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棉花学报 ›› 2019, Vol. 31 ›› Issue (1): 1-11.doi: 10.11963/1002-7807.ctzzbl.20190105

• 研究与进展 •    下一篇

GbVe1基因在棉花基因组中的整合与定位分析

陈天子(),凌溪铁,杨郁文,张保龙*()   

  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
  • 收稿日期:2018-03-30 出版日期:2019-01-15 发布日期:2019-01-15
  • 通讯作者: *张保龙 E-mail:actzi0503@gmail.com;zhbl2248@hotmail.com
  • 作者简介:陈天子(1981―),男,博士, actzi0503@gmail.com
  • 基金资助:
    国家重点研发计划(2016YFD0101418)

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

Chen Tianzi(),Ling Xitie,Yang Yuwen,Zhang Baolong*()   

  1. Jiangsu Provincial Key Laboratory of Agrobiology/Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China
  • Received:2018-03-30 Online:2019-01-15 Published:2019-01-15
  • Contact: *Zhang Baolong E-mail:actzi0503@gmail.com;zhbl2248@hotmail.com

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。

关键词: 侧翼序列; GbVe1; 棉花; 插入位点

Abstract:

[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.

Key words: flanking sequence; GbVe1; Gossypium hirsutum; insertion site