高亲和K+转运载体SsHKT1的抗体制备及表达分析

利用RT-PCR及RACE技术从盐生植物盐地碱蓬(Suaeda salsaL.)根中克隆了高亲和K 转运载体SsH-KT1的基因。以SsHKT1基因的cDNA为模板,扩增了cDNA中的亲水片段(403~612 bp),连接至原核表达载体pGEX-6P-3中并转化大肠杆菌BL21(DE3),IPTG诱导表达了融合蛋白。该蛋白主要以包涵体的形式存在,纯化后免疫新西兰兔,得到了特异性较高的SsHKT1多克隆抗体。利用此抗体进行了W estern b lot分析,表明SsHKT1可能主要存在于质膜上。进一步研究了K 饥饿及盐胁迫条件下SsHKT1蛋白表达量的变化,结果表明K 饥饿及盐胁迫条件下SsHKT1的表达量都明显增加,表明SsHKT1在盐地碱蓬K 吸收特别是高盐条件下K 营养中有重要作用。

Polyclonal Antibody Preparation and Expression Analysis of High-affinity K+ Transporter SsHKT1

We isolated a cDNA homologous to HKT1 from Suaeda salsa L.(SsHKT1) roots using RT-PCR and RACE methods.The hydrophilic fragment(403-612 bp) was amplified by PCR from SsHKT1 cDNA of halophyte S.salsa L.PCR product was inserted into prokaryotic expression vector pGEX-6P-3.The recombinant vector was transformed into Escherichia coli BL21(DE3) and the GST-SsHKT1 fusion protein expression was induced by IPTG.The fusion protein was mainly in inclusion bodies.The purified protein was injected into a New Zealand rabbit and the rabbit's anti-serum against SsHKT1 with higher specificity was prepared.Western-blot analysis using this polyclonal antibody demonstrated that SsHKT1 may be localized to the plasma membrane.Further study shows that level of SsHKT1 proteins increase under conditions of K~ starvation or salt stress.These datas suggest that SsHKT1 is important for K~ uptake of S.salsa L.and particularly for K~ nutrition under high salt conditions in this plant.