本文關(guān)鍵詞： 球孢白僵菌 BbeapA 基因敲除 生長(zhǎng) 毒力　出處：《西南大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：球孢白僵菌(Beauveria bassiana)是一種重要的昆蟲病原真菌,不僅在農(nóng)業(yè)害蟲防治方面有著極其重要的應(yīng)用價(jià)值,同時(shí)也可作為研究昆蟲病原真菌與宿主昆蟲互作的模式菌種。因此,研究球孢白僵菌的生長(zhǎng)及其與昆蟲互作的機(jī)制具有理論與實(shí)踐的雙重意義。球孢白僵菌通過(guò)體表入侵感染宿主,因此降解宿主昆蟲表皮是其侵染致病的關(guān)鍵環(huán)節(jié)。蛋白質(zhì)是昆蟲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過(guò)程中發(fā)揮了重要的作用。本課題組在前期研究中通過(guò)轉(zhuǎn)錄組分析獲得了球孢白僵菌在家蠶體表侵染過(guò)程中的部分差異表達(dá)基因,本研究從中選擇了天冬氨酸類蛋白酶-Endothiapepsin的表達(dá)基因BbeapA作為研究對(duì)象,通過(guò)同源重組方法構(gòu)建基因敲除突變株對(duì)該基因進(jìn)行了功能分析,探究該基因?qū)η蜴甙捉┚L(zhǎng)的影響以及在感染過(guò)程中的作用,為進(jìn)一步研究球孢白僵菌致病機(jī)制提供了理論基礎(chǔ)和參考。本文主要結(jié)果概述如下:1.球孢白僵菌BbeapA基因的克隆和生物信息學(xué)分析以球孢白僵菌GXsk1101基因組為模板,克隆得到BbeapA基因片段。生物信息學(xué)分析顯示,BbeapA基因ORF全長(zhǎng)1131bp,編碼376個(gè)氨基酸,分子量約為39.9kDa,理論等電點(diǎn)為5.00。BbeapA蛋白較為穩(wěn)定,肽鏈富含親水集團(tuán),可能是一種可溶性蛋白。BbeapA蛋白N端含有17個(gè)氨基酸的信號(hào)肽序列,無(wú)明顯跨膜區(qū)。在該蛋白二級(jí)結(jié)構(gòu)中,無(wú)規(guī)則卷曲所占比例最高。結(jié)構(gòu)域分析,表明該蛋白含有一個(gè)天冬氨酸結(jié)構(gòu)域,屬于天冬氨酸家族胃蛋白酶類,具有兩個(gè)天冬氨酸殘基催化位點(diǎn),因此推測(cè)Bbeap A蛋白為天冬氨酸類蛋白酶。2.球孢白僵菌BbeapA基因敲除株和回補(bǔ)株的構(gòu)建以球孢白僵菌GXsk1101基因組為模板,設(shè)計(jì)引物克隆得到基因BbeapA兩側(cè)同源臂片段,分別與載體pk2-bar連接,構(gòu)建得到敲除表達(dá)載體。通過(guò)同源重組的方法將載體中的表達(dá)原件與球孢白僵菌BbeapA基因置換,使目的基因缺失。根據(jù)該方法,首先以球孢白僵菌GXsk1101為轉(zhuǎn)化受體,利用AGL-1根癌農(nóng)桿菌介導(dǎo)的遺傳轉(zhuǎn)化法進(jìn)行轉(zhuǎn)化。通過(guò)PCR和RT-PCR的方法篩選、驗(yàn)證獲得的敲除株。進(jìn)一步以球孢白僵菌GXsk1101基因組為模板克隆得到基因BbeapA的編碼區(qū)及上游和下游的部分區(qū)域的完整片段,與載體pk2-sur連接,構(gòu)建得到目的基因的回補(bǔ)載體。將構(gòu)建好的回補(bǔ)載體用農(nóng)桿菌介導(dǎo)的方法遺傳轉(zhuǎn)化BbeapA基因敲除突變體,經(jīng)篩選、驗(yàn)證得到回補(bǔ)株。3.球孢白僵菌BbeapA基因的功能研究基因BbeapA參與調(diào)節(jié)球孢白僵菌生長(zhǎng)。將基因敲除株和野生株分別接種于完全培養(yǎng)基SDAY平板和基礎(chǔ)培養(yǎng)基CZA平板上,結(jié)果顯示相對(duì)于野生株,敲除株生長(zhǎng)速率相對(duì)降低,生長(zhǎng)減緩,并且菌落形態(tài)發(fā)生了較大的變化。在SDAY培養(yǎng)基中,野生株菌落具有明顯的形狀規(guī)整的年輪狀,中間隆起呈球狀,而敲除株則形成白色雪花狀、中間下凹的菌落。以上結(jié)果表明基因BbeapA可能參與了球孢白僵菌生長(zhǎng)發(fā)育的調(diào)控。基因BbeapA影響球孢白僵菌分生孢子的產(chǎn)孢量。研究采用完全培養(yǎng)基SDAY平板培養(yǎng)菌株,結(jié)果顯示突變株的分生孢子產(chǎn)量顯著高于野生株。菌體生長(zhǎng)到八天時(shí)差異最顯著,其中敲除株的產(chǎn)孢量為3.1×105 conidia/mm~2,而野生株為0.5×105conidia/mm~2,敲除株的孢子產(chǎn)量平均增加了5.2倍。基因BbeapA影響球孢白僵菌對(duì)碳氮源的利用。通過(guò)比較敲除株和野生株在SDAY培養(yǎng)基、CZA培養(yǎng)基及具有10種不同碳氮源的CZA培養(yǎng)基平板上的生長(zhǎng)情況,研究發(fā)現(xiàn)在以橄欖油為唯一碳源的培養(yǎng)條件下,敲除株生長(zhǎng)速率加快。此外,在以明膠和脯氨酸分別為唯一氮源的培養(yǎng)條件下,敲除株相對(duì)于野生菌株生長(zhǎng)速率加快。上述實(shí)驗(yàn)結(jié)果表明,基因BbeapA影響球孢白僵菌對(duì)碳氮源的利用。基因BbeapA影響球孢白僵菌在不同脅迫條件下的應(yīng)激。研究選用6種金屬離子Zn~(2+)、Mg~(2+)、Mn~(2+)、Cu~(2+)、Fe3+、Ca~(2+)作為脅迫因子,結(jié)果顯示在Ca~(2+)和Mg~(2+)脅迫下,敲除株相對(duì)于野生株的生長(zhǎng)速率分別降低了15%(P0.05)和10.8%(P0.05)。在高滲條件(NaCl)脅迫下,敲除株相對(duì)于野生株的生長(zhǎng)速率降低。在氧化劑H_2O_2脅迫下,敲除株的生長(zhǎng)速率高于野生株。在細(xì)胞壁抑制劑剛果紅(CR)、熒光增白劑(CFW)和SDS脅迫下,敲除株相對(duì)于野生株的生長(zhǎng)速率分別降低了20%(P0.05)、8.3%和7.7%(P0.05)。脅迫實(shí)驗(yàn)結(jié)果表明基因BbeapA可能影響球孢白僵菌對(duì)外界環(huán)境因子的敏感性�；駼beapA影響球孢白僵菌的毒力。以家蠶為試驗(yàn)對(duì)象通過(guò)體壁接觸分別接種球孢白僵菌基因BbeapA的敲除株和野生株,結(jié)果顯示,在孢子接種濃度為1×108conidia/m L的條件下,敲除株對(duì)家蠶的毒力明顯低于野生株,其半致死時(shí)間延長(zhǎng)了t=1.39 d。感染后的家蠶均表現(xiàn)出取食減少、活動(dòng)緩慢等典型病癥。進(jìn)一步觀察發(fā)現(xiàn)敲除株和野生株的菌絲生長(zhǎng)情況在死亡蠶體上存在較大差異,家蠶死亡2 d后,BbeapA敲除株的氣生菌絲生長(zhǎng)旺盛遠(yuǎn)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敓闀塊，

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