转GbVe1基因在棉花基因组中的整合与定位分析  

The integration and insertion site of GbVe1 gene in the genome of transgenic cotton(Gossypium hirsutum)

作　　者：陈天子[1] 凌溪铁 杨郁文[1] 张保龙[1] Chen Tianzi;Ling Xitie;Yang Yuwen;Zhang Baolong(Jiangsu Provincial Key Laboratory of Agrobiology/Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China)

机构地区：[1]江苏省农业科学院/江苏省农业生物学重点实验室,江苏南京210014

出　　处：《棉花学报》2019年第1期1-11,共11页Cotton Science

基　　金：国家重点研发计划(2016YFD0101418)

摘　　要：【目的】明确转GbVe1基因棉花的插入位点序列特征。【方法】Southern杂交筛选低拷贝基因插入的转基因棉花株系,以hiTAIL-PCR(Polymerase chain reaction)获取其T-DNA侧翼序列,然后根据获得的T-DNA侧翼序列设计特异PCR引物,验证插入位点的准确性。【结果】Southern杂交候选了T-DNA低拷贝插入的3个转基因棉花株系,hiTAIL-PCR分离到RB端侧翼序列(119~1 018 bp)、LB端侧翼序列(243~516 bp);侧翼序列的AT碱基含量在63%以上。转基因株系7/100826-152和12/100826-393插入位点都位于Gohir.D01G157600.1内含子上。转基因株系1/w-ch14插入位点分别位于Gohir.D01G157600.1内含子和A12染色体的基因间隔区中。T-DNA在Gohir.D01G157600.1内含子的插入事件造成了21 bp碱基的基因组序列缺失。T-DNA到侧翼序列的PCR产物证明Gohir.D01G157600.1上的插入位点真实可靠。【结论】hiTAIL-PCR获取了转GbVe1基因棉花的T-DNA侧翼序列,提供了T-DNA插入位点位于Gohir.D01G157600.1基因内含子的特异性检测引物。[Objective]The aim of this study was to obtain the flanking sequences of T-DNA in the transgenic cotton containing a GbVe1 over-expression cassette.[Method]The T-DNA insertion copy number in the transgenic GbVe1 cotton was analyzed by southern blot.Flanking sequences of the transgenic lines with putative single T-DNA insertion copy were obtained using high-efficiency Thermal asymmetric interlaced polymerase chain reaction(hiTAIL-PCR).The T-DNA insertion sites were further confirmed by PCR with specific primers.[Result]RB-flanking sequences(119-1 018 bp)and LB-flanking sequences(243-516 bp)were obtained from three transgenic lines with low copy number of T-DNA insertion.The AT content was more than 63%in these flanking sequences.A same single insertion site in the intron of Gohir.D01G157600.1 was found in the two transgenic lines 7/100826-152 and 12/100826-393,while two separated insertion sites,one also in the intron of Gohir.-D01G157600.1 and the other in the intergenic region of A12 chromosome,were found in the transgenic line 1/w-ch14.A deletion of 21 bp was found in the insertion site in the intron of Gohir.D01G157600.1.The T-DNA insertion in the intron of Gohir.D01G157600.1 was further confirmed by the specific PCR.[Conclusion]The flanking sequences of T-DNA in the transgenic GbVe1 cotton were obtained and the specific transformation event in the intron of Gohir.D01G157600.1 was further confirmed by PCR.

关 键 词：侧翼序列 GbVe1 棉花 插入位点 

分 类 号：S562[农业科学—作物学]