转基因耐逆大豆FvC5SD-L05事件的T-DNA侧翼序列分析及其特异性检测  

T-DNA Flanking Sequence Analysis and Specific Detection of Transgenic Soybean FvC5SD-L05

作　　者：张维东[1] 袁翊新 李明月 孟凡立[2] 张玲[1] ZHANG Wei-dong;YUAN Yi-xin;LI Ming-yue;MENG Fan-li;ZHANG Ling(Institute of Agricultural Biotechnology,Jilin Academy of Agricultural Sciences,Changchun 130033;College of Agriculture,Northeastern Agricultural University,Harbin 150030)

机构地区：[1]吉林省农业科学院农业生物技术研究所,长春130033 [2]东北农业大学农学院,哈尔滨150030

出　　处：《植物遗传资源学报》2022年第5期1500-1507,共8页Journal of Plant Genetic Resources

基　　金：吉林省农业科学院基本科研经费定向委托和杰出青年项目(KYJF2021JQ001)。

摘　　要：转FvC5SD-L05基因大豆事件是利用农杆菌介导的大豆子叶节遗传转化法将从金针菇克隆到的FvC5SD基因导入大豆栽培品种沈农9号中获得的纯合耐旱转基因大豆事件,该材料具有良好的耐旱特性和农艺性状。为了明确该转基因大豆材料的分子特征及其检测方法,从而推进其生物安全性评价工作,本研究以转基因大豆FvC5SD-L05的T_(4)代纯合株系为研究对象,利用Southern杂交方法鉴定了外源基因的拷贝数;及基因组重测序技术分析外源基因FvC5SD在大豆基因组中插入位点的位置和方向。同时利用PCR扩增技术获得了外源T-DNA的左右侧翼序列,并基于左右旁侧序列,建立了转FvC5SD基因耐逆大豆L05事件的特异性定性PCR检测方法。该方法特异性强、灵敏度高,能够在FvC5SD-L05基因组DNA含量为100 ng的模板中检测出转基因成分,实现了对转FvC5SD基因耐逆大豆L05事件的特异性检测,进一步为该转基因大豆及其衍生产品的检测和身份识别提供技术依据。The FvC5SD gene from Flammulina velutipes was transformed into soybean cultivar Shennong 9 by Agrobacterium mediated genetic transformation of soybean cotyledon nodes.The transgenic soybean line FvC5SD-L05 showed good drought tolerance and agronomic performance.To clarify the characteristics of the transgenic soybean FvC5SD-L05 material meeting its biosafety evaluation,we analyzed the copy number of FvC5SD gene in T_(4) generation plant by Southern hybridization and identified the position and direction of the insert in soybean genome.Meanwhile,we established a specific qualitative PCR detection method for FvC5SD-L05 by PCR amplification of the flanking sequences of exogenous T-DNA.This method represents specificity with high sensitivity for being able to detect transgenic fragment in the template of 100 ng genomic DNA content of FvC5SD-L05.The specific detection of FvC5SD-L05 provided technical basis to identify the transgenic soybeans with the target gene.

关 键 词：转基因大豆 T-DNA旁侧序列 特异性检测 FvC5SD基因 

分 类 号：S565.1[农业科学—作物学]