石油污染胁迫下紫茉莉XTH基因克隆及表达分析

发布时间：2018-01-01 04:25

本文关键词：石油污染胁迫下紫茉莉XTH基因克隆及表达分析　出处：《沈阳农业大学》2017年硕士论文　论文类型：学位论文

【摘要】：随着经济的发展及对石油开采量的大量需求,在其运输和加工的过程中造成了周边土壤、水等环境严重的石油污染,成为环境科学领域域解决的问题,作为环境友好型的植物修复策略成为污染生态的研究热点。本研究以紫茉莉为供试植物,根据前期转录组测序结果显示紫茉莉的XTH基因在清洁组与石油污染组表达量有显著差异,而对其功能进行进一步研究,为揭示紫茉莉对石油的耐受或降解机制寻求证据。通过克隆该基因、生物信息学分析、定位表达鉴定及相关生理指标的测定,分析该基因功能。本试验研究结果如下:1.将紫茉莉栽培于石油浓度为 0 g/kg、5 g/kg、10 g/kg、20 g/kg、30 g/kg、40 g/kg 的土壤中12 h、24 h、36 h、48 h、72 h,对紫茉莉根部做了相关生理指标的测定。不同石油浓度下,有无木葡聚糖处理的植株的SOD、POD、CAT酶活性均在24 h达到最高,可溶性蛋白和MDA含量也在24h含量最高。当石油浓度为20 g/kg时,SOD、POD酶活性均高于其它石油浓度下相应时间点的SOD、POD酶活性;可溶性蛋白和MDA含量也均高于其它石油浓度下相应时间的可溶性蛋白和MDA含量。而在石油浓度为5 g/kg时,CAT酶活性大于其它石油浓度相应时间点的CAT活性。SOD、POD、CAT活性和可溶性蛋白含量有木葡聚糖处理高于无木葡聚糖处理,而MDA含量与其相反。2.将紫茉莉栽培于石油浓度为 0 g/kg、5 g/kg、10 g/kg、20 g/kg、30 g/kg、40 g/kg 土壤中0h、12h、24h、36h、48h、72h,对根部XTH基因进行实时荧光定量PCR分析。结果显示,在石油浓度为5 g/kg、10 g/kg、20 g/kg、30 g/kg、40 g/kg时,XTH基因的相对表达量均随着时间的增加呈现上调趋势。石油浓度为5 g/kg、10 g/kg时,XTH基因在12h-36h相对表达量上升,36h-72h相对表达量下降。石油浓度为20 g/kg、30 g/kg、40 g/kg时,XTH基因在12 h-24 h相对表达量增加,24 h-72 h相对表达量下降。3.通过生物信息学软件分析,该基因的编码区长度为887 bp,编码285个氨基酸,是一种分泌蛋白。通过MEGA 5.0软件构建系统进化树,可以看到它与PtXTH1亲缘关系最近,PtXTH1基因具有木葡聚糖内转苷酶(XET)活性。通过同源序列比对也发现MirXTH蛋白具有木葡聚糖内转苷酶(XET)活性,催化活性域DEIDFEDFLG。4.通过基因枪的方法将带有GFP标签的MirXTH基因转化到洋葱表皮细胞,通过共聚焦显微镜观察结果发现该基因是定位在外质体上,是一个与细胞壁代谢有关的基因,参与调控细胞壁的延展性。5.成功地构建了植物表达载体pCAMBIA 1302-MrXTH,将表达载体pCAMBIA 1302-MirXTH转入农杆菌(LBA4404)中,预将其转入到野生型拟南芥中,做转基因验证其功能。
[Abstract]:With the development of economy and the oil production of the large demand, causing the surrounding soil in the process of transportation and processing, water environment, serious oil pollution, become the field of environmental science domain to solve the problem, as the plant repair strategy of environment friendly has become a hot topic of pollution state. In this study, purple jasmine as tested plants, according to preliminary transcriptome sequencing showed that XTH gene mirabilis had significant differences in the expression of oil pollution and clean group group, and further study of its function, to seek evidence for revealing purple jasmine oil tolerance and degradation mechanism. Through the gene cloning, bioinformatics analysis, location determination expression and identification of related physiological indexes, the analysis of gene function. The results are as follows: 1. the purple jasmine cultivation in the oil concentration of 0 g/kg, 5 g/kg, 10 g/kg, 20 g/kg, 30 g/kg, 40 g/kg In the soil of 12 h, 24 h, 36 h, 48 h, 72 h, the root of Mirabilis was measured. The physiological indexes of different oil concentrations, there is no xyloglucan processing plant of SOD, POD, CAT enzyme activity reached the highest at 24 h, the contents of soluble protein and MDA in 24h was the highest. When the oil concentration was 20 g/kg, SOD, POD activity was significantly higher than the corresponding time point the other oil concentrations of SOD, POD enzyme activity; soluble protein content and MDA content were higher than other oil concentration under the corresponding time the contents of soluble protein and MDA. And in the oil concentration of 5 g/kg. The activity of CAT is greater than the activity of CAT.SOD, the other oil concentration at the same time POD, CAT activity and soluble protein content of xyloglucan is higher than that without xyloglucan treatment, while the content of MDA in contrast to.2. will be cultivated in purple jasmine oil concentration of 0 g/kg, 5 g/kg, 10 g/kg, 20 g/kg, 30 g/kg, 40 g/kg The soil 0h, 12h, 24h, 36h, 48h, 72h, were analyzed by real-time fluorescence quantitative PCR of root XTH gene. The results showed that the oil concentration was 5 g/kg, 10 g/kg, 20 g/kg, 30 g/kg, 40 g/kg, the relative expression of XTH gene was increased with the increase of time. Stone the oil concentration of 5 g/kg, 10 g/kg, XTH gene in 12h-36h expression increased, 36h-72h expression decreased relative. The oil concentration of 20 g/kg, 30 g/kg, 40 g/kg, XTH gene in 12 h-24 h 24 h-72 relative expression increased, H expression decreased relative.3. by bioinformatics software analysis. The length of the encoding regions of the gene is 887 BP, encoding 285 amino acids, is a secreted protein. The phylogenetic tree was constructed by MEGA 5 software, you can see it recently and PtXTH1 related, PtXTH1 gene has a xyloglucan endotransglucosylase glucosyltransferase (XET) activity. The sequence also found MirXTH protein With xyloglucan endotransglucosylase glucosyltransferase (XET) activity, MirXTH gene transformation method of catalytic domain of DEIDFEDFLG.4. by gene gun with GFP tag into onion epidermal cells by confocal microscopy. The results showed that the gene is localized in the apoplast, is a gene and cell wall metabolism, ductility of.5. participate in the regulation of cell wall was successfully constructed the plant expression vector pCAMBIA 1302-MrXTH expression vector into Agrobacterium pCAMBIA 1302-MirXTH (LBA4404), pre transformed into wild type Arabidopsis transgenic do verify its function.

【学位授予单位】：沈阳农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：X74;Q943.2

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