大豆耐盐基因GmHAL3a的克隆及RNAi载体的构建  

Cloning of Halotolerance 3 Gene and Construction of Its RNAi Vector in Soybean(Glycine max)

作　　者：王明霞[1] 崔晓霞[1] 薛晨晨[1] 徐筋燕[1] 薛冬[1] 郭娜[1] 邢邯[1] 

机构地区：[1]南京农业大学国家大豆改良中心,江苏南京210095

出　　处：《大豆科学》2013年第1期12-18,共7页Soybean Science

基　　金：国家转基因专项(2011ZX08004-002-001);公益性行业(农业)科研专项(CARS-004-PS10);国家重点基础研究发展计划"973计划"项目课题(2009CB118400);江苏省科学技术厅项目(BE2011305)

摘　　要：利用同源克隆方法从大豆中克隆到一个耐盐基因HAL3,命名为GmHAL3a,组织表达分析发现GmHAL3a基因在大豆根中表达量最高,荚中次之,叶中表达量最低;非生物胁迫实验表明该基因受NaCl、LiCl和山梨醇诱导表达;通过生物信息学的方法分析发现该基因具有2个跨膜螺旋区域,可能定位于叶绿体中;同源序列比对证实该基因氨基酸序列具有与其他物种相似的保守位点:黄素单核苷酸(FMN)结合位点和磷酸泛酰半胱氨酸(PPC)脱羧酶活性位点。同时扩增了这个基因的2个干扰片段,用Gateway技术将扩增的片段通过BP反应连接到入门载体pDONR221,测序后又通过LR反应分别将目的片段插入到RNAi载体pB7GWIWG2(Ⅱ),再转入根癌农杆菌EHA105中。这2个载体的构建对于进一步研究大豆中GmHAL3a基因的功能具有重要意义。Halotolerance 3 gene was isolated from soybean（ Glycine max）by homology cloning, designed as GmttAL3a. Tissue specific analysis found that GmHAL3a expressed mainly in root, relatively lower in pod and lowest in leaf. RT-PCR revealed that the expression of GmttAL3a was induced by salt, LiC1 and sorbitol. Bioinfollnatics analysis indicated that GmHAL3a had two transmembrane helices regions and two conserved domains,flavin mononucleotide（FMN） binding sites and 4＇ -phosphopan- tothenoylcysteine（PPC） decarboxylase activity sites, and was possibly localized in chloroplast. We cloned two segments of Gm- HAL3a,ligased into entry clone vector （pDONR221）by BP reaction, and then ligased into RNAi transformation vector pB7 GWIWG2 （ Ⅱ ）by LR reaction. Finally we transformed the two vectors into Agrobacterium tumefaciens EHA105. The results provide foundation for further research the function of GmHAL3a gene in soybean.

关 键 词：GATEWAY技术 大豆 RNA干扰 GmHAL3a基因 

分 类 号：S565.1[农业科学—作物学]