转Cry1Ac-2A-gna基因甘蔗BCG-17转化体特异性检测方法的建立  

Establishment of a Transformant-specific Detection Method for Cry1Ac-2A-gna Transgenic Sugarcane BCG-17

作　　者：冯翠莲[1] 万玥 王俊刚[1] 冯小艳[1] 赵婷婷[1] 王文治[1] 沈林波[1] 张树珍[1,2] FENG Cui-lian;WAN Yue;WANG Jun-gang;FENG Xiao-yan;ZHAO Ting-ting;WANG Wen-zhi;SHEN Lin-bo;ZHANG Shu-zhen(Institute of Tropical Bioscience and Biotechnology,CATAS,Sugarcane Research Center,Ministry of Agriculture and Rural Affairs Key Biotechnology Laboratory for Tropical Crops,Haikou 571101;College of Life Science,Nanjing Agriculture University,Nanjing 210095)

机构地区：[1]中国热带农业科学院热带生物技术研究所甘蔗研究中心,农业农村部热带作物生物技术重点开放实验室,海口571101 [2]南京农业大学生命科学学院,南京210095

出　　处：《生物技术通报》2021年第5期248-258,共11页Biotechnology Bulletin

基　　金：中国热带农业科学院基本科研业务费专项(1630052020005);国家糖料产业技术体系“甘蔗病毒病防控岗位科学家”经费(CARS-170301)。

摘　　要：转基因作物外源T-DNA插入的侧翼序列是转基因事件检测的关键组分,也是转基因作物生物安全评价和监管所必需提供的重要信息。为了明确抗虫转基因甘蔗BCG-17的分子特征和建立其事件特异性检测方法,推进其生物安全性评价工作及未来的监管工作,以其T2代为研究材料,通过Southern 杂交检测外源基因在甘蔗基因组内的拷贝数;利用染色体步移技术分离外源基因在甘蔗基因组中插入位点的侧翼序列,并建立该转化体的高效灵敏的特异性PCR检测方法。结果表明:Southern杂交检测证明外源T-DNA以单拷贝方式插入BCG-17株系;经过3-4次的热不对称PCR扩增,分离到510 bp的T-DNA左侧翼序列和915 bp的右侧翼序列;以此为基础,分别设计左右侧翼检测引物,建立了BCG-17株系的转化事件特异性PCR检测方法,左侧翼的PCR检测方法特异性强、灵敏度高,检出极限为0.1%,相当于9个单倍体甘蔗基因组拷贝数。转基因株系BCG-17的分子特征及其转化事件特异性检测的完成,为该转基因甘蔗及其衍生产品的检测和身份识别提供了技术依据。The flanking sequence of T-DNA inserted into the plant’s genome is a key component of transgenic event detection,and it is also important information that must be provided for biosafety evaluation and supervision of transgenic crops.In order to clarify the molecular characteristics of insect-resistant transgenic sugarcane BCG-17 and establish its event-specific detection method,as well as to promote its biosafety assessment and future regulatory work,the T2 generation of BCG-17 was used as the study material,and the copy number of foreign genes was detected by Southern hybridization,and the flanking sequence of the insertion site were isolated by the chromosome walking technology.An efficient and sensitive specific PCR method for detecting this transformant was established.The results showed that the foreign T-DNA inserted into the BCG-17 strain was a single copy.After 3-4 times amplification of thermal asymmetric nested PCR,the left flanking sequence was 510 bp and the right flanking sequence was 915 bp;and detection primers were designed respectively based on these sequences,then the transformation event-specific PCR detection system of BCG-17 strain was established.This method presented strong specificity and high sensitivity to the left flanking,and the limit of detection was 0.1%(approximates to 9 haploid genome copies).The completion of the molecular characteristics and the establishment of the transformation event-specific detection in the transgenic strain BCG-17 provides a technical basis for the detection and identification of the transgenic sugarcane and its derivatives.

关 键 词：抗虫转基因甘蔗 T-DNA侧翼序列 转化事件特异性检测 染色体步移 

分 类 号：S566.1[农业科学—作物学] Q943.2[生物学—植物学]