球孢白僵菌天冬氨酸类蛋白酶-Endothiapepsin基因BbeapA的克隆及功能研究
发布时间：2018-02-05 21:22
本文关键词： 球孢白僵菌 BbeapA 基因敲除 生长 毒力 出处：《西南大学》2017年硕士论文 论文类型：学位论文
【摘要】：球孢白僵菌(Beauveria bassiana)是一种重要的昆虫病原真菌,不仅在农业害虫防治方面有着极其重要的应用价值,同时也可作为研究昆虫病原真菌与宿主昆虫互作的模式菌种。因此,研究球孢白僵菌的生长及其与昆虫互作的机制具有理论与实践的双重意义。球孢白僵菌通过体表入侵感染宿主,因此降解宿主昆虫表皮是其侵染致病的关键环节。蛋白质是昆虫表皮的主要成分之一,球孢白僵菌分泌的蛋白酶在表皮降解过程中发挥了重要的作用。本课题组在前期研究中通过转录组分析获得了球孢白僵菌在家蚕体表侵染过程中的部分差异表达基因,本研究从中选择了天冬氨酸类蛋白酶-Endothiapepsin的表达基因BbeapA作为研究对象,通过同源重组方法构建基因敲除突变株对该基因进行了功能分析,探究该基因对球孢白僵菌生长的影响以及在感染过程中的作用,为进一步研究球孢白僵菌致病机制提供了理论基础和参考。本文主要结果概述如下:1.球孢白僵菌BbeapA基因的克隆和生物信息学分析以球孢白僵菌GXsk1101基因组为模板,克隆得到BbeapA基因片段。生物信息学分析显示,BbeapA基因ORF全长1131bp,编码376个氨基酸,分子量约为39.9kDa,理论等电点为5.00。BbeapA蛋白较为稳定,肽链富含亲水集团,可能是一种可溶性蛋白。BbeapA蛋白N端含有17个氨基酸的信号肽序列,无明显跨膜区。在该蛋白二级结构中,无规则卷曲所占比例最高。结构域分析,表明该蛋白含有一个天冬氨酸结构域,属于天冬氨酸家族胃蛋白酶类,具有两个天冬氨酸残基催化位点,因此推测Bbeap A蛋白为天冬氨酸类蛋白酶。2.球孢白僵菌BbeapA基因敲除株和回补株的构建以球孢白僵菌GXsk1101基因组为模板,设计引物克隆得到基因BbeapA两侧同源臂片段,分别与载体pk2-bar连接,构建得到敲除表达载体。通过同源重组的方法将载体中的表达原件与球孢白僵菌BbeapA基因置换,使目的基因缺失。根据该方法,首先以球孢白僵菌GXsk1101为转化受体,利用AGL-1根癌农杆菌介导的遗传转化法进行转化。通过PCR和RT-PCR的方法筛选、验证获得的敲除株。进一步以球孢白僵菌GXsk1101基因组为模板克隆得到基因BbeapA的编码区及上游和下游的部分区域的完整片段,与载体pk2-sur连接,构建得到目的基因的回补载体。将构建好的回补载体用农杆菌介导的方法遗传转化BbeapA基因敲除突变体,经筛选、验证得到回补株。3.球孢白僵菌BbeapA基因的功能研究基因BbeapA参与调节球孢白僵菌生长。将基因敲除株和野生株分别接种于完全培养基SDAY平板和基础培养基CZA平板上,结果显示相对于野生株,敲除株生长速率相对降低,生长减缓,并且菌落形态发生了较大的变化。在SDAY培养基中,野生株菌落具有明显的形状规整的年轮状,中间隆起呈球状,而敲除株则形成白色雪花状、中间下凹的菌落。以上结果表明基因BbeapA可能参与了球孢白僵菌生长发育的调控。基因BbeapA影响球孢白僵菌分生孢子的产孢量。研究采用完全培养基SDAY平板培养菌株,结果显示突变株的分生孢子产量显著高于野生株。菌体生长到八天时差异最显著,其中敲除株的产孢量为3.1×105 conidia/mm~2,而野生株为0.5×105conidia/mm~2,敲除株的孢子产量平均增加了5.2倍。基因BbeapA影响球孢白僵菌对碳氮源的利用。通过比较敲除株和野生株在SDAY培养基、CZA培养基及具有10种不同碳氮源的CZA培养基平板上的生长情况,研究发现在以橄榄油为唯一碳源的培养条件下,敲除株生长速率加快。此外,在以明胶和脯氨酸分别为唯一氮源的培养条件下,敲除株相对于野生菌株生长速率加快。上述实验结果表明,基因BbeapA影响球孢白僵菌对碳氮源的利用。基因BbeapA影响球孢白僵菌在不同胁迫条件下的应激。研究选用6种金属离子Zn~(2+)、Mg~(2+)、Mn~(2+)、Cu~(2+)、Fe3+、Ca~(2+)作为胁迫因子,结果显示在Ca~(2+)和Mg~(2+)胁迫下,敲除株相对于野生株的生长速率分别降低了15%(P0.05)和10.8%(P0.05)。在高渗条件(NaCl)胁迫下,敲除株相对于野生株的生长速率降低。在氧化剂H_2O_2胁迫下,敲除株的生长速率高于野生株。在细胞壁抑制剂刚果红(CR)、荧光增白剂(CFW)和SDS胁迫下,敲除株相对于野生株的生长速率分别降低了20%(P0.05)、8.3%和7.7%(P0.05)。胁迫实验结果表明基因BbeapA可能影响球孢白僵菌对外界环境因子的敏感性。基因BbeapA影响球孢白僵菌的毒力。以家蚕为试验对象通过体壁接触分别接种球孢白僵菌基因BbeapA的敲除株和野生株,结果显示,在孢子接种浓度为1×108conidia/m L的条件下,敲除株对家蚕的毒力明显低于野生株,其半致死时间延长了t=1.39 d。感染后的家蚕均表现出取食减少、活动缓慢等典型病症。进一步观察发现敲除株和野生株的菌丝生长情况在死亡蚕体上存在较大差异,家蚕死亡2 d后,BbeapA敲除株的气生菌丝生长旺盛远超于野生型菌株。
[Abstract]:Beauveria bassiana (Beauveria bassiana) is one of the most important entomopathogenic fungi, has an extremely important value not only in terms of agricultural pest control, but also can be used as a research model of strains of entomopathogenic fungi and insect host interactions. Therefore, research on the mechanism of double meaning of Beauveria bassiana fungus growth and insect interactions as with theory and practice. Through the surface intrusion of Beauveria bassiana infected host, thus degrade the host insect cuticle is the key to its pathogenicity. The protein is a major component of insect cuticle, Beauveria bassiana protease plays an important role in epidermal degradation. In previous studies our group by transcriptome analysis has some differences in the process of silkworm surface infection of Beauveria bassiana gene expression, the choice of the aspartic acid protease from -Endothi The expression of apepsin gene BbeapA as the research object, by constructing the gene knockout mutant of the gene function analysis of homologous recombination method, to explore the impact of this gene on the growth of Beauveria bassiana and its role in the infection process, provides a theoretical basis and reference for the study of pathogenic mechanism of Beauveria bassiana further. The main results are summarized in this paper cloning and bioinformatics are as follows: 1. BbeapA gene from Beauveria bassiana by analysis of Beauveria bassiana GXsk1101 genome as template, cloned BbeapA gene fragment. The bioinformatics analysis showed that BbeapA gene ORF full-length 1131bp encoding 376 amino acids, molecular weight of 39.9kDa and isoelectric point of 5.00.BbeapA protein is relatively stable. Peptide rich in hydrophilic group, may be the signal peptide sequence of a soluble protein.BbeapA N terminal protein containing 17 amino acids, no obvious transmembrane region in the egg. Two white random coil structure, the highest proportion of domain analysis showed that this protein contains an aspartic acid domain, belongs to the family of aspartic acid pepsin, with two aspartic acid residues in the catalytic sites, suggesting that Bbeap A protein is an aspartic acid protease of Beauveria bassiana BbeapA.2. construction of gene knockout strains and complemented strain to Beauveria bassiana GXsk1101 genome as template, primers were designed to clone BbeapA gene homologous arm fragments are respectively connected with the vector pk2-bar, constructed by knockout expression vector. Through the method of homologous recombination to original expression vector with BbeapA gene from Beauveria bassiana to replacement. The purpose of gene deletion. According to this method, first by Beauveria bassiana GXsk1101 as transformation receptor, transformation by genetic transformation mediated by Agrobacterium tumefaciens AGL-1. Through PCR and RT-PCR screening method The knockout strains, verified. Further to Beauveria bassiana GXsk1101 genome as template fragment cloned complete BbeapA gene encoding region and upstream and downstream parts, connected with the vector pk2-sur, to construct the gene covering vector. The constructed methods complement vector mediated with Agrobacterium genetic the transformation of the BbeapA gene knockout mutant, after screening, verify the function of BbeapA gene complemented strain.3. of Beauveria bassiana BbeapA gene involved in the regulation of growth of Beauveria bassiana. Gene knockout strains and wild strains were cultured in complete culture medium and culture medium SDAY tablet CZA tablet, the results showed that compared to the wild-type strain. Knockout strain growth rate is relatively low, slowing growth and colony morphology changed greatly. In the SDAY medium, the rings of wild strain has obvious colony shape, middle uplift From the spherical, and the knockout strain is the formation of white snowflake, concave middle colonies. These results showed that BbeapA gene may be involved in the growth of Beauveria bassiana sporulation genes. BbeapA effect of Beauveria bassiana conidia. Using complete medium SDAY agar showed mutant strains. The conidial yield was significantly higher than that of the wild strain. Cell growth to eight days when the most significant differences, the knockout sporulation was 3.1 * 105 conidia/mm~2, while the wild strain is 0.5 * 105conidia/mm~2, knockout strains of spore yield increased by an average of 5.2 times. The BbeapA gene effect of Beauveria bassiana on carbon and nitrogen source. By comparing the knockout strain and wild-type strain in SDAY medium, CZA medium and has 10 kinds of different carbon and nitrogen sources of the CZA culture medium on the tablet, found in culture with olive oil as the sole carbon source under, 鏁查櫎鏍敓闀块，
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