【作者】 张世泽；

【导师】 花保祯；

【作者基本信息】 西北农林科技大学 ， 农业昆虫与害虫防治， 2009， 博士

【摘要】 黑森瘿蚊Mayetiola destructor (Say)是广布于世界主要小麦产区的一种毁灭性害虫,防治实践证明抗虫品种是控制其危害的最经济有效的措施。但是,大范围、长期使用抗虫品种对黑森瘿蚊种群施加了强烈的选择压力,导致黑森瘿蚊种群新的生物型出现,而这又降低了抗性品种的使用寿命。由于昆虫的消化道是食物消化、营养吸收、解毒和植物抗性的主要位点,因此鉴定和研究黑森瘿蚊幼虫消化道的基因及其特性,不但能够提高对昆虫肠生理的理解,而且还能为抗性品种的培育及改善害虫的治理提供重要的理论指导。另外,由于黑森瘿蚊-小麦系统符合gene-for-gene关系,因此也提供了研究刺吸式害虫与植物关系的模式系统。本论文利用ESTs、Microarray、RT-PCR及Transcriptome等技术,首次系统研究了黑森瘿蚊肠转录组、小麦-黑森瘿蚊相容(compatible interaction)、不相容(incompatible interaction)关系中小麦蛋白抑制基因表达水平的变化。主要研究结果如下:1.对黑森瘿蚊10 051条Expressed sequenced tag(ESTs)进行聚类拼接得到2 024 cluster(s包括870 contigs和1 154 singletons)。BLASTx鉴定了1 216 clusters与GenBank数据库中序列有同源匹配。在这些clusters中,有809 clusters编码的蛋白与已知功能蛋白具有相似性或是少于250个氨基酸的小的分泌蛋白(Small secretory proteins,SSP)。这些clusters被分成9个类别,分别是:I为代谢有关的蛋白(Proteins involved in metabolism);II为结构蛋白(Structural proteins);III为调节者(Regulators);IV为运输者(Transporters);V为与蛋白合成和折叠有关蛋白(Proteins involved in protein synthesis and folding);VI为消化酶类(Digestive enzymes);VII为解毒酶类(Detoxification enzymes);VIII为小的分泌蛋白(Small secretory proteins, SSP);IX其它类(Others)。前5类代表的基因参与看家功能(House-keeping functions),其余3类具有肠功能特征。2.转录编码的多样性的消化酶类与吮吸式口器昆虫明显不同。消化酶类包括的clusters编码10个trypsins(6个新基因,分别是MDP6A、MDP6B、MDP7A、MDP8C、MDP9A和MDP10A), 7个chymotrypsins(4个新基因,分别是MDP11A、MDP12B、MDP13A和MDP14B), 2个cysteine proteases(新基因), 1个aspartic protease(新基因), 1个endo-oligopeptidase(新基因), 3个aminopeptidases(新基因), 10个carboxypeptidases(1个新基因), 2个α-amylases(新基因)。3.解毒酶类包括的clusters编码13个cytochrome P450s(新基因), 3个glutathione S-transferases (GSTs)(1个新基因), 3个peroxidases(1个新基因), 3个ferritins(新基因), 1个catalase(新基因), 2个peroxiredoxins(新基因), 7个其它的酶类。转录编码多样性的抗氧化酶的存在可能是黑森瘿蚊克服毒性氧化剂和源于寄主植物次生代谢物质的分子基础。4.SSP包括111 clusters,其中22个编码结构类似于蛋白酶抑制剂(Protease inhibitors)(7个新基因),核糖核酸酶(Ribonuclease)(2个新基因),表皮蛋白(Cuticle protein)(4个新基因)和细胞调节者(Cellular regulator),其余的clusters编码未知的蛋白。大量转录编码SSP是黑森瘿蚊幼虫肠转录组独特的特征,占整个ESTs的25%,其中大约63%的SSP是黑森瘿蚊或瘿蚊昆虫特有的,表明这些SSP在黑森瘿蚊幼虫取食位点营养组织的建立、寄主生长抑制的效应器细胞(Effector)等方面发挥功能。5.鉴定了具有不同结构的4组编码蛋白的抑制基因。在小麦-黑森瘿蚊不相容关系中,大部分基因表达水平上调,而在相容关系中则下调。抗虫植物和感虫植物间,不相容关系中的基因上调水平和相容关系中的基因下调水平导致了4--30倍的差异。不相容关系中抑制基因表达量的提高表明这些基因是小麦抵抗黑森瘿蚊侵袭的防御机制的一部分,而相容关系中这些抑制基因表达量的降低则表明毒性黑森瘿蚊幼虫能够抑制植物的防御反应。另外,这些抑制基因表达水平的差异与伤口诱导途径无关。上述研究结果为更深入地注释黑森瘿蚊基因组的功能、理解黑森瘿蚊的进化地位以及揭示瘿蚊—植物的协同进化机制提供了重要的理论基础,对更进一步研究黑森瘿蚊功能基因组学具有重要的理论意义,而且在实践上可为抗性品种的培育及改善害虫的治理提供重要的理论指导。更多还原

【Abstract】 The Hessian fly, Mayetiola destructor (Say), is a destructive pest of wheat and is widely distributed throughout most wheat-growing areas of the world. Host plant resistance has been considered the most effective and economical means of controlling this pest in wheat. However, the widespread use of resistant cultivars has placed a strong selection pressure on Hessian fly populations, which has resulted in the evolution of new genotypes of the fly that can overcome previously resistant wheat. Since the gut is the primary site for food digestion, nutrient absorption, toxic degradation and plant resistance. Thus, identification and characterization of genes expressed in the Hessian fly larval gut will not only increase our understanding of insect gut physiology, but provide novel and specific targets for the development of strategies for pest management. On the other hand, the wheat-Hessian fly system follows a typical gene-for-gene interaction, and provides a good model for studying the scheme of attack and counter-attack between insects and plants. In the paper, the first global analysis of gut transcripts from a gall midge and systematic analysis of the expression patterns of inhibitor-like genes in wheat-Hessian fly system were conducted by means of methods and techniques of Expressed sequenced tag (ESTs), transcriptome, microarray and Real-Time PCR. The main results were as follows.1. More than 10 000 ESTs were assembled into 2 024 clusters (contigs and singletons). BLASTx identified 1 216 clusters with similarity to GenBank sequences. Among them, 809 clusters coded either for proteins with similarity to functionally known proteins or for small secretory proteins (SSP) (<250 amino acids). These clusters were grouped into nine categories: I. Proteins involved in metabolism, II. Structural proteins, III. Regulators, IV. Transporters, V. Proteins involved in protein synthesis and folding, VI. Digestive enzymes, VII. Detoxification enzymes, VIII. SSP, and IX. Others. The first five categories represent genes participating in house-keeping functions. The other three categories, including digestive enzymes, detoxification enzymes, and SSP, are characteristic of gut functions.2. There were transcripts coding for diverse putative digestive proteases, which is different from other plant-sucking insects such as aphids. The digestive enzymes included clusters coding for 10 trypsins (six represented novel genes, named MDP6A, MDP6B, MDP7A, MDP8C, MDP9A and MDP10A), seven chymotrypsins (four represented novel genes, named MDP11A, MDP12B, MDP13A and MDP14B), two cysteine proteases (novel genes), one aspartic protease (novel genes), one endo-oligopeptidase (novel genes), three aminopeptidases (novel genes), 10 carboxypeptidases (one novel gene), and twoα-amylases (novel genes).3. Detoxification enzymes included clusters coding for 13 cytochrome P450s (novel genes), three glutathione S-transferases (GSTs) (one novel gene), 3 peroxidases (one gene), three ferritins (novel genes), one catalase (novel gene), two peroxiredoxins (novel genes), and several other enzymes. The existence of transcripts coding for diverse antioxidant enzymes may be the molecular basis for Hessian fly to overcome toxic oxidants and secondary metabolites resulted from basal and induced defenses of host plants.4. The category of SSP contained 111 clusters, 22 of which coded for proteins structurally similar to protease inhibitors (seven novel genes), ribonucleases (two novel genes), cuticle proteins (four novel genes), and cellular regulators, whereas the other clusters coded for unknown proteins. The unique feature of Hessian fly larval gut transcriptome was the presence of a large number of transcripts coding for SSP, which represented nearly 25% of all the ESTs. Most (63%) of the putative SSP were unique to Hessian fly or gall midges. This strongly suggests that they probably perform functions characteristic of this insect, such as serving as effectors for the inhibition of host growth or in establishing nutritive tissue at the feeding site.5. Four groups of inhibitor-like genes encoding proteins with diverse structures were identified from wheat. The majorities of these genes were up-regulated by avirulent Hessian fly larvae during incompatible interactions, and were down-regulated by virulent larvae during compatible interactions. The upregulation during incompatible interactions and downregulation during compatible interactions resulted in 4 to 30 fold differences between the expression levels in resistant plants and those in susceptible plants. The increased expression of inhibitor-like genes during incompatible interactions suggested that these genes are part of defense mechanisms in wheat against Hessian fly attacks, whereas the downregulation of these genes during compatible interactions suggested that virulent larvae can suppress plant defenses. Both the upregulation of the inhibitor-like genes during incompatible interactions by avirulent larvae and the downregulation during compatible interactions by virulent larvae were through mechanisms that were independent of the wound response pathway.更多还原