本文選題：球孢白僵菌　切入點(diǎn)：膽固醇氧化酶　出處：《吉林農(nóng)業(yè)大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：球孢白僵菌[Beauveria bassiana(Bals.-Criv.)Vuill.]是害蟲生物防治中極為重要的昆蟲病原真菌,但在多年應(yīng)用過程中一直存在致病周期長、致病力低等方面的局限。為改變這一局面,利用基因工程技術(shù),將毒力基因?qū)肭蜴甙捉┚?提高其致病能力,是目前高效菌株開發(fā)的主要手段之一,已成為該領(lǐng)域研究的熱點(diǎn)。膽固醇氧化酶(Cholesterol oxidase,EC 1.1.3.6)是膽固醇降解代謝過程中的關(guān)鍵酶,是細(xì)胞膜的毒力因子,可以通過降解細(xì)胞膜主要組成成分膽固醇來破壞細(xì)胞膜。本文以白僵菌BbOFDH1-5為出發(fā)菌株,引入克隆自公主嶺霉素產(chǎn)生菌769(Streptomyces ahygroscopicus 769)的細(xì)胞膜毒力基因769_ChOA,以提高其破壞玉米螟中腸而達(dá)到提高殺蟲毒力。首先,通過PCR方法從鏈霉菌769的基因組中擴(kuò)增得到769_ChOA基因(GenBank登錄號KF290994)。該基因全長有1578 bp,編碼一個由525 aa組成的蛋白質(zhì),其理論分子量為57031.4 Da,等電點(diǎn)是6.29。769_ChOA與來源于Streptomyces natalensis的膽固醇氧化酶基因的同源性為91%。通過構(gòu)建硫氧還蛋白769_ChOA融合的表達(dá)載體pET32a::769_ChOA,在大腸桿菌Escherichia coli Origami B(DE3)中獲得了可溶性表達(dá)的膽固醇氧化酶,經(jīng)Ni親和層析純化得到的酶蛋白比活力為5.90 U/mg。酶學(xué)性質(zhì)分析表明,該酶的底物譜較為廣泛,對膽固醇的催化活性最高,對測定的7種膽固醇類似物也都具有一定的催化能力;酶的最適溫度和pH分別是30℃和7.0,并且酶在低于40℃和弱堿性條件下具有很好的穩(wěn)定性。該酶對鱗翅目害蟲亞洲玉米螟[Ostrinia furnacalis(Guenée)]幼蟲和水稻二化螟[Chilo suppressalis(Walker)]幼蟲具有很強(qiáng)的殺蟲活性,半致死濃度分別為27.4μg/mL和17.1μg/mL。為進(jìn)一步驗證該酶對玉米螟致病的作用機(jī)理,利用透射顯微鏡對重組769_ChOA注射亞洲玉米螟幼蟲后中腸組織的病理變化進(jìn)行檢測,發(fā)現(xiàn)中腸細(xì)胞微絨毛發(fā)生明顯脫落,細(xì)胞核染色質(zhì)減少,內(nèi)質(zhì)網(wǎng)腫脹斷裂,杯狀細(xì)胞微絨毛也出現(xiàn)嚴(yán)重脫落,且病變程度隨時間的增加而加劇。在上述研究基礎(chǔ)上,通過PEG介導(dǎo)的原生質(zhì)體遺傳轉(zhuǎn)化方法,借助含有抗萎銹靈基因和目的基因的載體pHD3-hsp70::769_ChOA,成功獲得了基因工程菌株。經(jīng)RT-PCR驗證可知769_ChOA在重組菌株中實現(xiàn)了轉(zhuǎn)錄和表達(dá)。轉(zhuǎn)化子酶活力測定實驗表明,重組菌株的酶活力比出發(fā)菌株有了明顯提高,在誘導(dǎo)36 h時,二者相差3.74倍。過表達(dá)769_ChOA對出發(fā)菌株的產(chǎn)孢量,生長速率及分生孢子萌發(fā)率基本沒有影響。對亞洲玉米螟毒力測定試驗中,在供試的3株轉(zhuǎn)基因菌株中,有2株較出發(fā)菌株毒力顯著增強(qiáng),而1株毒力則沒有提高。上述結(jié)論說明,本文通過將來自公主嶺霉素產(chǎn)生菌769的ChoA基因?qū)氚捉┚?成功構(gòu)建了高毒力的球孢白僵菌基因工程菌株,但是其具體的致病機(jī)理、安全性和應(yīng)用潛力還需要進(jìn)一步的研究來評估。
[Abstract]:Beauveria bassiana Bals.-Criv. Vuill. is a very important insect pathogen fungus in pest biological control, but it has many limitations in many years of application, such as long pathogenic period and low pathogenicity. In order to change this situation, genetic engineering technology is used. The introduction of virulence gene into Beauveria bassiana to improve its pathogenicity is one of the main methods for the development of highly efficient strains, and has become a hotspot in this field. Cholesterol oxidase Cholesterol oxidase EC 1.1.3.6) is the key enzyme in the process of cholesterol degradation and metabolism. Is the virulence factor of the cell membrane, it can destroy the cell membrane by degrading the cholesterol, the main component of the cell membrane. In this paper, we take Beauveria bassiana BbOFDH1-5 as the starting strain, The cell membrane virulence gene 769ChOAA was cloned from Gongzhulingmycin producing strain 769 Streptomyces ahygroscopicus 769, in order to improve its ability to destroy the midgut of corn borer and to increase its insecticidal toxicity. 769 ChOA gene was amplified from the genome of Streptomyces 769 by PCR method. The GenBank accession number KF290994 is 769. The gene has a total length of 1578 BP and encodes a protein composed of 525aa. The theoretical molecular weight is 57031.4 Da.The isoelectric point is 6.29.769 and the homology of the cholesterol oxidase gene derived from Streptomyces natalensis is 91.The soluble table was obtained by constructing the expression vector pET32a: 769ChOAA of thioredoxin 769: 769ChOAA in Escherichia coli Escherichia coli Origami BDE3. Cholesterol oxidase, The specific activity of enzyme protein was 5.90 U / mg by Ni affinity chromatography. The results of enzymatic analysis showed that the enzyme had broad substrate spectrum and the highest catalytic activity to cholesterol, and it also had certain catalytic activity to the seven cholesterol analogues. The optimum temperature and pH of the enzyme were 30 鈩，

本文編號：1633985