【作者】 梁彦；

【导师】 高希武；

【作者基本信息】 中国农业大学 ， 农业昆虫与害虫防治， 2017， 博士

【摘要】 桃蚜 Myzus persicae(Sulzer),属半翅目Hemiptera,蚜科Aphididae,是一种几乎遍布全球的多食性害虫。桃蚜不仅可以吸取植物汁液,还可以作为介体传播多种植物病毒,是常见的媒介昆虫,给农业生产带来了严重的损失。桃蚜主要以非持久性的方式来传播黄瓜花叶病毒(Cucumber mosaic virus,CMV),这种方式主要是指病毒在昆虫体内仅停留几秒至几分钟,病毒不会随昆虫的繁殖而传递给下一代。目前,关于桃蚜传播CMV的传毒机制尚不清楚。本研究一方面根据文献的报道,利用qPCR、酵母双杂交、RNAi等分子生物学方法,研究桃蚜表皮蛋白基因在传毒过程中的作用;另一方面利用RNA-seq和iTRAQ等组学研究技术,进一步挖掘与传毒相关的潜在传毒因子,为全面理解桃蚜的传毒机制奠定理论基础,同时也为生产中桃蚜和病毒病的防治策略提供参考依据。在研究表皮蛋白基因的作用方面,前期筛选出用于扩增MPCP1、MPCP2、MPCP4、MPCP5的引物,并优化了反应条件;用整个虫体和口针扩增的各表皮蛋白基因序列是一致的,故桃蚜整个虫体可以作为后续的研究对象。在基因水平上,CMVCP的相对表达量与各表皮蛋白基因的变化趋势相似,并且MPCP1、MPCP2和MPCP4的相对表达量明显高于MPCP5;在酵母双杂交系统里,只有基因MPCP4编码的蛋白质与CMVCP是相互作用的。在功能研究上,基因MPCP4的沉默可以降低桃蚜获得病毒的效率。综上所述,桃蚜表皮蛋白相关基因参与了传毒过程,且基因MPCP4在其中起主要作用。在挖掘与传毒相关的潜在传毒因子方面,对带毒的和不带毒的桃蚜分别进行了转录组和蛋白质组研究。在转录组测序中,一共找到20550个差异表达的基因。其中,9731个差异基因是上调表达的,10818个差异基因是下调表达的。在KEGG数据库中,差异表达的基因共注释到501个通路,其中有32个是富集分布的(p-value≤0.05),多集中在cAMP信号通路(cAMP signaling pathway)和药物代谢通路(drug metabolism)。在蛋白质组测序中,共找到744个差异表达的蛋白。其中,上调表达的差异蛋白有437个,下调表达的差异蛋白有307个。在KEGG数据库中,差异表达蛋白富集(p-value≤0.05)分布在了 14个通路里,其中包括脂肪酸代谢途径(fatty acid metabolism)、碳代谢途径(carbon metabolism)和各种氨基酸代谢途径等。最后,对桃蚜转录组和蛋白质组进行联合分析,发现两者存在一定的相关性,并且有282个蛋白质在转录组数据中可以找到联系,这其中表达模式(均上调表达或均下调表达)一致的有207个蛋白质,包括表皮蛋白、核糖体蛋白和细胞色素P450等。更多还原

【Abstract】 Myzus persicae(Sulzer)(Homoptera:Aphididae),is one of the most important agricultural pests worldwide.In addition to sucking phloem sap,M.persicae also transmits plant viruses as a vector.This could lead to a big loss in crop yield.Aphids transmit cucumber mostaic virus(CMV)in a non-persistent manner.The time of aphids acquiring and inoculating virus is extremely rapid(seconds to minutes)and the ability to transmit virus is also short-lived(minutes to hours).However,little is known about the transmission mechanism of CMV.On one hand,the roles of M.persicae cuticle proteins in the transmission mechanism were studied with qPCR,Y2H and RNAi.On the other hand,potential receptors were found between RNA-seq and iTRAQ database.These results lay a theoretical foundation for the transmission mechanism of CMV.Moreover,they can offer the important reference for M.persicae and CMVcontrolled in agricultural industry.In this study,primers and conditions were firstly optimized for MPCP1,MPCP2,MPCP4,and MPCP5.The body and stylet templates were used to amplify MpCuPs and the gene sequences are consistent,so the body can be used as follow-up studies.Then we measured the relative amount of the gene encoding Cucumber mosaic virus capsid protein(CMV CP)and the transcript levels of four cuticle protein genes.The results show that the expression patterns of the four cuticle protein genes were almost identical,but the relative expression of MPCP1,MPCP2 and MPCP4 were higher than MPCP5.Yeast two-hybrid assays demonstrated that the protein encoded by MPCP4 gene was closely associated with the CMV CP through the direct interaction.Moreover,the ability of M.persicae to acquire CMV was suppressed by RNA interference of MPCP4.All these lines of evidence indicate that MPCP4,as a viral putative receptor in the stylet of aphid,plays an important role in aphid acquisition of CMV.In this study,an integrative analysis of the transcriptome and proteome was performed to identify important receptors involved in CMV transmission.At the transcript level,a total of 20550 genes were identified as differentially expressed genes(DEGs)at 24 h after transferred to infected tobacco plants using RNA-seq approach.Of these,9732 were up-regulated and 10,818 were down-regulated.In the KEGG database,the DEGs were mapped to 501 pathways.Among these,32 pathways were substantially enriched(p-value<0.05)between the up-regulated and the down-regulated DEGs.Examples of enriched pathways include cAMP signaling pathway and drug metabolism.At the protein level,a total of 744 proteins were classified as being differentially expressed between viral and nonviral M.persicae using iTRAQ(isobaric tags for relative and absolute quantitation)analysis.Of these,437 were up-regulated and 307 were down-regulated.In this KEGG database,differentially expressed proteins(DEPs)were substantially enriched to 14 pathways(p-value<0.05),including fatty acid metabolism,carbon metabolism,and amino acid metabolism.The combined transcriptome and proteome analysis was shown some relevance.Specifically,it revealed 282 proteins had corresponding transcripts in the RNA-seq data.Of these,207 had the same direction of change(up-or down-regulation)in the two omics data sets,including cuticular proteins,ribosomal proteins and Cytochrome P450s.更多还原