中国农业科学2016，Vol.49Issue(17)：3452-3463,12.DOI:10.3864/j.issn.0578-1752.2016.17.018
家蚕感染二分浓核病毒（镇江株）的数字基因表达谱分析
Digital Gene Expression Analysis of Silkworm Infected by Bombyx mori Bidensovirus Zhenjiang Strain
摘要
Abstract
Objective] The objective of this study is to screen differentially expressed genes in theBombyx mori larvae infected with BmBDV-ZJ (B. mori bidensovirus Zhenjiang strain) and identify regulatory genes related to the virus infection and the host response so as to provide important clues for better understanding of the mechanism ofB. mori resistance against BmBDV-ZJ infection.[Method] The differential gene expression profiles in JSB. mori larvae after oral infection with BmBDV-ZJ were constructed using Illumina Genome Analyzer platform. In order to exclude the effects of individual differences, 10 larvae were dissected and pooled as one sample for digital gene expression (DGE) analysis, respectively. The differential expression detection of genes across samples was performed using a rigorous algorithm method. False discovery rate (FDR) was used to determine theP value threshold in multiple tests and analyses. The significance of the gene expression difference was obtained through a FDR≤0.001 and the absolute value of log2 ratio≥1. The gene ontology (GO) classification system was used to determine the possible functions of all differentially expressed genes.P value was calculated by GO (http://www.geneontology. org/) and corrected by Bonferoni. A correctedP value≤ 0.05 was selected as a threshold for significant enrichment of the gene sets. WEGO (web gene ontology annotation plot) software was used for visualizing, comparing and plotting GO annotation results. Pathway enrichment analysis was conducted to further identify the significantly enriched metabolic pathways or signal transduction pathways by using the KEGG database. Pathways with aQ value≤0.05 were designated as significantly enriched pathways in DGEs. Then some of the differentially expressed genes were verified by quantitative real-time PCR (qRT-PCR).[Result] Totally, 4 850 663 and 4 875 307 raw tags were generated in the control and BmBDV-ZJ infected DGE (digital gene expression) libraries, respectively. There were 4 757 934 and 4 788 406 clean tags corresponding to 62 436 and 63 680 distinct clean tags were filtered from the raw tags. The distribution of the total and distinct tags over the different tag abundance categories showed highly similar patterns in each DGE library. The sequencing depths reached approximately 3.5 and 3.7 million in the two DGE libraries, respectively, which satisfied the requirement for the experiment. So the two DGE libraries were reliable. The tag sequences of the two DGE libraries were mapped to the reference database ofB. mori. In the control and BmBDV-ZJ-infected DGE library, 36.39% and 45.30% of the clean tags were mapped to a gene in the reference database, 50.02% and 43.34% of the clean tags could be mapped to genome ofB. mori, while 13.59% and 12.35% of the clean tags were unknown tags. A total of 447 differentially expressed genes were detected, of which 306 were upregulated and 141 were downregulated. There were 218, 147, 179 differentially expressed genes have GO categories according to molecular function, cellular component and biological process, respectively. KEGG (http://www.genome.jp/kegg) ontology assignments were used to classify the functional annotations of the identified genes. Among the differentially expressed genes, 330 were mapped to 151 pathways in the KEGG database. Nineteen terms was significantly enriched (Qvalue≤0.05) and the cytosolic DNA-sensing pathway was significantly enriched. Moreover, 24 differentially expressed genes were verified using qRT-PCR, showing that 20 genes were concordant in the expression with DGE. Among the 9 differentially expressed genes related to cytosolic DNA-sensing pathway, BGIBMGA009408-TA, BGIBMGA004913- TA, BGIBMGA011753-TA, which were the DNA-directed RNA polymerase III genes inB. mori, were all up-regulated in the BmBDV-ZJ infectedB. mori with 4.3, 2.3, 3.4-fold change, respectively.[Conclusion] The results of this study may serve as a basis for future research not only on the molecular mechanism of BmBDV-ZJ invasion but also on the mechanism ofB. mori resistance against BmBDV-ZJ infection.关键词
家蚕/数字基因表达谱/二分浓核病毒/qRT-PCRKey words
Bombyx mori/digital gene expression/Bombyx mori bidensovirus/quantitative real-time PCR引用本文复制引用
高坤,尚梦珂,钱荷英,覃光星,郭锡杰..家蚕感染二分浓核病毒（镇江株）的数字基因表达谱分析[J].中国农业科学,2016,49(17):3452-3463,12.基金项目
国家自然科学基金（31402141）、江苏省自然科学基金 （）
