松墨天牛转录组及球孢白僵菌、苏云金芽孢杆菌感染下的基因表达谱

Monochamus alternatus, a key vector of Bursaphelenchus xylophilus, is a serious pest management concern in pine stands. In recent years, next generation sequencing techniques such as Solexa/Illumina (Illumina), 454 (Roche), and SOLID (ABI) platforms have emerged as the useful tool for transcriptome analysis. These tools have been widely used in detecting the gene expression, discovering the novel transcripts, and testing the differentially expressed genes. In the present study, we plan to obtain and characterize the transcriptome of M. alternatus with special emphasis on pathogen-induced genes using the Illumina sequencing platform, from which a large scale of transcript database and new unigenes for M. alternatus would be produced.Transcriptome de novo assembly is carried out with short reads assembling program - Trinity.Unigenes are aligned by blastx to protein databases, so both the nucleotide sequences and amino sequences of the unigene coding region are acquired. With NR annotation, we get GO, COG annotation of unigenes. With the help of KEGG database, we further study genes' biological complex behaviors and get pathway annotation for unigenes. We map out the gene expression profiles of M. alternatus in response to Beauveria bassiana and Bacillus thuringiensis,respectively to explore the interactions between M. alternatus and entomopathogens, and the molecular mechanism by which M. alternates adapt to environment. Results obtained from gene expression profiles provide insight into potential immune components that play significant role(s) in the host response subsequent to entomopathogens,discover the genes involved in insecticide resistance,insecticide targets,detoxification with potential influence in the insect population dynamics and the larval interactions with the microbial pesticides. To the best of our knowledge, this study is the first try to characterize the complete transcriptome of M. alternates. This total pool of sequences from the transcriptome will serve as a comprehensive resource for further research in M. alternates at molecular level, and provide an invaluable resource in the ultimate annotation of the full genome sequence which with the falling cost of sequencing may be achieved sooner than anticipated.Moreover,data mining allows the discovery of novel genes with potential application in the development of new strategies for the microbial control of this pest.

松墨天牛（Monochamus alternatus Hope）是重大森林检疫害虫，也是松材线虫的主要传播昆虫。第二代测序技术是转录组分析的有力工具，广泛用于发现新基因和基因表达谱分析。本研究用第二代测序技术中的Illumina研究松墨天牛转录组，克隆新基因，预测基因编码蛋白框，用GO、COG、KEGG注释基因功能，从组学水平解释其生命现象，扩展了松墨天牛分子生物学研究领域，丰富了现有的鞘翅目昆虫基因数据库；研究球孢白僵菌和Bt感染下松墨天牛的基因表达谱，发现靶标基因、抗性基因和免疫相关基因等重要功能基因，解析松墨天牛和昆虫病原微生物的相互作用关系，有助于阐明昆虫病原作用于宿主昆虫的分子毒理和松墨天牛适应于环境的分子机制；探索球孢白僵菌、Bt防治松墨天牛的潜力和可持续性,为研究新型、有效的昆虫病原微生物制剂防治松墨天牛提供分子依据。

松墨天牛在我国主要危害马尾松，是重大森林检疫害虫，也是松材线虫的主要传播昆虫。. 构建了松墨天牛cDNA文库，得到了48,787个unigene，平均长度为712 bp。78.1%最先与赤拟谷盗序列比对上。本项目鉴定出重要的解毒酶GSTs、 CarEs,和 P450。一些重要的杀虫剂靶标，如AChE、 GABA钠离子通道和nAChR也被鉴定出来。这些数据可用于发现与杀虫剂解毒和杀虫剂靶标酶有关的新基因。建立松墨天牛转录组数据库，可以在更广泛的组学水平上增强对松墨天牛的认知，解释其生命现象；丰富了现有的鞘翅目昆虫基因数据库；通过基因注释，以及预测编码蛋白框，为深入研究免疫相关、抗性相关、靶标相关等重要基因的功能奠定了基础。.Bt作用下，松墨天牛分别有947和8895个基因上调和下调。细胞色素P450基因上调表达与缓解免疫相关的自我损伤有关。组织蛋白酶基因表达下调可以扰乱松墨天牛的发育历程。另一种与Bt密切相关的途径是类固醇激素生物合成，有53个表达差异基因富集于这一途径中，解毒酶基因表达水平上调，可能是对宿主对Bt实施化学预防的机制之一。. 球孢白僵菌作用下，松墨天牛分别有1204 和 7297条基因上调和下调。这些基因涉及发育过程、酶调控、免疫系统、生物学过程的负调控等。昆虫信息素合成途径与球孢白僵菌胁迫有关， JHEH 的表达上调，激发松墨天牛保幼激素代谢，使幼虫变小，蛹期也会缩短。蜕皮激素氧化酶参与松墨天牛蜕皮激素的失活，调控松墨天牛发育。蜕皮激素基因表达下降，表明松墨天牛感染后未产生足够高的蜕皮激素滴度。. 松墨天牛对杀虫剂的转录组学反应不仅提供了Bt、球孢白僵菌分子毒理机制的认识，而且也鉴定了新的靶标，有望提高微生物杀虫剂的杀虫效果和安全性。我们鉴定了大量功能基因，这些基因与松墨天牛的毒理和基础生物学是相联系的，有利于理解Bt、球孢白僵菌及其潜在靶标之间网络关系，为研究新型、有效的昆虫病原微生物制剂防治松墨天牛提供了分子依据。