携带与未携带松材线虫的松墨天牛转录组差异表达基因分析

Transcriptome analysis of differentially expressed genes in Monochamus alternatus(Coleoptera:Cerambycidae)carrying and without carrying pine wood nematode(Bursaphelenchus xylophilus)

【目的】本研究旨在建立携带与未携带松材线虫Bursaphelenchus xylophilus的松墨天牛Monochamus alternatus成虫各组织的转录组数据库,揭示松墨天牛对松材线虫响应的转录组整体表达特征。【方法】以携带和未携带松材线虫的松墨天牛成虫的表皮、气管和脂肪体为材料,采用Illumina HiSeq TM 2000测序平台开展转录组测序,利用Trinity软件对RNA-Seq数据进行从头组装,利用NCBI数据库进行基因注释。通过DEG-Seq分析携带和未携带松材线虫的松墨天牛成虫中的差异表达基因,对上调表达基因进行GO和KEGG代谢途径富集分析,并通过实时荧光定量PCR技术对部分差异表达的基因包括ENV,CDK1,HSP70-C,HSP70,HSP75,DUO,PABPN1和IGFP基因的表达水平进行验证。【结果】测序过滤后共获得55059条单基因簇(unigene),平均长度为1536 bp。将unigene与数据库中的序列进行BLASTX比对,成功注释24354条unigenes,其中4022条unigene在GO数据库中获得注释,6098条unigene在KEGG数据库中获得注释。经过GO与KEGG富集分析显示,松墨天牛成虫携带松材线虫后,差异表达基因主要为与其气管中压力调节、组织与DNA修复、激素响应方面相关的基因。实时荧光定量PCR分析结果显示CDK1,HSP70,HSP75,DUO,PABPN1和IGFP基因的表达量在携带松材线虫的松墨天牛气管中相比未携带松材线虫的松墨天牛气管中的显著上调。【结论】本研究初步阐明松墨天牛携带松材线虫后转录组的整体表达模式,为进一步研究松墨天牛的基因功能及对松材线虫的胁迫响应过程奠定了基础。

【Aim】The objective of this study is to establish the transcriptome database of adult tissues of the pine sawyer beetle,Monochamus alternatus,carrying and without carrying the pine wood nematode,Bursaphelenchus xylophilus,and to reveal the overall expression characteristics of M.alternatus in response to pine wood nematode.【Methods】The transcriptomes of epidermis,trachea and fat body of M.alternatus adults carrying and without carrying B.xylophilus were sequenced using the Illumina HiSeq TM 2000 platform,the transcriptome data were de novo assembled using Trinity software,and gene annotation was conducted by using the NCBI database.The differentially expressed genes in M.alternatus adults carrying and without carrying B.xylophilus were screened by DEG-Seq,and the up-regulated genes were subjected to GO and KEGG metabolic pathway enrichment analysis.The expression levels of some differentially expressed genes including ENV,CDK1,HSP70-C,HSP70,HSP75,DUO,PABPN1 and IGFP genes were verified by real-time quantitative PCR(RT-qPCR).【Results】After sequencing and filtering,a total of 55059 unigenes were obtained with an average length of 1536 bp.A total of 24354 unigenes were annotated using BLASTX searches against the databases,of which 4022 unigenes were assigned in GO database and 6098 unigenes were assigned in the KEGG database.The enrichment analysis of GO and KEGG showed that the main differentially expressed genes in the trachea of M.alternatus adults carrying B.xylophilus are related with pressure regulation,tissue and DNA repair,and hormone response.The RT-qPCR analysis showed that the expression levels of CDK1,HSP70,HSP75,DUO,PABPN1,and IGFP genes were significantly higher in the trachea of M.alternatus carrying pine wood nematodes than in the trachea of M.alternatus without carrying pine wood nematodes.【Conclusion】This study preliminarily clarified the overall expression pattern of the transcriptome of M.alternatus carrying B.xylophilus,providing a foundation for further study of the gene function of M.alternatus and the stress response of M.alternatus to B.xylophilus.