多位点整合的转基因棉花后代分子鉴定  

Molecular Characterization on Offsprings from a Multiple-sites Integration Transgenic Cotton(Gossypium hisutum L.)

作　　者：马小波[1,2] 王芙蓉[1] 张传云[1] 刘国栋[1] 高俊平[1,2] 张军[1] 

机构地区：[1]国家棉花遗传改良中心山东分中心,山东棉花研究中心,济南250100 [2]山东师范大学,济南250014

出　　处：《棉花学报》2008年第1期9-13,共5页Cotton Science

基　　金：国家转基因专项基金项目(JY03-B-05-02);山东省“三O工程”种质资源项目棉花子专题

摘　　要：GFP47是一个利用双元载体pPZP111构建的绿色荧光蛋白基因的简单质粒载体pPZP-GFP经农杆菌介导转化陆地棉(Gossypiumhirsutum L.)获得的T0株系,PCR和Southern杂交分析证实其不仅整合了多个拷贝的正常的T-DNA,而且有紧邻T-DNA左边界的载体骨架序列的整合。对来源于GFP47的33个T1个体的PCR分析表明,T1后代中可以分离只有正常T-DNA整合的个体。对T1群体中不同基因型个体的Southern杂交分析结果显示,该多拷贝整合的T0转化体至少在棉花基因组中有四个插入位点,不同的Southern带型暗示不同插入位点的T-DNA结构和排布有很大的不同。至少有一个位点整合了只包括目的基因和选择标记基因在内的正常T-DNA结构。To investigate the pattern and organization of foreign gene integration in transgenic cotton, PCR and Southern blot analysis on To plant,and the corresponding T1 population of a transgenic cotton （Gossypium hirsutum L. ） with multiplentegration sites derived from a simple construct pPZP- GFP via Agrobacteriurn-mediated transformation, and the corresponding T1 population were carried out in this paper. The seeds harvasted from GFP47 To and genomic DNA isolated from To plants were introduced from Plant Biotechnology lab of Temasek Life Sciences Laboratory, National University of Singapore. T1 plants were planted in the greenhouse for DNA isolation and thereafter molecular analysis. PCR results showed that the To plant was integrated not only with normal T-DNA including target gene （gfp） and selection marker gene （nptII）, but also with vector backbone fragment outside the left border of the T-DNA. Besides confirmed the PCR results, Southern blot analysis on To plant indicated that the To plant was integrated with multi-copy of T-DNA. PCR characterization on 33 T1 individuals showed that some individuals with only normal T-DNA structures were segregated from the population. Further analysis with Southern blot on these individuals with different genotype identified by PCR showed that there were, at least, 4 loci with different integration patterns in the cotton genome. The desirable transgenic event with only single copy of normal T-DNA integration could be segregated from the offsprings of this multiple-sites integration transgenic cotton.

关 键 词：转基因棉花 多位点整合 T-DNA 载体骨架 PCR Southern杂交分析 

分 类 号：S562[农业科学—作物学]