胆固醇氧化酶基因769_ChOA鉴定及其提高球孢白僵菌毒力研究

发布时间：2018-03-19 11:05

  本文选题：球孢白僵菌　切入点：胆固醇氧化酶　出处：《吉林农业大学》2015年硕士论文　论文类型：学位论文

【摘要】：球孢白僵菌[Beauveria bassiana(Bals.-Criv.)Vuill.]是害虫生物防治中极为重要的昆虫病原真菌,但在多年应用过程中一直存在致病周期长、致病力低等方面的局限。为改变这一局面,利用基因工程技术,将毒力基因导入球孢白僵菌中,提高其致病能力,是目前高效菌株开发的主要手段之一,已成为该领域研究的热点。胆固醇氧化酶(Cholesterol oxidase,EC 1.1.3.6)是胆固醇降解代谢过程中的关键酶,是细胞膜的毒力因子,可以通过降解细胞膜主要组成成分胆固醇来破坏细胞膜。本文以白僵菌BbOFDH1-5为出发菌株,引入克隆自公主岭霉素产生菌769(Streptomyces ahygroscopicus 769)的细胞膜毒力基因769_ChOA,以提高其破坏玉米螟中肠而达到提高杀虫毒力。首先,通过PCR方法从链霉菌769的基因组中扩增得到769_ChOA基因(GenBank登录号KF290994)。该基因全长有1578 bp,编码一个由525 aa组成的蛋白质,其理论分子量为57031.4 Da,等电点是6.29。769_ChOA与来源于Streptomyces natalensis的胆固醇氧化酶基因的同源性为91%。通过构建硫氧还蛋白769_ChOA融合的表达载体pET32a::769_ChOA,在大肠杆菌Escherichia coli Origami B(DE3)中获得了可溶性表达的胆固醇氧化酶,经Ni亲和层析纯化得到的酶蛋白比活力为5.90 U/mg。酶学性质分析表明,该酶的底物谱较为广泛,对胆固醇的催化活性最高,对测定的7种胆固醇类似物也都具有一定的催化能力;酶的最适温度和pH分别是30℃和7.0,并且酶在低于40℃和弱碱性条件下具有很好的稳定性。该酶对鳞翅目害虫亚洲玉米螟[Ostrinia furnacalis(Guenée)]幼虫和水稻二化螟[Chilo suppressalis(Walker)]幼虫具有很强的杀虫活性,半致死浓度分别为27.4μg/mL和17.1μg/mL。为进一步验证该酶对玉米螟致病的作用机理,利用透射显微镜对重组769_ChOA注射亚洲玉米螟幼虫后中肠组织的病理变化进行检测,发现中肠细胞微绒毛发生明显脱落,细胞核染色质减少,内质网肿胀断裂,杯状细胞微绒毛也出现严重脱落,且病变程度随时间的增加而加剧。在上述研究基础上,通过PEG介导的原生质体遗传转化方法,借助含有抗萎锈灵基因和目的基因的载体pHD3-hsp70::769_ChOA,成功获得了基因工程菌株。经RT-PCR验证可知769_ChOA在重组菌株中实现了转录和表达。转化子酶活力测定实验表明,重组菌株的酶活力比出发菌株有了明显提高,在诱导36 h时,二者相差3.74倍。过表达769_ChOA对出发菌株的产孢量,生长速率及分生孢子萌发率基本没有影响。对亚洲玉米螟毒力测定试验中,在供试的3株转基因菌株中,有2株较出发菌株毒力显著增强,而1株毒力则没有提高。上述结论说明,本文通过将来自公主岭霉素产生菌769的ChoA基因导入白僵菌,成功构建了高毒力的球孢白僵菌基因工程菌株,但是其具体的致病机理、安全性和应用潜力还需要进一步的研究来评估。
[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，

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