【作者】 张伟；

【导师】 夏玉先；

【作者基本信息】 重庆大学 ， 生物医学工程， 2007， 博士

【摘要】 农业害虫是造成农作物减产的主要原因,同时也给林业和牧业造成了巨大的灾害。飞蝗是一种洲际性农业重大害虫,在我国,以东亚飞蝗[Locusta migratoria manilensis (Meyen)]分布面积广,危害最为严重。因而加强对飞蝗等农业害虫的研究和灾害的综合防治,已成为关系我国经济发展和社会稳定的重要因素。长期以来,化学农药在害虫的防治中起着主导作用,这将直接导致三大环境问题:(1)污染环境,残毒积累,最终危害人类健康;(2)害虫抗药性直线上升,使化学农药用量不断增加,导致恶性循环;(3)杀伤天敌,破坏生态平衡,从而致使害虫再猖獗和次要害虫上升为主要害虫。为了降低环境污染,减少或禁用化学农药势在必行,开发高效、稳定的生物农药已成为当务之急。微生物杀虫剂具有无公害、无残留、害虫不易产生抗性等优点,日益受到人们的广泛关注。而杀虫真菌是昆虫病原微生物(细菌、真菌和病毒)中最大的一个类群,具有直接穿透寄主体壁和持续控制等优点,是其它微生物杀虫剂不可比拟的。绿僵菌是一种非常有效防治蝗虫的杀虫真菌,但是同其它杀虫真菌一样,存在着致死时间长等不足,影响其广泛应用。昆虫的体壁是抵御真菌侵染的第一道物理屏障,因而杀虫真菌穿透寄主体壁的成功率和速度是影响其杀虫时间的重要因素之一。杀虫真菌依靠酶解作用和机械压力穿透寄主体壁,在侵染过程中产生一系列的酶来降解昆虫表皮,其中蛋白酶起着主要作用。因而,来源于昆虫病原真菌的这类蛋白酶基因,已成为利用基因工程手段,构建高效杀虫真菌的重要毒力因子。同时,除了昆虫病原真菌能产生降解昆虫体壁的蛋白酶外,昆虫自身也能产生降解自身表皮的蛋白酶。在昆虫蜕皮过程中,昆虫会分泌大量蜕液,其中包含以蛋白酶为主的一系列水解酶,来降解老表皮,并回收利用促进新表皮的合成。因此,本研究以昆虫病原真菌绿僵菌和东亚飞蝗为材料,分离、克隆具有壳降解功能的蛋白酶基因,并进行相关的功能验证、分析。为发展高效、安全的基因工程杀虫真菌提供毒力基因和前期的基础研究工作,这对于发展真菌杀虫剂及提高我国真菌农药产业的核心竞争力都具有重要的意义。同时,昆虫蜕液中的壳降解蛋白酶,与昆虫的蜕皮具有重要的关系,这类蛋白酶的缺损,将导致昆虫不能蜕皮,并最终导致死亡,是潜在的杀虫靶标。因此,研究昆虫壳降解蛋白酶基因及功能,对于研究昆虫的蜕皮生理发育过程,以及以这类蛋白酶为靶标,建立新型、高效、安全的杀虫剂筛选模型都具有重要的意义。主要研究结果如下:(1)根据已报道的绿僵菌PR1A基因保守序列设计引物,从绿僵菌CQMa102中克隆PR1A基因,其DNA序列、mRNA序列和编码蛋白的GenBank登录号分别为:AY839935,EF627449和ABR20899。其DNA序列全长1571 bp,cDNA序列全长1319 bp,其中包含一个1173 bp的开放阅读框和一个146 bp的3’端非编码区,开放阅读框编码一个含390个氨基酸序列的蛋白。根据同源序列比对以及序列分析结果,表明PR1A属于类枯草杆菌丝氨酸蛋白酶(subtilisin-like serine protease,SLSP)家族。通过与来源于丝状真菌、酵母、细菌、蓝细菌、无脊椎动物及脊椎动物的16种SLSP构建同源树和系统发育树分析,表明CQMa102 PR1A与丝状真菌的SLSP具有非常高的同源性和很近的进化关系,而且与绿僵菌Metarhizium anisopliae var. acridum strain ARSEF 324 PR1A同源性高达99%。同时,我们用CQMa102 PR1A基因构建毕赤酵母分泌表达载体,并成功实现在毕赤酵母P. pastoris KM71中的分泌表达。用KM71原始菌株和pPIC9K空载体转化菌株作为对照,对重组菌株KM71/pPIC9K-PR1A表达产物进行分析。SDS-PAGE和Western blot都显示,在目标位置26 kDa大小附近新增加一条带,且与来源于绿僵菌的天然蛋白大小相同。另外,表达产物具有显著的对PR1特异底物活性、总蛋白酶活性以及对蝗虫壳蛋白的降解活性。这些研究结果表明,我们成功克隆到CQMa102 PR1A基因,且通过功能表达证明该基因编码蛋白具有降解蝗虫壳蛋白的功能。(2)对来源于昆虫的类胰蛋白酶和丝氨酸蛋白酶氨基酸序列进行比对分析,根据保守区域设计两对嵌套简并引物。选取东亚飞蝗五龄幼虫MFP活性增加最快时期的虫体提取mRNA,进行反转录,用所设计的简并引物进行PCR扩增,克隆到一种新的蜕液蛋白酶基因,命名为Lm-TSP,其cDNA序列及推导的氨基酸序列GenBank登录号分别为:EF081255和ABN13876。cDNA序列全长1587 bp,其中包含一个735 bp的开放阅读框和一个852 bp的3’端非编码区,开放阅读框编码一个含244个氨基酸序列的蛋白。根据同源性搜索比对以及序列分析,表明Lm-TSP属于类胰蛋白酶丝氨酸蛋白酶(trypsin-like serine protease,TLSP)家族。通过与来源于昆虫、蛛形纲动物、哺乳动物和细菌的14种TLSP构建同源树和系统发育树分析,表明Lm-TSP与来源于昆虫的TLSP有较高的同源性和很近的进化关系,而且与来源于昆虫赤拟谷盗(Tribolium castaneum)的TLSP同源性高达82%。RNAi研究结果表明,Lm-TSP基因的沉默将引起东亚飞蝗从四龄幼虫到五龄幼虫,和从五龄幼虫到成虫蜕皮功能的丧失,这说明Lm-TSP在东亚飞蝗蜕皮过程中起着重要作用。而且Lm-TSP基因的沉默同时会引起蜕液中总蛋白酶活性、特异蛋白酶活性和壳蛋白降解活性的急剧降低,继而引起对昆虫老表皮降解功能的缺失,最终导致昆虫不能蜕皮而死亡。这些结果表明Lm-TSP与东亚飞蝗蜕皮过程相关,是与昆虫蜕皮功能相关的第一个蛋白酶基因。(3)建立起了比较完善的东亚飞蝗幼虫RNAi(larvae RNAi)体系。更多还原

【Abstract】 The migratory locust is a widespread agriculture pest in China with a comparatively broad geographic distribution that includes both temperate and tropical climatic zones. It can cause signicant damage to pastures and crops and is a major threat for food safety, social stability and economic development. So we should strengthen on the research and pest control of agriculture pest.For a long time, the use of chemical insecticides for the control of insect pests was proven very effective at increasing agriculture and forestry productivities. However, the abuse of poisonous chemical substances directly led to three main environmental problems (residue, resistance and resurgence). To reduce environmental pollution, the usage of chemical pesticide should be reduced. Our immediate concern is to develop efficient, stable and safe bio-pesticides. Microbial pesticides have many advantages such as: safety, no-residue, hard to appear resistances and so on, and therefore are applied extensively. Entomopathogenic fungi are a big group of entomopathogenic microorganisms (bacteria, fungi and virus). Unlike entomopathogenic bacteria and viruses that invade insects though the alimentary canal, fungi can actively penetrate the host cuticle. Metarhizium anisopliae is an effective fungus for locust control. However, like other enfomopathogenic fungi, Metarhizium anisopliae needs a long time to kill pest insects, which impedes its wide application. To the invading fungus, insect cuticle represents the first barrier to penetration. In common with other entomopathogenic fungi the initial stage of infection involves penetration of the insect cuticle, so the success rate and speed of cuticle-penetration have a great effect on time of pest killing. Mechanical pressure plays a role in this process but of major importance is enzymatic degradation of cuticle within the immediate vicinity of infection hyphae. Cuticle-degrading proteases play a great role in the cuticle-degrading activity. Thus, this kind of proteases has been used as virus factors for developing high effective fungi pesticide by genetic engineering.Moreover, we found that besides entomopathogenic fungi, insects could also generate proteases to degrade their own cuticle. During the molting process, degradation and recycling of the old cuticle are brought about by enzymes present in the molting fluid filling the space between the old cuticle and the new cuticle. So, this study took entomopathogenic fungi Metarhizium anisopliae and Locusta migratoria as research targets, isolated and cloned cuticle-degrading protease genes and then verified their functions. This research could provide virus genes and basic work for developing high effective fungi pesticide by genetic engineering, which would speed the process of industrialization of fungi pesticides and promote their core competitive capability. Furthermore, cuticle-degrading proteases in the molting fluid of insects play an important role in molting. Silencing these proteases caused molt defects and finally led to death in locust. So this study is very important for the research on ecdysis of locust and for the development of novel, high effective and safe pesticides by taking these kinds of proteases as screening target. The main results are as follows:(1) PR1A gene was cloned from Metarhizium anisopliae strain CQMa102 using primers designed according conserved region of other Metarhizium anisopliae PR1A genes. The DNA, cDNA and and its deduced protein sequences were deposited in GenBank (accession numbers AY839935, EF627449 and ABR20899, respectively). The DNA sequences were 1571 bp. The 1319-bp cDNA sequence contained an 1173-bp single open reading frame encoded a protein of 390 amino acids and a 146-bp 3’untranslated region. Analysis of the amino acid sequences by computer using the NCBI database and BLAST revealed that PR1A belonged to subtilisin-like serine protease family. Amino acid sequences of PR1A from CQMa102 with other 16 subtilisin-like proteases from filamentous fungi, yeast, bacteria, cyanobacteria, vertebrate and invertebrate were used for the multiple sequence alignment, homology tree and phylogenetic tree. The results showed that PR1A had significant homology and close relationship with subtilisin-like proteases from filamentous fungi, and had the closest relationship to the protein from Metarhizium anisopliae var. acridum strain ARSEF 324 with 99% identity. Furthermore, the gene was cloned into and secretively expressed in Pichia pastoris KM71 under the control of the AOX1 promoter. Compared with controls, the product of PR1A recombinant (KM71/pPIC9k-PR1A) added a new band at about 26 kDa by SDS-PAGE and Weatern blot, and was good accordance with the nature peotein from CQMa102. The recombinant protein showed significant specific protease activity, total protease activity and cuticle-degrading activity. From these results, we could conclude that we cloned PR1A gene from Metarhizium anisopliae strain CQMa102 and its encoded protein had cuticle-degrading function.(2) Trypsin-like proteases and serine proteases from insects were used for multi-sequence alignment. Two pairs of degenerate primers were designed according to the conserved region. For RT-PCR cloning experiments, mRNA was prepared from locusts at the time when the molting fluid protease activity increased sharply. We cloned a novel molting fluid protease gene from Locusta migratoria manilensis,designated as Lm-TSP. The cDNA and its deduced protein sequences were deposited in GenBank (accession numbers EF081255 and ABN13876, respectively). The 1587-bp cDNA sequence contained a 735-bp single open reading frame encoded a protein of 244 amino acids and an 852-bp 3’untranslated region. Analysis of the amino acid sequences by computer using the NCBI database and BLAST revealed that Lm-TSP belonged to trypsin-like serine protease family. Amino acid sequences of Lm-TSP with other 14 trypsin-like serine proteases from insects, arachnid, mammals and bacteria were used for the multiple sequence alignment, homology tree and phylogenetic tree. The results showed that Lm-TSP had significant homology and close relationship with trypsin-like serine proteases from insects, and had the closest relationship to the protein from Tribolium castaneum with 82% identity. Furthermore, RNA interference (RNAi) studies showed that the silencing of Lm-TSP caused dramatic reduction in specific protease activity and cuticle degrading activity of the molting fluid, and caused cuticle degrading defects as a consequence, which led to molt defect from the fifth instar larvae (L5) to adult and molt defect from the fourth instar larvae (L4) to L5 and finally led to death. The results described herein suggested that Lm-TSP plays a critical role in controlling L. migratoria manilensis ecdysis(3) A relative mature larvae RNAi system of Locusta migratoria was established.更多还原