松材线虫山梨醇脱氢酶基因的克隆与功能性研究

发布时间：2018-05-29 13:27

  本文选题：松材线虫 + 山梨醇脱氢酶　； 参考：《青岛大学》2016年硕士论文

【摘要】：由松材线虫(Bursaphelenchus xylophilus)引起的松树萎蔫病(pine wilt disease)是一种极具毁灭性的松林病害,导致包括中国在内的世界范围松林资源出现巨大的损失。在前期研究中,本实验室通过对松材线虫转录组进行高通量测序,获得了与其致病性相关的差异表达基因sodh-1的部分序列。在本研究中,根据sodh-1的部分序列,利用PCR技术首次从松材线虫的cDNA文库中克隆出松材线虫编码山梨醇脱氢酶(SODH-1)的完整开放阅读框(ORF),序列分析表明,该ORF全长是1059 bp,编码352个氨基酸。将松材线虫sodh-1基因与表达载体pET-15b连接构建重组表达质粒pET-15b-sodh-1。将构建好的重组表达载体转化大肠杆菌BL21(DE3)中,构建了工程菌。工程菌扩大培养后经异丙基-β-D硫代半乳糖苷(IPTG)诱导,SDS-PAGE分析表明,重组山梨醇脱氢酶在大肠杆菌中实现了高效表达,表达产物主要是以包涵体形式存在。包涵体溶于8 mol/L尿素溶液,通过稀释复性法并经Ni2+树脂亲和层析得到可溶性的重组山梨醇脱氢酶。SDS-PAGE分析表明,重组蛋白为41 kDa。对重组SODH-1蛋白催化反应的底物特异性进行分析,结果表明,纯化的重组山梨醇脱氢酶对供试的8种醇都具有一定的催化作用,但对正丙醇的催化作用明显高于其他7种醇类。在以正丙醇为底物时,纯化的重组松材线虫山梨醇脱氢酶最适温度是30°C,最适pH是pH8.0。应用实时荧光定量PCR(qRT-PCR)技术检测了松材线虫携带的荧光假单胞菌(Pseudomonas fluorescens GcM5-1A)以及乙醇对松材线虫山梨醇脱氢酶基因sodh-1表达水平的影响,结果显示荧光假单胞菌和乙醇皆可引起松材线虫sodh-1基因的上调表达,且荧光假单胞菌处理组sodh-1基因表达水平是无菌松材线虫对照组的2.65倍。松材线虫经4%和6%浓度乙醇处理后,其sodh-1基因表达水平明显高于对照组,分别是对照组的1.11倍和1.45倍。本研究首次分析了松材线虫sodh-1基因的特征和功能,并证明了松材线虫所携带的荧光假单胞菌、环境乙醇浓度与sodh-1基因表达水平之间存在明显的相关性,这为从分子水平上阐明松材线虫的致病机制奠定了基础。
[Abstract]:Pine wilt disease caused by Bursaphelenchus xylophilus) is a devastating pine forest disease, which causes great loss of pine forest resources in the world, including China. In previous studies, we obtained some sequences of differentially expressed gene sodh-1 related to the pathogenicity of pine wood nematodes by high-throughput sequencing. In this study, according to the partial sequence of sodh-1, the complete open reading frame of pine wood nematode encoding sorbitol dehydrogenase (SODH-1) was cloned from the cDNA library of pine wood nematode by PCR technique for the first time. The full length of the ORF is 1059 BP, encoding 352 amino acids. The recombinant expression plasmid pET-15b-sodh-1 was constructed by ligating the sodh-1 gene of pine wood nematodes with the expression vector pET-15b. The recombinant expression vector was transformed into Escherichia coli BL21 DE3) and the engineering strain was constructed. SDS-PAGE analysis showed that the recombinant sorbitol dehydrogenase was highly expressed in Escherichia coli and the expression product was mainly in the form of inclusion body. The inclusion body was dissolved in 8 mol/L urea solution. The soluble recombinant sorbitol dehydrogenase. SDS-PAGE analysis showed that the recombinant protein was 41 kDa by dilution renaturation and Ni2 resin affinity chromatography. The substrate specificity of the catalytic reaction of recombinant SODH-1 protein was analyzed. The results showed that the purified recombinant sorbitol dehydrogenase could catalyze the eight alcohols tested, but the catalytic effect on n-propanol was significantly higher than that on the other seven alcohols. When n-propanol was used as substrate, the optimum temperature and pH of purified sorbitol dehydrogenase from recombinant pine wood nematode were 30 掳C and 8.0, respectively. The effects of Pseudomonas fluorescens GcM5-1A and ethanol on the expression of sorbitol dehydrogenase gene sodh-1 in pine wood nematode were detected by real-time fluorescence quantitative PCRQRT-PCRR technique. The results showed that both Pseudomonas fluorescens and ethanol could induce the up-regulation of sodh-1 gene expression in pine wood nematode, and the expression level of sodh-1 gene in the treated group was 2.65 times higher than that in the control group. The sodh-1 gene expression of pine wood nematode treated with 4% and 6% ethanol was 1.11 and 1.45 times higher than that of the control, respectively. In this study, the characteristics and functions of sodh-1 gene of pine wood nematode were analyzed for the first time, and it was proved that there was a significant correlation between the environmental ethanol concentration and the expression level of sodh-1 gene in Pseudomonas fluorescein carried by pine wood nematode. This laid a foundation for elucidating the pathogenic mechanism of pine wood nematode at molecular level.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S763.18
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本文编号：1951043