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补体C3a受体在尿路感染小鼠膀胱和肾脏中的表达及意义分析*邵静1，陈婉冰1，曹博1，赵淑娟2，韩锦2，李可1，武坤毅1△（1西安交通大学第二附属医院科研中心实验室，陕西西安 710004；2西安交通大学第二附属医院肾病科，陕西西安 710004）［摘要］目的：研究补体C3a受体（C3aR）在尿路感染模型小鼠膀胱和肾脏的表达与分布。

方法：将雌性C57BL/6小鼠随机分为正常对照组，以及3、6、24和48 h感染组，每组8~10只。

采用逆行推注人型大肠杆菌J96建立小鼠尿路感染模型。

平板涂布计数法检测小鼠膀胱和肾脏细菌负荷量；HE染色评估膀胱和肾脏病理损伤程度；ELISA检测肾组织肾脏损伤分子1（KIM-1）和尿液中C3a水平；蛋白印迹和免疫荧光检测C3aR在膀胱和肾脏的表达与分布。

结果：感染24 h时小鼠膀胱细菌负荷量达到峰值，48 h时细菌量下降；感染6 h时小鼠肾脏中细菌负荷量最高，随着感染时间延长逐渐减少。

感染后，膀胱病理表现为移行上皮细胞脱落，黏膜和固有层炎症细胞浸润；肾脏病理表现为肾小球萎缩，肾小管上皮细胞损伤，肾小管间质炎症细胞浸润及肾盂处组织脱落。

感染后肾脏KIM-1显著增加，尿液中C3a水平升高。

蛋白印迹结果显示，感染后膀胱和肾脏C3aR蛋白水平升高。

免疫荧光分析显示，C3aR在正常膀胱上皮细胞有少量表达，感染后在上皮细胞大量表达，在深层肌肉组织有少量表达；C3aR在正常肾皮质的肾小球和髓质的实质细胞有少量表达，感染后在肾小球、肾小管间质以及髓质的炎症浸润细胞中大量表达。

结论：小鼠尿路感染过程中C3a-C3aR信号激活，C3a在尿液中表达升高，C3aR在膀胱和肾脏中表达上调，炎症细胞表达的C3aR参与小鼠尿路感染进程并发挥作用。

［关键词］尿路感染；过敏毒素；C3a受体；炎症［中图分类号］R691.3； R392； R378.2+1 ［文献标志码］A doi： 10.3969/j.issn.1000-4718.2023.08.005 Analysis of expression and significance of C3aR in bladder and kidney of mice with urinary tract infectionsSHAO Jing1，CHEN Wanbing1，CAO Bo1，ZHAO Shujuan2，HAN Jin2，LI Ke1，WU Kunyi1△（1Core Research Laboratory， The Second Affiliated Hospital， Xi'an Jiaotong University， Xi'an 710004， China；2Department of Nephrology， The Second Affiliated Hospital， Xi'an Jiaotong University， Xi'an 710004， China. E-mail： wky6135@）［ABSTRACT］AIM： To investigate the expression and distribution of C3a receptor （C3aR） in the bladder and kidney of mice with urinary tract infections （UTIs）.METHODS： Female C57BL/6 mice were randomly divided into 5 groups， including normal group， and 3， 6， 24 and 48 h infection group， with 8 to 10 mice in each group. The model of UTIs in the female mice was established by bladder inoculation with uropathogenic Escherichia coli J96. Bacterial load in bladder and kidney tissues was measured by plate assay. Bladder and kidney histopathology were assessed by hematoxylin-eosin （HE） staining. Levels of kidney injury molecule-1 （KIM-1） in the kidney supernatants and C3a in the urine were de‑termined using ELISA kits. The expression and distribution of C3aR were assessed by Western blot and immunofluores‑cence.RESULTS： The maximum bacterial load in the bladder was achieved at 24 h and the bacterial load decreased at48 h. Meanwhile， the bacterial load in the kidney peaked at 6 h and gradually declined from 24 to 48 h with the time exten‑sion. After infection， the histopathology of the bladder showed shedding of transitional epithelial cells and infiltration of in‑flammatory cells in mucosa/lamina propria. The histopathology of the kidney showed injury of renal tubule epithelial cells，infiltration of renal tubule interstitial cells and shedding of renal pelvis tissue. Expression of KIM-1 in kidney was signifi‑cantly increased and C3a in urine was increased after infection.Western blot results revealed a significant increase in ［文章编号］1000-4718（2023）08-1383-07［收稿日期］ 2023-02-28 ［修回日期］ 2023-07-23*［基金项目］国家自然科学基金资助项目（No. 81900620）；陕西省自然科学基础研究计划项目（No. 2020JQ-536）△通讯作者Tel：166****2838； E-mail： wky6135@ ··1383C3aR expression in infected bladder and kidney tissues compared with normal tissues. Immunofluorescence analysis re‑vealed that the C3aR protein was observed in a limited quantity within normal bladder epithelial cells. However， there was a significant increase in C3aR expression after infection， which could be detected in numerous bladder epithelial cells as well as deep muscle tissue. Expression of C3aR was presented in a limited number of glomerular and medullary parenchy‑mal cells in normal kidney. However， C3aR expression was up-regulated in a significant proportion of inflammatory infil‑trating cells within the glomeruli， tubulointerstitial region and medulla after infection.CONCLUSION： The C3a-C3aR signaling pathway is activated in UTIs， with increased C3a level found in urine and up-regulated expression of C3aR ob‑served in bladder and kidney tissues. The C3aR expressed by inflammatory cells is involved in the process of UTIs in mice.［KEY WORDS］urinary tract infections； anaphylatoxin； C3a receptor； inflammation尿路感染（urinary tract infections， UTIs）是常见感染性疾病之一，主要由尿路致病性大肠杆菌（uro‑pathogenic Escherichia coli， UPEC）引起，可导致膀胱炎、急慢性肾盂肾炎、尿道炎、肾脓肿等。

microRNA过表达载体构建技术的实验研究姜彬【摘要】目的：构建microRNA的表达载体，再转染生物体细胞观察该microRNA的转录或表达情况，以获知它的作用机理及对生物体的影响。

方法：以microRNA-21（简化为miR-21）为例说明构建表达载体的方法。

根据microRNA的成熟序列以及附近约200多碱基共约470个碱基序列，设计PCR 引物，PCR扩增，PCR产物和pcDNA3.1（＋）质粒双酶切后，用T4连接酶进行连接反应，并转化入感受态细胞，筛选后对重组质粒进行双酶切、琼脂糖凝胶电泳鉴定及测序分析，并将其转染 Hela细胞，经 RT-PCR实验检测其表达情况。

结果：pcDNA3.1（＋）/miR-21过表达载体转染细胞后使得miR-21在细胞中过表达。

结论：成功构建了miR-21的真核表达载体，为进一步研究miR-21功能及作用机制奠定实验基础。

%This article introduces how to construct miRNA-21 overexpression vector and transfect it into cell ( Hela). Then the level of expression of miR-21 was observed. The miR-21 precursor is amplified by PCR method ,then the recombinant vector pcDNA3.1 (+ ) and miR-21 are constructed. After screening and identifying the recombinant ,it is transfected into cancer cells (Hela).The miR-21 expressing level is detected by RT-PCR.The result shows that the level of the miR-21 expression increases significantly in the cells that are transfected the recombinant. The miR-21 overexpression vector is successfully constructed ,and a foundation for studying the function of miR-21 is established .【期刊名称】《实验技术与管理》【年(卷),期】2014(000)001【总页数】5页(P41-44,48)【关键词】微小RNA;miR-21;pcDNA3.1(+)【作者】姜彬【作者单位】北京大学医学部生化系，北京 100191【正文语种】中文【中图分类】Q5221 microRNA概述1993年，Lee等［1］在线虫中发现了小分子单链非编码RNA，其长度只有22nt，当时命名为1in－4。

作物学报ACTA AGRONOMICA SINICA 2008, 34(7): 1290−1293/zwxb/ ISSN 0496-3490; CODEN TSHPA9E-mail: xbzw@基金项目: 中国博士后科学基金项目(20060390773); 河南省教育厅自然科学基金项目(2006210007)作者简介: 康国章(1971–), 男, 从事作物分子生理研究; 王永华(1973–), 男, 从事作物生理研究。

**共同第一作者。

*通讯作者(Corresponding author): 郭天财(1953–), 博士生导师, 从事作物生理研究。

E-mail: gmzx-guo@ Received(收稿日期): 2007-10-22; Accepted(接受日期): 2008-01-24.DOI: 10.3724/SP.J.1006.2008.01290小麦AGPase胞质型大亚基基因的克隆与表达分析康国章**王永华**刘超沈丙权郭天财*朱云集(河南农业大学国家小麦工程技术研究中心, 河南郑州450002)摘要: 从大粒型小麦品种豫教2号中克隆出调控小麦ADPG焦磷酸化酶(AGPase)胞质型大亚基基因(LSU I)cDNA全长。

在花后15 d的小麦植株中, 通过半定量PCR法, 发现LSU I基因在叶、籽粒、叶鞘和颖壳中表达量较高, 但在根和茎中表达量较低, 表明在绿色光合功能器官和淀粉合成器官中, LSU I基因量表达较高。

比较了LSU I基因在千粒重不同的豫教2号和兰考矮早八两小麦品种籽粒发育过程中的表达差异后, 发现在籽粒形成期, LSU I基因在两品种籽粒内表达相似, 但在籽粒灌浆期和成熟期间, LSU I基因在千粒重较高的豫教2号内表达量明显高于千粒重较低的兰考矮早八, 这个表达差异与两品种间淀粉积累差异趋势相似, 表明LSU I基因的表达量可能与淀粉的积累量密切相关。

关键词: 小麦; AGPase胞质型大亚基; 基因克隆; RT-PCR; 基因表达Cloning and Expression of AGPase Cytoplasmic Large Subunit Gene in Common WheatKANG Guo-Zhang**, WANG Yong-Hua**, LIU Chao, SHEN Bing-Quan, GUO Tian-Cai*, and ZHU Yun-Ji (Engineering Research Center for Wheat, Henan Agricultural University, Zhengzhou 450002, Henan, China)Abstract: The 1941-bp full cDNA sequence (GenBank No. DQ839506) of LSU I, the cytoplasmic large subunit gene I of adenosine 5' diphosphate glucose pyrophosphorylase (AGPase), was cloned from a high 1000-grain weight wheat (Triticum aestivum L.) cultivar Yujiao 2. Semi-quantitive PCR analysis showed that LSU I expressed higher in leaves, glumes, sheaths, and grain than in roots and stems, indicating that LSU I expressed actively in photosynthetic and starch synthesis-related organs. During the primal period of grain development (5–10 d after thesis), there was no difference in transcript levels of LSU I gene between Yujiao 2 and Lankao Aizao 8 (low 1000-grain weight), but the transcript amounts in grains of Yujiao 2 was markedly higher than that of Lankao Aizao 8 at grain filling and maturity stages. The changes of LSU I gene expression were similar to the trend of starch accumulation in the two cultivars, suggesting that LSU I gene may be associated with starch accumulation in common wheat.Keywords:Triticum aestivum L.; Cytoplasmic large subsuit gene I of AGPase; Gene cloning; RT-PCR; Gene expression小麦的产量由单位面积穗数、穗粒数和千粒重三要素构成。

JNK2、TIMP-1及Collagen Ⅲ在胆道闭锁肝纤维化中的作用研究高婷;詹江华;陈扬;张爱华;卫园园【摘要】目的研究c-Jun氨基末端激酶2(c-Jun N-terminal kinase,JNK2)、金属蛋白酶组织抑制剂-1(tissue inhibitor of metalloproteinase 1,TIMP-1)及Collagen Ⅲ在胆道闭锁(BA)肝组织中的表达情况及在肝纤维化进程中的作用. 方法选取胆管扩张症肝活检病例10例,为胆扩组;BA肝活检病例15例,为BA组;BA 晚期因肝功能衰竭而行肝移植患者自体肝活检10例,为肝移植组.采用HE染色观察并评价肝标本纤维化程度;免疫组化染色检测JNK2、TIMP-1及Collagen Ⅲ在肝组织中的表达;实时荧光定量聚合酶链式反应(qRT-PCR)检测肝组织中JNK2、TIMP-1及Collagen Ⅲ基因表达情况. 结果①HE染色:胆扩组偶可见少许纤维细胞增生;BA组汇管区增宽,纤维组织增生、桥接纤维化现象普遍,可见假小叶形成;肝移植组汇管区明显增宽,纤维组织增生较重,广泛桥接纤维组织形成,假小叶显著.②免疫组化:胆扩组JNK2、TIMP-1及Collagen Ⅲ表达为弱阳性,BA组及肝移植组JNK2、TIMP-1及CollagenⅢ蛋白均在肝细胞、汇管区胆管上皮细胞及血管内皮细胞胞质中阳性表达.③半定量分析:三组JNK2(0.122±0.008、0.182±0.017和0.198±0.033),TIMP-1(0.123 ±0.009、0.185±0.012和0.201 ±0.017)和Collagen Ⅲ(0.126±0.012、0.194±0.008和0.208±0.033)表达比较,差异有显著统计学意义(P＜0.001);三组中,BA组及肝移植组JNK2、TIMP-1及CollagenⅢ表达明显高于胆扩组(P＜0.05);肝移植组JNK2、TIMP-1及Collagen Ⅲ蛋白含量与BA组比较,差异无统计学意义(P值均＞0.05).④qRT-PCR:三组JNK2、TIMP-1和Collagen Ⅲ mRNA表达水平比较,差异有统计学意义(0.221(0.17)vs1.395(1.22)vs 1.095 (1.21),H=17.686,P=0.003;0.439(0.31)vs1.404(0.85)vs 1.571(0.66),H=20.648,P =0.000;0.917(0.09)vs 1.802(1.35)vs 1.957(1.30),H=15.555,P =0.007),BA组及肝移植组肝内JNK2、TIMP-1及Collagen Ⅲ mRNA表达含量比胆扩组高(P值均小于0.017). 结论 JNK2、TIMP-1及Collagen Ⅲ在BA患儿随着肝纤维化加重而表达升高,表明其可能参与并促进BA肝纤维化进程.%Objective To explore the expressions of c-Jun N-terminal kinase (JNK2),tissue inhibitor of metalloproteinase-1 (TIMP-1) and collagen Ⅲ in liver tissues and elucidate their functions in the process of liver fibrosis of biliary atresia (BA).Methods Liver biopsy specimens were collected from congenital biliary dilatation (CBD group,n =10),BA liver biopsy (BA group,n =15),BA children undergoing liver transplantation due to liver failure (liver transplantation group,n =10).Hematoxylin and eosin (HE) staining was used for evaluating the degree of liver fibrosis.And the expressions of JNK2,TIMP-1 and collagen Ⅲ in liver tissue were detected by immunohistochemical staining.Quantitative real-time polymerase chain reaction (qRT -PCR) was used to test the gene expressions of JNK2,TIMP-1 and collagen Ⅲ.Results ①HE staining:Fiber cell hyperplasia in CBD group;Portal area expansion,fibrous tissue proliferation,bridging fibrosis and little few pseudo lobules in Kasai group;Portal area became widened obviously,fibrous tissue proliferation was heavier,bridging fibrosis generally formed,pseudo-lobular was remarkable in liver transplantation group.② Immunohistochemistry:The expressions of JNK2,TIMP-1 and collagen Ⅲ were weakly positive in CBD group.The positive expression of JNK2,TIMP-1 and co llagen Ⅲ protein in hepatocytic cytoplasm,portal area of bile duct epithelial cells and vascular endothelial cells in BA andtransplantation groups.Semi-quantitative analysis:The expression levels of JNK2,TIMP-1 and collagen Ⅲ protein had significant diffe rences among three groups (0.122 ± 0.008 vs 0.182 ± 0.017 vs 0.198 ± 0.033,F =75.687,P =0.000;0.123 ± 0.009 vs 0.185 ±0.012 vs 0.201 ± 0.017,F =99.418,P=0.000;0.126 ± 0.012 vs 0.194 ± 0.008 vs 0.208 ± 0.033,F =54.001,P=0.000);BA and liver transplantation groups were significantly higher than that in CBD group (P ＜ 0.05).No significant differences existed in protein level between BA and transplantation groups (P ＞ 0.05);qRT-PCR:The mRNA expression levels of JNK2,TIMP-1 and collagen Ⅲ had significant differences among three groups (0.221 (0.17) vs 1.395 (1.22) vs 1.095 (1.21),H =17.686,p =0.003;0.439(0.31) vs 1.404(0.85) vs1.571(0.66),H=20.648,P=0.000;0.917(0.09) vs 1.802 (1.35) vs 1.957 (1.30),H =15.555,P =0.007);The mRNA expression of JNK2,TIMP-1 and coll agen Ⅲ were higher in BA group and liver transplantation group than those of CBD group (P ＜ 0.017).Conclusions The expressions of JNK2,TIMP-1 and Collagen Ⅲ increased in liver of BA during fibrosis.It hints that the expressions of JNK2,TIMP-1 and collagen Ⅲ may promote the process of liver fibrosis in BA.【期刊名称】《临床小儿外科杂志》【年(卷),期】2017(016)002【总页数】6页(P127-132)【关键词】胆道闭锁;肝硬化;转化生长因子B1;免疫组织化学【作者】高婷;詹江华;陈扬;张爱华;卫园园【作者单位】天津医科大学研究生院天津市,300070;天津市儿童医院,天津市儿科研究所天津市,300134;天津医科大学研究生院天津市,300070;天津市儿童医院,天津市儿科研究所天津市,300134;天津医科大学研究生院天津市,300070【正文语种】中文胆道闭锁（biliary atresia，BA）是危及婴幼儿生命的肝胆系统疾病之一，以肝内外胆管进行性炎症、纤维性梗阻及胆汁淤积为特点，导致进行性肝纤维化和肝硬化，是婴幼儿时期肝移植的主要指征［1-2］。

SREBP-1c基因过表达细胞模型的构建及对HepG2细胞脂滴含量的影响刘芳;秦双红;赵燕霞【摘要】OBJECTIVE:Construction of a recombinant lentiviral vector overexpressed SREBP-1c,its transfection into human hepatocellular carcinoma cell lines (HepG2) with high transfection efficiency and its effect on cellular lipid storage.METHODS:SREBP-1c gene was cloned from pCMV5-HA-SREBP-1c by polymerase chain reaction and identified by gene sequencing.The SREBP-1c gene was cloned to lentiviral expression vector (pLenti6.3/V5) by recombing DNA technology.The 293T cells were co-transfected with lentiviral packaged systems and the SREBP-1c plasmid by Lipofectamine2000 reageant to package the lentiviral particles,viral supernatant was collected,HepG2 cells were then infected by pLenti6.3-SREBP-1c.After puromycin screening,HepG2 cells were constructed.Real time PCR and Western blot assays were performed to analyzed SREBP-1c expression levels.Cellular lipid storage was detected under fluorescent microscopy and the triacylglyceride(TAG) content by TLC analysis in SREBP-1c-overexpressing HepG2 cells.RESULTS:pLenti6.3-SREBP-1c was successfully constructed and verified by restriction analysis and sequencing.SREBP-1c gene expression level in HepG2 cells with SREBP-1c gene transfection was 11.4 times higher than the control.SREBP-1c protein level in HepG2 cells with SREBP-1c gene transfection was 3.2 times higher than the control.The number and size of lipid droplets (LDs) wasincreased,and a higher concentration of TAG was found in SREBP-1coverexpressing cells compared with the control (P＜0.01).CONCLUSION:Recombinant lentiviral vector pLenti6.3-SREBP-1c and SREBP-1c-overexpressing cell strain was successfully constructed.The high protein and mRNA levels of SREBP-1c were assessed after HepG2 cells were transfected.SREBP-1c increased the lipid storage of HepG2 cells.The constructed cells can become a useful tool for lipid metabolism research of liver carcinoma.%目的:构建SREBP-1c过表达HepG2细胞模型并观察其对脂滴含量的影响,为进一步深入研究肝癌脂滴代谢异常机制提供细胞模型.方法:采用DNA重组技术,将SREBP-1c基因克隆至慢病毒表达载体pLenti 6.3/V5中,并用脂质体介导法将慢病毒包装系统和带目的基因的质粒pLenti6.3-SREBP-1c共转染293T细胞,收集上清,感染HepG2细胞.通过Real-time PCR和Western blot对细胞株进行鉴定.采用免疫荧光检测HepG2细胞中脂滴的数量、大小及细胞内甘油三酯的含量.结果:成功构建了过表达SREBP-1c基因的慢病毒载体pLenti6.3-SREBP-1c,转染后HepG2细胞中可见明显的SREBP-1c蛋白表达.Real-time PCR 检测结果显示转染SREB P-1c基因的HepG2细胞中SREBP-1c mRNA水平较对照组升高了约11.4倍.Western blot结果显示SREBP-1c蛋白水平较对照组提高3.2倍.免疫荧光显示过表达SREBP-1c能够明显增加HepG2细胞内脂滴的数量和大小.细胞内甘油三酯定量结果显示甘油三酯含量较对照组明显升高(P＜0.01).结论:成功构建了,REBP-1c基因过表达的重组慢病毒载体及其稳定转染细胞株.高表达SREBP-1c的HepG2细胞增加了细胞中脂滴的数量、大小和甘油三酯的含量,可用于从脂滴代谢方面对肝癌发病分子机制的研究.【期刊名称】《癌变·畸变·突变》【年(卷),期】2018(030)001【总页数】6页(P14-19)【关键词】SREBP-1基因;慢病毒载体;人肝癌细胞HepG2;基因转染;脂滴【作者】刘芳;秦双红;赵燕霞【作者单位】第四军医大学西京医院皮肤科,陕西西安710032;第四军医大学西京医院病理科,陕西西安710032;解放军第518医院,陕西西安710043;解放军第518医院,陕西西安710043【正文语种】中文【中图分类】Q291肝细胞肝癌(hepatocellular carcinoma，HCC)在全球最常见的恶性肿瘤中排名第5，每年有超过70万的新发病例，年死亡人数高达25万人 [1-2]。

小麦TaTVP 1基因的克隆与表达分析马燕斌1,李换丽1,文晋1,王霞2,周仙婷1,王新胜1(1.山西农业大学/山西省农业科学院棉花研究所,山西运城044000;2.运城学院,山西运城044000)摘要㊀[目的]发掘并研究小麦TaTVP 1基因响应耐盐㊁耐旱等非生物胁迫的功能与机制,为小麦抗逆育种提供新的基因资源㊂[方法]利用PCR 技术克隆小麦TVP 1基因,扩增获得cDNA 全长序列,并对其进行生物信息学分析,进一步利用qRT -PCR 技术分析其表达模式㊂[结果]小麦TVP 1基因全长2289bp ,编码762个氨基酸;其氨基酸序列具有12个跨膜结构,该结构中疏水氨基酸具有高度保守性,且肽链N 末端与C 末端均位于膜外,预测该跨膜结构可形成具有明显孔道的高级3D 结构蛋白,推测与其具有的H +离子泵通道功能密切相关;系统发育树分析表明,TaTVP1蛋白与单子叶植物TVP1蛋白的同源性较高,该类蛋白在进化过程中单子叶和双子叶植物可形成2个明显的类群Ⅰ和Ⅱ;定量PCR 分析表明,小麦中TVP 1基因在根㊁茎中的表达量要明显高于叶㊁穗中,具有组织特异性,且不同材料间同一组织中该基因的表达趋势也存在差异,这可能与不同材料间的耐旱等抗逆性存在差异有关㊂[结论]克隆并分析了小麦TaTVP 1基因,为进一步阐明该基因参与抵御非生物胁迫的机理奠定一定的研究基础㊂关键词㊀小麦;TVP 1基因;生物信息分析;表达模式分析中图分类号㊀S 512.1㊀㊀文献标识码㊀A㊀㊀文章编号㊀0517-6611(2023)17-0087-05doi :10.3969/j.issn.0517-6611.2023.17.019㊀㊀㊀㊀㊀开放科学(资源服务)标识码(OSID):Cloning and Expression Analysis of TaTVP 1Gene in WheatMA Yan-bin ,LI Huan-li ,WEN Jin et al㊀(Shanxi Agriculture University /Institute of Cotton Research,Shanxi Academy of Agricultural Sciences,Yuncheng,Shanxi 044000)Abstract ㊀[Objective]To explore and investigate the functions and mechanism of TaTVP 1genes in wheat in response to abiotic stresses such as salt,drought tolerance,and provide a new genetic resources for wheat stress resistance breeding.[Method]In this study,we cloned the wheat TVP 1gene by PCR,amplified the full-length cDNA sequence,and performed the bioinformatic analysis,and further analyzed the ex-pression pattern by qRT-PCR.[Result]TVP 1gene in wheat is 2289bp and encodes 762amino acids which containing 12transmembrane structures,in these structures,hydrophobic amino acids are highly conserved,and both the peptide chain N and C terminal are located outside the membrane,it is predicted that the transmembrane structure could form a high-level 3D structure protein with obvious pore,which may be closely related to its H +pump channel function;phylogenetic tree analysis indicates high homology of TaTVP 1to monocot TVP 1proteins,and two distinct groups I and II could be classified between monocotyledons and dicotyledons during the evolution of the type of TVP 1genes;quanti-tative PCR analysis showed that the expression of TVP 1gene in roots and stems was significantly higher than that in leaves and ears,with tis-sue specificity,and the expression trend of this gene in the same tissue was also different between different materials,this may be related to the difference in drought resistance and other stress resistance among different materials.[Conclusion]In this study,the TaTVP 1gene was cloned and analyzed,which laid a foundation for further clarifying the mechanism of this gene involved in protection against abiotic stresses.Key words ㊀Triticum aestivum ;TVP 1gene;Bioinformatic analysis;Expression pattern analysis基金项目㊀山西省应用基础研究面上项目(202103021224145);山西省农业科学院农业科技创新研究课题(YGJPY2007);运城市科技局项目(YCKJ -2021023)㊂作者简介㊀马燕斌(1978 ),男,山西清徐人,副研究员,博士,从事作物分子遗传育种研究㊂收稿日期㊀2023-03-21㊀㊀土壤盐碱化等非生物胁迫是导致小麦等农作物减产的主要因素之一,土壤中过高的钠离子浓度会破坏胞内的离子平衡,造成作物体内的代谢途径紊乱,从而抑制其生长㊂为降低盐碱逆境对农作物产量的影响,除改良土壤外,培育耐盐碱农作物品种也可有效抵御土壤盐碱化等不利影响,进而在一定程度上稳定农作物产量㊂多年来,传统遗传育种与分子改良相结合极大地促进了研究人员选育具有耐盐碱㊁耐旱等特性种质资源的效率[1-2]㊂近年来,作物中多种耐旱㊁耐盐碱的基因被克隆鉴定并证实有助于改良作物的耐逆特性㊂H +转运无机焦磷酸酶(H +-PPase,TVP)是一种广泛存在于生物体内的H +转运酶,可作为调解细胞酸度的质子泵,驱动各种离子在液泡内的区隔化,同时也参与了调解植物生长素的运输,在植物环境耐受性㊁生长发育过程中都可能起到了作用[3]㊂有报道称,大豆GmVP 1基因能提高转基因大豆发状根在一定条件下对盐胁迫的反应[4];TPSP 融合基因在小麦中的表达可表现出较强的耐旱性[5];烟草中敲除NtMYB 68转录因子能改善烟叶中紫黄质的堆积,促进脱落酸的合成,从而改善烟株的抗旱性[6];拟南芥中过表达白菜Bp -NFYA 5㊁番茄sly -miR 397基因可以提高其干旱耐受性[7-8];外源基因玉米C4型磷酸烯醇式丙酮酸羧化酶的过表达,可使玉米耐旱性增强[9];GhPNP 1可通过cGMP 信号途径正向调解棉花对干旱的耐受性[10];大豆中的BADH 基因也表现出较强的抗旱性[11];此外,植物耐旱密切相关的还有DREB 类等基因[12-13];细胞质膜蛋白SOS1和液泡膜蛋白NHX 协同作用,使胞质内维持低浓度盐离子,从而提高了植物的抗盐性[14-16]㊂综上所述,各类不同基因的研究极大地丰富了耐旱㊁盐碱基因研究的理论基础,同时也说明作物耐旱㊁盐碱机理可能具有更复杂的多途径调控网络㊂因此,还需对植物的耐盐㊁耐旱性进一步探索,从更多的抗逆性作物着手,挖掘更多相关基因,从细胞㊁组织和整体水平上阐释植物耐干旱㊁盐胁迫的信号调控机制,进而为作物的遗传改良奠定基础㊂现有的研究已有很多关注小麦抗逆材料的创制及其分子机制的解析,笔者以小麦耐旱品种晋麦47和舜麦1718为材料,对其TVP 1基因进行克隆鉴定,并利用生物信息学对该蛋白进行理化性质㊁跨膜结构㊁保守安徽农业科学,J.Anhui Agric.Sci.2023,51(17):87-91㊀㊀㊀结构域和发育进化等方面进行分析,旨在为进一步研究该基因的功能与机理打下基础,同时也为利用该类基因进行植物耐逆优良种质选育提供科学参考㊂1㊀材料与方法1.1㊀试验材料㊀供试材料晋麦47(JM47)㊁舜麦1718 (SM1718)均为山西省农业科学院棉花研究所国审品种,种植于山西省农业科学院棉花研究所试验田㊂1.2㊀材料处理㊀将晋麦47(JM47)㊁舜麦1718(SM1718)2种品种小麦籽粒浸泡在经过高压灭菌的蒸馏水中,过夜萌发,次日挑选生长状态一致饱满的种子,播种于土壤条件相同的花盆中,在22ħ恒温培养箱中进行培养,长日照7d,取苗样液氮速冻,于-80ħ保存,供DNA提取之用㊂小麦在温室盆栽中生长至幼穗阶段时,取根㊁茎㊁叶㊁穗4个组织样品于无RNA酶离心管中,液氮速冻以备RNA提取㊂1.3㊀总DNA提取、RNA提取和cDNA制备㊀DNA按照GENEOUTTM Plant DNA Isolation Kit(LABGENE,上海)试剂盒的说明书进行提取,-20ħ保存㊂小麦材料及其不同组织样品在液氮低温下冷冻磨成样品,分别称取约0.1g样品粉末,加入Trizol试剂(Invitrogen,USA)进行RNA提取,整个试验过程严格按照操作要求进行,避免RNase污染㊂利用分光光度计测定各样品RNA浓度㊁纯度后,以RNA为模板,参考RNA反转录试剂盒GoScriptTM Reverse Transcription Mix, Oligo(dT)(Promega,上海)说明书,合成cDNA第1链,于-20ħ冰箱保存㊂1.4㊀TaTVP1基因的克隆㊀从NCBI数据库下载TaTVP1 (AY296911.1)基因CDS序列,利用Primer Premier6.0软件根据该基因保守序列设计克隆特异性引物,引物由北京奥科生物科技公司合成,序列分别为TaTVP1-F:5ᶄ-CATG-GCGATCCTCGGG-3ᶄ,TaTVP1-R:5ᶄ-CTAGATGTACTTGAAC -3ᶄ㊂以晋麦47和舜麦1718幼苗的cDNA分别为模板,利用Ex Taq酶(Takara,上海)扩增TaTVP1基因的全长序列㊂反应条件为:94ħ预变性4min,94ħ变性40s,60ħ退火40s, 72ħ延伸2min,终延伸10min,30个循环㊂PCR产物经浓度1%琼脂糖凝胶电泳检测,按照DNA纯化回收试剂盒(Omega Bio-Tek,美国)说明书回收片段,进行克隆㊂1.5㊀蛋白结构分析㊀利用SOSUI软件预测分析TaTVP1蛋白的跨膜结构域;利用SWISS-MODEL(https://swissmodel. /interactive)对TaTVP1蛋白的三维结构进行在线预测㊂1.6㊀进化关系分析㊀从NCBI网站blastp分析并下载与TaTVP1氨基酸序列同源性较高的其他物种的氨基酸序列,利用Clustal工具进行多序列比对,MEGA5.0软件分析进化关系,利用Neighbor-Joining算法构建进化树,Bootstrap分析1000次㊂1.7㊀TaTVP1基因的组织表达分析㊀利用Beacon Designer 软件,参照TaTVP1基因序列,选择基因保守区设计特异性的荧光定量引物,内参基因为小麦Actin基因引物(表1)㊂按照SYBR Premix Ex Taq TMⅢ(Tli RNaseH Plus)试剂盒(TaKaRa)说明书,采用Applied Biosystems Step OnePlusTM仪扩增, PCR体系25μL,扩增程序为:95ħ预变性4min,95ħ变性5s,60ħ退火20s,72ħ延伸10s,循环40次㊂设置3次重复㊂数据处理采用2-ΔΔCt法计算相对表达量㊂表1㊀定量PCR分析使用引物序列Table1㊀The sequence of primers used for real-time PCR引物Primers序列Sequences(5ᶄ 3ᶄ)预期大小Expected sizeʊbp TaActin-F GGTGATGAGGCGCAGTCCAAG386 TaActin-R CGACCAGCGAGATCCAAACGATaTVP-F TCAGACTGCCACAAGGC160 TaTVP-R CTAGATGTACTTGAAC2㊀结果与分析2.1㊀TaTVP1基因全长的克隆㊀以晋麦47(JM47)㊁舜麦1718(SM1718)的cDNA为模板,扩增克隆均得到全长2000余bp的TaTVP1基因片段(图1),纯化回收产物,连接克隆载体pMD19-T转化至Trans-T1感受态细胞,随机挑取长势较好的菌落,经菌液PCR检测,筛选出阳性克隆,送至北京奥科生物科技公司测序㊂结果显示,TaTVP1基因序列全长2289bp,推测编码762个氨基酸㊂注:M.5000DNA Marker;1.晋麦47材料扩增的TaTVP1片段;2.舜麦1718扩增的TaTVP1片段㊂Note:M.5000DNA Marker;1:TaTVP1amplified fragment from JM47;2.TaTVP1amplified fragment from SM1718.图1㊀TaTVP1基因的PCR扩增Fig.1㊀PCR amplification of TaTVP1gene2.2㊀小麦TaTVP1的系统进化树分析㊀通过NCBI网站中blastp在线工具对TaTVP1氨基酸序列进行同源性搜索,选取相似性较高的多个作物TVP1氨基酸序列与TaTVP1进行比较分析,利用MEGA软件构建系统进化树,结果显示(图2),各作物TVP1蛋白在进化中分化成两大类,一类是大豆㊁拟南芥㊁棉花㊁烟草聚为一簇的双子叶植物TVP1Ⅰ类;另一类是小麦㊁玉米㊁水稻㊁青稞㊁大麦草TVP1聚为一簇的Ⅱ类,其中小麦TVP1蛋白与水稻㊁玉米等禾本科单子叶植物TVP1蛋白的遗传距离最近㊂这说明TVP1蛋白在单双子叶植物中的遗传分化明显㊂88㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀安徽农业科学㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀2023年注:作物分别为拟南芥(AB015138)㊁油菜(JN940184㊁JN940185)㊁大豆(AK286008)㊁棉花(HM370494)㊁烟草(DQ630713)㊁甜菜(L32792)㊁番茄(AB300442)㊁葡萄(AJ5572565)㊁短芒大麦草(AY255181)㊁青稞(AB032839)㊁玉米(NM001111910㊁BT066389)㊁水稻(D45384)㊁小麦(AAP55210)㊂Note:The crops are Arabidopsis (AB015138),Rape (JN940184,JN940185),Cotton (HM370494),Soybean(AK286008),Tobacco (DQ630713),Sugar beet (L32792),Tomato (AB300442),Grape(AJ5572565),Short awn barley grass(AY255181),Highland bar-ley(AB032839),Corn(NM001111910,BT066389),Rice(D45384),Wheat(AAP55210).图2㊀小麦TaTVP1与其他植物TVP1的系统进化树分析Fig.2㊀Phylogenetic tree of predicted a mino acid sequences fromwheat TaTVP1and other plant TVP12.3㊀TaTVP1蛋白的跨膜结构预测分析㊀利用SOSUI 软件对TaTVP1蛋白进行跨膜结构预测分析,表明TaTVP1蛋白的氨基酸序列包含12个疏水跨膜结构域(图3)㊂亚结构域分析显示,小麦TVP1蛋白分为2个独立的结构,一个是N 端,包含4个疏水的跨膜区域,另一端是C 端,包含8个疏水跨膜区域,其多肽链的N 端和C 端均在膜外㊂2.4㊀TaTVP1跨膜氨基酸序列比对分析㊀小麦TVP1编码762个氨基酸,由该氨基酸序列(图4)包含的12个跨膜结构域(I -XII)序列的分析和比较可知,小麦TVP1与拟南芥㊁水稻各跨膜区中保守氨基酸一致的分别为10㊁13㊁15㊁19㊁21㊁20㊁13㊁19㊁21㊁19㊁25及17个,各跨膜区疏水氨基酸的个数依次为16㊁15㊁12㊁13㊁11㊁13㊁14㊁15㊁17㊁13㊁11及14个㊂2.5㊀TaTVP1蛋白三级结构域预测㊀根据前述预测的不同疏水氨基酸跨膜结构,进一步预测TaTVP1蛋白在细胞内形成的功能结构,分别利用不同长度的氨基酸序列进行模型匹配比较分析可知,全长TaTVP1氨基酸可折叠形成膜结构孔道蛋白高级结构(图5A),该孔道具有较清晰的管状结构;其中N 段300氨基酸(图5B)与C 端301~762aa(图5C)能够形成独立的孔道结构组成模型㊂2.6㊀TaTVP 1在小麦不同组织的表达分析㊀选用耐旱品种晋麦47和水地品种舜麦1718为材料,分析TaTVP 1基因在根㊁茎㊁叶㊁穗4个不同组织的表达水平,并比较该基因在2个品种中不同组织的表达水平㊂定量PCR 结果显示(图6),TaTVP 1在不同组织中的表达水平存在明显差异,在根与茎中的表达量较高,叶㊁穗组织中表达量较低㊂在2个品种间,晋麦47的茎中表达量最高,高出根中表达量的1.63倍,穗中表达量最低;而舜麦1718茎中TaTVP 1表达量与根中的表达量差异很小,叶㊁穗中的表达量较低㊂图3㊀TaTVP1跨膜结构分析Fig.3㊀Characteristics of transmembrane structure of TaTVP13㊀结论与讨论植物对逆境非生物胁迫的响应过程十分复杂,涉及生理㊁生化特性的多种过程㊂在植物中,H +转运无机焦磷酸酶与NHX 逆向转运蛋白可以稳定胞质内的酸碱度,使胞质内的Na +维持在较低浓度,进而增强植物的耐盐㊁耐旱性[15-16]㊂该研究中克隆所获得的小麦TVP 1基因的核苷酸序列与参考基因序列存在一些单核苷酸差异,这可能是不同品种小麦之间单核苷酸多态性造成的㊂而且,六倍体小麦中含有A㊁B㊁D3个染色体组,TVP 1在不同染色体组中还存有同源基因[17]㊂TaTVP1蛋白中12个跨膜区域中的疏水氨基酸序列具有较高的一致性,表明该蛋白在进化过程中,结构域遗传保守性较高,也预示着其在细胞中的功能不可替代㊂有研究报道,9851卷17期㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀马燕斌等㊀小麦TaTVP 1基因的克隆与表达分析注:I~XII 为分析跨膜序列结果所标示的氨基酸序列,疏水性氨基酸有A 丙氨酸㊁Y 酪氨酸㊁F 苯丙氨酸㊁W 色氨酸㊁V 缬氨酸㊁I 异亮氨酸㊁L 亮氨酸和P 脯氨酸㊂Note:I -XII is the amino acid sequence marked by the analysis of transmembrane sequence results.Hydrophobic amino acids include A alanine,Y tyro-sine,F phenylalanine,W tryptophan,V valine,I isoleucine,L leucine and P proline.图4㊀TaTVP1的跨膜氨基酸保守序列Fig.4㊀Amino acid sequence of TaTVP1transmembrane注:A㊁B㊁C㊁D 分别表示氨基酸序列为全长㊁N 端1~300aa㊁C 端301~762aa 及241~762aa㊂Note:A,B,C and D represent the full length,N-terminal 1-300aa,C-terminal 301-762aa and 241-762aa respectively.图5㊀TaTVP1蛋白的3级结构分析Fig.5㊀Tertiary structure analysis of TaTVP1protein图6㊀TaTVP 1在小麦不同组织中的表达量Fig.6㊀Expression of TaTVP 1in different tissues of wheat9㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀安徽农业科学㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀㊀2023年拟南芥NHX肽链C末端的缺失可引起Na+/H+交换活性发生明显变化[18],表明C末端与Na+/H+交换活性密切相关,分析显示TVP1的C末端位于膜外的氨基酸序列较长,且亲水氨基酸含量较高,推测该序列可能参与胞质内的离子交换,影响Na+/H+的交换活性,同时,TVP1蛋白三级结构具有明显的孔道结构,也预示与其具有的质子泵作用密切相关,但具体的行使功能过程需要进一步探究㊂㊀㊀研究表明,植物耐盐性与耐旱性具有一定的相关性,耐旱材料往往也表现出较好的耐盐性㊂过表达液泡H+焦磷酸酶的转基因植物对高浓度NaCl的耐受性更强,对干旱的耐受性也更强,由此可知该基因有助于提高植株的耐旱和耐盐性[19-20]㊂而在各种非生物胁迫下,许多转H+焦磷酸酶基因植物的茎和根生物产量增加[21-24]㊂该研究组织特异性表达结果中,该基因在根㊁茎中的相对表达量明显高于叶㊁穗中,这可能与TVP1在不同组织中抗逆性的作用特征有关;耐旱品种晋麦47与舜麦1718相比,茎部表达量又明显高于根部,这种表达模式可能与晋麦47表现出优良的耐旱性特征相关,后续可以通过转化该基因等方法对其功能进行进一步阐释㊂该研究获得并分析了小麦TVP1基因的核苷酸与氨基酸序列,对其表达模式也进行了研究㊂后期将在小麦中过量表达TVP1基因,探究其耐旱㊁耐盐功能,并进一步阐释其机制,从而为培育耐逆优良种质的植物提供理论基础㊂参考文献[1]FLEURY D,JEFFERIES S,KUCHEL H,et al.Genetic and genomic tools to improve drought tolerance in wheat[J].Journal of experimental botany, 2010,61(12):3211-3222.[2]MIR R R,ZAMAN-ALLAH M,SREENIVASULU N,et al.Integrated ge-nomics,physiology and breeding approaches for improving drought toler-ance in crops[J].Theoretical and applied genetics,2012,125(4):625-645.[3]MAESHIMA M.Vacuolar H+-pyrophosphatase[J].Biochimica biophysica acta(BBA):Biomembranes,2000,1465(1/2):37-51.[4]赵甜甜,范会芬,孙天杰,等.GmVP1基因克隆与转GmVP1/GmNHX1双价基因的大豆发状根耐盐性分析[J].河北农业大学学报,2020,43(2): 19-25,33.[5]李金花,孙敏善,张春艳,等.转TPSP融合基因小麦的耐旱相关特性[J].植物生理学报,2012,48(1):81-84.[6]程泽华,王召军,单玉静,等.NtMYB68基因对烟草苗期紫黄质和脱落酸生物合成及烟株耐旱性的影响研究[J].中国烟草学报,2022,28(2): 122-130.[7]黄人卉,王桂香,刘凡.大白菜耐旱相关基因BpNFYA5的克隆及功能初步分析[J].园艺学报,2012,39(8):1501-1510.[8]向娟,林鹏,李兴盛,等.过表达番茄Sly-miR397基因增强拟南芥的耐旱性[J].中国农业大学学报,2016,21(10):51-58.[9]宋凝曦,李霞,王净,等.大环内酯类和高表达玉米C4-PEPC基因对水稻耐旱性的影响[J].作物学报,2021,47(10):1927-1940.[10]刘小双,刘廷利,袁洪波,等.棉花钠尿肽基因GhPNP1的耐旱功能分析[J].中国农业科学,2015,48(12):2306-2316.[11]秦迪,赵翠兰,郑成忠,等.转BADH基因大豆耐旱性分析[J].中国油料作物学报,2015,37(6):752-758.[12]LATA C,PRASAD M.Role of DREBs in regulation of abiotic stress re-sponses in plants[J].Journal of experimental botany,2011,62(14):4731 -4748.[13]AKHTAR M,JAISWAL A,TAJ G,et al.DREB1/CBF transcription fac-tors:Their structure,function and role in abiotic stress tolerance in plants [J].Journal of genetics,2012,91(3):385-395.[14]ZHU J K.Salt and drought stress signal transduction in plants[J].Annual review of plant biology,2002,53:247-273.[15]JIANG X Y,LEIDI E O,PARDO J M.How do vacuolar NHX exchangers function in plant salt tolerance?[J].Plant signaling&behavior,2010,5(7):792-795.[16]XU K,HONG P,LUO L J,et al.Overexpression of AtNHX1,a vacuolar Na+/H+antiporter from Arabidopsis thalina in Petunia hybrida enhances salt and drought tolerance[J].Journal of plant biology,2009,52(5):453-461.[17]MENADUE D J,RIBONI M,BAUMANN U,et al.Proton-pumping pyro-phosphatase homeolog expression is a dynamic trait in bread wheat(Triti-cum aestivum)[J].Plant direct,2021,5(10):1-16.[18]YAMAGUCHI T,APSE M P,SHI H Z,et al.Topological analysis of a plant vacuolar Na+/H+antiporter reveals a luminal C terminus that regu-lates antiporter cation selectivity[J].Procssdings of the national academy of sciences of the United States of America,2003,100(21):12510-12515.[19]GAXIOLA R A,LI J,UNDURRAGA S,et al.Drought-and salt-tolerant plants result from overexpression of the AVP1H+-pump[J].Procssdings of the national academy of sciences of the United States of America,2001, 98(20):11444-11449.[20]SCHILLING R K,TESTER M,MARSCHNER P,et al.AVP1:One pro-tein,many roles[J].Trends in plant of science,2017,22(2):154-162.[21]SCHILLING R K,MARSCHNER 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文章编号:1001-2486(2008)02-0056-05东乡野生稻NBS 2L RR 类抗性基因同源序列的分离与鉴定Ξ王正华1,杲修杰1,曹孟良2(1.国防科技大学计算机学院,湖南长沙　410073;　2.国家杂交水稻工程技术研究中心,湖南长沙　410125)摘　要:基于植物的抗性基因具有保守序列,根据已克隆基因的保守序列设计探针,利用菌落杂交的方法,对东乡野生稻大片段基因组文库进行筛选,得到两个候选克隆。

序列比对发现其中含有N B 2ARC 的保守序列,可用于后续转基因鉴定。

关键词:NBS 2LRR ;抗性基因;菌落杂交;基因组文库中图分类号:Q94-33 文献标识码:AIsolation and Character ization of NBS 2L RR Resistance G eneH omology Sequences fr om Dongxiang Wild RiceWA NG Z heng 2hua 1,G A O X iu 2jie 1,CA O Meng 2liang 2(1.C oll ege of C om puter ,National Univ.of De fense Technology ,Changsha 410073,China ;2.Nati onal Hybrid R ice R&D Cent er ,C hangs ha 410125,Chi na)Abstract :T he resis tan t g ene o f plants has conserv ative sequence.Probes w ere designed in terms of the sequences of kn own resistance gene.With th e colony hybridization meth od ,the g enomic library of D ong xiang wild rice w as screened and tw o cand idate clones w ere ob tained.T he sequences w ere analy zed w ith the blast program to the G enBank databas e and a conservative N B 2ARC domain was found in both of the sequences.T he clon es w ould be trans ferred in to rice for a f urther analysis.K ey w or ds :N BS 2LRR ;resistance gene ;colony h ybridization ;gen omic library植物病虫害是导致农作物减产的重要原因,每年的损失估计约数亿美元[1]。