基因枪法介导的转KN2基因小麦的获得及鉴定

Acquisition and identification of transgenic wheat with KN2 gene by biolistics

【目的】获得转KN2基因的小麦植株,为研究KN2基因对提高转基因小麦抗寒性的作用,以及为利用基因工程提高小麦抗逆性奠定基础。【方法】以弱冬性小麦品种小偃22为供试材料,通过基因枪法,用含有KN2基因的植物表达载体和筛选标记基因bar共转化小麦幼胚愈伤组织,经过除草剂(Phosphinothricin,草丁膦)筛选和愈伤组织分化获得再生植株,并对得到的再生小麦植株进行PCR检测。【结果】采用基因枪法共轰击小偃22的幼胚愈伤组织1 680个,共获得再生植株17株,移栽到花盆中成活15株;根据目标基因序列设计特异引物,对成活的小麦植株进行PCR检测,结果表明获得含有bar基因和KN2基因的转基因阳性植株分别为6株和4株,转化率分别为0.36%和0.24%,bar和KN2的共转化率为0.24%。【结论】KN2基因成功地转入到小麦品种小偃22中。

[Objective] This study aimed to obtain transgenic wheat with KN2 gene,which would im- prove future investigation for effect of KN2 on wheat cold resistance. [Method] Using the wheat variety Xiaoyan 22 as the acceptor,KN2 gene and bar gene were co-transformed into immature callus by biolistics. After herbicide screening and callus differentiation,the regenerated plants were tested using PCR. [Result] 1 680 callus of Xiaoyan 22 were bombarded by biolistic particle and 17 regenerated plants were obtained. After being transplanted to flower pots, 15 plants survived. The regenerated plants were tested by PCR with primers based on target gene sequence,and 6 transgenic plants with bar gene and 4 transgenic plants with KN2 gene were identified in the To generation. The transformation frequencies of bar gene and KN2 gene were 0.36% and 0.24% ,respectively. The co-transformation frequency was 0.24%. [Conclusion] The KN2 gene was successfully integrated into Xiaoyan 22.