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【目的】筛选家蚕Bombyx mori应对白僵菌Beauveria bassiana侵染的应答基因,以进一步研究家蚕抵御真菌侵染的分子机制。【方法】采用新一代Solexa高通量测序技术对感染白僵菌及未感染白僵菌的对照组家蚕进行了测序分析,筛选差异表达基因;结合生物信息学工具分析差异表达基因的功能注释、分类及涉及的信号通路等;应用荧光定量PCR技术验证10个基因的差异表达。【结果】通过测序和生物信息学分析共获得377个差异表达基因,其中表达上调基因236个,下调基因141个;KEGG通路分析表明,各通路中既有表达上调的基因,也有下调基因;12个上调基因、26个下调基因参与3个显著性富集的KEGG通路,即核糖体、氨酰tRNA生物合成和剪接体通路。定量PCR与测序结果显示,溶菌酶、热激蛋白、谷胱甘肽S-转移酶、肽聚糖识别蛋白等与免疫应激相关的蛋白基因均呈现表达上调。【结论】本研究筛选获得的差异表达基因,特别是上调表达的基因可能与家蚕应对白僵菌侵染的应答机制有关,其中与免疫应激相关的蛋白基因如溶菌酶、热激蛋白、谷胱甘肽S-转移酶、肽聚糖识别蛋白基因等可能直接参与了家蚕对白僵菌的免疫识别和防御,研究结果为从分子水平阐明家蚕抵御真菌侵染的防御机制和白僵菌对家蚕的致病机理提供新的依据。
【Objective】 The objective of this study was to screen the response genes of Bombyx mori in response to Beauveria bassiana infection in silkworm so as to further study the molecular mechanism of silkworm resistance to fungal infection. 【Method】 A new generation of Solexa high-throughput sequencing was used to sequence and analyze the control silkworms infected with Beauveria bassiana and those not infected with Beauveria bassiana. The differentially expressed genes were screened by bioinformatics tools. Functional annotation of differentially expressed genes was analyzed, Classification and signaling pathways involved. Fluorescent quantitative PCR was used to verify the differential expression of 10 genes. 【Result】 A total of 377 differentially expressed genes were obtained by sequencing and bioinformatics analysis. Among them, 236 genes were up-regulated and 141 genes were down-regulated. KEGG pathway analysis showed that both genes were up-regulated and down-regulated in all pathways. Up-regulated genes and 26 down-regulated genes are involved in three significantly enriched KEGG pathways, ribosomal, aminoacyl-tRNA biosynthesis and spliceosome pathways. Quantitative PCR and sequencing results showed that lysozyme, heat shock protein, glutathione S-transferase, peptidoglycan recognition protein and other immune stress-related protein genes were up-regulated. 【Conclusion】 The differentially expressed genes selected in this study, especially the up-regulated genes, may be related to the response mechanism of silkworm to Beauveria bassiana infection. Among them, the protein genes related to immune stress such as lysozyme, heat shock protein, The results indicated that the molecular mechanism of silkworm defense against fungal infection and the effect of Beauveria bassiana on silkworm (Bombyx mori) could be directly involved in the immune recognition and defense of B. bassiana. Provide a new basis for the pathogenesis.