【作者】 张烨；

【导师】 雷仲仁；

【作者基本信息】 中国农业科学院 ， 农业昆虫与害虫防治， 2013， 博士

【摘要】 随着生物防治理念的提出，一些具有潜在应用价值的昆虫病原真菌被广泛应用于农林害虫的防治。昆虫病原真菌资源丰富、杀虫谱广、致病力强且安全无污染，已被研发成各种剂型并投入到田间应用，已取得一定成效。但目前真菌杀虫剂的市场份额还远远低于常规杀虫剂，主要缘于真菌杀虫剂的田间防效不稳定，易受环境因素影响。解决这一尴尬境地的重要途径就是通过对虫生真菌致病机理的研究，借助分子生物学技术和遗传学手段，以提升虫生真菌的毒力并增强其抗逆性。本论文以国内外广泛报道的球孢白僵菌为研究对象，从其侵染过程中的致病基因入手，探索该基因所行使的功能，以期为球孢白僵菌的分子致病机理研究及菌株的遗传改良提供一些理论基础。本文的研究内容如下：1cDNA克隆和DNA全序列的获得，及HsbA基因的生物信息学分析利用RACE（Rapid Amplification of cDNA Ends）法和Genome Walking法分别获得了该菌疏水表面结合蛋白（Hydrophobic surface binding protein）基因的cDNA序列及DNA全序列，并将该基因命名为HsbA。结合两条序列信息，对其进行生物信息学分析，主要结果如下：该基因无内含子区，其开放阅读框总计564bp，编码187个氨基酸；其预测的启动子核心区大小为50bp，位于起始密码子上游99bp处；转录终止信号AATAAA位于终止密码子下游164bp处。该推测蛋白隶属于疏水表面结合蛋白家族，分子量为19.374kD，等电点为5.07，总疏水性参数为-0.045；其信号肽序列的剪切位点位于第17个氨基酸处，成熟蛋白的分子量约为17.6kD；其二级结构富含α-螺旋（α-helix）结构，无跨膜区存在。2球孢白僵菌HsbA基因对热胁迫和诱导物的相对表达量分析利用荧光定量PCR技术对球孢白僵菌HsbA基因在不同环境下的相对表达量进行分析，结果表明：热胁迫环境下，处理组中HsbA基因的相对表达量要显著高于对照组，且该基因的相对表达量与处理时间呈显著的正相关；不同诱导环境下，HsbA基因在西花蓟马表皮中的相对表达量显著高于几丁质和对照组。据此推断，HsbA基因与球孢白僵菌对高温的耐受性tolerence有关，并有可能参与侵染过程中的吸附阶段。3HsbA基因的原核表达纯化、抗体制备及抗原特异性分析利用原核表达系统获得了约22.68kD的HsbA融合蛋白，通过Ni柱、酶切纯化及透析等步骤，得到了约17.6kD的目的蛋白，并成功制备了兔抗HsbA多克隆抗体。Western杂交结果显示，该多抗具有较好的免疫原性，可用于免疫定位分析。4HsbA蛋白在球孢白僵菌和感染虫体中的免疫电镜观察利用免疫染色和电镜技术观察BbHsbA蛋白的定位情况，结果表明：HsbA蛋白在菌丝和孢子中呈随机分布。正常生长状态下，菌丝中该蛋白被标记的数目显著大于孢子；侵染过程中，菌丝和孢子中的该蛋白均被大量标记；孢子在感染前后该蛋白被标记的数目也有显著性差异，感染中被标记的数目更多；菌丝在感染前后该蛋白均被大量标记；感染虫体中该蛋白被标记的数目集中在昆虫体壁以及孢子和体壁的接触部位。我们推断，该蛋白在侵染过程及菌丝生长中具有一定作用，且侵染时的作用部位在昆虫的体壁。5HsbA基因的敲除及功能验证利用基因敲除技术获得突变体并进行一系列功能分析，结果表明：在进行高温敏感性测定，生物量与孢子产量测定，菌丝和孢子的疏水能力与附着能力测定，对西花蓟马毒力测定，以及气生菌丝与形成及穿透试验中，突变体的整体性能均显著低于野生型和空载转化子。我们推断HsbA基因的功能主要体现在以下几个方面：（1）气生菌丝的生长；（2）对热的耐受性；（3）与疏水性和附着能力相关。更多还原

【Abstract】 Potential entomo-pathogenic fungi are widely used in pest control, as the developing ofbiological control concept. Since the abundant resource, broad insecticidal spectrum, strongvirulence, and eco-friendly, entomo-pathogenic fungi have been, to some extent, successfullyapplied in the field works. However, due to the unstable effects and flexibility to environmentalfactors of the entomo-pathogenic fungi pesticide, its application is far less than chemical pesticide.This problem can be solved by enhancing the toxicity and tolerance of entomo-pathogenic fungithrough molecular and/or genetics techniques to change the pathnogenesis of entomo-pathogenicfungi.Here, by making use of Beauveria bassiana, a gene that is crucial for the pathnogenesis ofBeauveria bassiana was cloned and the functions of this gene were analysis. Our works pave away for illustrating the mechanism of infection and genetically improving the B. bassiana strain.1. Bioinformatics analysis of HsbA geneUsing rapid amplification of cDNA ends (RACE) and genome walking, full length of cDNAand DNA of a hydrophobic surface binding protein of B. bassiana were sequenced, respectively,named HsbA. The full length of HsbA DNA was564bp without any intron and encodes a predictprecursor protein containing187amino acids. The50bp predicted promoter located100bpupstream from the initiation codon and consensus polyadenylation signal (AATAAA) located at164bp downstream from the termination codon. The calculated molecular weight and theisoelectric point of this premature protein were19.374and5.07, respectively. Cutting out thededuced signal peptides at the17thamino acid, the molecular weight of the mature protein was17.6kD. The secondary structure of HsbA was α-helix rich, no trans-membrane domain was found.Its hydrophobicity value is-0.045.2. The expression pattern of HsbA under heat stress and certain inducerThe expression pattern of HsbA in different environment was examined by using quantitativePCR (qPCR). When B. bassiana exposed to heat stress, the expression of HsbA spores wassignificant higher than control in a strikingly positive correlated to treatment time. When exposedto different inducers, HsbA was expressed higher than Chitin in cuticle of western flower thrips,Frankliniella occidentalis. It indicated that HsbA gene may improve the tolerance of B. bassianato heat stress and may facilitate the absorption of B. bassiana to insect cuticle.3. The antibody generation and the specificity analysisUsing the prokaryotic expression vector pET-30a, we got a22.68kD protein and thenpurified by Ni ion affinity chromatography. Eventually, we got our target protein at17.6kD. Thena multiple-clone anti-rabbit antibody was produced by Huada Protein Institute. Western blotshowed that the antibody had high affinity to our target protein and it was sufficient forimmuno-staining.4. Immunolocalization of HsbA protein in B. bassiana and B. bassiana-infected thrips. The immune-staining work showed that the HsbA protein randomly localized in aerialmycelium and spores. Before infection occurred, there was more HsbA protein in aerial myceliumthan in spores. These observations indicated that HsbA was important for the growth of aerialmycelium. During infection, HsbA was up-regulated in both aerial mycelium and spores.Interestingly, HsbA located specifically in the insect cuticle that conjunct to fungi. These meantthat HsbAmay involve in spores infection at the conjunction region.5. Verification of HsbA gene functionBy using gene knock out, we obtained HsbA deletion mutation of B. bassiana. Thecomparation between HsbA deletion mutation and wild type (WT) showed that all the parameterswe tested of HsbA deletion mutation were significantly inferior than WT, including the biomassand reproduction of spores, the ability of affinity, and the virulence of B. bassiana.Based on all the results above, we predicted that HsbA gene may function in three respects:1)improving the growth of aerial mycelium,2) enhancing the tolerance of heat stress, and3)influencing the hydrophobic and ability of affinity of B. bassiana.更多还原