拟南芥热激因子cDNA的克隆及转化烟草研究

CLONING OF THE cDNA ENCODING THE Arabidopsis HEAT SHOCK FACTOR AND TRANSFORMATION IN TOBACCO

拟南芥热激因子是一重要的耐热基因。从拟南芥幼苗中提取总RNA ,根据报道设计引物 ,利用RT PCR的方法扩增出大约 1.4kb的拟南芥热激因子基因Athsfc。将其克隆入pBluescriptSK载体的XbaI和SacI位点 ,测序结果显示核苷酸序列与国外H櫣ｂｅｌ(1994)报道的Athsf1基因间 99.8%同源 ,只有 3个核苷酸不同。序列分析显示 ,编码相同的氨基酸。将该基因构建入含内含子Km筛选标记的植物双元表达载体pYP12 0 3E中 ,构成了拟南芥热激因子基因 Athsfc植物表达载体pYP12 0 3E(Athsfc)。利用根癌农杆菌 (Agrobacteriumtumefaciens)对烟草进行Athsfc基因的转化 ,获得了GUS染色显示阳性的烟草转基因植株 。

Arabidopsis heat shock factor is an important central regulator of the heat stress response. Total RNA was isolated from seedling of Arabidopsis thaliana, and a 1.4kb fragment (Athsfc) was amplified using RT PCR method. The fragment was digested with Xba I and Sac I, then it was inserted into the pBluescript SK vector. The nucleotide sequence showed 99.8% homology between the Athsfc gene cloned in this lab and the reported by Hübel in 1994. Only three nucleotides were different between them but their amino acid sequences were the same. Athsfc was cloned into the plant expression vector pYP1203E containing intron kanamycin gene. Athsfc gene was successfully introduced into tobacco plant by Agrobacterium mediated transformation, and the transgenic plants of tobacco were obtained, which was proved by GUS reporter gene analysis, PCR, and PCR southern blot method.