基于重测序鉴定GbTMEM214基因在陆地棉基因组的插入位点

dentification of GbTMEM214 insertion site in Gossypium hirsutum genome based on resequencing

【目的】利用农杆菌介导法获得了转GbTMEM214基因陆地棉品系,旨在明确转基因棉花株系中T-DNA插入位点的序列特征及检测方法,为其生物安全评价提供依据。【方法】基于基因组重测序技术,利用BLASTn将测序数据与陆地棉TM-1基因组序列进行比对分析,设计特异性引物通过聚合酶链式反应(polymerase chain reaction,PCR)验证插入位点。【结果】携带目标基因GbTMEM214的T-DNA整合在陆地棉基因组D13号染色体57019068~57019106 bp,T-DNA插入导致受体基因组37 bp的片段缺失;通过PCR扩增获得携带GbTMEM214基因的T-DNA插入位点的侧翼序列,由此建立了转GbTMEM214基因棉花的特异性检测方法。【结论】基于基因组重测序技术获得了转GbTMEM214基因棉花的T-DNA插入位点及侧翼序列信息,可为转基因棉花的生物安全评价提供技术参考。

[Objective]GbTMEM214 transgenic Gossypium hirsutum line,obtained using Agrobacterium-mediated method,was used to clarify the sequence characteristics and detection methods of the T-DNA insertion site,and further promote its biosafety evaluation.[Methods]Based on the genome resequencing technology,the sequencing data was compared with the G.hirsutum standard line TM-1 genome sequence by BLASTn,and specific primers were designed to verify the insertion site by polymerase chain reaction(PCR).[Results]The T-DNA carrying the target gene GbTMEM214 was integrated into the position of 57019068-57019106 bp on chromosome D13 of G.hirsutum genome,resulting in 37 bp deletion of cotton genome.Combined with the flanking sequence of T-DNA insertion site obtained by PCR amplification,the specific detection method for GbTMEM214 transgenic cotton was established.[Conclusion]The T-DNA insertion site and flanking sequence of GbTMEM214 transgenic cotton was obtained based on genome resequencing technology,which can provide technical reference for biosafety evaluation of the transgenic cotton.