盐穗木HcPS_H基因真核表达载体的构建及转化

Construction and Transformation of Eukaryotic Express Vector of HcPS _ H Gene from Halostachys caspica

【目的】盐穗木(Halostachys caspica)属藜科盐生植物,是广泛分布于新疆干旱或半干旱荒漠盐碱环境中的一种极端耐盐的多年生稀盐多汁半灌木,对盐分适应性极强。PS_H基因是一个参与光合反应光系统的基因,在植物光合作用中起着重要的作用。构建盐穗木HcPS_H基因真核表达载体对于了解该基因在盐穗木耐盐过程中的作用有着重要意义。【方法】利用PCR技术扩增得到大小为441 bp的盐穗木HcPS_H基因,经限制性内切酶SalI和NotI进行消化,将目的基因与真核表达质粒pREP-5N连接,获得重组真核表达质粒HcPS_H-5N。【结果】通过菌落PCR、双酶切及测序等方法进行鉴定,重组真核表达质粒HcPS_H-5N构建成功。制备酵母感受态,将重组质粒电击转入酵母中,酵母菌落PCR结果显示电击转化成功。【结论】成功构建盐穗木HcPS_H基因真核表达载体,并且能在真核细胞中正常表达。

[ Objective ] Halostachys caspica is a leaf - succulent euhalophytic plant capable of surviving under high salinity, and a genus of Chenopodiaceae plants, widely distributed in Xinjiang arid or semi - arid desert saline environments. PS _ H gene is a part of photosynthetic response to light system gene, which plays an important role in Photosynthesis in plants. [ Method] It is helpful to learn the function of HcPS _ H in salt tolerance procession of Halostachys caspica by constructing the eukaryotic express system of this gene. We cloned the gene of HcPS_ H from Halostachys caspica by PCR. The target gene fragments and the plasmid pREP -5N were digested with the restriction enzymes Salt and NotI, and then were linked together by the T4 DNA ligase. Finally, the recombinant eukaryotic expression plasmid HcPS_ H- 5N was obtained. [Result] The identification results of the individual bacterial colonies PCR, double enzymes digestion and sequencing showed that the recombinant expressing plasmid HcPS_ H- 5N was constructed successfully. [ Conclusion] The recombinant plasmid was transformed into the yeast competent cells by electro -transformation, and the yeast single colonies PCR ：indicted that the recombinant expressing plasmid HcPS _ H - 5N was successfullytransferred.