苦荞植物络合素合酶（FtPCS）基因克隆与功能研究

发布时间：2018-03-16 23:18

本文选题：苦荞　切入点：植物络合素合酶　出处：《西北农林科技大学》2017年硕士论文　论文类型：学位论文

【摘要】：植物络合素(Phytochelatins,PCs)是在植物细胞内广泛存在的一种可以有效螯合Cd2+、Pb2+、Cu2+等重金属离子的多肽类化合物,其具有高度保守的一级结构:(γ-Glu-Cys)n-Gly(n=2~11)。在植物和酵母细胞内,PCs对游离重金属离子的富集和解毒过程具有重要作用,而植物络合素合酶(Phytochelatin synthase,PCS)是催化其合成的关键酶。本实验研究以Pb2+胁迫条件下的苦荞叶片转录组数据为基础,通过RT-PCR克隆了苦荞植物络合素合酶(FtPCS)基因CDs序列和DNA序列,并对其进行了一系列的生物信息学分析。随后,将获得的苦荞FtPCS基因CDs序列分别转化E.coli BL21 Star(DE3)、酿酒酵母YK44突变体菌株、以及拟南芥植株进行异源表达,并对其在重金属Cd2+胁迫条件下的功能和表达特点进行分析,以期为进一步揭示FtPCS在苦荞植物重金属富集和解毒过程中的机制奠定基础。本研究得到的主要结论有:(1)苦荞植物络合素合酶(FtPCS)基因的CDs序列长1 485 bp,编码494个氨基酸,预测分子量为55.10 kDa;FtPCS基因的DNA序列长5 456 bp,由8个外显子和7个内含子组成,且内含子两侧的剪接位点均满足GT-AG规则。生物信息学分析表明,FtPCS基因氨基酸序列N-端序列高度保守,包括5个保守的Cys-残基特征位点,是FtPCS蛋白活性中心;C-端序列包含12个可变的Cys-残基位点,是主要的重金属离子结合位点。(2)通过NEBuilder HiFi DNAAssembly技术,构建pET28a-PCS重组表达载体,转化E.coli BL21 Star(DE3)并通过IPTG诱导表达。可溶性分析结果表明,FtPCS在E.coli内以包涵体的形式大量表达。通过Co2+螯合层析结合梯度透析复性,获得了纯化的FtPCS蛋白;通过反向-HPLC结合DTNB[5,5'-二硫代双(2-硝基苯甲酸)]柱后衍生的方法,对FtPCS重组蛋白在Pb2+存在条件下的催化活性进行分析。结果表明,纯化的FtPCS蛋白具有催化活性,能将还原型谷胱甘肽(GSH)络合生成PC化合物,而低浓度的Pb2+对其活性具有激活作用。(3)通过NEBuilder HiFi DNAAssembly技术,构建pYES2-PCS酵母表达载体,转化对重金属离子Zn2+/Cd2+/Ni2+/Co2+敏感的酿酒酵母YK44突变体菌株,并通过半乳糖诱导表达。酵母功能互补实验结果表明,在不同浓度Cd2+胁迫条件下,转化pYES2-PCS重组质粒的酵母菌株比转化空载的酵母菌株对重金属Cd2+的耐受性明显提高。在Cd2+胁迫条件下的酵母生长曲线同样表明,FtPCS基因在酵母细胞中的表达,能够弥补其重金属抗性基因的缺陷,从而提高其对Cd2+的抗性。(4)构建pCAMBIA3301-PCS植物表达载体,转化农杆菌GV3101,并通过花序浸染法转化拟南芥植株。通过Basta筛选和PCR扩增鉴定,目前已获得部分T2代阳性转基因株系。该研究结果为进一步筛选T3代纯合转基因株系奠定基础,为进一步研究植物在重金属富集和解毒方面的机制奠定基础。
[Abstract]:Phytochelatinsus (PCS) is a polypeptide compound which can effectively chelate heavy metal ions such as Cd2 Pb2 and Cu2 in plant cells. It has a highly conserved primary structure:: (纬 -Glu-CysCysn Glynus 211C). It plays an important role in the enrichment and detoxification of free heavy metal ions in plants and yeast cells. Phytochelatin synthase (Phytochelatin synthase) is the key enzyme to catalyze its synthesis. Based on the transcriptional data of Tartary buckwheat leaves under Pb2 stress, the CDs and DNA sequences of FtPCS gene of Tartary buckwheat were cloned by RT-PCR. After a series of bioinformatics analysis, the obtained CDs sequence of Tartary buckwheat FtPCS gene was transformed into E. coli BL21 Starder DE3, Saccharomyces cerevisiae YK44 mutants and Arabidopsis thaliana for heterologous expression. The function and expression characteristics of heavy metal Cd2 stress were analyzed. In order to further reveal the mechanism of FtPCS in the process of heavy metal enrichment and detoxification in Tartary buckwheat plants, the main conclusions of this study are: 1: 1) the CDs sequence of FtPCS gene of Tartary buckwheat is 1 485 BP, encoding 494 amino acids. The predicted molecular weight of 55.10 kDa FtPCS gene DNA sequence is 5 456 BP, which consists of 8 exons and 7 introns, and the splicing sites on both sides of intron meet the GT-AG rule. Bioinformatics analysis shows that the amino acid sequence of FtPCS gene is highly conserved. There are five conserved Cys-residues characteristic sites. The Cys-terminal sequence of FtPCS protein contains 12 variable Cys-residue sites, which is the main heavy metal ion binding site. The recombinant expression vector of pET28a-PCS is constructed by NEBuilder HiFi DNAAssembly technique. The results of soluble analysis showed that the FtPCS protein was expressed in the form of inclusion body in E. coli. The purified FtPCS protein was obtained by Co2 chelation chromatography combined with gradient dialysis renaturation. The catalytic activity of FtPCS recombinant protein in the presence of Pb2 was analyzed by reverse HPLC and post-column derivatization of DTNB. The results showed that the purified FtPCS protein had catalytic activity. The reduced glutathione glutathione (GSH) can be complexed to form PC compounds, but the low concentration of Pb2 can activate its activity. PYES2-PCS expression vector can be constructed by NEBuilder HiFi DNAAssembly technique. The strain of Saccharomyces cerevisiae YK44 sensitive to heavy metal ions Zn2 / CD _ 2 / Ni _ 2 / Co _ 2 was transformed and expressed by galactose. The results of yeast functional complementation test showed that under different concentration of Cd2 stress, The tolerance of yeast strain transformed with recombinant plasmid of pYES2-PCS to heavy metal Cd2 was significantly higher than that of non-loaded yeast strain. The growth curve of yeast under Cd2 stress also indicated the expression of FtPCS gene in yeast cells. It can make up for the defect of heavy metal resistance gene, thus improve its resistance to Cd2, construct pCAMBIA3301-PCS plant expression vector, transform Agrobacterium tumefaciens GV3101, and transform Arabidopsis thaliana plants by inflorescence soaking. Basta screening and PCR amplification were used to identify Arabidopsis thaliana plants. At present, some T2 generation positive transgenic lines have been obtained. The results laid a foundation for further screening of T3 generation homogenized transgenic lines and for further study on the mechanism of heavy metal enrichment and detoxification in plants.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：X173;S517;Q943.2

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