【作者】 张传博；

【导师】 夏玉先；

【作者基本信息】 重庆大学 ， 生物医学工程， 2009， 博士

【摘要】 金龟子绿僵菌(Metarhizium anisopliae)是一类重要的昆虫病原真菌,在害虫生物防治中起着重要作用。与化学农药相比,昆虫病原真菌开发的真菌杀虫剂作用时间持久、无环境污染、对非靶标生物安全,但存在致死时间长,杀虫效率低,防效不稳定等缺点,严重阻碍了真菌杀虫剂的广泛应用,为解决这一问题,在分子水平上揭示昆虫病原真菌致病机理尤为重要。金龟子绿僵菌侵染寄主昆虫时,分生孢子在体表萌发形成附着胞,并分泌多种蛋白酶类降解昆虫体壁,附着胞的形成与分化在侵染过程中发挥着重要作用。金龟子绿僵菌直接穿透寄主体壁进入体内,在血淋巴定殖,以掠夺寄主营养或分泌毒素使寄主死亡,能否在体内成功定殖决定着侵染的最终结果。筛选附着孢形成时期和血淋巴定殖阶段的特异表达基因和基因表达特征分析,对于研究昆虫病原真菌致病机理具有重要意义。EST序列分析和微阵列技术是研究昆虫病原真菌致病机理的成功策略,国内外许多研究者通过这种策略对绿僵菌侵染昆虫致病机理进行了深入研究,但采用的方法均是离体条件下,在各种液体培养基中加入诱导物培养,然后构建绿僵菌cDNA文库,大规模测序,进行EST序列分析。很明显,这些技术路线有一定的局限性,绿僵菌在离体液体培养环境下处于腐生生长阶段,而绿僵菌侵染昆虫过程属于寄生生活阶段,离体条件下诱导培养和事实的侵染过程差别明显,很难对绿僵菌穿透寄主体壁机制,定殖寄主血淋巴适应机制作出完整的诠释,从而阐明病原真菌和寄主的相互作用关系。本研究以金龟子绿僵菌(Metarhizium anisopliae)菌株CQMa102和东亚飞蝗(Locusta migratoria)为研究对象,从寄主血淋巴中分离出无寄主核酸污染的绿僵菌菌体,以已降解寄主核酸的东亚飞蝗翅膀诱导附着孢形成与分化,利用抑制消减杂交技术(SSH)筛选绿僵菌在附着胞形成时期和定殖血淋巴阶段的特异表达基因。DSN均一化和SMART技术相结合构建了金龟子绿僵菌CQMa102定殖东亚飞蝗血淋巴阶段的全长均一化cDNA文库,大规模测序,进行EST序列分析,分析绿僵菌定殖寄主血淋巴阶段的基因表达特征,以阐明寄主和病原物之间的相互作用关系。主要研究结果如下:1.观察了金龟子绿僵菌CQMa102在实验条件下侵染东亚飞蝗的致病过程:发现24 h左右分生孢子穿透寄主表皮进入血淋巴,接触血细胞,引起血细胞聚集;3天～6天大量的芽生孢子被血细胞吞噬、包囊;在寄主昆虫死亡前24 h左右,各种血细胞数量急剧减少,芽生孢子开始迅速繁殖,血淋巴内悬浮着大量酵母状虫菌体(hyphal body)。2.根据寄主血细胞和病原真菌细胞结构差别,建立和完善了利用蛋白酶K和SDS迅速裂解寄主血细胞的方法,整个流程时间约40 min,RT-PCR检测表明,利用寄主血细胞裂解释放的内源性RNases和后续PBS洗涤可高效去除血淋巴分离绿僵菌菌体中的寄主核酸污染,这一结论也被后续EST序列分析所证明。3.以寄主体内分离,去除寄主核酸污染的绿僵菌菌体提取mRNA为实验组(tester),用RNA fragmentation buffer处理东亚飞蝗翅膀,降解寄主核酸,并诱导附着孢形成与分化,以该时期的菌体提取mRNA为驱动组(driver),进行抑制消减杂交(suppression subtractive hybridization, SSH),筛选出350个阳性克隆,测序,获得的EST序列经聚类分析、共拼接成120 unique ESTs,其中100条singletons,20条contigs,经基因注释和功能分类,29条(24.17%) ESTs和NR数据库有同源性,其功能参与各种细胞代谢和应答过程,包括细胞代谢、蛋白代谢、细胞结构与功能以及参与胁迫和抵抗等生物学过程。4.为验证消减效率,随机选择5个预测基因,以半定量RT-PCR分析了在绿僵菌侵染东亚飞蝗不同阶段的表达水平,包括碗豆素脱甲基酶(pisatin demethylase)基因(Mpd1),α-烯醇化酶(α-enolase)基因(Me1),异戊烯基转移酶(prenyl transferase)基因(Mpt),α-1,2甘露糖基转移酶(α-1,2-mannosyltransferase)基因(Mmnt1),胆碱脱氢酶(cholin dehydrogenase)基因(Mcd)。结果表明5个基因均在定殖寄主血淋巴阶段有较高的表达量。5.为筛选附着孢形成阶段特异表达或上调表达基因,以无寄主核酸污染的蝗虫翅膀诱导附着孢形成,提取mRNA为实验组(tester),以去除寄主核酸污染的血淋巴分离绿僵菌菌体提取mRNA为驱动组(driver),消减杂交筛选出460个阳性克隆,测序,获得329条高质量ESTs,聚类分析、拼接成78 unique ESTs。其中46条ESTs与数据库中已知蛋白序列有明显的同源性,24条己知功能的同源ESTs涉及多种代谢和应答过程,包括细胞代谢、蛋白代谢、细胞结构与功能以及胁迫和抵抗等生物学过程。6.随机选择12条ESTs,分别提取绿僵菌在蝗虫翅膀生长8 h、15 h、24 h、30 h,以及体内定殖阶段(第2天、第3天、第4天、第5天、第6天、第7天)血淋巴的总RNA,DNaseⅠ处理后,以3-磷酸甘油醛脱氢酶基因(G3pdh)为参照基因,进行半定量分析,结果表明12条ESTs均在附着孢形成时期上调表达。7. DSN均一化和SMART技术相结合构建了金龟子绿僵菌CQMa102定殖东亚飞蝗血淋巴阶段的全长均一化cDNA文库,并对整个文库进行了EST序列测定,共挑取5222个克隆进行测序,获高质量ESTs 4606条,聚类、拼接成1863个unique ESTs,其中1237条singletons,626条contigs。经基因注释和功能分类,同源ESTs涉及基础代谢,能量产生,维持细胞功能与结构,调节细胞周期、分裂或生长,对抗胁迫及防御等各种生物学功能。8.利用SMART技术构建了金龟子绿僵菌CQMa102定殖东亚飞蝗血淋巴时期的全长cDNA文库,挑克隆,测序获得187条unigenes,这些ESTs均是绿僵菌定殖寄主血淋巴阶段的高丰度表达基因。更多还原

【Abstract】 Metarhizium anisopliae is an economically important insect pathogenic fungus widely used as an insect biocontrol agent. Mycoinsecticides have many advantages compared with chemical insecticides, including low toxicity to non-targeted organisms, high insect specificity, and low environmental impact, but the slow killing speed has been considered a potential drawback which prevents the utilization of these fungi against insects. M. anisopliae adheres to insects cuticle and germinates to form short germ tubes, then elaborates a specialized infection cell called appressorium that forms a thin penetration peg to initiate penetration. M. anisopliae produces a mumber of cuticle-degrading proteases to facilitate host penetration. The formation of appressorium paly an important role in fungal pathogenesis. M. anisopliae traverses insect cuticle and enters host hemolymph, then colonizes the host hemolymph. It utilizes the nutrients available in hemolymph, and produces insecticidal secondary metabolites, which cause insect death. Identification genes differentilly expressed by M. anisopliae during appressorium formation and colonization of host hemolymph would reveal the mechanisms of fungal pathogenesis.EST analysis and microarray approaches are useful and successful for studying insect fungal pathogenesis. Many researchers adopted these strategies to study the developmental and transcriptional events during M. anisopliae infection. The methodologies of their studies are that M. anisopliae were cultured in liquid minimum medium supplemented with various inducer, followed by cDNA library construction and ESTs analysis. The life of the fungus cultured in liquid medium is saprophytic, while the life of the fungus infecting insect is parasitical. There are large difference between the life of the fungus cultured in vitro with the fact that M. anisopliae naturally infect insects. It is impossible to elucidate the mechanism underlying fungal pathogenesis and adaptation to host hemolymph, furthermore to understand the host-pathogen interactions.In this study, Metarhizium anisopliae var. acridum strain CQMa102 and Locusta migratoria were used, we prepared the hyphal bodies from infected insect hemolymph without host nucleic acid contamination, and employed a reagent called RNA fragmentation buffer to degrade the locust wing nucleic acid before inoculation with M. anisopliae conidia. This study was aimed at identifying genes were up regulated during colonization of hemolymph when hyphal bodies occur, and during appressorium formation on locust wings. A normalized cDNA library of M. anisopliae during colonization of host hemolymph was successfully constructed utilizing DSN (duplex-specific nuclease) normalization method combined with SMART (switching mechanism at 5′end of RNA transcript) library construction method, ESTs analysis of cDNA library revealed new insight on the host-pathogen interactions.The main results were as follows:1. The process of M. anisopliae strain CQMa102 infect locust L. migratoria was observed with a digital microscope at experimental conditions. M. anisopliae enters and colonizes the host hemolymph within 24 h post inoculation, hyphal bodies (short hyphal lengths and yeast-like blastospores) had attached to hemocytes and appeared to stimulate hemocytes aggregation. At the time point 3-6 days, hyphal bodies become phagocytosed and encapsulated by hemocytes. Before time 24-48 h of insect death, hyphal bodies have an increase of the concentration accompanied by reduction in hemocyte counts, and freely floating in the hemolymph.2. We perfectly establish a method to lyse host hemocytes for isolating hyphal body by utilization of proteinase K and SDS according to the different cell structure between fungus and host hemocyte. The procedure takes about 40 min, and RT-PCR analysis indicates that endogenous RNases that released from membrane-bound organelles upon disruption and sequent simple washing steps could rapidly remove locust nucleic acid before fungal mRNA extraction. This assertion was further confirmed by sequent EST analysis.3. Suppression subtractive hybridization was performed here using cDNA generated from purified hyphal bodies in hemolymph as tester, and cDNA generated during conidia germination and appressorium formation on locust wing as driver. 350 positive clones were obtained by cDNA array dot blotting, sequenced, resulted in 120 unique expressed sequence tags (ESTs) that were up-regulated during colonization of hemolymph, including 100 singletons and 20 contigs. Among these 120 ESTs, 29 (24.17% of total) were significantly similar to known proteins involved in various cell and molecular processes, such as general metabolism, energy production, maintenance of cell function and structure, cell cycle and control, stress response etc.4. To confirm the reliability of SSH and dot blotting, the expression levels of five randomly chosen genes in different stages of pathogenesis were analyzed using semi-quantitative RT-PCR analysis. These ESTs included those with homology to α-1,2-mannosyltransferase,α-enolase, pisatin demethylase, prenyl transferase and choline dehydrogenase. All the five genes confirmed by RT-PCR analysis were up regulated during colonization of host hemolymph.5. To identify the genes were up-regulated during appressorium formation, suppression subtractive hybridization was also performed here using cDNA generated during conidia germination and appressorium formation on locust wing as tester and cDNA generated from purified hyphal bodies in hemolymph as driver. 460 positive clones were obtained by cDNA array dot blotting, and sequenced, resulted in 329 high quality ESTs which represented 78 uniESTs. Among these 78 ESTs, 46 with significant similarity to NCBI hypothetical proteins or known proteins, 24 ESTs represented a broad spectrum of biological functions, including protein metabolism proteins, cell metabolism proteins, cell sturctrue and function proteins and stress response proteins, etc.6. A total of 12 ESTs predicted to be up regulated expressed by SSH were tested by semi-quantitative reverse transcription PCR. For time-course experiments, total RNA were extracted from the fungi collected after 8 h, 15 h, 24 h, 30 h from locust wings respectively and the mixture of host hemocytes and hyphal bodies ( 2 days, 3 days, 4 days, 5 days, 6 days, 7 days). Total RNA were treated by DNAseⅠbefore RT-PCR. The glyceraldehyde-3-phosphate dehydrogenase gene (gpd) was selected as the endogenous control. The results demonstrated that these genes all were up-regulated during appressorium formation on locust wing.7. A normalized cDNA library of M. anisopliae during colonization of host hemolymph was successfully constructed utilizing DSN (duplex-specific nuclease) normalization method combined with SMART (switching mechanism at 5′end of RNA transcript) library construction method. 5222 clones were sequenced, and a total of 4606 ESTs of high quality was obtained, resulted in 1863 unique ESTs, including 1237 singletons and 626 contigs. The ESTs had significant similarity to known proteins involved in various cell and molecular processes, such as general metabolism, energy production, maintenance of cell function and structure, cell cycle and control, stress response etc.8. A full length cDNA library of M. anisopliae during colonization of host hemolymph was successfully constructed utilizing SMART (switching mechanism at 5′end of RNA transcript) library construction method. Clones were sequenced, and a total of 187 unique ESTs was obtained, which represented the high abundant genes during the colonization of host hemolymph.更多还原