东南景天镉超积累相关基因SaPCS的克隆和功能研究

发布时间：2018-02-28 09:54

本文关键词： 超积累型东南景天植物螯合肽合成酶镉超表达　出处：《山东农业大学》2017年硕士论文　论文类型：学位论文

【摘要】：全球土壤面临重金属污染威胁,而对解决这一问题,目前新兴的技术之一就是植物修复技术。超积累型东南景天是浙大杨肖娥教授于浙江省的一个古老铅锌矿区中发现的,对Cd、Pb和Zn有较强的超积累作用。早有研究表明PCS基因在植物参与重金属螯合过程有重要作用,本文以超积累型东南景天为实验材料,克隆获得SaPCS基因,对其进行功能研究,结论如下:1、从已获得的超积累型东南景天的转录组数据筛选获得一个显著受镉胁迫影响的基因片段,经分析该片段含有完整的CDS区,利用PCR进行扩增得到SaPCS基因。SaPCS基因ORF区长1668 bp,共编码555个氨基酸,分子量大小为62.078 kD。通过生物信息学分析软件序列分析发现,SaPCS蛋白含有PCS的完整结构域,无跨膜结构。分析SaPCS基因组全长4555 bp,对比ORF区序列可得该基因含有9个外显子和8个内含子。2、利用qRT-PCR检测了东南景天SaPCS基因在不同组织部位及镉胁迫不同时间下的表达情况。实验结果表明,在正常条件下,SaPCS基因在根部表达最高,茎部略高于叶部。胁迫处理后,根部表达量上升,从24 h后一直保持显著增长,在96 h的表达量约为0 h的13倍。叶部表达量先下降后上升,在48 h达到最大值,约为初始值的1.5倍,随后又显著下降。这表明胁迫初期SaPCS基因主要表达部位是根部,随后是叶部。3、将SaPCS基因构建酵母表达载体并转化镉敏感型酵母菌株ycf1,通过点板实验、生长曲线和酵母镉积累量的测定研究SaPCS对ycf1菌株的耐镉性影响。结果显示:在含镉平板上,转SaPCS酵母转化子生长状况明显好于转空载酵母;生长曲线结果表明了转SaPCS酵母转化子长势明显优于转空载酵母,说明SaPCS基因能够提高对镉的抗性。转SaPCS酵母转化子积累的镉含量显著高于转空载酵母,说明SaPCS基因能增强酵母对镉的耐性和积累。4、将SaPCS基因在拟南芥中超表达后,鉴定并筛选出阳性、表达量高的三个转基因株系。对这三个株系和野生型拟南芥植株进行镉处理,观察表型变化、分别测定根部和叶部的各项生理指标。结果表明转基因拟南芥生长状况明显好于野生型。并且镉积累量结果为,转基因株系镉积累量显著高于野生型。对拟南芥幼苗根部进行Cd~(2+)流速和方向测定,发现Cd~(2+)始终是流入细胞的,且转基因植株流速显著高于野生型植株。这三个实验均表明SaPCS基因能够增强植株对镉的抗性和积累量。
[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.

【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：X173;Q943.2

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