转GmWRKY70基因大豆的PCR检测及其T-DNA侧翼序列分析  

PCR Detection and T-DNA Flanking Sequence Analysis of GmWRKY70 in Transgenic Soybean(Glycine max)

作　　者：杨清华[1] 董德坤[1] 操晶 梅娜 陈芬 胡兴旺 朱丹华[1] YANG Qing-Hua;DONG De-Kun;CAO Jing;MEI Na;CHEN Fen;HU Xing-Wang;ZHU Dan-Hua(Institute of Crop and Nuclear Technology Utilization,Zhejiang Academy of Agricultural Sciences,Hangzhou 310021,China;School of Horticulture and Gardening,Yangtze University,Jinzhou 434025,China;College of Chemistry and Life Sciences,Zhejiang Normal University,Jinhua 321004,China)

机构地区：[1]浙江省农业科学院作物与核技术利用研究所,杭州310021 [2]长江大学园艺园林学院,荆州434025 [3]浙江师范大学化学与生命科学学院,金华321004

出　　处：《农业生物技术学报》2020年第7期1203-1210,共8页Journal of Agricultural Biotechnology

基　　金：国家转基因生物新品种培育重大专项(2016ZX08004002-010)。

摘　　要：对转基因株系的PCR检测以及对T-DNA侧翼基因组序列的深入分析,可以充分了解转基因株系的特异性分子特征。本研究利用农杆菌(Agrobacterium)介导的大豆(Glycine max)子叶转化方法,获得了3个转GmWRKY70基因的大豆株系。本研究对除草剂抗性基因(bialaphos resistance,Bar)、目标基因GmWRKY70以及载体构建特异性片段进行PCR检测,利用高效热不对称交错PCR(high-efficient thermal asymmetric interlaced PCR,Hi-TAIL PCR)对T-DNA侧翼序列进行整合位点分析。结果表明,3个转GmWRKY70基因的大豆株系均为阳性株系。T-DNA在43119699~43119712 bp之间反向整合到大豆染色体Chr07中。T-DNA整合导致大豆染色体Chr07插入位点12 bp缺失。对于引入的T-DNA,发现左边界(left border,LB)的80 bp序列和右边界(right border,RB)的22 bp序列被截短。在导入的LB端和大豆基因组DNA的整合位点上发现了两个碱基对的微同源性,而RB未发现同源性。本研究设计新的整合位点特异性引物,并验证这种转化事件,为促进分子辅助选择在育种计划中的应用提供了资料基础。The PCR detection of transgenic lines and the in-depth analysis of T-DNA flanking genome sequence can help to understand the specific molecular characteristics of transgenic lines.Three GmWRKY70 transgenic soybean(Glycine max)lines were developed using Agrobacterium-mediated soybean cotyledonary nod transformation method.Transformation events were confirmed by PCR detections of selective marker bialaphos resistance(Bar)gene,target gene GmWRKY70,as well as vector-construction-specific fragments and the integration site of T-DNA flanking sequence was analyzed by high-efficient thermal asymmetric interlaced PCR(Hi-TAIL PCR).The results showed that all 3 GmWRKY70 transgenic soybean lines were positive.T-DNA was reversely integrated into soybean chromosome Chr07 between 43119699 and 43119712 bp.The T-DNA integration led to a 12 bp deletion at the insertion site of soybean chromosome Chr07.For the introduced T-DNA,it was found that the 80 bp sequence at the end of the left border(LB)and the 22 bp sequence at the end of the right border(RB)were truncated.Micro-homologies of 2 base pairs were found between the introduced LB end and soybean genomic DNA at the integration site,while no homology was found for RB.New integration-site-specific primers were designed to validate this transformation events and could further facilitate molecular assisted selections in breeding program.

关 键 词：大豆转基因株系 GmWRKY70 高效热不对称交错PCR(Hi-TAIL PCR) T-DNA 

分 类 号：S565.1[农业科学—作物学]