The Gene Expression Of Na～+/H～+ Antiporters From Karelinia Caspica & Kalidium Foliatum And Preparation Of Their Antisera

There is a large area of soil with salt in Xinjiang. Halophytic vegetation, which are distributed over the salt soil, play a important role in maintaining the balance of the complex eco-system salt-soil-hungriness-oasis, boosting the regional economy. As the representative of halophytic vegetation in this area, Karelinia caspica and Kalidium foliatum are also distributed in the salt meadow and abandoned ground in Northwest China. These two species grows in the extremely salty sand, withstanding the salt-stress of sulfate, chloride and soda. Salt would hurt vegetation though the toxicity of the excessively acquired salt ions ,especially Na~+, in water-absorbing. The survival of halophytic vegetation is attributed to a important compartmentation that makes the redundant Na~+ inflood into the vacuole. In this mechanism, Na~+/H~+ antiporter(NHX) plays a important role.With the more and more researches on the mechanism that cause the halophytic vegetation'withstanding the salt toxicity, NHX genes of more and more plants have been cloned. My workmates had cloned two Na ~+/H~+ antiporter genes Kcnhx1 and Kfnhx1 on researching on the salt-tolerant mechanism on Karelinia caspica and Kalidium foliatum.Aiming at detecting the expression of Kcnhx1 and Kfnhx1 and preparing the antisura against KcNHX1 and KfNHX1, we did the experiment: the 285bp fragment of the 3'-end of Kcnhx1 and the 472bp fragment of the 3'-end of Kfnhx1 were cloned and ligated to the prokaryotic expressive vector pMAL-p2x.The recombinant plasmid pMAL-p2x- Kcnhx1-c285 and pMAL-p2x- Kfnhx1-c471 wrere transformed into E.coli BL21 and TB1 then induced by IPTG.. The fusion proteins M?BP-KcNHX1-C94 and M?BP-KfNHX1-C157 were purified. The ORF of the two Na ~+/H~+ antiporter genes were ligated to the eukaryotic expressive vector pcDNA3.1,then transfected into the mouse liver by hydrodynamics-based transfection method. The genes were detected to express in the hepatocytes of mouse by RT-PCR. Then, pcDNA3.1-Kcnhx1 and pcDNA3.1-Kfnhx1 were used as DNA vaccine to immunize Kuming White mice and to prepare the antisera against KcNHX1 and KfNHX1 with MBP-KcNHX1-C94 and MBP-KfNHX1-C157 protein-boost repectively. Meanwhile, a simple and cheap method for testing the gene-expressing products was established. It will help the plant functional gene researches in the future.