为研究沙冬青脱水素的功能,根据沙冬青cDNA文库中脱水素基因序列设计引物,采用PCR技术进行了脱水素基因的扩增,扩增产物与载体pGM-T载体连接,转化大肠杆菌Top10,筛选阳性克隆,测序结果表明,已成功克隆的阳性重组子经测序鉴定其片段大小与已知沙冬青cDNA文库中脱水素基因大小一致,为760 bp,说明此序列无内含子.将成功克隆的阳性重组子提取质粒与pCAMBIA3301质粒同样进行酶切反应,再以TDNA连接酶连接,成功构建了脱水素基因的植物表达载体,为下一步的抗逆性研究打下了基础.
Dehydrin is a LEA protein in plant, it can substantial expression in the late embryo development period and stress environment in plants. First, the primers were designed according to the sequence of Ammopiptanthus mongolicus dehydrin cDNA library, the dehydrin gene was amplified with PCR, then cloned into E. coli Top10 by using pGM-T vector. Sequence analysis shows that the cloned 760 bp,which the same size as the known cDNA library Ammopiptanthus mongolicus dehydrin gene size. It note that this gene sequence without intron. the recombinant plasmid of cloned digested by Bgl ??and Best ??same as the pCAMBIA3301 plasmid, then linked by T4DNA ligase. Now, we successfully constructed the expression vector of dehydrin gene,To lay the foundation for the research of resistance to stress environment.
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