摘要
[目的]为Brazzein基因在酵母SMD1168中转化和表达奠定基础。[方法]利用重叠延伸PCR(SOE-PCR)合成Brazzein基因,将其连接到pMD18-T载体上,构建克隆载体pMD18-T-Bra。分别用XhoI和XbaI限制内切酶对克隆质粒pMD18-T-Bra和酵母表达载体pGAPZαA进行酶切,并在T4DNA连接酶作用下,将回收的目的基因连接到pGAPZαA载体上,构建重组质粒pGAPZαA-Bra。[结果]通过PCR扩增获得了约188 bp的Brazzein类似物的编码序列,将其克隆到pMD18-T质粒,用XhoI和XbaI双酶切后,连接到pGAPZαA载体,成功构建了重组表达载体pGAPZαA-Bra。Brazzein基因其中各碱基未发生突变,整个表达载体阅读框正确无误。[结论]利用基因工程的方法生产Brazzein是可行的。
[Objective] The research aimed to lay the foundation for the transformation and expression of Brazzein gene in yeast SMD1168.[Method] Brazzein gene was synthesized by using splicing overlap extension PCR（SOE-PCR）.It was connected with pMD18-T vector to construct cloning vector pMD18-T-Bra.Enzyme digestion with restriction endonuclease Xho I and Xba I was made on cloning vector pMD18-T-Bra and yeast expression vector pGAPZαA.Under the action of T4 DNA ligase,the reclaimed target gene was connected with pGAPZαA vector to construct plasmid pGAPZαA-Bra.[Result] A coding sequence of Brazzein analog with the size of about 188 bp was obtained through PCR amplification.After it was cloned pMD18-T plasmid and digested with double enzymes Xho I and Xba I,it was connected with pGAPZαA vector.And the recombinant expression vector pGAPZaA Bra was successfully constructed.No mutation was found in each base in Brazzein gene and the reading frame in the whole expression vector was exact.[Conclusion] It was feasible to produce Brazzein by the method of genetic engineering.
出处
《安徽农业科学》
CAS
北大核心
2008年第15期6230-6232,共3页
Journal of Anhui Agricultural Sciences
基金
黑龙江省自然基金项目(C200618)
黑龙江省农垦总局科技攻关项目(HNKXIY-08-11A)
