基因枪介导的转PYL5基因小麦的获得与鉴定

Transformation of wheat with PYL5 gene by biolistic particles and its molecular identification

【目的】通过基因枪介导共转化,获得转拟南芥抗旱基因PYL5的小麦植株,为转基因小麦的抗旱功能研究奠定基础。【方法】以小麦品种西农889、绵阳19和宁春16为供试材料,通过基因枪介导法将诱导型启动子Rd29A启动的PYL5基因和筛选标记基因Bar共转化到小麦幼胚愈伤组织中,经过除草剂PPT(Phosphinothricin)筛选和愈伤组织分化,获得再生植株。根据目标基因PYL5和Bar基因序列分别设计特异引物,对移栽成活的小麦T0代再生植株进行基因特异性PCR检测。【结果】采用基因枪分别轰击西农889的1800个、绵阳19的800个和宁春16的800个幼胚愈伤组织,经过筛选和分化分别获得了9,5和14株小麦再生植株;对转基因小麦T0代再生植株的基因特异性PCR检测结果表明,西农889、绵阳19和宁春16 Bar基因的转化率分别为0.280%,0.500%和0.750%,PYL5基因的转化率分别为0.110%,0.125%和0.500%,Bar和PYL5基因的共转化率分别为0.110%,0.125%和0.500%。【结论】PYL5基因成功转入到了小麦品种西农889、绵阳19和宁春16中。

【Objective】The purpose of this study was to obtain transgenic wheat with PYL5 gene based on by biolistic particles.【Method】Wheat cultivars including Xinong 889,Mianyang 19 and Ningchun 16 were used as acceptors,PYL5 and Bar genes were co-transformed into immature callus by biolistic particles.After herbicide selection(Phosphinothricin,PPT)and callus differentiation,the regenerated plants were obtained.Then,allele specific PCR(AS-PCR)was conducted to analyze T0 generation of regenerated plants using primers designed according to sequences of target genes.【Result】1 800 callus of Xinong 889,800 callus of Mianyang 19 and 800callus of Ningchun 16 were bombarded by biolistic particles,and 9,5and14 regenerated plants were obtained,respectively.The regenerated plants were tested using allele specific PCR(AS-PCR)and the obtained transformation frequencies of Bar,PYL5 gene for Xinong 889,Mianyang19 and Ningchun 16 were 0.280%,0.500%,and 0.750%;0.110%,0.125%,0.500%,respectively,The co-transformation frequencies of Bar and PYL5 were 0.110%,0.125% and 0.500%,respectively.【Conclusion】PYL5gene was successfully transformed into wheat cultivars Xinong 889,Mianyang 19 and Ningchun16.