蜜蜂螺原体几丁质酶、几丁质脱乙酰酶基因的研究及螺原体对意蜂抗氧化相关酶的影响

发布时间：2018-05-06 05:02

本文选题：蜜蜂螺原体 + 几丁质酶基因　；参考：《南京农业大学》2016年硕士论文

【摘要】：螺原体(spiroplasma)是柔膜菌纲中一种螺旋状无细胞壁的原核微生物,能与节肢动物和植物相互作用。在系统发育学上,螺原体与芽孢杆菌纲及其他营自由生活的厚壁菌门的细菌亲缘关系较近。我国蜜蜂螺原体病发生非常普遍,Spiroplasma melliferum是蜜蜂“爬蜂病”主要病原之一,基本生物学特性已被详细研究,但对其致病机制了解很少。通过比较基因组学和蛋白质组学研究发现,蜜蜂螺原体S.melliferum存在2种特有的基因——几丁质酶和几丁质脱乙酰酶基因,而其他几种借助昆虫传播但对昆虫无致病性的螺原体(如S.citri和S.kunkelli)没有发现这2种基因,推测这2个基因与致病性直接相关,在螺原体入侵蜜蜂过程中起作用。本论文克隆表达了蜜蜂螺原体几丁质酶和几丁质脱乙酰酶基因并测定了几丁质脱乙酰酶活力和酶学性质。另一方面,蜜蜂在受到螺原体侵染后会产生一系列抗氧化相关的防御反应,本文分析了感染螺原体后蜜蜂抗氧化应激系统中相关酶活变化。主要研究结果有:第一、应用生物信息学方法和在线预测工具,对蜜蜂螺原体CH-1几丁质脱乙酰酶的理化性质、功能域等进行预测分析,揭示蜜蜂螺原体几丁质脱乙酰酶与其他细菌该酶的进化关系。结果显示:(1)S.melliferum CH-1中的几丁质脱乙酰酶基因相对分子质量为26 kD;(2)酸碱性氨基酸比例较其他细菌小,主要表现为亲水性,无明显的跨膜区和信号肽,整个肽链位于细胞膜外,可能是外分泌蛋白;(3)与其他细菌几丁质脱乙酰酶基因序列比对显示该目的蛋白含有3个保守的基序(motif),可能是重要的功能域。对该蛋白进行生物信息学分析能够预先了解目的蛋白的基本性质和假设的空间结构,这对后续表达目的蛋白和功能探索起前导作用,并与后续的研究结果相互验证。第二、由于密码子TGA在多数柔膜菌纲细菌中编码色氨酸而非终止密码子,因此柔膜菌相关基因在大肠杆菌中的表达受到限制。为研究蜜蜂螺原体CH-1几丁质酶和几丁质脱乙酰酶的致病性,本文利用重叠延伸PCR定点突变技术克隆蜜蜂螺原体CH-1中可能与致病相关的几丁质酶(chitinase)基因chiA以及几丁质脱乙酰酶(chitin deacetylase,CDA)基因chid,利用表达载体pET-28a(+)进行蛋白表达,NTA-Ni2+柱纯化,Western blot验证,测定其活力和酶学性质。结果显示:(1)成功克隆到chiA、chid基因全长,并得到有活性的外源表达的完整几丁质脱乙酰酶蛋白和以包涵体形式表达的几丁质酶蛋白。几丁质酶及几丁质脱乙酰酶基因编码区分别为768 bp、672bp,分别编码255、223个氨基酸组成的约32 kD、28 kD的蛋白质;(2)研究中采用两种方法对几丁质酶包涵体蛋白进行复性,由于瞬间表达的蛋白质量太多以至于未能进行正确的折叠而未能成功获得有活性的目的蛋白,后续研究中可以尝试选择以毕赤酵母为载体的真核表达系统进行外源表达。第三、利用紫外分光光度法测定了外源表达几丁质脱乙酰酶的酶活力和部分酶学性质。结果显示:(1)测得外源表达的该酶活力最高可达10.14 U.mL-1;(2)最适温度为50℃左右,最适pH值为7.0-7.5,该酶对温度较不敏感,各个温度梯度对该酶处理30 min后,在35℃-60℃之间仍能保持60%以上活力,pH值稳定性试验研究表明该酶是一种较耐碱性的酶;(3)一价金属离子Na+和K+均对CDA酶活性表现出促进趋势;二价金属离子对CDA酶活力影响表现出三种趋势,分别是Ca2+、Mg2+主要表现为抑制作用,Zn2+对CDA酶活力无明显影响,Fe2+能促进酶活性。三价金属离子Fe3+和A13+离子能够在很低的离子浓度影响CDA酶活性,主要表现为促进作用。第四、通过饲喂对数期螺原体菌液感染意蜂,研究了接种后不同时间段蜜蜂体内抗氧化相关酶活性变化。结果表明:(1)在蜜蜂螺原体致病菌S.melliferum CH-1感染后,第3天蜜蜂表现出“爬蜂病”症状,第5天死亡率达到最大值。(2)感染第3天时,抗氧化应激系统中过氧化氢酶(catalase/CAT)活力先下降,随着感染时间的延长,在第5、7天表现为上升趋势,但与未感染螺原体的对照组差别不明显。(3)超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-Px)在感染螺原体初期酶活性就明显上升,第5天时酶活性达到最大,随后略微下降。研究结果说明蜜蜂在被螺原体侵染后,能引起体内清除自由基的保护系统中酶活力发生改变,机体本身的抗氧化相关酶活性变化具有一定的抵抗外界微生物毒害的作用。本研究结果为了解几丁质酶和几丁质脱乙酰酶基因在蜜蜂螺原体的致病过程中的作用以及对该基因的功能研究提供了重要信息。
[Abstract]:Spiroplasma is a kind of prokaryotic microorganism of helix cell without cell wall, which can interact with arthropods and plants. In phylogenetics, the mycoplasma is closely related to the bacteria of Bacillus spore and other free living thick wall bacteria. The occurrence of the disease is very common in our country, Spiroplasma me Lliferum is one of the main pathogens of bee crawling disease. The basic biological characteristics have been studied in detail, but there are few understanding of its pathogenesis. Through comparative genomics and proteomics research, it is found that there are 2 specific genes, chitinase and chitin deacetylase gene in the S.melliferum of honeybee stub, and other kinds of genes. The 2 genes were not found to help insects, such as S.citri and S.kunkelli, which were not pathogenic to insects. It was speculated that the 2 genes were directly related to pathogenicity and played a role in the invasion of the honeybee. This paper cloned and expressed the chitinase and the chitin deacetylase gene of the honeybee and measured the chitin deacetylation. On the other hand, honeybees produce a series of antioxidant related defense responses after the infection of the spiral mycoplasma. This paper analyses the changes in the activity of related enzymes in the antioxidant stress system of the infected stub. The main results are as follows: first, the application of bioinformatics and online prediction tools to the honeybee stub The physicochemical properties and functional domains of the CH-1 chitin deacetylase were predicted and analyzed. The evolutionary relationship between the chitin deacetylase and other bacteria was revealed. The results showed: (1) the relative molecular mass of the chitin deacetylase gene in S.melliferum CH-1 was 26 kD; (2) the proportion of acid alkali amino acids was smaller than that of other bacteria. The expression is hydrophilic, there is no obvious transmembrane region and signal peptide, the whole peptide chain is outside the cell membrane and may be exocrine protein; (3) the comparison with other bacterial chitin deacetylase gene sequences shows that the target protein contains 3 conservative motif (motif), which may be an important functional domain. Bioinformatics analysis of the protein can be used in advance. Understanding the basic properties of the target protein and the spatial structure of the hypothesis, which plays a leading role in the subsequent expression of target protein and function, and verifies the results of subsequent studies. Second, because codon TGA encodes tryptophan rather than terminating codon in most of the bacteria, so the gene related to the bacteria is in Escherichia coli. In order to study the pathogenicity of the CH-1 chitinase and chitin deacetylase of the honeybee, this paper uses the overlapping extended PCR point mutation technique to clone the CH-1 gene chiA and the chitin deacetylase, CDA gene chid, which may be related to the pathogenicity of the pathogen, and use the table. PET-28a (+) was expressed as protein, NTA-Ni2+ column purification and Western blot verification to determine its vitality and enzymatic properties. The results showed that: (1) the full length of chiA, chid gene was cloned successfully and the chitinase protein expressed in the form of inclusion body, chitinase and several chitinase proteins expressed in the form of inclusion body were obtained. The coding region of the gene deacetylase gene is 768 BP and 672bp, respectively, which encodes about 32 kD and 28 kD protein of 255223 amino acids, respectively. (2) two methods have been used to reactivate the chitinase inclusion body protein. In the follow-up study, we could try to choose the eukaryotic expression system with Pichia pastoris as the carrier for exogenous expression. Third, the enzyme activity and some enzymatic properties of the exogenous chitin deacetylase were measured by UV spectrophotometry. The results showed that: (1) the activity of the exogenous expression of the enzyme was up to 10.14 U.mL-1; (2 ) the optimum temperature is about 50 C and the optimum pH value is 7.0-7.5. The enzyme is not sensitive to the temperature. After the enzyme treatment is 30 min, the enzyme can still maintain more than 60% activity between 35 and -60 C. The pH value stability test shows that the enzyme is a more alkaline resistant enzyme, and (3) the monovalent metal ions Na+ and K+ promote the activity of CDA enzyme. Trend; two valence metal ions showed three trends on the activity of CDA enzyme, respectively, Ca2+, Mg2+ mainly showed inhibition, Zn2+ had no obvious effect on CDA enzyme activity, Fe2+ could promote enzyme activity. Trivalent metal ions Fe3+ and A13+ ions could affect the activity of CDA enzyme at very low ionic concentration, mainly as promoting effect. Fourth, pass The changes of antioxidant related enzyme activities in honeybees after inoculation were studied. The results showed that: (1) after third days of S.melliferum CH-1 infection, the bees showed "crawling disease" and the fifth day death rate reached the maximum. (2) after third days of infection, the antioxidant should be found. The activity of catalase (catalase/CAT) in the excitation system decreased first. With the prolongation of the infection time, it showed an upward trend on the 5,7 day, but there was no obvious difference between the control group and the non infected control group. (3) the activity of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in the early stage of infection of the Mycoplasma was obviously increased, at the time of fifth days. The enzyme activity reached the maximum and then decreased slightly. The results showed that the enzyme activity in the protection system that could cause the free radical scavenging in the body was changed after the infection of the screw, and the activity of antioxidant related enzymes in the body itself had a certain resistance to the external microbial toxicity. The role of chitin deacetylase gene in the pathogenesis of honeybee spiraplasma and provides important information for the study of the function of the gene.

【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S895

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