家蚕感染二分浓核病毒（镇江株）的数字基因表达谱分析

doi:10.3864/j.issn.0578-1752.2016.17.018

摘要/Abstract

摘要： 【目的】筛选家蚕与二分浓核病毒（Bombyx mori bidensovirus Zhenjiang strain，BmBDV-ZJ）感染相关的差异表达基因，鉴定家蚕与病毒感染有关的调控基因，为进一步阐明家蚕抗BmBDV-ZJ的分子机制提供理论依据。【方法】采用Illumina高通量测序技术，构建家蚕品种JS口服感染BmBDV-ZJ的数字基因表达谱，为排除个体间差异，以10头蚕作为一个样本用于DGE检测。样本中基因的差异表达检测通过严格的运算法则进行，对差异检验的P值（P value）作多重假设检验校正，通过控制FDR（false discovery rate）来决定P值的域值。本研究中，差异表达基因定义为FDR≤0.001且差异倍数在2倍及以上（|log₂ratio|≥1）的基因。采用基因本体论（GO）分类体系确定所有差异表达基因可能的功能。用GO计算P值和bonferoni校正。选用校正P值≤0.05作为基因组显著富集的阈值。WEGO软件用来视化、比较和绘制GO注释结果。利用KEGG数据库进行通路富集分析,进一步确定显著富集代谢途径或信号传导途径，Q值≤0.05的通路指定为DGEs中的显著富集通路。通过qRT-PCR方法对部分差异表达基因进行验证。【结果】感染组和对照组分别得到4 850 663和4 875 307个原始标签,去除低质量标签后，分别得到4 757 934和4 788 406个清洁标签，对应的标签种类数量分别为62 436和63 680种。两个文库间的清洁标签和清洁标签种类的数量在不同拷贝区间分布类似，感染组和对照组样本的测序量分别为3.5 M和3.7 M，测序深度符合试验的要求，两样本的DGE数据是可信的。将这两个DGE数据库的所有清洁标签与家蚕参考基因库进行比对，在对照组与感染组中，分别有36.39%和45.30%的清洁标签可以比对到基因。另有50.02%和43.34%的清洁标签可以比对到家蚕参考基因组，剩余的未知标签分别占清洁标签总数的13.59%和12.35%。共发现了447个差异表达基因，其中306个上调表达，141个下调表达。分别有218、147和179个差异表达基因涉及GO 3个本体中的分子功能、细胞组分和生物过程。利用KEGG公共数据库进行Pathway显著性富集分析，注释到的基因总数为8 473个。447个差异表达基因经鉴定后，其中的330个基因被归类到151个KEGG路径中。差异表达基因显著富集的Pathway（Q值≤0.05）有19个，其中最显著富集的是细胞质中DNA识别通路。挑选了24个差异表达基因进行qRT-PCR验证，其中20个基因的差异表达趋势与DGE的结果一致。其中在DNA识别通路中共检测到9个差异表达基因，BGIBMGA009408-TA、BGIBMGA004913-TA、BGIBMGA011753-TA均为编码RNA聚合酶III的基因，表达量均上调，是对照组的4.3、2.3、3.4倍。【结论】构建了3龄家蚕JS感染BmBDV-ZJ后28 h感染组及对照组幼虫的数字基因表达谱，Pathway显著性富集分析和qRT-PCR验证显示，家蚕感染BmBDV-ZJ后可能通过启动胞质内DNA识别通路来感应入侵病毒的异源DNA成分并迅速启动天然免疫抵御BmBDV-ZJ病毒感染,为研究BmBDV-ZJ侵染家蚕和家蚕抵御BmBDV感染的分子机制打下了基础。

关键词: 家蚕, 数字基因表达谱, 二分浓核病毒, qRT-PCR

Abstract: 【Objective】 The objective of this study is to screen differentially expressed genes in the Bombyx 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 of B. mori resistance against BmBDV-ZJ infection. 【Method】 The differential gene expression profiles in JS B. 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 the P 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 log₂ratio≥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 corrected P 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 a Q 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 of B. 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 of B. 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 (Q value≤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 in B. mori, were all up-regulated in the BmBDV-ZJ infected B. 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 of B. mori resistance against BmBDV-ZJ infection.

Key words: Bombyx mori, digital gene expression, Bombyx mori bidensovirus, quantitative real-time PCR

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): 3453-3464.

GAO Kun, SHANG Meng-ke, QIAN He-ying, QIN Guang-xing, GUO Xi-jie. Digital Gene Expression Analysis of Silkworm Infected by Bombyx mori Bidensovirus Zhenjiang Strain[J]. Scientia Agricultura Sinica, 2016, 49(17): 3453-3464.

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