[1]马阔,仲晓芳,牛陆,等.耐盐转基因大豆事件FA8015旁侧序列分离及定性PCR检测 [J].大豆科学,2018,37(06):854-859.[doi:1011861/jissn1000-98412018060854]
　MA Kuo,ZHONG Xiao-fang,NIU Lu,et al.Flanking Sequence Isolation of Salt-Tolerant Transgenic Event FA8015 of Soybean and Quantitative PCR Test [J].Soybean Science,2018,37(06):854-859.[doi:1011861/jissn1000-98412018060854]
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耐盐转基因大豆事件FA8015旁侧序列分离及定性PCR检测

《大豆科学》[ISSN:1000-9841/CN:23-1227/S] 卷: 第37卷 期数: 2018年06期 页码: 854-859 栏目: 出版日期: 2018-11-20
Title:
Flanking Sequence Isolation of Salt-Tolerant Transgenic Event FA8015 of Soybean and Quantitative PCR Test
作者:
马阔1; 2; 仲晓芳2; 牛陆2; 赵倩倩2; 杨向东2
（1吉林师范大学 生命科学学院，吉林 四平 136000；2吉林省农业科学院 农业生物技术研究所/吉林省农业生物技术重点实验室，吉林 长春 130033）
Author(s):
MA Kuo1; 2;  ZHONG Xiao-fang2;  NIU Lu2;  ZHAO Qian-qian2;  YANG Xiang-dong2
（1 School of Life Science, Jilin Normal University, Siping 136000, China; 2 Agro-Biotechnology Institute, Jilin Academy of Agricultural Sciences / Jilin Provincial Key Laboratory of Agricultural Biotechnology, Changchun 130033, China）
关键词:
大豆; 耐盐; 转基因事件; 重测序; 旁侧序列; 定性PCR检测; 耐盐
Keywords:
Soybean;  Salt tolerance;  Transgenic event;  Re-sequencing;  Flanking sequence;  Qualitative PCR detection
DOI:
1011861/jissn1000-98412018060854
文献标志码:
A
摘要:
本研究前期利用转基因技术，将菠菜（Atriplex hortensis）甜菜碱醛脱氢酶编码基因AhBADH导入栽培大豆Williams 82中，获得耐盐性较强的转基因大豆事件FA8015。为进一步加快该转基因事件生物安全评价，本研究分离了FA8015外源T-DNA旁侧序列，并依据其序列特征，建立事件特异性检测方法。Southern杂交结果表明，转基因事件FA8015外源T-DNA为单拷贝插入。利用基因组重测序技术，获得转基因大豆事件FA8015的基因组信息，并与参考基因组Williams 82做比较，初步确定转基因大豆事件FA8015外源T-DNA片段整合位点。然后根据整合位点，设计融合大豆基因组和T-DNA序列的引物，获得了1 160 bp的左边界旁侧序列，包括405 bp的大豆基因组序列和750 bp的T-DNA序列；右边界获得了1 254 bp的旁侧序列，其中601 bp为T-DNA片段，653 bp为大豆基因组序列；序列分析证明转基因事件FA8015的T-DNA整合位点为Chr15染色体的25129882位点，整合方式为正向单拷贝插入，与Southern杂交和重测序结果一致。依据PCR结果设计事件特异性引物，建立了转基因大豆事件FA8015转化体特异性检测方法。该方法特异性强、灵敏度高，可快速识别该转基因大豆事件的身份，为该转化事件及其衍生品的安全性和监管提供技术支撑。
Abstract:
In our previous study, we introduced the betaine-aldehyde dehydrogenase encoding gene AhBADH from Atriplex hortensis into the soybean cultivar Williams 82 to generate the salt-tolerant transgenic event FA8015 To advance safety assessment of the transgenic event, in this study, we further analyzed the flanking sequences of the foreign T-DNA insertion and established an event-specific detection method based on these flanking sequences Southern blot analysis confirmed the single-copy insertion of the T-DNA in the genome of the transgenic event FA8015 Genome re-sequencing method was used to obtain the genomic sequences of transgenic event FA8015 The integration site of T-DNA in FA8015 was preliminary determinated compared with Williams 82 genome information Then the primers were designed according to the flanking sequences of integration site for PCR amplification After sequencing these fragments, a 1 160 bp fragment was amplified from left border of T-DNA, including 405 bp fragment from soybean genome and 750 bp sequence from T-DNA, and a 1 254 bp product was obtained from right border with 601 bp T-DNA fragment and 653 bp soybean genome sequence These results indicated that one copy of T-DNA in FA8015 was integrated into soybean genome at 2512882 on Chr15 in accord with Southern blot and genome re-sequencing Event-specific primers were designed base on fragment sequencing and then event-specific PCR was constructed This method could identify this transgenic event quickly and specifically And it also provided support for the safety and supervision of the transgenic event FA8015 and its derivatives

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备注/Memo

收稿日期：2018-07-18

基金项目：国家转基因生物新品种培育科技重大专项(2016ZX08004-004)；吉林省农业科学院创新工程项目（c6215000220，c7208000307）。
第一作者简介：马阔（1993-），男，硕士，主要从事大豆生物技术育种研究。E-mail：448586253@qqcom。
通讯作者：杨向东（1976-），男，博士，研究员，主要从事大豆生物技术育种研究。E-mail：xdyang020918@126com。

更新日期/Last Update: 2018-12-04