【作者】 胡月；

【导师】 沈昕；

【作者基本信息】 北京林业大学 ， 生物化学与分子生物学， 2013， 硕士

【摘要】 本研究将从胡杨中克隆的果糖-1,6-二磷酸醛缩酶基因(PeALD)构建到原核表达载体pGEX-4T-1上,经IPTG诱导成功获得融合蛋白GST:PeALD,并将蛋白进行纯化,得到目的条带大小约为52KD。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情况,我们将基因的Open Reading Frame (ORF)区构建到定位表达载体pMDC85上,该载体带有GFP标签,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。T1代烟草种子在盐培养基上的萌发实验表明转基因烟草获得了更高的耐盐性；烟草水培苗经200mMNaCl盐处理一周后,其叶片可溶性糖的质谱检测结果显示转基因烟草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。更多还原

【Abstract】 The fructose-1,6-bisphosphate aldolase gene (PeALD) of Populus euphratica was cloned and constructed to an expression vector pGEX-4T-1. And then the recombinant plasmid pGEX-PeALD was transformed into Escherichia 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 fusion protein was about52KDa.The E. coli strains expressing the PeALD 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 Arabidopsis mesophyll protoplast. The green fluorescence detected by fluorescence microscope showed that the protein encoded by PeALD localized in the cytoplasm. Seed germination experiment under200mmol· L-1NaCl showed that genetically modified tobacco seeds exhibited a higher salt tolerance. The content of soluble sugars in wile-type and transgene lines was analyzed after salt treatment. Results showed 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.更多还原