本文選題：松材線蟲 + 山梨醇脫氫酶�。� 參考：《青島大學(xué)》2016年碩士論文

【摘要】：由松材線蟲(Bursaphelenchus xylophilus)引起的松樹萎蔫病(pine wilt disease)是一種極具毀滅性的松林病害,導(dǎo)致包括中國在內(nèi)的世界范圍松林資源出現(xiàn)巨大的損失。在前期研究中,本實驗室通過對松材線蟲轉(zhuǎn)錄組進行高通量測序,獲得了與其致病性相關(guān)的差異表達基因sodh-1的部分序列。在本研究中,根據(jù)sodh-1的部分序列,利用PCR技術(shù)首次從松材線蟲的cDNA文庫中克隆出松材線蟲編碼山梨醇脫氫酶(SODH-1)的完整開放閱讀框(ORF),序列分析表明,該ORF全長是1059 bp,編碼352個氨基酸。將松材線蟲sodh-1基因與表達載體pET-15b連接構(gòu)建重組表達質(zhì)粒pET-15b-sodh-1。將構(gòu)建好的重組表達載體轉(zhuǎn)化大腸桿菌BL21(DE3)中,構(gòu)建了工程菌。工程菌擴大培養(yǎng)后經(jīng)異丙基-β-D硫代半乳糖苷(IPTG)誘導(dǎo),SDS-PAGE分析表明,重組山梨醇脫氫酶在大腸桿菌中實現(xiàn)了高效表達,表達產(chǎn)物主要是以包涵體形式存在。包涵體溶于8 mol/L尿素溶液,通過稀釋復(fù)性法并經(jīng)Ni2+樹脂親和層析得到可溶性的重組山梨醇脫氫酶。SDS-PAGE分析表明,重組蛋白為41 kDa。對重組SODH-1蛋白催化反應(yīng)的底物特異性進行分析,結(jié)果表明,純化的重組山梨醇脫氫酶對供試的8種醇都具有一定的催化作用,但對正丙醇的催化作用明顯高于其他7種醇類。在以正丙醇為底物時,純化的重組松材線蟲山梨醇脫氫酶最適溫度是30°C,最適pH是pH8.0。應(yīng)用實時熒光定量PCR(qRT-PCR)技術(shù)檢測了松材線蟲攜帶的熒光假單胞菌(Pseudomonas fluorescens GcM5-1A)以及乙醇對松材線蟲山梨醇脫氫酶基因sodh-1表達水平的影響,結(jié)果顯示熒光假單胞菌和乙醇皆可引起松材線蟲sodh-1基因的上調(diào)表達,且熒光假單胞菌處理組sodh-1基因表達水平是無菌松材線蟲對照組的2.65倍。松材線蟲經(jīng)4%和6%濃度乙醇處理后,其sodh-1基因表達水平明顯高于對照組,分別是對照組的1.11倍和1.45倍。本研究首次分析了松材線蟲sodh-1基因的特征和功能,并證明了松材線蟲所攜帶的熒光假單胞菌、環(huán)境乙醇濃度與sodh-1基因表達水平之間存在明顯的相關(guān)性,這為從分子水平上闡明松材線蟲的致病機制奠定了基礎(chǔ)。
[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 擄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.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：S763.18
，

本文編號：1951043