was supported by the SSP Research and Development Corporation Program of Iran under grant no. 2012-Persian Gulf-2B.Reference[1]Heywood, J., “Internal Combustion EngineFundamentals”, McGraw-Hill, New York, 1988. [2]J. G. 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Jalali,“Colonial Competiti ve Optimization Sliding Mode Controller with Application to Robot Manipulator”, International Journal of Intelligent Systems andApplications, 5(7), 2013.[21]Farzin Piltan, Amin Jalali, N. Sulaiman, AtefehGavahian & Sobhan Siamak, “Novel Artificial Control of Nonlinear Uncertain System: Design a Novel Modified PSO SISO Lyapunov Based Fuzzy Sliding Mode Algorithm”, International Journal of Robotics and Automation, 2 (5): 298-316, 2011.[22]Farzin Piltan, N. Sulaiman, Iraj Asadi Talooki &Payman Ferdosali, “Control of IC Engine: Designa Novel MIMO Fuzzy Backstepping AdaptiveBased Fuzzy Estimator Variable Structure Control”, International Journal of Robotics and Automation, 2 (5):360-380, 2011.[23]Farzin Piltan, N. Sulaiman, S.Soltani, M. H.Marhaban & R. Ramli, “An A daptive Sliding Surface Slope Adjustment in PD Sliding ModeFuzzy Control For Robot Manipulator”, International Journal of Control and Automation, 4(3): 65-76, 2011.[24]Farzin Piltan, N. 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星星草脂氧合酶基因家族鉴定及生物信息学分析冯慧婷李跃跃齐家兴李莹*（东北林业大学生命科学学院/东北盐碱植被恢复与重建教育部重点实验室，黑龙江哈尔滨150040）摘要星星草具有较强的耐盐碱特性，是改良土壤盐碱化的优良牧草。

脂氧合酶（lipoxygenase，LOXs）广泛存在于动植物中，催化多不饱和脂肪酸的双加氧反应，最终生成各种氧脂素，从而在植物生长发育、响应逆境胁迫中发挥重要的生物学功能。

目前，星星草中的LOXs家族基因仍未见报道。

本研究利用生物信息学方法对PutLOXs基因家族成员进行鉴定，并对其理化性质、亚细胞定位和系统进化进行了分析。

结果表明，星星草基因组中共有8个LOX基因，它们均具有lipoxygenase homology2（简称LH2）和lipoxygenase结构域，均不包含跨膜结构域，预测定位于细胞质或叶绿体。

系统发生分析表明PutLOXs蛋白分为9-LOX和13-LOX两个亚家族，并且与水稻和玉米这类单子叶植物的LOXs蛋白系统发生关系更近。

本研究将为后期深入分析星星草耐盐碱分子机制提供理论依据。

关键词脂氧合酶；基因家族；星星草；生物信息学分析中图分类号S562文献标识码A文章编号1007-5739（2024）03-0157-05DOI：10.3969/j.issn.1007-5739.2024.03.036开放科学（资源服务）标识码（OSID）：Identification and Bioinformatics Analysis of Lipoxygenase Gene Family inPuccinellia tenuifloraFENG Huiting LI Yueyue QI Jiaxing LI Ying*(School of Life Sciences,Northeast Forestry University/Key Laboratory of Northeast Saline-alkali Vegetation Restoration and Reconstruction,Ministry of Education,Harbin Heilongjiang150040) Abstract Puccinellia tenuiflora has strong salt-alkali resistance characteristics and is an excellent forage for improving soil salinization.Lipoxygenase(LOXs)is widely present in plants and animals,which catalyzes the double oxy-genation reaction of polyunsaturated fatty acids,ultimately generates various oxylipins,thereby plays an important biological role in plant growth and development and response to stress.At present,the LOXs family genes in Puccinellia tenuiflora have not been reported.This paper utilized bioinformatics methods to identify members of the PutLOXs gene family numbers and analyzed their physicochemical properties,subcellular localization,and phylogenetic evolution.The results showed that there were a total of8LOX genes in the genome of Puccinellia tenuiflora,all of which had lipoxy-genase homology2(LH2)and lipoxygenase domains,but did not contain transmembrane domains.They were predicted to be located in the cytoplasm or chloroplast.Phylogenetic analysis showed that PutLOXs proteins were divided into two subfamilies(9-LOX and13-LOX),and had a closer phylogenetic relationship with LOXs proteins in monocotyledonous plants such as rice and corn.This study will provide a theoretical basis for further analysis of the molecular mechanism of salt-alkali tolerance in Puccinellia tenuiflora.Keywords lipoxygenase(LOXs);gene family;Puccinellia tenuiflora;bioinformatics analysis脂氧合酶（lipoxygenase，LOXs）广泛分布于动、植物中，是一种具有非血红素铁作为活性中心的氧化还原酶[1]，它能够催化具有顺，顺-1，4戊二烯结构的多不饱和脂肪酸的双加氧反应，最终生成各种氧脂素（oxylipin），这个过程被称为脂氧合酶途径[1-3]。

I.J.Mathematical Sciences and Computing,2018, 2, 12-21Published Online April 2018 in MECS ()DOI: 10.5815/ijmsc.2018.02.02Available online at /ijmscA Systematic Expository Review of Schmidt-Samoa CryptosystemQasem Abu Al-Haija a*, Mohamad M.Asad b, Ibrahim Marouf a,b, a,b c Department of Electrical Engineering, King Faisal University, Hufof 31982, Saudi Arabia Received: 21 November 2017; Accepted: 13 February 2018; Published: 08 April 2018AbstractPublic key cryptographic schemes are vastly used to ensure confidentiality, integrity, authentication and non-repudiation. Schmidt-Samoa cryptosystem (SSC) is a public key cryptosystem, which depends on the difficulty of large integer factorization problem. The implementation of SSC to secure different recent communication technologies such as cloud and fog computing is on demand due to the assorted security services offered by SSC such as data encryption/decryption, digital signature and data integrity. In this paper, we provide a systematic review of SSC public key cryptosystem to help crypto-designers to implement SSC efficiently and adopt it in hardware or software-based applications. According to the literature, the effective utilization and design SSC can place it as a viable alternative of RSA cryptosystems and many others.Index Terms: Information Security, Public Key Cryptography, Schmidt-Samoa Cryptosystem, Integer Factorization.© 2018 Published by MECS Publisher. Selection and/or peer review under responsibility of the Research Association of Modern Education and Computer Science1.IntroductionIn the last decades, the communication system over the world has been extremely enlarged where millions of computers were connected to networks and internet to exchange a huge amount of information. This information is vulnerable to interrupt, change, or even seen by unwanted people (i.e. unauthorized). Because of that, secure communication channels were introduced to prevent any third party from reading or changing information. Such prevention is accomplished by setting rules for accessing the confidential data known collectively as Cryptography. Cryptography is the science that concern with encrypting and decrypting data to provide secure transactions between communication parties. Cryptography provides the secure communication networks by a means of cryptographic primitives [1] (listed in table 1) which contributed along with the crypto-* Corresponding author. Tel.: +966-13-589-5400; fax: +966-13-581-7068E-mail address: Qalhaija@.saalgorithms to provide many services such as: confidentiality: To help protect a user's identity or data from being read, data integrity: To help protect data from being changed, authentication: To ensure that data is originated from a certain user, and non-repudiation: To prevent a certain party from being denied of sending messages [1].Table 1. Cryptographic Primitive and Their UseCryptographic primitive UseSecret-key encryption (symmetric cryptography) Performs a transformation on data to keep it from being read by third parties. This type of encryption uses a single shared, secret key to encrypt and decrypt data.Public-key encryption (asymmetric cryptography) Performs a transformation on data to keep it from being read by third parties. This type of encryption uses a public/private key pair to encrypt and decrypt data.Cryptographic signing (Digital Signatures) Helps verify that data originates from a specific party by creating a digital signature that is unique to that party. This process also uses hash functions.Cryptographic hashes (Fixed Size Digesting) Maps data from any length to a fixed-length byte sequence. Hashes are statistically unique; a different two-byte sequence will not hash to the same value.Based on encryption/decryption process, cryptographic algorithms are categorized as Symmetric key algorithms and Public key algorithms (Asymmetric key). Symmetric Key Cryptography (SKC) is a field of cryptography where the same key is shared between both sender and receiver to be used for encryption and decryption processes. SKC ciphers can either be stream cipher which encrypt and decrypts data as bit-by-bit process using bit operations (such as XOR) or block cipher which deals with blocks of fixed length of bits encrypted/decrypted with a key. An examples of stream cipher is LFSR encryption [2] and examples of block cipher are DES, 3DES, Blowfish, and AES. Modern symmetric algorithms such as AES or 3DES are very secure. However, there are several drawbacks associated with symmetric-key scheme like key distribution problem, number of keys or the lack of protection against cheating [3]. In symmetric key algorithms, the key must be established in a secure channel which does not exist in communication channels. Even if this problem solved, substantial number of keys will be needed when each pair needs a separate key in a network. Moreover, any party can cheat and accuse the other party. Hence, asymmetric key algorithms are needed to solve these problems.Public Key Cryptography (PKC) where the two parties (sender and receiver) have two different keys; one public shared key for encryption and one private key for decryption. Public-key algorithms are used mainly for Key Establishment, Identification and Encryption. Diffie-Hellman Key Exchange (DHKE) [4] is an example of an asymmetric key algorithm used for key exchange and RSA is an encryption public-key algorithm [5]. PKC algorithms are fundamental security component in many cryptosystems, applications and such as Transport Layer Security (TLS) protocol [6]. Public key algorithms provide data encryption, key exchange, and digital signatures [7].PCK algorithms can be categorized based on the mathematical problem used in the scheme into [4]: Integer-factorization based schemes such RSA and McEliece [8] algorithms and discrete logarithm-based schemes such as Diffie–Hellman key exchange and ELGamal encryption scheme [4]. Integer factorization is the process where an integer is decomposed to the product of smaller numbers. If these numbers are prime numbers, then it is called prime factorization. The complexity in this method arises when factoring a very large number because there no such known efficient algorithm. However, not all number with the same length are equal in complexity. When the number is the product of two coprime numbers, it is infeasible to factor this kind of numbers using the current technology [9]. Most non-RSA public-key algorithms with practical relevance are based on another one-way function, the discrete logarithm problem [3]. The security of many cryptographic schemes relies on the computational intractability of finding solutions to the Discrete Logarithm Problem (DLP). The discrete logarithm problem is defined in what are called cyclic groups. However, there are four families of alternative public key schemes [10] that are potentially interesting for use in practice: hash based, code-based, lattice-based and multivariate quadratic (MQ) public-key algorithms.Practically, public key schemes are preferred to use due to many reasons such as the non-exitance of thesecure communication channels. Therefore, the efficient implementation of public key cryptosystems is on demand especially if its implemented with appropriate technology with high precision design. In this paper, Schmidt-Samoa Cryptosystem (SSC) [11] will be used analyzed as efficient and comparable alternative to RSA which is a well-known secure and practicable public key scheme that can be used to protect information during the transmission over the insecure channels. SSC Cryptosystem is heavily based on modular arithmetic involving large prime numbers.The remaining of this paper is organized as follows: Section 2 discusses the Schmidt-Samoa Cryptosystem (SSC) in details including SSC crypto-algorithm, the SSC factoring, numerical example of how SSC works, some possible attacks of SSC, and the underlying design issues and requirements followed by conclusions.2.Schmidt-Samoa Cryptosystem (SSC)Schmidt-Samoa Cryptosystem (SSC) is an asymmetric cryptographic technique (public key algorithm) in which security depends on the difficulty of integer factorization problem used for data encryption and decryption. Just like RSA, SSC uses very large prime numbers and modular arithmetic to provide different security services such as conditionality, integrity, and non-repudiation.2.1.SSC AlgorithmTo start the secure communication session, the receiver, who is Alice in this case, starts by choosing two large prime numbers (p, q) and then compute her public key 2=. Alice then share the public key (N) withN p qBob (and even other senders) who will use it to encrypt the plaintext messages communicated with Alice. Again, Alice computes her private key (d) to be used for decryption processes 1=. Next, using the privated N-key, Alice decrypts the ciphertext.Fig.1. Complete Diagram of Schmidt-Samoa Algorithm.Fig.1, shows the complete SSC algorithm diagram which is divided into three stages: key generation stage, Encryption stage, and Decryption stage. The challenge in SSC is the ability to factor out the public key which is the product of two very large primes. As the size of the key is increases, the factorization problem becomes even more complicated [9]. Factoring a number means defining that number as a product of prime numbers. InSSC, factoring the public key (N ) means as breaking the cryptosystem. If an attacker can factor out the public key, he can easily calculate the private key (d ) and decrypt any data. As public key 2N p q =, is known to everyone, therefore factoring (N ) leads to compute p and q . Then the private key can be computed using congruent (1) (where LCM is the least common multiple of two numbers):1mod (1,1)d N LCM p q -≡-- (1) For better understanding, we provide the following simplified numerical example. Let’s assume that the plaintext message m = 2 and the domain parameters (p = 11, q = 17, m = 2), then we run SSC (11,17,2) as follows:22057N p q ==11mod (10,16)2057mod8073d N LCM --≡==2057mod 20571855c m ==731855mod1872m ==2.2. Possible Attacks of SSCReasonably, there is no such a perfect system, but there are systems hard to be attacked. SSC is proved to be very secure [11], however, it is vulnerable to some known attacks such as Brute-force attack, Man-in-the-Middle attack, and Side Channel attack. Generally, all public key cryptography algorithms suffer from these attacks [3].∙ Exhaustive search of SSC: In computer science, brute-force search or exhaustive search, also known asgenerate and test, is a very general problem-solving technique that consists of systematically generating all possible candidates for the solution and checking whether each candidate satisfies the problem's statement. For instance, finding the factorization of a very large number by trying all the numbers less than the asked number. In cryptography, an exhaustive search attack involves checking all possible keys until the correct key is found [12]. This strategy theoretically can be used against any cryptosystem by an attacker who is unable to take advantage of any weakness in the system that would make breaking the system easier. The length of the used key in the encryption process determines the practical feasibility of performing a brute force attack, with larger keys exponentially more difficult to break than smaller ones. One of the measures of the strength of an encryption system is how long it would theoretically take an attacker to successfully mount a brute force attack against it. In Schmidt-Samoa cryptosystem, as the bit size of the key is increased, the time needed to perform an exhaustive search would increase exponentially. It is believed that a 1024-bit key can be factored in period of 10-15 years, where it is possible for some intelligence agencies to compute the key earlier [12]. However, for 2048- bit or more, it is not feasible to factor out SSC key relying on the current technology (computers). Sample example of exhaustive search algorithm (brute force) is illustrated in figure 2 as it shows the possible trial values of simple 4-bit key.Fig.2. Example of Brute Force Attack of 4 bit KeyMan-in-the-Middle Attack [13]: it is a type of cyberattack where a malicious actor inserts him/herself into a conversation between two parties, impersonates both parties and gains access to information that the two parties were trying to send to each other. It allows a malicious actor to intercept, send and receive data meant for someone else, or not meant to be sent at all, without either outside party knowing until it is too late. Man-in-the-middle attacks can be abbreviated in many ways, including MITM, MitM, MiM or MIM. An example of MITM by using SSC scheme is shown in Fig.3 where Alice generates her public and private keys and sends the public key over unsecure channel. However, Trudy interrupts the communication and generates new public key then sends it to Bob. Bob now encrypts data and sends it back to Alice on the unsecure channel, however, only Trudy who can decrypt the message. Trudy can generate new false message and send it to Alice, pass the original message, or just block it where Alice and Bob thinking they are communicating with each other securely.a = K prAA = αa mod(n) = K pubAb = K prBB = αb mod(n) = K pubBA’ = αT1 mod(n)B’ = αT2 mod(n)A BK AT = (B’)a = (αT2)amod(n)K BT = (A’)a = (αT1)bmod(n)A’B’K AT = A T2 = (αa)T2 mod(n)K BT = B T1 = (αb)T1 mod(n) Fig.3. MITM Attack Scheme for SSC.Side Channel Attack: In cryptography, a side-channel attack is an attack based on analyzing the physical implementation gained information of a cryptosystem, rather than a brute-force of any theoretical weakness [12]. They exploit information about the private key which is leaked through physical channels such as the power consumption or the timing behavior. However, to observes such channels, an attacker must have access to the cipher implementation, e.g., in cell phones or smart card. Fig.4 shows the power trace of an RSA implementation on a microprocessor [12], or the drown electric power by the processor to be more precise. The attacker goal is to extract the private key d which is used during the RSA decryption. It can be differentiated between the high and low activity from the graph, this behavior is explained by the square-and-multiply algorithm. If an exponent bit has the value 0, only a squaring is per formed. If an exponent bit has the value 1, a squaring together with a multiplication is computed.Fig.4. The Power Trace of an RSA Implementation.2.3.SSC ServicesSSC is very flexible and can provide the four main cryptographic services: confidentiality, integrity, authentication, and non-repudiation. As for RSA algorithm, SSC algorithm can be used to encrypt and decrypt private message providing, confidentiality and non-repudiation. Also, SSC can be implemented to be used as digital signature (DSA-SSC) as shown in Fig.5, providing integrity. PKI and alternative schemes; hashed-based, coded-based, etc., can be implemented using SSC.2.4. several digital arithmetic and modular arithmetic algorithms as well as different number theory schemes. It employs the properties of prime numbers alongside the congruent to produce a very secure hard to break cryptosystem. Arithmetic operation like multiplication and squaring, and modular exponentiation and modular inverse are involved in the algorithm to add complexity to the cipher. Thus, implementing a SSC coprocessor requires the contribution of many design components as seen in the diagram of figure 6.Fig.6. SSC Underlying Design Requirements Diagram.Number Theory Algorithms: Because of the modular factors (p, q) must be prime, therefore, twocomponents are contributing here generate test a prime number with desired length: a random number generator (RNG) [2] and a prime number tester PNT) [14]. Also, to test the co-prime relativity, a greatest common devisor (GCD) unit [15] is required in Schmidt-Samoa. In addition, to generate the private key modulus, a Least common multiple (LCM) [15] unit is needed.∙Digital Arithmetic Algorithms: in order to compute the public key (N) which is also used as the encryption algorithm modulus, efficient arithmetic digital multiplier (used for squaring as well) unit is required to generate N, such as Karatsuba multiplier [16]. The multiplier is built from fast two operand adder units such as Kogge Stone adder (KSA) [17] as an efficient Parallel prefix adder [18], fast three operand adder such as Carry save adder [18] and multi-operand addition trees such as Wallace trees [18]. ∙Modular Arithmetic Algorithms: As for SSC encryption and decryption processes, an efficient modular expatiation such as [19] should be carefully selected as this operation consumes most of the time in the SSC system. Similarly, another costly operation is needed in the generation of decryption key which is the modular inverse (division by modulus) operation [9] which is well known to be one of the long-time operations performed by the Cryptoprocessor.∙Hardware/Software design tools: SSC Cryptoprocessor can be implemented either in software environment or in hardware platform. However, it’s noted that building Cryptoprocessor via hardware is more secure and efficient than in software [20]. Nowadays, reconfigurable hardware devices are commonly spread to implement various digital applications such as cryptographic coprocessor and embedded systems design. It’s largely recommended to implement SSC using the field programmable gate arrays (FPGA) [21] which provide wide range of flexibility and dynamic control of several design factors such as delay, area and power consumption. The reconfigurability feature of FPGA devices attracted many cryptographic researchers to implement their designs using FPGA devices benefiting from the spacious libraries and modules offered by Computer Aided Design (CAD) [22] tools as well as the flexibility of Hardware description languages (HDLs) [23].Eventually, the adequate adoption of the efficient accelerated built-in units and component along with affordable high technology design platform will result in undoubtedly robust SSC cryptosystem that is comparable and competitive with RSA and many other well-known secure cryptosystems. It can replace RSA Cryptosystem in many applications such as in design of the cryptography system with multi-level crypto-algorithms [24], in the design an effective parallel digital signature algorithm for GPUs [25], in the design of robust image Steganography [26], in the design of an alternative equations for Guillou-Quisquater Signature scheme which is based originally on RSA [27], or many other valid applications.3.Conclusions and RemarksSchmidt-Samoa cryptosystem public key cryptosystem (SSC) with numerical example and sample possible attacks as well as the cryptosystem's design issues has been methodologically analysed and investigated in this paper. Thus, even if you use the best possible random number generators to create candidates for the primes that are needed to make SSC secure, the security of SSC encryption/decryption depends critically on the difficulty of factoring large integers which become easier for shorter key sizes due the existence of powerful computers. Therefore, SSC cryptography has had to rely on increasingly larger values for the integer modulus and, Hence increasingly longer encryption keys. As for RSA, these days you are unlikely to use a key whose length is shorter than 1024 bits for SSC as many people recommended to use 2048 or even 4096-bit keys. References[1]Denning, D.E.R.E, “Cryptography and data security”, Reading, MA: Addison-Welsey.[2]Q. A. Al-Haija, N. A. Jebril, and A. AlShua'ibi. (2015). Implementing variable length Pseudo RandomNumber Generator (PRNG) with fixed high frequency (1.44 GHZ) via Vertix-7 FPGA family. Network Security and Communication Engineering, CRC press, Pp. 105 -108.[3] C. Paar, J. Pelzl, (2010) ‘Understanding Cryptography’. Springer-Verlag Berlin Heidelberg Publisher.https:///10.1007/978-3-642-04101-3.[4]Menezes, A.J., van Oorschot, P.C. and Vanstone, S.A., (1996), 'Handbook of applied cryptography',CRC Press, http://cacr.uwaterloo.ca/hac/[5]Q. Abu Al-Haija, et. al, (2014) 'Efficient FPGA Implementation of RSA Coprocessor using ScalableModules', 9th International Conference on Future Networks & Communications (FNC), Elsevier, Canada. https:///10.1016/j.procs.2014.07.092[6]Dierks and Rescorla, (2008), Standards Track: The Transport Layer Security (TLS) Protocol Version1.2', The IETF Trust, RFC 5246.[7]Developer Network (2017). 'Cryptographic Services', Microsoft. https:///en-us/dotnet/standard/security/[8]H. Sun. Enhancing the Security of the McEliece Public-Key Cryptosystem. Journal of InformationScience and Engineering 16, pages 799-812, 2000.[9]W. Trappe and L. C. Washington, (2002) 'Introduction to Cryptography with Coding Theory', PrenticeHall, vol. 1: p.p. 1-176, /citation.cfm?id=560133[10]Daniel J. Bernstein, Johannes Buchmann, Erik Dahmen, (2009), 'Post-Quantum Cryptography',Springer-Verlag Berlin Heidelberg, DOI: 10.1007/978-3-540-88702-7[11]Katja Schmidt-Samoa, (2006) ‘A New Rabin-type Trapdoor Permutation Equivalent to Factoring’,Electronic Notes in Theoretical Computer Science, Elsevier, vol.157, issue 3, p.p.79-94.https:///2005/278.pdf[12]Mark Burnett, (2007), ‘Blocking Brute Force Attacks', UVA Computer Science, University of Virginia(UVA). /~csadmin/gen_support/brute_force.php[13]Desmedt, Y. Man in the middle attack. In: van Tilborg, H.C.A. (ed.) Encyclopedia of Cryptography andSecurity, p. 368. Springer, Heidelberg (2005) Xx[14]M. M. Asad, I. Marouf, Q. Abu Al-Haija, " Investigation Study of Feasible Prime Number TestingAlgorithms", Acta Technica Napocensis Electronics and Telecommunications, 58 (3), Pp. 11– 15, 2017 [15]I. Marouf, M. M. Asad, Q. Abu Al-Haija, " Reviewing and Analyzing Efficient GCD/LCM Algorithmsfor Cryptographic Design", International Journal of New Computer Architectures and their Applications (IJNCAA), By Society of Digital Information and Wireless Communications (SDIWC), 7(1), Pp. 1-7, 2017.[16]M. M. Asad, I. Marouf, Q. Abu Al-Haija, Qasem Abu Al-Haija, " Review of Fast MultiplicationAlgorithms for Embedded Systems Design ", International Journal of Scientific & Technology Research (IJSTR), 6 (8), Pp., 238 – 242, 2017.[17]Kogge, P. & Stone, H. "A Parallel Algorithm for the Efficient Solution of a General Class of RecurrenceEquations". IEEE Transactions on Computers, 1973, C-22, 783-791Xx[18]M. D. Ercegovac and T. Lang, “Digital Arithmetic," Morgan Kaufmann Publishers, Elsevier, Vol1, Ch2,pages (51-136), 2004.[19]I. Marouf, M. M. Asad, Q. Abu Al-Haija, "Comparative Study of Efficient Modular ExponentiationAlgorithms", COMPUSOFT, An international journal of advanced computer technology, 6 (8), Pp.2381– 2389, 2017[20]L. Tawalbeh and Q. Abu Al-Haija," Enhanced FPGA Implementations for Doubling Oriented andJacobi-Quartics Elliptic Curves Cryptography,” Journal of Information Assurance and Security (JIAS), By Dynamic Publishers Inc., Vol 6 (3), Pp. 167-175, 2010[21] C. Maxfield, " The Design Warrior’s Guide to FPGAs: Devices, Tools and Flows", Mentor GraphicsCorporation and Xilinx, Elsevier, 2004.[22]Nicos Bilalis, (2000), 'Computer Aided Design CAD', INNOREGIO Project: dissemination ofinnovation and knowledge management techniques, Technical University of Crete.[23]David Harris Sarah Harris, (2012), ‘Digital Design and Computer Architecture’, Imprint: MorganKaufmann, ISBN: 9780123944245, Elsevier.[24]Surinder Kaur, Pooja Bharadwaj, Shivani Mankotia,"Study of Multi-Level Cryptography Algorithm:Multi-Prime RSA and DES", International Journal of Computer Network and Information Security(IJCNIS), Vol.9, No.9, pp.22-29, 2017.DOI: 10.5815/ijcnis.2017.09.03.[25]Sapna Saxena, Neha Kishore," PRDSA: Effective Parallel Digital Signature Algorithm for GPUs ",International Journal of Wireless and Microwave Technologies(IJWMT), Vol.7, No.5, pp. 14-21, 2017.DOI: 10.5815/ijwmt.2017.05.02.[26]M.I.Khalil,"Medical Image Steganography: Study of Medical Image Quality Degradation whenEmbedding Data in the Frequency Domain", International Journal of Computer Network and Information Security(IJCNIS), Vol.9, No.2, pp.22-28, 2017.DOI: 10.5815/ijcnis.2017.02.03[27]J. Ettanfouhi, O. Khadir," Alternative Equations for Guillou-Quisquater Signature Scheme ",International Journal of Computer Network and Information Security, 2016, 9, 27-33, DOI:10.5815/ijcnis.2016.09.04/Authors’ ProfilesQasem Abu Al-Haija is a senior lecturer of Electrical and Computer Engineering at KingFaisal University. Eng. Abu Al-Haija received his B.Sc. in ECE from Mu’tah University inFeb-2005 and M.Sc. in computer engineering from Jordan University of Science &Technology in Dec-2009. His current research Interests: Information Security &Cryptography, Coprocessor & FPGA design, Computer Arithmetic, Wireless SensorNetworks.Muhammad M. Asad is a senior student of Electrical Engineering Department at KingFaisal University. He is a Syrian resident born on Jan-01-1994 and excellent in bothlanguages Arabic and English. His research interests include (but not limited to): PublicKey Cryptography, FPGA Design, Digital Arithmetic, Microcontroller Design, ElectronicDesign.Ibrahim A. Marouf is a senior student of Electrical Engineering Department at KingFaisal University. He is a Syrian resident born on Aug -15-1995 and excellent in bothlanguages Arabic and English. His research interests include (but not limited to): PublicKey Cryptography, FPGA Design, Digital Arithmetic, Microcontroller Design, ElectronicDesign.How to cite this paper: Qasem Abu Al-Haija, Mohamad M.Asad, Ibrahim Marouf,"A Systematic Expository Review of Schmidt-Samoa Cryptosystem", International Journal of Mathematical Sciences and Computing(IJMSC), Vol.4, No.2, pp.12-21, 2018.DOI: 10.5815/ijmsc.2018.02.02。

I.J. Intelligent Systems and Applications, 2012, 4, 33-39Published Online April 2012 in MECS (/)DOI: 10.5815/ijisa.2012.04.05Analysis towards Mobile IPV4 and Mobile IPV6in Computer NetworksSeyedeh Masoumeh AhmadiSchool of Information Technology, Guilan University, Irans_m_a57@Abstract—With the rapid growth in the number of mobile devices like cellular phones, personal digital assistants (PDAs), and laptop computers, the demand for “anywhere, anytime, and any way” high-speed Internet access is becoming a primary concern in our lives. Mobile IP has been designed with the Internet Engineering Task Force (IETF) to serve the needs of growing population of mobile computer users who wish to connect to the internet and maintain communication as they move from place to place. Mobile IP enables a wireless network node to move freely from one point of connection to the Internet to another, without disrupting the end-to-end connectivity. The goals of this paper are to define the fundamentals of mobile IPV4, elaborate the problems of mobile IPV4, give a brief overview of some of the literature that deals with the fundamentals of mobile IPV6, explain the problems of mobile IPV6, compare Mobile IPv6 and Mobile IPv4, discuss the advantages of mobile IP, and review the application of mobile IP for vehicular networks.Index Terms—fundamentals, mobile IPV4, mobile IPV6, advantages, application.I.I NTRODUCTIONWith increasing popularity of mobile devices such as PDA’s, internet ready cell phones, PC Tablets, etc, there is a need to provide access to the Internet that may be always in motion or wireless access to the Internet.[1] Mobile devices can be connected to the Internet by using wireless network interfaces. However, a mobile device may change its network attachment each time it moves to a new link. The necessity for uninterrupted communication when the mobile device moves from one location to another one calls for a new technology. This kind of communication can be effectively implemented using Mobile IP [2]. Mobile IP, an extension to standard Internet Protocol proposed by the Internet Engineering Task Force (IETF), aims to make mobile computing a reality and to solve the mobility problem of network node. Mobile IP refers to the mobility aspect of IP that allows nodes to move to different networks all over the world and change their point-of-attachment while maintaining all existing communications and using the same IP address. This is not to be confused with ‘portability’ that allows nodes to move to different networks all over the world and remain reachable, but upper-layer connections must be disrupted each time the node relocates because it has to be addressed by a new address at each location [2]. The main advantage of Mobile IP is that it frees the user from a fixed location. It makes invisible the boundaries between attachment points [3]. Mobile IP is focused on providing clear connectivity to mobile nodes in an IP based network environment while transparent mobility support is necessary for compatibility with applications [4]. Therefore, the aim of Mobile IP is to allow a mobile node to keep the same IP address in order to maintain existing connections, while remaining reachable at any new location in the Internet. It is likely the most widely known IP mobility support protocol. Two versions of34Analysis towards Mobile IPV4 and Mobile IPV6 in Computer NetworksMobile IP have been standardized for supporting host-based mobility on the Internet: MIPv4 and MIPv6 [5].Mobile IPv4 is the most promising solution for mobility management in the current IPV4 network but it suffers from certain problems. Mobile IPv6 is designed for the next generation wireless networks and it overcomes the problems of MIPv4. Although MIPv6 shares many features with MIPv4, there exist some differences. The integration of mobile cell phones with Internet-based multimedia services will force a move to the next generation version of IP (IPv6)[3]. They support the mobility of IP hosts by allowing them to use two IP addresses: a Home Address (HoA) that shows the fixed address of a Mobile Node (MN) and a Care-of- Address (CoA) that changes with the IP subnet to which an MN is currently attached. However, there are minor differences with regard to some important details. MIPv6 consists of three components: the MN, the home agent (HA), and the correspondent node (CN). The role of the foreign agent (FA) in MIPv4 was replaced by the access router (AR) in MIPv6. In addition, although route optimization extensions were proposed for both MIPv4 and MIPv6, they were only standardized for MIPv6 [5].When a Mobile Node moves far away from its home network at foreign network, it will receive new address called Care-of-address (CoA). To the Correspondent Node can continue communicate to Mobile Nodes when it only knows home address of MN, there must be a home agent in MN's home network that will forward the packets to CoA of MN [6]. The rest of this paper is organized as follows. Section 2 defines the fundamentals of mobile IPV4. Section 3 elaborates the problems of mobile IPV4. Section 4 deals with the fundamentals of mobile IPV6. Section 5 explains the problems of mobile IPV6. Section 6 compares the mobile IPV4 with mobile IPV6. Section 7 discusses the advantages of mobile IP. Section 8 reviews the application of mobile IP for vehicular networks.II. FUNDAMENTALS OF MOBILE IPV4Since IPv4 was not built with mobility in mind, Mobile IPv4 was designed as an extension to the base IPv4 protocol to support mobility. Mobile IP resolves the issue of mobility by assigning the mobile node a temporary address at each new location, maintaining the MN’s original IP address, and creating and storing a covering between the two addresses with a router in the mobile node’s original network.[3]. MIPv4 uses foreign agent routers functionality. MIPv4 involves a Mobile Node (MN), Home Agent (HA) that exists in home network and keeps mobility information on a MN, and Foreign Agent (FA) that controls its network having a moving MN and acts as Care-of-Address (COA) for the MN. And Corresponding Node (CN) is a node that wants to connect or is communicating with the MN [7] Three main mechanisms of Mobile IP are agent discovering, registration, and tunnelling. The function of the three mentioned mechanisms of Mobile IP is as follows: [8]Agent Discovery: Mobility agents announce their availability on each link for which theyprovide service.Registration: When the mobile node is away from home, it registers its care-of address withits home agent.Tunnelling: In order for datagrams to be delivered to mobile node when it is away fromhome, the home agent has to tunnel thedatagrams to the care-of address.The operation of MIPv4 is shown in Figure 1.Figure1. The operation of MIPv4As shown in Figure 1, for FA periodically sends out the agent advertisement message, MN receives such themessage if MN enters the range controlled by FA. After receiving the message, MN starts location registration procedure by sending the registration request message with COA being FA to HA through FA. Then, HA updates mobility information on the MN and send the registration response and binding message. Whenever MN moves from a network to another network, HA tracks the MN's location by registration request and response. If a CN wants to communicate with MN, the CN sends data to MN. Because of standard internet routing, data from CN is arrived at the home network of MN, specially HA. HA controls the binding list with the data's destination address and then transfers the data to its FA if MN is not in the home network. FA controls the visiting list with the destination address of the data arrived from HA by tunnelling. And then, FA directly sends the data to MN if MN is in the foreign network[3].III. PROBLEMS OF MOBILE IPV4Mobile IPv4 has many problems that need to be worked. They maybe cause communication involving Mobile IP inefficient. They have security, triangulation problems, duplication field in “IP within IP”, and reliability issues. In t his section, these problems are explained below.∙Security: Security is the most outstanding problem with Mobile IP. A great deal of attention is being focused on making Mobile IPv4 coexist with the security features coming into use within the internet.Firewalls, especially cause problem for Mobile IPv4because they prevent all types of incoming packets that do not meet identified criteria. Enterprise firewalls are specifically configured to prevent packets from entering through the Internet that seemto come out from internal computers. Although this allows management of internal Internet nodes without great attention to security, it causes problems for mobile nodes within their home enterprise networks. Such communications carry themobile node’s home address and would therefore beprevented by firewall[3].∙Triangulation Problems: Triangle Routing is likely the second largest problem of Mobile IP. Themain idea behind triangle routing is as follows. Amobile node wants to send a packet to another nodethat is on the same network. The receiver nodeoccurs to be the far side of the Internet, far awayfrom the mobile node’s home network. Then thesending node directs all the packets to the homenetwork. They pass through the Internet to reach thehome agent and then tunnel them back across theInternet to reach the foreign agent. It would beoptimal if the sending node could find out that themobile node is on the same network and deliver thepacket directly. The aim is to deliver packets asdirectly as possible from sending node to mobilenode without passing through a home agent. Thisplan is called triangle routing because the single“leg” of the triangle goes from a mobile node to thecorresponding node, and the home agent shapes thethird vertex, controlling the path taken by the datafrom the correspondent node to the mobile node.This is clearly a problem since route from sender tomobile node by way of the home agent takes twosides of the triangle, rather than the third side, which is the direct path.∙Duplication field in “IP within IP”: To express the datagram, we put the original datagram insideanother IP envelope, then the whole packetinvolves the outer IP header plus the originaldatagram. The fields in the outer IP header add toomuch overhead to the final datagram several fieldsare copied from the inner IP header. This waste ofunnecessary space is not producing profit.∙Reliability Issues: Some other issues are faced in Mobile IP. One concerns the perception ofreliability, which refers to the premise of Mobile IPconnections are based on TCP surviving cellchanges. Many people assume that most communication systems are bursty and an increasein reliability is not needed. Another concernregards issues in IP addressing. Mobile IP creates a perception that a mobile node is always attached to its home network. Inefficiencies can branch from this. For example, many of communications are short lived and don’t depend on the identity of the node, this does not take advantage of the idea of having a mobile node home address.IV. FUNDAMENTALS OF MOBILE IPV6MIPv4 has not been used widely enough to provide much mobility and has several major shortcomings, including a communications process and a limited number of IP addresses. The latter is a key problem because the number of mobile devices that need their own IP address to access the Internet is increasing rapidly. To overcome these shortcomings and introduce new capabilities, the IETF has been developing MIPv6. MIPv6 makes many more IP addresses available and lets mobile users stay connected to the Internet as they move between networks [9]. Mobile IPv6 was designed based on the experiences gained with Mobile IPv4 and as such solves many of the problems identified with the previous version. The key advantage in MIPv6 is that it is based on the new IPv6 protocol. In MIPv6, IP addresses are 128 bits long integers, mobility support in MIPv6 solves many problems of basic Mobile IP (tunnelling, ingress filter). Mobile IPv6 is in the next generation IPv6 network for supporting mobility [3].There is no FA functionality in MIPv6 because MN obtained its new IPv6 address within a foreign network by address auto-configuration method. The operationalprocedure of MIPv6 is shown in Figure 2.[10]Figure 2. The operational procedure of MIPv6In MIPv6, the MN's mobility is shown by the router advertisement message and node's neighbour unreachable detection mechanism. That is, a MN can cause a router to send its advertisement message by solicitation if necessary. After showing its mobility, the MN gets COA unlikely in MIPv4. The MN sends the binding update message to the HA and the communicated CN. The HA and CN update its binding list and send their acknowledgement messages. If a CN wants to communicate with MN, the CN sends data to MN's original IP address because of not knowing MN's COA. As a result, data from CN is arrived at the home network through standard IP routing. The HA controls its binding list with the data's destination address and then tunnels the data to the MN's COA. The MN that tunnelled data is received send the binding update message to CN with understanding that the CN has no its own COA. After receiving the update message, the CN sends only data to MN's COA [10].V. PROBLEMS OF MOBILE IPV6One of the most representative efforts on the way toward next generation all-IP mobile networks is Mobile IPv6 (MIPv6) (1). Although MIPv6 is a well-known mature standard for IPv6 mobility support and solves many problems seen in Mobile IPv4 (MIPv4) (2), it has still revealed some problems such as handover latency, packet loss, and signalling overhead. Furthermore,despite the reputation of this protocol, it has been slowly used in real implementations over the past years, and does not seem to receive widespread acceptance in the market [10].VI. COMPARISON BETWEEN MOBILE IPV6 AND MOBILE IPV4There are two versions of Mobile IP: Mobile IP for IPv4 and IPv6. The major differences are summarised as follows[8]:TABLE I. COMPARISON BETWEEN MOBILE IPV6 ANDMOBILE IPV4Key Features MobileIPv4 Mobile IPv6Special router as foreignagentYes NoSupport for route optimization Part of theprotocolInExtensionsEnsure symmetricreachability between mobilenodes and its router atcurrent locationNo YesRouting bandwidth overhead More Less Decouple from Link Layer No Yes Need to manage Tunnel softstateYes No Dynamic home agentaddress discoveryNo YesVII. ADVANTAGES OF MOBILE IPMobile IP is a set of standards used to adapt networking to an increasingly mobile environment. The advantages of Mobile IP are as follows:∙Convenience: Mobile IP's biggest advantage is a sustained connection to the Internet while travelling amongst a number of network connection points. In the past, a device would have to reconnect with each node as it moved along, but Mobile IP provides a simple hand-off solution that permits a device to move around seamlessly within a larger network, comprised of many connection points, without once losing their connection.∙Scalability: Since Mobile IP is built for large, expansive networks with thousands of wireless users, networks can be easily expanded to include alarger area and allow for greater connectivity for users. Whether this is on cellular networks or specific wireless LANs, a Mobile IP setup can expand to include new connection points with relative ease.∙Consistency: When a user connects to a network, they are given an IP address that identifies them on that network and permits a router connected to that portion of the network, or subnet, to send them their requested data. With Mobile IP, a user keeps the same IP address across all of the subnets they travel between while the router on their home subnet intercepts and forwards all incoming information to them across the network through a network tunnel.This provides the user with a seamless and consistent mobile connection, even if they are hosting services while moving around inside the network schema.∙Standardized: Since Mobile IP's inception, it has been adopted as a standard and is included with many advanced networking tools. Companies, such as Cisco, provide Mobile IP solutions with a number of their products, making the setup and expansion of a network using Mobile IP even easier. ∙Integration: Mobile IP solutions do not need an additional network, but rather mix with a current network schema. This makes the implementation ofa Mobile IP setup in any situation much easier thanhaving to shape an additional mobile or wireless setup for interaction with the main, fixed position network.∙Uniqueness: A mobile IP address permits users to connect to the Internet without a normal static or dynamic IP address through the use of a unique mobile IP address. This unique address lets the computer connect through a network to a home IP address but still uses and communicate with the network's protocol. Having a unique IP is important for routing information to the correct computer. In short, if it were not for mobile IPs, informationwould continue to be routed to the last known IP address at which that computer was located, and a seamless connection would be impossible .Portability: IP creation is the only way that users can access the Internet while away from a traditional modem/router setup. Mobile IP addresses create a tunnel to a host server, which allows an access point to the Internet from any location where a signal can be received. Mobile IPs can create connection protocols to connect to the Internet through multiple servers and networks. A mobile IP address allows users to travel through multiple networks and keep an IP address. This option is useful for employees who travel throughout a building and cross into multiple wireless areas.VIII. APPLICATION OF MOBILE IP FORVEHICULAR NETWORKSVehicular ad hoc networks are gaining importance for inter-vehicle communication, because they allow for the local communication between vehicles without any infrastructure, configuration effort, and without the high costs of cellular networks [11]. Besides local data exchange, vehicular applications may be extended by accessing Internet services. In VANETs, Internet gateways (IGWs) installed at the roadside can provide a temporary Internet access, which opens up the Internet for the VANET and vice versa. This communication scenario is shown in fig. 3 where an IGW provides Internet access for the passing vehicles organized in a VANET. Vehicular applications can also considerinformation from the global Internet.Figure 3. Internet access for the VANETThe Internet integration needs a respective mobility support of the vehicular ad hoc network. Mobile IPv6 (RFC 3775) cannot be used for the mobility support of multihop VANETs since it always needs a direct link layer connection between gateway and mobile node [12]. For this reason, several approaches were proposed to integrate multihop ad hoc networks into the Internet using Mobile IP [13].A mobility management protocol called MMIP6 is developed in order to control the mobility of vehicles. MMIP6 is based on the principles of Mobile IPv4, but was designed to support IPv6-based mobile nodes organized in ad hoc networks. In contrast to existing approaches, MMIP6 is optimized for scalability and efficiency. The key concept is a proactive service discovery protocol for Internet gateways providing connectivity to the Internet. This protocol is combined with an optimized mobility management protocol to control the mobility of the vehicles. Due to the proactive nature of the foreign agent discovery, MMIP6 scales well with the size of the ad hoc network. Whereas existing approaches are focused on small-scale ad hoc networking scenarios, MMIP6 is highly optimized for scalability and efficiency. Also MMIP6 is a suitable solution providing a scalable mobility support with an acceptable performance characteristic [14].IX. CONCLUSIONTypical mobile devices change networks several times throughout the day. When the original set of Internet protocols was designed, mobility was not an issue that was taken into account. With the rise of smart phones and increasingly more mobile devices, networking has had to adapt to accommodate the difference in how we interact with and exchange information with our network devices. Mobility support in the IP protocol has been developed by the IETF leading to the Mobile IP protocol. Mobile IP has gained attention as a technology that can provide mobility to universal users independently of the access network. Currently, two versions of Mobile IP are available,versions 4 (MIPv4) and 6 (MIPv6). Vehicular ad hoc networks become very important for inter-vehicle communication. For this reason, there is a growing need to integrate them seamlessly into the Internet, which is not possible using standard Mobile IP. The MMIP6 protocols proposed in this paper are able to integrate vehicles organized in IPv6-based multihop VANETs into the Internet.REFERENCES[1] C. E. 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Min Young, "Comparative Evaluation of TCP Performances on MIPv4 and MIPv6Protocols in Mobile Mesh Networks," in BroadbandConvergence Networks, 2007. BcN '07. 2nd IEEE/IFIPInternational Workshop on, 2007, pp. 1-9. [11] W. Enkelmann, "FleetNet - applications for inter-vehiclecommunication," in Intelligent Vehicles Symposium, 2003. Proceedings. IEEE, 2003, pp. 162-167.[12] S. William and M. Gerla, "IPv6 flow handoff in ad hocwireless networks using mobility prediction," in GlobalTelecommunications Conference, 1999. GLOBECOM '99, 1999, pp. 271-275 vol.1a.[13] C. Yuh-Shyan, C. Ching-Hsueh, H. Chih-Shun, and C.Ge-Ming, "Network Mobility Protocol for Vehicular AdHoc Networks," in Wireless Communications and Networking Conference, 2009. WCNC 2009. IEEE, 2009, pp. 1-6.[14] M. Bechler and L. Wolf, "Mobility management forvehicular ad hoc networks," in Vehicular TechnologyConference, 2005. VTC 2005-Spring. 2005 IEEE 61st,2005, pp. 2294-2298 Vol. 4.Seyedeh Masoumeh Ahmadi received her B.A. degree from Islamic Azad University of Lahijan, Iran. She is M.A. student in Information Technology at the Guilan University, Iran.。

J. Microbiol. Biotechnol. (2010),20(4), 678–682doi: 10.4014/jmb.0910.10031First published online 29 January 2010Two pHZ1358 Derivative V ectors for Efficient Gene Knockout in Streptomyces He, Yunlong, Zhijun Wang, Linquan Bai, Jingdan Liang, Xiufen Zhou, and Zixin Deng*Laboratory of Microbial Metabolism, and School of Life Sciences and Biotechnology, Shanghai Jiaotong University, Shanghai 200030, ChinaReceived:October 22, 2009 / Revised:November 26, 2009 / Accepted:November 28, 2009The deletion of sti from the Streptomyces plasmid pIJ101 made its derivative pHZ1358 an efficient vector for gene disruption and replacement. Here, pHZ1358 was further optimized by the construction of a derivative plasmid pJTU1278, in which a cassette carrying multiple cloning sites and a lacZ selection marker were introduced for convenient plasmid construction in E. coli. In addition, the ori T region of pJTU1278 was also deleted, generating a vector (pJTU1289) that can be used specifically for PCR-targ eting. The efficient usag e of these vectors was demonstrated by the deletion of the g ene involved in avermectin biosynthesis in S.avermitilis.Keywords: Shuttle vector, pHZ1358, pJTU1278, pJTU1289, conjugation, PCR-targetingStreptomyces, soil-inhabiting Gram-positive bacteria, occupya significant niche in biotechnology research because oftheir ability to produce a wide variety of bioactive compounds. Streptomyces are also unusual among prokaryotes as theyundergo a complex cycle of morphological differentiation,forming spores, vegetative substrate hyphae, and aerial hyphaeduring different stages of their life cycle [4]. Although newtechniques are continually being developed for differentresearch purposes in this area, gene deletion/disruptionremains one of the most efficient and indispensable means.For gene disruption and replacement in Streptomycesspecies, pHZ1358 [7] has been developed as an efficientpIJ101 [3] derivative vector by the removal of a 574-bpDNA region containing an sti (strong incompatibility locus)that causes the accumulation of single-strand plasmids inthe host [1,7]. Further deletion of 36-bp direct repeatsmakes the plasmid stably replicate as a shuttle vector in E. coli.Here, we report on the construction of two pHZ1358derivatives, pJTU1278 and pJTU1289, where the former contains the additional virtues of multiple cloning sites anda LacZ selection marker (for convenient construction in E. coli), and the latter has a deleted ori T, allowing it to beused specifically for PCR-targeting. The usage of pJTU1289was also demonstrated by the deletion of an avermectinbiosynthetic gene.M ATERIALS AND M ETHODSBacterial Strains and Plasmids Used in This StudyDH10B (GIBCO-BRL) and ET12567 (pUZ8002) [5] were used as the E. coli host for cloning and conjugation, respectively. S. avermitilis NRRL 8165 was used for the gene deletion.The fosmid vector pCC2FOS TM (EPICENTRE Biotechnologies) was used to construct the genomic library of S. avermitilis NRRL 8165 [2]. Plasmid pJTU412 was a gift from Sun et al. [7].The DNA isolation and plasmid manipulation from E. coli were performed according to Sambrook and Russell[6]. Construction of pJTU1278 and pJTU1289For the construction of pJTU1278, pJTU412 was first digested with Kpn I, blunted with the Klenow fragment of E. coli DNA polymerase I, self-ligated to generate pJTU1275, and further digested with Bgl II and Sph I. The resulting 8.7-kb Bgl II-Sph I fragment from pJTU1275 was then treated with the Klenow fragment and self-ligated to generate pJTU1276, which was digested with Xba I, treated with the Klenow fragment, and self-ligated to generate pJTU1277. A 585-bp PCR fragment from pBluescript II SK(+) using primers L acZ-P1 (5'-AACAA TTGCCA TTCGCCA TTCAGG-3') and LacZ-P2 (5'-AA-CAA TTGCCCAA TACGCAAACC-3') was digested with Mun I and inserted into the Eco RI site of pJTU1277 to generate pJTU1278. The LacZ was in the same direction as the resistant gene bla in pJTU1278.For the construction of pJTU1289, pJTU1278 was digested with Sse8387I, and the 8.4-kb Sse8387I fragment from pJTU1278 was then self-ligated to generate pJTU1289.Gene Deletion of aveA4 (S. avermetilis HYL16)The 4,671-bp Bam HI fragment containing the portion of aveA4 from cosmid 14A7 of the S. avermitilis NRRL 8165 genome library was cloned into pJTU1289 to generate pJTU1314. The aac(3)IV and*Corresponding authorPhone: +86-21-62933404; Fax: +86-21-62932418;E-mail: zxdeng@679He et al.Fig. 1. Construction of pJTU1278 and pJTU1289.pJTU1278 and pJTU1289 were optimized from pJTU412. After two rounds of digestion, blunting with polymerase, and re-ligation using Kpn I and Bgl II-Sph I, respectively, Kpn I and Hin dIII were eliminated from the vector. A multiple cloning site (Sac I, Xba I, Spe I, Bam HI, Eco RI, Hin dIII, Kpn I) and lacZ gene (for clone selection in E. coli) were then inserted. Eco RV and Xho I can also be used for cloning. ori pIJ101, ori ColE1, and ori T were kept for the shuttle between Streptomyces and E. coli. The vector can also be used for cosmid library construction.P JTU1278 AND P JTU1289 FOR G ENE K NOCKOUT IN S TREPTOMYCES680ori T cassettes were amplified from the pIJ733 disruption cassette using the primers aveA4-T1 (5'-ACTCCCGCCTGCACGACGCC-ACTCCCCCAGCCCACAAGGA TTCCGGGGA TCCGTCGACC-3') and aveA4-T2 (5'-GCGGAGCCA TGGTGGCCA TCGAGGCGTCC-GAGGACGAGA TGTAGGCTGGAGCTGCTTC-3'). The resulting PCR product was then used to replace the 1,150-bp DNA region of aveA4 in pJTU1314 to generate pJTU1317, and then in strain NRRL 8165 to generate S. avermetilis HYL16. A comparison of the wild-type and mutant S. avermetilis HYL16 was conducted through fermentation, extraction, and an LC-MS analysis.Cultivation of S. avermitilisThe Streptomyces culture conditions were as described by Kieser et al.[4]: An SFM solid medium was used for the S. avermitilis sporulation, fermentation, and conjugation between E. coli and Streptomyces. A TSB culture supplemented with 10.3% sucrose and 1% yeast extract was used for the growth of mycelia for the isolation of total DNA. Thiostrepton and apramycin were used at a concentration of 12.5µg/ml and 15µg/ml in the solid and liquid medium, respectively, when necessary for the screening of conjugants.Analysis of AvermectinAfter 7 days of growth on an SFM agar plate, the mycelia of S. avermitilis and the S. avermetilis HYL16 mutants were collected and extracted with ethanol. The extract was concentrated under reduced pressure to yield an oily substance, which was further extracted with 1ml of methanol. The methanol extract was analyzed directly using an HPLC-MS Agilent 1100 equipped with an Agilent ZORBAX SB-C18 (2.1×150mm) column and using a linear gradient program of acetonitrile/H2O: 60% acetonitrile over 3min, 60%-90% over 5min, 90% over 2min, 90%-100% over 2min, and constant 100% acetonitrile over 3min at a flow rate of 0.5ml/min. An iontrap mass spectrometer was operated with an electrospray ionization source in the positive-ion mode, where the drying gas flow was 8l/min, the nebulizer pressure was 30psi, the drying gas temperature was 325o C, and the fragmentation amplitude was varied between 1.0 and 1.8V.R ESULTS AND D ISCUSSIONConstruction of pJTU1278 and pJTU1289Derived from pIJ101, pHZ1358 is an efficient Escherichia coli/Streptomyce s shuttle vector for targeted gene deletion and disruption. However, a further improvement of pHZ1358 would be removing the 36-bp direct repeats, which often cause a problem of plasmid recombination in E. coli [7]. For the convenience of gene cloning, a multiple cloning site is needed. Therefore, the vector optimization was started from the pHZ1358 derivative pJTU412. By digesting with Kpn I and then blunt-ending by filling-in to eliminate the Kpn I site in pJTU412, followed by further digestion with Bgl II-Sph I, blunt-ending by filling-in, and re-ligation to eliminate the Hin dIII site, a cassette carrying multiple cloning sites was introduced, along with the selection marker lac Z gene with a PCR fragment from pBluescript II SK(+) (Fig.1). Successful cloning was confirmed by restriction digestion and sequencing (Fig.2).Fig. 2. Confirmation of successful cloning of pJTU1278 and pJTU1289.The successful construction of pJTU1275 was indicated by the loss of the Kpn I restriction site at 2,999-bp (lane 2), whereas pJTU412 was linearized by Kpn I (lane 1). The loss of the Bgl II and Hin dIII sites confirmed the successful cloning of pJTU1276 (lanes 4, 6), whereas pJTU1275 was linearized by Bgl II and Hin dIII (lanes 3, 5). The cloning of pJTU1276, pJTU1277, pJTU1278, and pJTU1289 was confirmed with Xba I (B). The loss of the Xba I site confirmed the successful cloning of pJTU1277 (lane 8), whereas pJTU1267, pJTU1278, and pJTU1289 were linearized by Xba I (lanes 7, 9, 10). All the clones were confirmed with Pvu II (C). La nes 11-16 show the digestion of pJTU412, pJTU1275, pJTU1267, pJTU1277, pJTU1278, a nd pJTU1289 with Pvu II, separately.681He et al.Based on the above manipulations, the pHZ1358 derivative pJTU1278 contained the following characteristics: ori , rep from pIJ101, and resistant gene tsr for operation in Streptomyces ;ori (ColE1), lac Z, and resistance gene bla for manipulation in E. coli ; and ori T for conjugation from E. coli to Streptomyce s. In addition, owing to its segregational instability from its progenitor pHZ1358, it can be used for efficient gene deletion/disruption in Streptomyces . Since a single cos was included, it can also be used for cosmid library construction.For the specific purpose of PCR-targeting, an additional optimization of pJTU1278 was carried out to generate pJTU1289, where the 0.8-kb Sse83871 fragment containing ori T was deleted.Deletion of aveA4The usage of pJTU1278 and pJTU1289 as gene disruption vectors was demonstrated by the deletion of a gene essential for avermectin biosynthesis in S. avermitilis MA-4680. As shown in Fig.3, the 1,150-bp DNA region of aveA4 was replaced by the ori T -aac(3)IV cassette to generate S.avermetilis HYL 16. The successful construction of S.avermetilis HYL16 was confirmed using a PCR (Fig. 3B),and the successful deletion of aveA4 was confirmed by a comparison of the production of metabolites between the wild-type and the mutant S. avermetilis HYL16 using an HPLC analysis. Clearly, the production of avermectin “a”was completely abolished in S. avermetilis HYL16, in sharp contrast to the wild-type strain, whereas the oligomycin “o” was still produced in S. avermet ilis HYL 16 at the same level as that in the wild-type strain NRRL 8165(Fig. 3C).In addition to the S. avermitilis tested in this study, the two plasmids pJTU1278 and pJTU1289 were also successfully used for gene deletion in several other Streptomyces strains,including S. nanchangensis , S. hygroscopicus , Streptomyces sp. FR-008, and Actinosynnema pretiosum , confirming their efficiency as gene deletion/disruption vectors.AcknowledgmentsThe authors are very grateful to Dr. Tobias Kieser for hiscritical and patient editing of the manuscript. The authorsFig. 3. Deletion of the aveA4 gene.The 1.2-kb region of aveA4 was replaced with the ori T-aac(3)IV cassette using a double crossover (A ). The successful construction of the S. avermetilis HYL16 was confirmed by PCR (B ) with a 1.5-kb PCR product as expected, in contrast to the 1.2-kb DNA fragment from the wild type strain. Comparison of metabolites between the wild-type strain and the mutant S. avermetilis HYL16 showed that the avermectin corresponding to “a” peaks of the wild-type strain NRRL 8165 disappeared in S. avermetilis HYL16. Oligomycin corresponding to “o” peaks still produced in HYL16 (C ).P JTU1278 AND P JTU1289 FOR G ENE K NOCKOUT IN S TREPTOMYCES682also wish to thank the National Science Foundation of China, the Ministry of Science and Technology (973 and 863 programs), the Ministry of Education of China, the Shanghai Municipal Council of Science and Technology and Shanghai L eading Academic Discipline Project, and the State Key Laboratory of Bio-organic and Natural Products Chemistry (CAS) for their research support.R EFERENCES1.Deng, Z. X., T. Kieser, and D. A. Hopwood. 1988. “Strongincompatibility” between derivatives of the Streptomyces multi-copy plasmid pIJ101. Mol. Gen. Genet. 214: 286-294.2.He, Y., D. Zhu, L. Bai, and Z. Deng. 2009. Cloning ofantibiotic biosynthetic gene clusters from genomic library by narrow-down polymerase chain reaction. J. Shanghai Jiaotong Univ.43: 5-8.3.Kendall, K. J. and S. N. Cohen. 1988. Complete nucleotidesequence of the Streptomy ces lividans plasmid pIJ101 and correlation of the sequence with genetic properties. J. Bacteriol. 170: 4634-4651.4.Kieser, T., M. J. Bibb, M. J. Buttner, K. F. Chater, and D. A.Hopwood. 2000. Practical Streptomy ces Genetics. John Innes Centre.5.Paget, M. S., L. Chamberlin, A. Atrih, S. J. Foster, and M. J.Buttner. 1999. Evidence that the extracytoplasmic function sigma factor sigmaE is required for normal cell wall structure in Streptomyces coelicolor A3(2). J. Bacteriol.181: 204-211.6.Sambrook, J. and D. Russell. 2000. Molecular Cloning: ALaboratory Manual, 3rd Ed. Cold Spring Harbor L aboratory Press, NY, U.S.A.7.Sun, Y., X. He, J. Liang, X. Zhou, and Z. Deng. 2009. Analysisof functions in plasmid pHZ1358 influencing its genetic and structural stability in S treptomyces lividans 1326. Appl. Microbiol.Biotechnol.82: 303-310.。

第43 卷第 5 期2024 年5 月Vol.43 No.5674~681分析测试学报FENXI CESHI XUEBAO（Journal of Instrumental Analysis）氮掺杂Ti3C2 MXene量子点荧光探针用于α-葡萄糖苷酶活性检测李军1，杨新杰1，罗焰1，李泉1，宗玉红1，张艳丽1*，王红斌1，杨文荣1，2，庞鹏飞1*（1．云南民族大学化学与环境学院，云南省教育厅环境功能材料重点实验室，云南昆明650504；2．迪肯大学生命与环境科学学院，澳大利亚维多利亚州吉朗3217）摘要：α-葡萄糖苷酶是生物体糖代谢途径中不可或缺的一类酶，发展简单、灵敏、准确的α-葡萄糖苷酶活性检测及抑制剂筛选方法对于糖尿病的治疗与预防具有重要意义。

该文基于氮掺杂Ti3C2 MXene量子点（N-Ti3C2 MQDs）荧光探针和内滤效应（IFE），构建了一种“开-关-开”型荧光传感α-葡萄糖苷酶活性检测及抑制剂筛选的新方法。

研究发现，N-Ti3C2 MQDs发射蓝色荧光（λem = 440 nm），荧光量子产率为15.7%。

其检测机理为：α-葡萄糖苷酶水解底物对硝基苯基-α-D-吡喃葡萄糖苷，水解产物对硝基苯酚通过内滤效应导致N-Ti3C2 MQDs荧光猝灭；而抑制剂阿卡波糖可使α-葡萄糖苷酶的活性受到抑制，水解产物减少，N-Ti3C2 MQDs 荧光恢复。

结果显示，N-Ti3C2 MQDs探针荧光强度与α-葡萄糖苷酶浓度在5~300 U/L范围内呈良好线性关系，检出限为2.5 U/L（S/N = 3），对阿卡波糖的半最大抑制浓度（IC50）为178.5 μmol/L。

该方法具有成本低、操作简单、灵敏度高、选择性好等特点，已成功用于人血清中α-葡萄糖苷酶活性的测定。

关键词：α-葡萄糖苷酶；氮掺杂Ti3C2 MXene；量子点；荧光探针中图分类号：O657.3；O629.8文献标识码：A 文章编号：1004-4957（2024）05-0674-08 Fluorescent Probe for Detection of α-Glucosidase Activity Based onN-Doped Ti3C2MXene Quantum DotsLI Jun1，YANG Xin-jie1，LUO Yan1，LI Quan1，ZONG Yu-hong1，ZHANG Yan-li1*，WANG Hong-bin1，YANG Wen-rong1，2，PANG Peng-fei1*（1．Key Laboratory of Environmental Functional Materials of Yunnan Province Education Department，School ofChemistry and Environment，Yunnan Minzu University，Kunming　650504，China；2．School of Life andEnvironmental Sciences，Deakin University，Geelong　VIC 3217，Australia）Abstract：α-Glucosidase is an indispensable enzyme in the glucose metabolism pathway of organ⁃isms. The development of simple，sensitive and accurate detection methods for α-glucosidase activi⁃ty and inhibitors screening are of great significance for the treatment and prevention of diabetes. In this work，a novel fluorescent "on-off-on" method for detection of α-glucosidase activity and its in⁃hibitors screening was developed based on N-doped Ti3C2 MXene quantum dots（N-Ti3C2 MQDs） and internal filtration effect（IFE）. The prepared N-Ti3C2 MQDs emitted blue fluorescence（λem = 440 nm）with a fluorescence quantum yield of 15.7%.α-Glucosidase can hydrolyze substrate p-nitrophenyl-α-D-glucopyranoside to produce p-nitrophenol，which causes fluorescence quenching of the N-Ti3C2 MQDs through IFE.After adding the acarbose inhibitor，the activity of α-glucosidase is inhibited and the produced p-nitrophenol by hydrolysis is reduced，resulting in fluorescence recovery of N-Ti3C2 MQDs. Fluorescent intensity of N-Ti3C2 MQDs probe is proportional to α-glucosidase concentra⁃tion in the range of 5-300 U/L，with a detection limit of 2.5 U/L（S/N = 3）. The half-maximal inhibi⁃tory concentration of inhibitor（IC50） value is calculated to be 178.5 μmol/L for acarbose. This pro⁃posed method has the advantages of low cost，simple operation，high sensitivity，and good selectivi⁃ty，which has been successfully used for determination of α-glucosidase activity in human serum.doi：10.12452/j.fxcsxb.24010503收稿日期：2024－01－05；修回日期：2024－02－18基金项目：国家自然科学基金资助项目（21665027，21565031）；云南省科技厅应用基础研究项目（202001AT070012）∗通讯作者：张艳丽，博士，教授，研究方向：化学与生物传感，E-mail：ylzhang@庞鹏飞，博士，教授，研究方向：荧光传感分析，E-mail：pfpang@第 5 期李军等：氮掺杂Ti3C2 MXene量子点荧光探针用于α-葡萄糖苷酶活性检测Key words：α-glucosidase；N-doped Ti3C2 MXene；quantum dots；fluorescent probe糖尿病作为一种影响多种慢性病的重要因素，因发病率高，引起了广泛的关注。