基于重测序鉴定SbHKT基因在陆地棉基因组中的插入位点  

Identification of SbHKT insertion site in Gossypium hirsutum genome based on re-sequencing technology

作　　者：徐鹏[1] 郭琪[1] 徐珍珍[1] 孟珊 陈天子[1] 沈新莲[1] Xu Peng;Guo Qi;Xu Zhenzhen;Meng Shan;Chen Tianzi;Shen Xinlian(Institute of Industrial Crops,Jiangsu Academy of Agricultural Sciences/Key Laboratory of Cotton and Rapeseed,Ministry of Agriculture,Nanjing 210014,China)

机构地区：[1]江苏省农业科学院经济作物研究所/农业部长江下游棉花与油菜重点实验室,南京210014

出　　处：《棉花学报》2021年第4期377-383,共7页Cotton Science

基　　金：国家科技重大专项——转基因生物新品种培育(2016ZX08005-004-002)。

摘　　要：【目的】明确SbHKT基因棉花HKT-1株系的插入位点序列特征。【方法】对HKT-1株系重测序,本地BlastN比对获得插入位点处的侧翼序列,设计聚合酶链式反应(Polymerase chain reaction,PCR)特异引物验证插入位点的准确性。【结果】获得SbHKT基因插入位点处107 bp的左边界(Left border,LB)端侧翼序列HKT1_LSEQ,122 bp的右边界(Right border,RB)端侧翼序列HKT1_RSEQ;锚定基因组后显示SbHKT基因的插入引发了陆地棉染色体结构变异。分别根据LB和RB端侧翼序列设计PCR特异引物扩增HKT-1株系T-DNA全长,目的条带含有完整的T-DNA骨架序列以及SbHKT基因序列,证实HKT1_LSEQ和HKT1_RSEQ是同一个插入位点的左右侧翼序列。【结论】基于重测序技术获得了SbHKT基因在陆地棉基因组中的侧翼序列,建立了SbHKT基因转化体特异性检测方法。[Objective]The purpose of this study was to identify the SbHKT insertion site in Gossypium hirsutum genome.[Method]The flanking sequences at the insertion sites were obtained by local BlastN alignment.The T-DNA insertion site was validated by specific primer polymerase chain reaction(PCR)assay.[Result]A 107 bp left border(LB)flanking sequence named HKT1_LSEQ and a 122 bp right border(RB)flanking sequence named HKT1_RSEQ were obtained.It was shown that the insertion of SbHKT gene caused chromosomal structural variation in upland cotton after anchoring HKT1_LSEQ and HKT1_RSEQ to reference genome of G.hirsutum.Specific PCR primers were designed in the flanking sequences of LB and RB to amplify the intact T-DNA of the HKT-1 strain.The sequencing of the target band revealed that it contained complete T-DNA skeleton sequence and SbHKT gene sequences.HKT1_LSEQ and HKT1_RSEQ were proved to be the flanking sequences of the same insertion.[Conclusion]The flanking sequences of SbHKT insertion site were obtained based on re-sequencing technology.A method for specific detection of SbHKT gene transformation events was established.

关 键 词：陆地棉 重测序 SbHKT 插入位点 侧翼序列 

分 类 号：S562[农业科学—作物学]