不同转化方法获得的转基因棉花外源基因拷贝数分析  

Comparative Analysis of Exogenous Gene Copy Numbers in Transgenic Cotton Transformed by Different Methods

作　　者：杨晓杰[1,2] 刘传亮[1] 张朝军[1] 武芝霞[1] 张雪妍[1] 刘坤[1] 房卫平[2] 李付广[1] 

机构地区：[1]中国农业科学院棉花研究所农业部棉花遗传改良重点开放实验室,安阳455000 [2]河南省农业科学院院经济作物研究所,郑州450002

出　　处：《农业生物技术学报》2011年第2期221-229,共9页Journal of Agricultural Biotechnology

基　　金：国家重点基础研究发展规划(973)项目(2010CB126005)资助

摘　　要：转基因植物中外源基因拷贝数是影响其自身表达水平与遗传稳定性的重要因素。本研究旨在比较农杆菌介导法、基因枪轰击法和花粉管通道法等3种常用方法转化同一植物表达载体的转基因棉花(Gossypium hirsutum)中外源基因拷贝数的差异。本文主要采用实时定量PCR技术及相应的PfaffI分析法检测转基因棉花单株拷贝数,并与Southern杂交做了相互验证。实时定量PCR研究结果显示,农杆菌介导法、基因枪轰击和花粉管通道注射法获得的T0代转化体中,外源基因拷贝数为1～2的单株占群体总数的百分比分别为26.2%、60%和42.8%。外源基因拷贝数为3～5的单株比例分别为47.5%、40%和57.1%。另外,农杆菌介导获得的转基因群体中有高达27.3%的单株整合有5拷贝以上的外源基因,而基因枪轰击和花粉管通道注射法获得的转基因群体中没有发现5拷贝以上的转基因单株。研究结果表明,基因枪轰击和花粉管通道注射法均能获得较高比例低拷贝(1～3拷贝)的转化体。本研究结果对棉花转基因育种中转化方法的选择有一定的理论指导意义。Identifying the copy number of foreign gene in transgenic primary plants is the basic step in genetic engineering application.In this paper,Real-time PCR and the related PfaffI method,usually applied to studying transgenic expression,was used to analyze comparatively the differences of exogenous gene copy number of transgenic cotton（Gossypium hirsutum） T0 plants transformed with one same vector by three different methods,Agrobacterium-mediated,gene-gun bombardment and pollen tube injection,respectively.Results showed that in Agrobacterium-mediated T0 generation,the transgenic plants integrated one or two copy number accounted for only 26.2% of the total plants,respectively;In contrast,that of transgenic plants transformed by gene-gun bombardment and pollen tube injection were 60% and 42.8%,respectively.Transgenic lines with three to five copies accounted for 47.5%,40% and 57.1%,respectively.In addition,in Agrobacterium-mediated T0 generation,transgenic lines with more than 5 copies accounted for 27.3%,while there was no transgenic plant with more than 5 copies in genetically modified T0 generation transformed by gene gun bombardment or pollen tube methods.These results provide some rationale for selection of transformation methods in crops transgene breeding.

关 键 词：遗传转化方法 转基因棉花 实时定量PCR 转基因拷贝数差异 

分 类 号：S562[农业科学—作物学]