本文關(guān)鍵詞： 超積累型東南景天 植物螯合肽合成酶 鎘 超表達(dá)　出處：《山東農(nóng)業(yè)大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：全球土壤面臨重金屬污染威脅,而對解決這一問題,目前新興的技術(shù)之一就是植物修復(fù)技術(shù)。超積累型東南景天是浙大楊肖娥教授于浙江省的一個(gè)古老鉛鋅礦區(qū)中發(fā)現(xiàn)的,對Cd、Pb和Zn有較強(qiáng)的超積累作用。早有研究表明PCS基因在植物參與重金屬螯合過程有重要作用,本文以超積累型東南景天為實(shí)驗(yàn)材料,克隆獲得SaPCS基因,對其進(jìn)行功能研究,結(jié)論如下:1、從已獲得的超積累型東南景天的轉(zhuǎn)錄組數(shù)據(jù)篩選獲得一個(gè)顯著受鎘脅迫影響的基因片段,經(jīng)分析該片段含有完整的CDS區(qū),利用PCR進(jìn)行擴(kuò)增得到SaPCS基因。SaPCS基因ORF區(qū)長1668 bp,共編碼555個(gè)氨基酸,分子量大小為62.078 kD。通過生物信息學(xué)分析軟件序列分析發(fā)現(xiàn),SaPCS蛋白含有PCS的完整結(jié)構(gòu)域,無跨膜結(jié)構(gòu)。分析SaPCS基因組全長4555 bp,對比ORF區(qū)序列可得該基因含有9個(gè)外顯子和8個(gè)內(nèi)含子。2、利用qRT-PCR檢測了東南景天SaPCS基因在不同組織部位及鎘脅迫不同時(shí)間下的表達(dá)情況。實(shí)驗(yàn)結(jié)果表明,在正常條件下,SaPCS基因在根部表達(dá)最高,莖部略高于葉部。脅迫處理后,根部表達(dá)量上升,從24 h后一直保持顯著增長,在96 h的表達(dá)量約為0 h的13倍。葉部表達(dá)量先下降后上升,在48 h達(dá)到最大值,約為初始值的1.5倍,隨后又顯著下降。這表明脅迫初期SaPCS基因主要表達(dá)部位是根部,隨后是葉部。3、將SaPCS基因構(gòu)建酵母表達(dá)載體并轉(zhuǎn)化鎘敏感型酵母菌株ycf1,通過點(diǎn)板實(shí)驗(yàn)、生長曲線和酵母鎘積累量的測定研究SaPCS對ycf1菌株的耐鎘性影響。結(jié)果顯示:在含鎘平板上,轉(zhuǎn)SaPCS酵母轉(zhuǎn)化子生長狀況明顯好于轉(zhuǎn)空載酵母;生長曲線結(jié)果表明了轉(zhuǎn)SaPCS酵母轉(zhuǎn)化子長勢明顯優(yōu)于轉(zhuǎn)空載酵母,說明SaPCS基因能夠提高對鎘的抗性。轉(zhuǎn)SaPCS酵母轉(zhuǎn)化子積累的鎘含量顯著高于轉(zhuǎn)空載酵母,說明SaPCS基因能增強(qiáng)酵母對鎘的耐性和積累。4、將SaPCS基因在擬南芥中超表達(dá)后,鑒定并篩選出陽性、表達(dá)量高的三個(gè)轉(zhuǎn)基因株系。對這三個(gè)株系和野生型擬南芥植株進(jìn)行鎘處理,觀察表型變化、分別測定根部和葉部的各項(xiàng)生理指標(biāo)。結(jié)果表明轉(zhuǎn)基因擬南芥生長狀況明顯好于野生型。并且鎘積累量結(jié)果為,轉(zhuǎn)基因株系鎘積累量顯著高于野生型。對擬南芥幼苗根部進(jìn)行Cd~(2+)流速和方向測定,發(fā)現(xiàn)Cd~(2+)始終是流入細(xì)胞的,且轉(zhuǎn)基因植株流速顯著高于野生型植株。這三個(gè)實(shí)驗(yàn)均表明SaPCS基因能夠增強(qiáng)植株對鎘的抗性和積累量。
[Abstract]:Facing the global soil heavy metal pollution threat, and to solve this problem, the current technology is one of the emerging phytoremediation technology. Hyperaccumulator Sedum alfredii is found in Zhejiang University professor Yang Xiaoe an ancient lead-zinc mine in Zhejiang province in the Cd, Pb and Zn have strong super accumulation. Early studies have shown that PCS the gene has an important role in plants involved in heavy metal chelating process, the hyperaccumulator Sedum alfredii as experimental materials, SaPCS gene was cloned, function research, the conclusions are as follows: 1, obtained a significant influence of cadmium stress by gene fragment from hyperaccumulator Sedum type days of transcriptome data have been obtained through the analysis, the fragment containing the complete CDS region were amplified SaPCS gene.SaPCS gene ORF region of 1668 BP by PCR, encoding 555 amino acids, the molecular weight is 62.078 kD. by Bioinformatics Software analysis of sequence analysis showed that the complete SaPCS domain containing PCS protein, no transmembrane structure. Analysis of SaPCS genome is 4555 BP, the ORF sequences can be compared to the gene contains 9 exons and 8 introns of.2 were detected s.alfredii SaPCS expression in different tissue parts and cadmium stress at different time with qRT-PCR. The experimental results show that, under normal conditions, SaPCS gene expression was the highest in roots and stems is slightly higher than that of leaf. After the stress treatment, the root was increased from 24, h has maintained a remarkable growth, in 96 the expression of h was about 13 times the 0 leaf H. The expression increased after the first drop in 48, H reached the maximum value is about 1.5 times of the initial value, then decreased. This indicates that SaPCS gene is mainly expressed in the initial stress is the root, followed by leaf.3, SaPCS gene to construct the yeast expression vector and transformation of cadmium sensitive yeast Strain ycf1, through plate test, impact resistance of SaPCS determination of cadmium and cadmium accumulation in yeast growth curve of strain ycf1. The results showed that the cadmium plate, SaPCS yeast transformant growth condition was better than to load yeast; growth curve indicated that the transgenic SaPCS yeast transformants were obviously better than turn the no-load yeast, indicating that the SaPCS gene can improve the resistance to cadmium. The cadmium content of transgenic SaPCS yeast transformants accumulation was significantly higher than that in turn no-load yeast, indicating that SaPCS gene can enhance the tolerance of yeast on cadmium accumulation and.4, SaPCS gene expression in Arabidopsis super, identified and positive, high content of three transgenic strain expression. Of the three lines and the wild type Arabidopsis plants to cadmium treatment, observe phenotypic changes, the physiological indexes of leaves and roots were measured. The results showed that the growth of transgenic Arabidopsis like Kuang Mingxian Better than the wild type. And the accumulation of cadmium, cadmium accumulation in transgenic lines were significantly higher than that of wild type. The Cd~ of Arabidopsis root (2+) determination of velocity and direction, found that Cd~ (2+) is always flowing into the cell, and the flow rate of transgenic plants was significantly higher than that of wild type plants. These three experiments show that the SaPCS gene can enhance resistance to cadmium and accumulation in plants.

【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：X173;Q943.2

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