胡杨果糖-1,6-二磷酸醛缩酶的定位及功能研究

Subcellular Localization and Functional Analysis of Fructose-1,6-Bisphosphate Aldolase Gene from Populus euphratica

将从胡杨中克隆的果糖-1,6-二磷酸醛缩酶基因(PeALD)构建到pGEX-4T-1载体上,经IPTG诱导成功获得融合蛋白GST:PeALD,并将蛋白进行纯化,其大小约为52 kD,转化大肠杆菌的耐盐性实验表明,PeALD基因的成功表达有利于提高大肠杆菌菌株的耐盐性。为研究该基因编码蛋白在植物细胞中的定位,将基因的ORF区构建到定位表达载体pMDC85上,通过PEG介导的拟南芥瞬时转化法观察融合蛋白PeALD:GFP在细胞中的定位情况,结果显示该蛋白定位于胞质中,由此说明实验克隆得到的该胡杨果糖-1,6-二磷酸醛缩酶基因编码的是胞质蛋白。烟草种子在盐培养基上的萌发实验结果显示转基因烟草具有更高的耐盐性;烟草水培苗经200 mmol/L NaCl处理一周后,可溶性糖的质谱检测结果显示转基因植株中葡萄糖的含量有很大提高。表明胡杨果糖-1,6-二磷酸醛缩酶通过促进糖酵解和有氧呼吸途径来提高植物对盐胁迫的适应性。

The fructose- 1,6-bisphosphate aldolase gene （PeA LD） of Pop ulus e uphratic a was cloned and constructed to an expression vector pGEX-4T-1. And then the recombinant plasmid pGEX-PeA LD was transformed into E. coli competent cell BL21 （DE3） to identify the expression of fusion protein GST： PeALD that induced by IPTG. The SDS-PAGE gel electrophoresis showed that the molecular weight of protein coded by PeA LD gene was about 52 kD. The E. coli strains expressing the PeA LD showed a higher salt tolerance compared to control strains that expressing the null vector. We constructed the fusion expression vector PeALD： GFP and identify the localization of PeALD in A rabidopsis mesophyll protoplast. The green fluorescence detected by fluorescence microscope showed that the protein encoded by PeA LD localized in the cytoplasm. Seed germination experiment showed that genetically modified tobacco seeds exhibited a higher salt tolerance. The content of soluble sugars in wild-type and transgene lines was analyzed after salt treatment. Results show that the content of glucose in transgenic plants was greatly elevated under salt stress. Our data suggest that PeALD enhanced plant salt adaptation through up-regulation of glycolysis and aerobic respiration.