超积累型东南景天SaCAD基因的克隆及其功能分析

发布时间：2018-05-16 17:01

  本文选题：超积累型东南景天 + 肉桂醇脱氢酶　； 参考：《中国林业科学研究院》2016年硕士论文

【摘要】：植物细胞壁具有阻隔、吸附和运输重金属的作用。木质素在填充在次生壁的骨架内,能阻止重金属离子的吸收和运输。肉桂醇脱氢酶(cinnamyl alcohol dehydrogenases,CAD)是植物细胞壁木质素苯丙烷合成途径中第一个研究的酶,在植物木质素合成和逆境胁迫响应中起着关键作用。本论文以镉超积累型东南景天为实验材料,从中分离出受镉胁迫诱导的SaCAD基因并对其功能进行分析。主要结论如下:1、从本实验室现有的东南景天转录组数据中筛选得到一个在镉胁迫下发生显著变化的SaCAD基因片段,利用RACE技术扩增出SaCAD的cDNA全长。该基因cDNA全长1371 bp,ORF长1089 bp,编码362个氨基酸,蛋白分子量38.65 kD。利用PCR技术扩增出全长2275 bp、含有5个外显子和4个内含子的基因组DNA序列。2、生物信息学分析及亚细胞定位实验显示SaCAD蛋白定位在细胞质中,SaCAD-GFP融合蛋白主要在细胞质内聚集,这与CELLO v.2.5和Plant-mPLoc软件预测的结果一致。进化树分析表明SaCAD与拟南芥AtCAD7、AtCAD8,毛白杨的PtCAD7和野茶树CsCAD3亲缘关系最近。多序列比对发现,SaCAD氨基酸序列具有醇脱氢酶典型的保守结构域:Zn1结合位点(Zn1 binding motif)、Zn2结合位点(Zn2 binding motif)和富含甘氨酸的NADPH结合位点(NADPH binding motif)。3、利用实时荧光定量(qRT-PCR)对SaCAD进行分析发现:SaCAD在根部表达量最高,茎部和叶片的表达量较低。镉胁迫后根部的SaCAD的表达量呈先上升后降低的趋势,而茎部和叶部的表达量随着胁迫时间的延长而升高,胁迫72 h后表达量下降。说明该基因主要在东南景天根部发挥作用,随着镉胁迫时间的延长,茎和叶中的SaCAD也开始参与应答。4、构建SaCAD酵母表达载体转化镉敏感型酵母菌株ycf1,研究SaCAD对ycf1耐镉性的影响。不同镉浓度的点板实验结果显示,超表达SaCAD的酵母转化子和空载的长势没有明显差异,说明SaCAD不能提高酵母突变体的耐镉性。对转基因拟南芥纯合株系进行镉耐性分析后发现:镉胁迫后转基因植株的长势和生理状况都优于野生型植株且转基因株系最高能积累1079.3 mg·kg-1的镉,说明SaCAD基因能够增加拟南芥的镉抗性和镉积累能力。5、利用qRT-PCR对SaCAD与拟南芥AtCAD基因家族成员的表达情况进行分析后发现,SaCAD在转基因拟南芥中具有较高的转录丰度,镉胁迫下SaCAD较AtCADs具有明显的表达优势。对拟南芥酶活、细胞壁结构组分进行分析后发现:超表达Sa CAD后的拟南芥具有更高的CAD酶活性,细胞壁木质化加重,木质素和果胶含量也相应提高。此外,镉胁迫后转基因植株的上述各个指标都明显优于野生型拟南芥。
[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.
【学位授予单位】：中国林业科学研究院
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q943.2

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