本文選題：超積累型東南景天 + 肉桂醇脫氫酶�。� 參考：《中國(guó)林業(yè)科學(xué)研究院》2016年碩士論文

【摘要】：植物細(xì)胞壁具有阻隔、吸附和運(yùn)輸重金屬的作用。木質(zhì)素在填充在次生壁的骨架內(nèi),能阻止重金屬離子的吸收和運(yùn)輸。肉桂醇脫氫酶(cinnamyl alcohol dehydrogenases,CAD)是植物細(xì)胞壁木質(zhì)素苯丙烷合成途徑中第一個(gè)研究的酶,在植物木質(zhì)素合成和逆境脅迫響應(yīng)中起著關(guān)鍵作用。本論文以鎘超積累型東南景天為實(shí)驗(yàn)材料,從中分離出受鎘脅迫誘導(dǎo)的SaCAD基因并對(duì)其功能進(jìn)行分析。主要結(jié)論如下:1、從本實(shí)驗(yàn)室現(xiàn)有的東南景天轉(zhuǎn)錄組數(shù)據(jù)中篩選得到一個(gè)在鎘脅迫下發(fā)生顯著變化的SaCAD基因片段,利用RACE技術(shù)擴(kuò)增出SaCAD的cDNA全長(zhǎng)。該基因cDNA全長(zhǎng)1371 bp,ORF長(zhǎng)1089 bp,編碼362個(gè)氨基酸,蛋白分子量38.65 kD。利用PCR技術(shù)擴(kuò)增出全長(zhǎng)2275 bp、含有5個(gè)外顯子和4個(gè)內(nèi)含子的基因組DNA序列。2、生物信息學(xué)分析及亞細(xì)胞定位實(shí)驗(yàn)顯示SaCAD蛋白定位在細(xì)胞質(zhì)中,SaCAD-GFP融合蛋白主要在細(xì)胞質(zhì)內(nèi)聚集,這與CELLO v.2.5和Plant-mPLoc軟件預(yù)測(cè)的結(jié)果一致。進(jìn)化樹分析表明SaCAD與擬南芥AtCAD7、AtCAD8,毛白楊的PtCAD7和野茶樹CsCAD3親緣關(guān)系最近。多序列比對(duì)發(fā)現(xiàn),SaCAD氨基酸序列具有醇脫氫酶典型的保守結(jié)構(gòu)域:Zn1結(jié)合位點(diǎn)(Zn1 binding motif)、Zn2結(jié)合位點(diǎn)(Zn2 binding motif)和富含甘氨酸的NADPH結(jié)合位點(diǎn)(NADPH binding motif)。3、利用實(shí)時(shí)熒光定量(qRT-PCR)對(duì)SaCAD進(jìn)行分析發(fā)現(xiàn):SaCAD在根部表達(dá)量最高,莖部和葉片的表達(dá)量較低。鎘脅迫后根部的SaCAD的表達(dá)量呈先上升后降低的趨勢(shì),而莖部和葉部的表達(dá)量隨著脅迫時(shí)間的延長(zhǎng)而升高,脅迫72 h后表達(dá)量下降。說明該基因主要在東南景天根部發(fā)揮作用,隨著鎘脅迫時(shí)間的延長(zhǎng),莖和葉中的SaCAD也開始參與應(yīng)答。4、構(gòu)建SaCAD酵母表達(dá)載體轉(zhuǎn)化鎘敏感型酵母菌株ycf1,研究SaCAD對(duì)ycf1耐鎘性的影響。不同鎘濃度的點(diǎn)板實(shí)驗(yàn)結(jié)果顯示,超表達(dá)SaCAD的酵母轉(zhuǎn)化子和空載的長(zhǎng)勢(shì)沒有明顯差異,說明SaCAD不能提高酵母突變體的耐鎘性。對(duì)轉(zhuǎn)基因擬南芥純合株系進(jìn)行鎘耐性分析后發(fā)現(xiàn):鎘脅迫后轉(zhuǎn)基因植株的長(zhǎng)勢(shì)和生理狀況都優(yōu)于野生型植株且轉(zhuǎn)基因株系最高能積累1079.3 mg·kg-1的鎘,說明SaCAD基因能夠增加擬南芥的鎘抗性和鎘積累能力。5、利用qRT-PCR對(duì)SaCAD與擬南芥AtCAD基因家族成員的表達(dá)情況進(jìn)行分析后發(fā)現(xiàn),SaCAD在轉(zhuǎn)基因擬南芥中具有較高的轉(zhuǎn)錄豐度,鎘脅迫下SaCAD較AtCADs具有明顯的表達(dá)優(yōu)勢(shì)。對(duì)擬南芥酶活、細(xì)胞壁結(jié)構(gòu)組分進(jìn)行分析后發(fā)現(xiàn):超表達(dá)Sa CAD后的擬南芥具有更高的CAD酶活性,細(xì)胞壁木質(zhì)化加重,木質(zhì)素和果膠含量也相應(yīng)提高。此外,鎘脅迫后轉(zhuǎn)基因植株的上述各個(gè)指標(biāo)都明顯優(yōu)于野生型擬南芥。
[Abstract]:Plant cell walls can block, absorb and transport heavy metals. Lignin can prevent the absorption and transport of heavy metal ions by filling in the skeleton of secondary wall. Cinnamyl alcohol dehydrogenes (cad) is the first enzyme in plant cell wall lignin phenylpropane synthesis pathway, which plays a key role in plant lignin synthesis and stress response. In this paper, SaCAD gene induced by cadmium stress was isolated and its function was analyzed. The main conclusions are as follows: 1. A SaCAD gene fragment with significant changes under cadmium stress was obtained from the existing transcriptional data of Sedum southeastern China in our laboratory, and the total cDNA of SaCAD was amplified by RACE technique. The full length of the cDNA is 1371 BP and the length is 1089 BP, which encodes 362 amino acids and the molecular weight of the protein is 38.65 kD. The genomic DNA sequence of 2275 BP with 5 exons and 4 introns was amplified by PCR technique. Bioinformatics analysis and subcellular localization experiments showed that the SaCAD protein was located in the cytoplasm of SaCAD-GFP fusion protein. This is consistent with the results predicted by CELLO v. 2.5 and Plant-mPLoc software. Phylogenetic tree analysis showed that SaCAD had the closest relationship with Arabidopsis thaliana AtCAD7 / AtCAD8, PtCAD7 of Populus tomentosa and CsCAD3 of wild tea tree. Multiple sequence alignment found that the amino acid sequence of SaCAD has a typical conserved domain of alcohol dehydrogenase, Zn1 binding motif2 binding site, Zn2 binding motif, and glycine rich NADPH binding site, NADPH binding motifl. 3. The SaCAD was studied by real-time fluorescence quantitative analysis with qRT-PCR. The analysis found that the highest level of expression was found in the root of the SaCAD. The expression of stem and leaf was low. After cadmium stress, the expression of SaCAD in root increased first and then decreased, while the expression of SaCAD in stem and leaf increased with the extension of stress time, and decreased after 72 h of stress. With the prolongation of cadmium stress time, SaCAD in stems and leaves began to participate in the response. The expression vector of SaCAD yeast was constructed to transform the cadmium sensitive yeast strain ycf1 to study the effect of SaCAD on the cadmium tolerance of ycf1. The results of dot plate experiments with different cadmium concentrations showed that there was no significant difference between yeast transformants and no-load growth of overexpression of SaCAD, indicating that SaCAD could not improve the cadmium tolerance of yeast mutants. After cadmium tolerance analysis of transgenic Arabidopsis thaliana homozygous lines, it was found that the growth and physiological status of transgenic plants under cadmium stress were superior to those of wild type plants, and the highest accumulation of cadmium in transgenic lines was 1079.3 mg kg-1. The results showed that SaCAD gene could increase cadmium resistance and cadmium accumulation ability of Arabidopsis thaliana. QRT-PCR was used to analyze the expression of SaCAD and AtCAD gene family members in Arabidopsis thaliana, and it was found that SaCAD had a high transcription abundance in transgenic Arabidopsis thaliana. The expression of SaCAD was superior to that of AtCADs under cadmium stress. The enzyme activity and cell wall structure of Arabidopsis thaliana were analyzed. The results showed that Arabidopsis thaliana had higher activity of CAD enzyme, more lignin and pectin, and higher lignin and pectin content in the cell wall of Arabidopsis thaliana after overexpression of sa CAD. In addition, the above indexes of transgenic plants under cadmium stress were obviously superior to those of wild type Arabidopsis thaliana.
【學(xué)位授予單位】：中國(guó)林業(yè)科學(xué)研究院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：Q943.2

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 安婧;宮曉雙;魏樹和;;重金屬污染土壤超積累植物修復(fù)關(guān)鍵技術(shù)的發(fā)展[J];生態(tài)學(xué)雜志;2015年11期

2 邢艷帥;喬冬梅;朱桂芬;齊學(xué)斌;;土壤重金屬污染及植物修復(fù)技術(shù)研究進(jìn)展[J];中國(guó)農(nóng)學(xué)通報(bào);2014年17期

3 黃益宗;郝曉偉;雷鳴;鐵柏清;;重金屬污染土壤修復(fù)技術(shù)及其修復(fù)實(shí)踐[J];農(nóng)業(yè)環(huán)境科學(xué)學(xué)報(bào);2013年03期

4 張正慶;鮑美娥;陳嘉斌;郝海艷;;植物對(duì)重金屬的耐性機(jī)制[J];甘肅科技;2013年05期

5 張玉秀;金玲;馮珊珊;劉金光;柴團(tuán)耀;;鎘對(duì)鎘超累積植物龍葵抗氧化酶活性及基因表達(dá)的影響[J];中國(guó)科學(xué)院研究生院學(xué)報(bào);2013年01期

6 孫福金;;重金屬“鎘”對(duì)動(dòng)植物、人體的危害及應(yīng)對(duì)措施[J];現(xiàn)代農(nóng)業(yè);2012年05期

7 崔斌;王凌;張國(guó)印;孫世友;耿暖;茹淑華;陳貴今;;土壤重金屬污染現(xiàn)狀與危害及修復(fù)技術(shù)研究進(jìn)展[J];安徽農(nóng)業(yè)科學(xué);2012年01期

8 丁艷菲;朱誠(chéng);王珊珊;劉海麗;;植物microRNA對(duì)重金屬脅迫響應(yīng)的調(diào)控[J];生物化學(xué)與生物物理進(jìn)展;2011年12期

9 任珊珊;趙艷玲;白華;蔣湘寧;蓋穎;;楊樹肉桂醇脫氫酶基因序列和表達(dá)模式分析及CAD4酶活性檢測(cè)[J];華中農(nóng)業(yè)大學(xué)學(xué)報(bào);2011年05期

10 陳婧;林振景;孟媛媛;王博;宋瑜;;土壤重金屬污染的植物修復(fù)及超富集植物的研究進(jìn)展[J];中國(guó)環(huán)境管理干部學(xué)院學(xué)報(bào);2011年01期

相關(guān)博士學(xué)位論文 前2條

1 張杰;超積累植物東南景天Cd耐性和積累的分子機(jī)制[D];浙江大學(xué);2015年

2 田生科;超積累東南景天（Sedum alfredii Hance）對(duì)重金屬（Zn/Cd/Pb）的解毒機(jī)制[D];浙江大學(xué);2010年

相關(guān)碩士學(xué)位論文 前4條

1 方潔;超積累型東南景天SaNramp6基因的克隆及其功能分析[D];中國(guó)林業(yè)科學(xué)研究院;2015年

2 李真;東南景天銅/鋅超氧化物歧化酶基因的分離與功能分析[D];山東農(nóng)業(yè)大學(xué);2014年

3 桑健;基于轉(zhuǎn)錄組測(cè)序的超積累型東南景天內(nèi)參基因篩選與HSF基因家族初步分析[D];中國(guó)林業(yè)科學(xué)研究院;2014年

4 李志菜;蠟梅肉桂醇脫氫酶基因CpCAD的克隆、表達(dá)特性分析及功能驗(yàn)證[D];西南大學(xué);2013年

，

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