本文選題：蜜蜂螺原體 + 幾丁質(zhì)酶基因　； 參考：《南京農(nóng)業(yè)大學(xué)》2016年碩士論文

【摘要】：螺原體(spiroplasma)是柔膜菌綱中一種螺旋狀無細(xì)胞壁的原核微生物,能與節(jié)肢動物和植物相互作用。在系統(tǒng)發(fā)育學(xué)上,螺原體與芽孢桿菌綱及其他營自由生活的厚壁菌門的細(xì)菌親緣關(guān)系較近。我國蜜蜂螺原體病發(fā)生非常普遍,Spiroplasma melliferum是蜜蜂“爬蜂病”主要病原之一,基本生物學(xué)特性已被詳細(xì)研究,但對其致病機(jī)制了解很少。通過比較基因組學(xué)和蛋白質(zhì)組學(xué)研究發(fā)現(xiàn),蜜蜂螺原體S.melliferum存在2種特有的基因——幾丁質(zhì)酶和幾丁質(zhì)脫乙酰酶基因,而其他幾種借助昆蟲傳播但對昆蟲無致病性的螺原體(如S.citri和S.kunkelli)沒有發(fā)現(xiàn)這2種基因,推測這2個基因與致病性直接相關(guān),在螺原體入侵蜜蜂過程中起作用。本論文克隆表達(dá)了蜜蜂螺原體幾丁質(zhì)酶和幾丁質(zhì)脫乙酰酶基因并測定了幾丁質(zhì)脫乙酰酶活力和酶學(xué)性質(zhì)。另一方面,蜜蜂在受到螺原體侵染后會產(chǎn)生一系列抗氧化相關(guān)的防御反應(yīng),本文分析了感染螺原體后蜜蜂抗氧化應(yīng)激系統(tǒng)中相關(guān)酶活變化。主要研究結(jié)果有:第一、應(yīng)用生物信息學(xué)方法和在線預(yù)測工具,對蜜蜂螺原體CH-1幾丁質(zhì)脫乙酰酶的理化性質(zhì)、功能域等進(jìn)行預(yù)測分析,揭示蜜蜂螺原體幾丁質(zhì)脫乙酰酶與其他細(xì)菌該酶的進(jìn)化關(guān)系。結(jié)果顯示:(1)S.melliferum CH-1中的幾丁質(zhì)脫乙酰酶基因相對分子質(zhì)量為26 kD;(2)酸堿性氨基酸比例較其他細(xì)菌小,主要表現(xiàn)為親水性,無明顯的跨膜區(qū)和信號肽,整個肽鏈位于細(xì)胞膜外,可能是外分泌蛋白;(3)與其他細(xì)菌幾丁質(zhì)脫乙酰酶基因序列比對顯示該目的蛋白含有3個保守的基序(motif),可能是重要的功能域。對該蛋白進(jìn)行生物信息學(xué)分析能夠預(yù)先了解目的蛋白的基本性質(zhì)和假設(shè)的空間結(jié)構(gòu),這對后續(xù)表達(dá)目的蛋白和功能探索起前導(dǎo)作用,并與后續(xù)的研究結(jié)果相互驗(yàn)證。第二、由于密碼子TGA在多數(shù)柔膜菌綱細(xì)菌中編碼色氨酸而非終止密碼子,因此柔膜菌相關(guān)基因在大腸桿菌中的表達(dá)受到限制。為研究蜜蜂螺原體CH-1幾丁質(zhì)酶和幾丁質(zhì)脫乙酰酶的致病性,本文利用重疊延伸PCR定點(diǎn)突變技術(shù)克隆蜜蜂螺原體CH-1中可能與致病相關(guān)的幾丁質(zhì)酶(chitinase)基因chiA以及幾丁質(zhì)脫乙酰酶(chitin deacetylase,CDA)基因chid,利用表達(dá)載體pET-28a(+)進(jìn)行蛋白表達(dá),NTA-Ni2+柱純化,Western blot驗(yàn)證,測定其活力和酶學(xué)性質(zhì)。結(jié)果顯示:(1)成功克隆到chiA、chid基因全長,并得到有活性的外源表達(dá)的完整幾丁質(zhì)脫乙酰酶蛋白和以包涵體形式表達(dá)的幾丁質(zhì)酶蛋白。幾丁質(zhì)酶及幾丁質(zhì)脫乙酰酶基因編碼區(qū)分別為768 bp、672bp,分別編碼255、223個氨基酸組成的約32 kD、28 kD的蛋白質(zhì);(2)研究中采用兩種方法對幾丁質(zhì)酶包涵體蛋白進(jìn)行復(fù)性,由于瞬間表達(dá)的蛋白質(zhì)量太多以至于未能進(jìn)行正確的折疊而未能成功獲得有活性的目的蛋白,后續(xù)研究中可以嘗試選擇以畢赤酵母為載體的真核表達(dá)系統(tǒng)進(jìn)行外源表達(dá)。第三、利用紫外分光光度法測定了外源表達(dá)幾丁質(zhì)脫乙酰酶的酶活力和部分酶學(xué)性質(zhì)。結(jié)果顯示:(1)測得外源表達(dá)的該酶活力最高可達(dá)10.14 U.mL-1;(2)最適溫度為50℃左右,最適pH值為7.0-7.5,該酶對溫度較不敏感,各個溫度梯度對該酶處理30 min后,在35℃-60℃之間仍能保持60%以上活力,pH值穩(wěn)定性試驗(yàn)研究表明該酶是一種較耐堿性的酶;(3)一價金屬離子Na+和K+均對CDA酶活性表現(xiàn)出促進(jìn)趨勢;二價金屬離子對CDA酶活力影響表現(xiàn)出三種趨勢,分別是Ca2+、Mg2+主要表現(xiàn)為抑制作用,Zn2+對CDA酶活力無明顯影響,Fe2+能促進(jìn)酶活性。三價金屬離子Fe3+和A13+離子能夠在很低的離子濃度影響CDA酶活性,主要表現(xiàn)為促進(jìn)作用。第四、通過飼喂對數(shù)期螺原體菌液感染意蜂,研究了接種后不同時間段蜜蜂體內(nèi)抗氧化相關(guān)酶活性變化。結(jié)果表明:(1)在蜜蜂螺原體致病菌S.melliferum CH-1感染后,第3天蜜蜂表現(xiàn)出“爬蜂病”癥狀,第5天死亡率達(dá)到最大值。(2)感染第3天時,抗氧化應(yīng)激系統(tǒng)中過氧化氫酶(catalase/CAT)活力先下降,隨著感染時間的延長,在第5、7天表現(xiàn)為上升趨勢,但與未感染螺原體的對照組差別不明顯。(3)超氧化物歧化酶(SOD)和谷胱甘肽過氧化物酶(GSH-Px)在感染螺原體初期酶活性就明顯上升,第5天時酶活性達(dá)到最大,隨后略微下降。研究結(jié)果說明蜜蜂在被螺原體侵染后,能引起體內(nèi)清除自由基的保護(hù)系統(tǒng)中酶活力發(fā)生改變,機(jī)體本身的抗氧化相關(guān)酶活性變化具有一定的抵抗外界微生物毒害的作用。本研究結(jié)果為了解幾丁質(zhì)酶和幾丁質(zhì)脫乙酰酶基因在蜜蜂螺原體的致病過程中的作用以及對該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.

【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：S895

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 周丹霞;危蓉萍;康昕;錢正宗;紀(jì)燕玲;于漢壽;;蜜蜂螺原體細(xì)胞骨架相關(guān)基因mreB的克隆表達(dá)及在螺原體二種形態(tài)時的表達(dá)差異[J];微生物學(xué)通報;2014年09期

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本文編號：1850883