[1]王明霞,崔晓霞,薛晨晨,等.大豆耐盐基因GmHAL3a的克隆及RNAi载体的构建[J].大豆科学,2013,32(01):12-18.[doi:10.3969/j.issn.1000-9841.2013.01.004]
　WANG Ming-xia,CUI Xiao-xia,XUE Chen-chen,et al.Cloning of Halotolerance 3 Gene and Construction of Its RNAi Vector in Soybean (Glycine max)[J].Soybean Science,2013,32(01):12-18.[doi:10.3969/j.issn.1000-9841.2013.01.004]
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大豆耐盐基因GmHAL3a的克隆及RNAi载体的构建

《大豆科学》[ISSN:1000-9841/CN:23-1227/S] 卷: 第32卷 期数: 2013年01期 页码: 12-18 栏目: 出版日期: 2013-02-25
Title:
Cloning of Halotolerance 3 Gene and Construction of Its RNAi Vector in Soybean (Glycine max)
作者:
王明霞;崔晓霞;薛晨晨;徐筋燕;薛冬;郭娜;邢邯
南京农业大学国家大豆改良中心
Author(s):
WANG Ming-xia; CUI Xiao-xia; XUE Chen-chen; XU Jin-yan; XUE Dong; GUO Na; XING Han
National Center for Soybean Improvement,Nanjing Agricultural University,Nanjing 210095,Jiangsu,China
关键词:
Gateway技术; 大豆; RNA干扰; GmHAL3a基因
Keywords:
Gateway technology; Soybean; RNA interference; GmHAL3a gene
分类号:
S565.1
DOI:
10.3969/j.issn.1000-9841.2013.01.004
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。
Abstract:
Halotolerance 3 gene was isolated from soybean(Glycine max)by homology cloning,designed as GmHAL3a.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 GmHAL3a was induced by salt,LiCl and sorbitol.Bioinformatics analysis indicated that -GmHAL3a had two transmembrane helices regions and two conserved domains,flavin mononucleotide(FMN)binding sites and 4’-phosphopantothenoylcysteine(PPC)decarboxylase activity sites,and was possibly localized in chloroplast.We cloned two segments of GmHAL3a,ligased into entry clone vector(pDONR221)by BP reaction,and then ligased into RNAi transformation vector pB7GWIWG2(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.

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备注/Memo

基金项目：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）。

第一作者简介：王明霞（1986-），女，在读硕士，研究方向为大豆的分子生物学。E-mail:yuewang0122@126.com。
通讯作者：邢邯（1963-）,男，教授，博士生导师，从事大豆遗传育种研究。E-mail：hanx@njau.edu.cn。

更新日期/Last Update: 2013-02-20