植物细菌诱导型表达载体的构建

Cloning and Sequencing of Plant Bacteria-inducible Promoters

根据GenBank中细菌诱导型启动子的碱基序列设计一对引物,通过PCR扩增出启动子PPP3(AF469483,220bp).经分子操作将启动子和质粒pUC18连接后转化E.coliDH5α,经蓝白筛选和PCR检测筛选阳性菌落,测序结果与Genebank中的碱基序列完全相同.对扩增到的PPP3片段和含目的基因(hrap或pflp)的pBI121质粒进行双酶切,分别回收PPP3片段和含目的基因的pBI121大片段,连接后转化E.coliDH5α,通过卡那霉素抗性筛选和PCR检测,表明细菌诱导型启动子和目的基因已正确连接.用重组质粒转化农杆菌,经卡那霉素抗性筛选和PCR检测,证明植物细菌诱导型表达载体构建成功.

According to the sequence of plant bacteria-inducible promoters from GenBank, a pair of specific primers were designed. The promoter PPP3 was amplified from tobacco genomic DNA by PCR. The PPP3 was cloned into the vector pUC18 and transferred into the E. coli strain DH5α. The putative clones were selected by blue-white cloning qualified and PCR. The sequence of putative clones is the same as the PPP3 that has been reported in GenBank. The PPP3, Pflp-pBI and Hrap - pBI were digested with restriction enzymes HindIII and BamHI. The object DNA fragments were purified and ligated. The ligation product was transferred into GV3101. PCR detection and sequencing result show that the transferring was correct.