盐藻(Dunaliella viridis)细菌人工染色体文库的构建和鉴定

杜氏盐藻高盐诱导蛋白的分离和鉴定*柴玉荣1),刘国红1),崔柳青2),刘红涛2),李 杰2),薛乐勋2)#1)郑州大学基础医学院组织学与胚胎学教研室郑州450001 2)郑州大学生物工程系细胞生物学研究室郑州450001#通讯作者,男,1944年2月生,研究员,研究方向:肿瘤与生物工程,E-m ai:l xue l x@关键词 杜氏盐藻;双向电泳;质谱;高盐中图分类号 Q786摘要 目的:对高盐和低盐培养条件下杜氏盐藻蛋白质组的双向电泳(2-DE)图谱进行比较分析,筛选高盐诱导蛋白并鉴定。

方法:提取高盐(3.5mo l/L N aC l)和低盐(1.5mo l/L N aC l)培养条件下杜氏盐藻的总蛋白并进行2-DE,考马斯亮蓝染色后用I m age-Scanner扫描仪扫描,I m ag e M aster5.0图像分析软件分析,并利用基质辅助激光解析电离质谱法(M ALD I-TOF)对在高盐培养条件下增强表达达到3倍的蛋白进行鉴定。

结果:2-DE图谱上共分离出509个杜氏盐藻蛋白质斑点,主要分布在等电点4~8和相对分子质量20000~97000的范围内;高盐培养条件下有21个蛋白增强表达达到3倍并且通过M ALD I-TO F鉴定出了其中的12个。

结论:成功获得了分辨率和重复性较好的杜氏盐藻蛋白质组2-DE图谱,鉴定得到了12种高盐诱导增强表达达到3倍的蛋白。

Isolati on and i dentificati on of h i gh concentrati on sal-t i nduced protei ns fro m Dunaliell a s alinaC HAI Yurong1),LIU Guohong1),CUI L iuqing2),LIU H ong tao2),LI J ie2),XUE Lexun2)1)D epart m ent of H istology and Em bryology,College of Basic M edical S ciences,Zhengzhou Universit y,Zhengzhou450001 2)Laboratory for C ell B iology,D e p art m ent o f B ioeng i n eering,Zhengzhou Universit y, Zhengzhou450001K ey word s D una liella salina;t w o-di m ensiona l electrophoresi s;m ass spectru m;h i gh concen trati on sa ltAbstrac t A m i:T o ana l yze the t w o-di m ensi onal e l ec trophoresis(2-DE)m aps o f D unaliella salina(D.sali na)underd ifferen tN a+concentrati on(3.5m o l/L N aC l and1.5m ol/L N aC l)and screen differentl y expressed prote i ns bet weenthem.Me thods:2-DE was taken to separate t he t o ta l pro te i ns fro m D.sali na treated w ith high and low concentra ti on sa lt.P ro tei n patterns we re ana l yzed and identifi ed by I m age M aste r5.0so ft ware and MA LD I-TOF a fter sta i n i ng and scanni ng.Resu lts:A t o ta l o f509pro tein spo ts w ere found as diff e rentl y expressed pro te i ns,out o f wh i ch,the express i on l eve l of21 prote i ns cult ured i n h i gh concentra ti on sa lt condition w as t hree ti m es of t hose cu lt ured in low concentra tion sa lt condition.Conc lusion:2-DE pro fil es fro m D.sali na w ith h i gh reso luti on and reproduci b ility have been obtained and12up-regulated prote i ns i n h i gh sa lt concen trati on have been identifi ed.近年来,人们对生物耐盐机制有了深入的认识,但植物的整个耐盐体系及耐盐机制仍不清楚[1-2]。

研究报告Research Report盐藻胞质型3-磷酸甘油脱氢酶基因的克隆及性质鉴定蔡元适1甘嘉嘉1蓝海琳1贺庆华1张庆莲2*1西南民族大学生命科学与技术学院,青藏高原动物遗传资源保护与利用重点实验室,成都,610041;2成都医学院,检验医学院,成都,610500*通信作者,*****************摘要3-磷酸甘油脱氢酶(GPDH)是盐藻(Dunaliella salina )甘油合成途径上的关键酶，本研究首次报道了一条胞质型GPDH 基因的克隆(命名为DsGPDH1)，经大肠杆菌表达和融合蛋白的纯化及性质鉴定，也研究了在盐胁迫期间DsGPDH1和DsGPDH2的表达变化。

研究发现DsGPDH1和DsGPDH2相比，少了一段信号肽序列，但包含完整的SerB 结构域和GPDH 结构域。

通过大肠杆菌制备的融合DsGPDH1蛋白高度可溶，但却没有NADH 依赖的脱氢酶活性，其SerB 结构域是否具有磷酸化酶的活性还没有研究清楚。

在高盐胁迫时DsGPDH1和DsGPDH2都表现出诱导性高表达，在低盐胁迫时，DsGPDH1的表达被高度抑制，但DsGPDH2的表达变化却不太明显。

GPDH 同工酶在盐藻甘油合成中的具体分工仍然不清楚，但本研究迈出了坚实的一步。

关键词盐藻,GPDH,甘油,耐盐Cloning and Characterization of a Cytoplasmic Glycerol 3-phosphate Dehydrogenase Gene from Dunaliella salinaCai Yuanshi 1Gan Jiajia 1Lan Hailin 1He Qinghua 1Zhang Qinglian 2*1College of Life Sciences and Technology,Southwest Minzu University,Chengdu,610041;2Department of Laboratory Medicine,Chengdu Medical College,Chengdu,610500*Corresponding author,*****************DOI:10.13417/j.gab.039.004122Abstract Glycerol 3-phosphate dehydrogenase (GPDH)is the key enzyme on the glycerol synthesis pathway ofDunaliella salina .This paper reports for the first time the cloning,E.coli expression,recombinant protein purification and characterization of a cytoplasmic GPDH isoform (named DsGPDH1),the expression profile of DsGPDH1is also investigated under salt stress and compared to that of DsGPDH2.The results show that DsGPDH1lacks a signal peptide compare to DsGPDH2,but contains the entire SerB domain and the entire GPD domain.The fusion DsGPDH1protein prepared by Escherichia coli is highly soluble but does not have NADH-dependent dehydrogenase activity,and whether the phosphoenzyme activity in the SerB structure domain has not been studied.Under high salt stress,DsGPDH1and DsGPDH2show high induced expression;under low salt stress,DsGPDH1expression is highly inhibited,but the change of DsGPDH2is not obvious.The specific division of labor of GPDH isozymes in glycerol synthesis of salina is still unclear,but this study is a solid step forward.KeywordsDunaliella salina ,GPDH,Glycerol,Salt tolerance基金项目：本研究由西南民族大学中央高校项目(2020PTJS15)和国家级大创项目(201810656095)共同资助引用格式：Cai Y.S.,Gan J.J.,Lan H.L.,He Q.H.,and Zhang Q.L.,2020,Cloning and characterization of a cytoplasmic glycerol 3-phosphate dehydrogenase gene from Dunaliella salina ,Jiyinzuxue Yu Yingyong Shengwuxue (Genomics and Applied Biology),39(9):4122-4129(蔡元适,甘嘉嘉,蓝海琳,贺庆华,张庆莲,2020,盐藻胞质型3-磷酸甘油脱氢酶基因的克隆及性质鉴定,基因组学与应用生物学,39(9):4122-4129)杜氏盐藻(Dunaliella salina )是一种极耐盐的单细胞真核绿藻，能生存于含0.05mol/L 至饱和NaCl(~5.5mol/L)的溶液中(Ben-Amotz and Avron,1973)。

杜氏盐藻无菌体系的建立宋程飞;郝敬云;程蔚兰;史飞飞;季春丽;李润植【摘要】Abstr act:To establish the aseptic culture system of Dunaliella salina,six antibiotics ofkanamycin,cephalosporin,chloramphenicol,streptomycin,hygromycin and ampicillin were used to test the antibiotic tolerance of Dunaliella salina and the bactericidal effect of algae.The results showed that salt algae tolerance to chloramphenicol was low,cephalosporin and ampicillin tolerance was higher.Chloramphenicol,cephalosporin and ampicillin alone had significant inhibitory effect on the algae algae solution,and the bacteriostatic effect of kanamycin,hygromycin and streptomycin alone was poor.Antibiotic combination treatment of algae algae bacteria was more effective.The aseptic culture system of Dunaliella salina was establi shed by 50 μg/mL chloramphenicol,100 μg/mL ampicillin and 100 μg/mL cephalosporin.%为建立杜氏盐藻无菌培养体系,试验选用卡那霉素、头孢霉素、氯霉素、链霉素、潮霉素和氨苄青霉素6种抗生素,分别测试盐藻细胞的抗生素耐受性和对盐藻藻液的除菌效果.结果表明,盐藻对氯霉素耐受性低,对头孢霉素和氨苄青霉素的耐受性较高;氯霉素、头孢霉素和氨苄青霉素单独处理对盐藻藻液的抑菌效果明显,卡那霉素、潮霉素和链霉素单独处理的抑菌效果较差;抗生素组合处理对盐藻藻液的除菌效果更强;应用50 μg/mL氯霉素、100 μg/mL氨苄青霉素和100 μg/mL头孢霉素组合,经3次除菌处理和5次继代培养,建立了盐藻无菌培养体系.【期刊名称】《山西农业科学》【年(卷),期】2018(046)001【总页数】5页(P25-28,97)【关键词】杜氏盐藻;无菌体系;抗生素【作者】宋程飞;郝敬云;程蔚兰;史飞飞;季春丽;李润植【作者单位】山西农业大学分子农业与生物能源研究所,山西太谷030801;山西农业大学分子农业与生物能源研究所,山西太谷030801;山西农业大学分子农业与生物能源研究所,山西太谷030801;山西农业大学分子农业与生物能源研究所,山西太谷030801;山西农业大学分子农业与生物能源研究所,山西太谷030801;山西农业大学分子农业与生物能源研究所,山西太谷030801【正文语种】中文【中图分类】S917.3杜氏盐藻（Dunaliella sallina）是一种单细胞、无细胞壁的绿藻，在高盐环境下能够正常生长，是迄今为止发现的最耐盐的真核藻种之一[1-3]。