發(fā)布時間：2018-03-16 23:18

  本文選題：苦蕎　切入點：植物絡(luò)合素合酶　出處：《西北農(nóng)林科技大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：植物絡(luò)合素(Phytochelatins,PCs)是在植物細(xì)胞內(nèi)廣泛存在的一種可以有效螯合Cd2+、Pb2+、Cu2+等重金屬離子的多肽類化合物,其具有高度保守的一級結(jié)構(gòu):(γ-Glu-Cys)n-Gly(n=2~11)。在植物和酵母細(xì)胞內(nèi),PCs對游離重金屬離子的富集和解毒過程具有重要作用,而植物絡(luò)合素合酶(Phytochelatin synthase,PCS)是催化其合成的關(guān)鍵酶。本實驗研究以Pb2+脅迫條件下的苦蕎葉片轉(zhuǎn)錄組數(shù)據(jù)為基礎(chǔ),通過RT-PCR克隆了苦蕎植物絡(luò)合素合酶(FtPCS)基因CDs序列和DNA序列,并對其進(jìn)行了一系列的生物信息學(xué)分析。隨后,將獲得的苦蕎FtPCS基因CDs序列分別轉(zhuǎn)化E.coli BL21 Star(DE3)、釀酒酵母YK44突變體菌株、以及擬南芥植株進(jìn)行異源表達(dá),并對其在重金屬Cd2+脅迫條件下的功能和表達(dá)特點進(jìn)行分析,以期為進(jìn)一步揭示FtPCS在苦蕎植物重金屬富集和解毒過程中的機(jī)制奠定基礎(chǔ)。本研究得到的主要結(jié)論有:(1)苦蕎植物絡(luò)合素合酶(FtPCS)基因的CDs序列長1 485 bp,編碼494個氨基酸,預(yù)測分子量為55.10 kDa;FtPCS基因的DNA序列長5 456 bp,由8個外顯子和7個內(nèi)含子組成,且內(nèi)含子兩側(cè)的剪接位點均滿足GT-AG規(guī)則。生物信息學(xué)分析表明,FtPCS基因氨基酸序列N-端序列高度保守,包括5個保守的Cys-殘基特征位點,是FtPCS蛋白活性中心;C-端序列包含12個可變的Cys-殘基位點,是主要的重金屬離子結(jié)合位點。(2)通過NEBuilder HiFi DNAAssembly技術(shù),構(gòu)建pET28a-PCS重組表達(dá)載體,轉(zhuǎn)化E.coli BL21 Star(DE3)并通過IPTG誘導(dǎo)表達(dá)�？扇苄苑治鼋Y(jié)果表明,FtPCS在E.coli內(nèi)以包涵體的形式大量表達(dá)。通過Co2+螯合層析結(jié)合梯度透析復(fù)性,獲得了純化的FtPCS蛋白;通過反向-HPLC結(jié)合DTNB[5,5'-二硫代雙(2-硝基苯甲酸)]柱后衍生的方法,對FtPCS重組蛋白在Pb2+存在條件下的催化活性進(jìn)行分析。結(jié)果表明,純化的FtPCS蛋白具有催化活性,能將還原型谷胱甘肽(GSH)絡(luò)合生成PC化合物,而低濃度的Pb2+對其活性具有激活作用。(3)通過NEBuilder HiFi DNAAssembly技術(shù),構(gòu)建pYES2-PCS酵母表達(dá)載體,轉(zhuǎn)化對重金屬離子Zn2+/Cd2+/Ni2+/Co2+敏感的釀酒酵母YK44突變體菌株,并通過半乳糖誘導(dǎo)表達(dá)。酵母功能互補(bǔ)實驗結(jié)果表明,在不同濃度Cd2+脅迫條件下,轉(zhuǎn)化pYES2-PCS重組質(zhì)粒的酵母菌株比轉(zhuǎn)化空載的酵母菌株對重金屬Cd2+的耐受性明顯提高。在Cd2+脅迫條件下的酵母生長曲線同樣表明,FtPCS基因在酵母細(xì)胞中的表達(dá),能夠彌補(bǔ)其重金屬抗性基因的缺陷,從而提高其對Cd2+的抗性。(4)構(gòu)建pCAMBIA3301-PCS植物表達(dá)載體,轉(zhuǎn)化農(nóng)桿菌GV3101,并通過花序浸染法轉(zhuǎn)化擬南芥植株。通過Basta篩選和PCR擴(kuò)增鑒定,目前已獲得部分T2代陽性轉(zhuǎn)基因株系。該研究結(jié)果為進(jìn)一步篩選T3代純合轉(zhuǎn)基因株系奠定基礎(chǔ),為進(jìn)一步研究植物在重金屬富集和解毒方面的機(jī)制奠定基礎(chǔ)。
[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:: (緯 -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.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：X173;S517;Q943.2

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