【目的】通过RNA seq技术对纯培养的蜜蜂球囊菌Ascosphaera apis孢子和球囊菌侵染的蜜蜂幼虫肠道组织进行测序，de novo组装球囊菌的参考转录组，并对其进行功能与代谢通路注释，进而基于该转录组数据开发球囊菌的SSR分子标记。【方法】首先通过差速离心获得活化的球囊菌孢子，配制含1×107孢子/mL的饲料饲喂4, 5和6日龄的意大利蜜蜂Apis mellifera ligustica幼虫和中华蜜蜂Apis cerana cerana幼虫，通过Illumina HiSeqTM 2500平台同时对上述蜜蜂幼虫肠道及纯化球囊菌孢子进行深度测序，原始数据过滤后通过Trinity软件组装得到unigenes，进而通过BLASTX比对NCBI Nr, Swiss-Prot, KOG和KEGG数据库对unigenes进行功能与代谢通路注释。利用MISA软件对所有unigenes进行SSR搜索，并利用Primer Premier 5软件设计SSR特异性引物，通过PCR对不同来源的球囊菌SSR位点进行扩增。【结果】本研究共获得146 135 308条高质量reads，de novo组装得到42 609个unigenes。BLASTX比对结果显示，29 316个unigenes在上述公共数据库中具有功能和代谢通路注释。注释到法夫酵母Xanthophyllomyces dendrorhous上的unigenes最多，达6 050个。KEGG注释结果显示，unigenes可注释到117个代谢通路，其中富集在核糖体（ribosome）上的unigenes数量最多（529）。所有unigenes中共预测到7 968个SSRs，通过PCR开发出5个球囊菌SSR分子标记。【结论】本研究成功组装球囊菌的参考转录组，并进行了功能与代谢通路注释，可为在分子水平深入研究球囊菌提供重要的参考信息。基于该转录组信息开发的5个SSR分子标记可推动菌株鉴定、基因图谱构建及基因定位等研究。

Bombyx mori bidensovirus (BmBDV) was previously termed as Bombyx mori densovirus type 2 and later it was reclassified in the new genus bidensovirus of the new family Bidnaviridae. The genome of BmBDV Zhenjiang isolate (BmBDV-Z) consists of two non-homologous singlestranded linear DNA molecules VD1 and VD2 which are encapsidated into separate virion. To investigate the infectivity of BmBDV DNA, recombinant plasmids pGEM-VD1 inserted with VD1 genome were transfected into the BmN cells of silkworm. Structural proteins of BmBDV were detected with Western blot and immunofluorescence assay, which indicates pGEM-VD1 replicated in the transfected BmN cells and viral proteins were also expressed. Through TEM observation, we identified about 20 nm BmBDV-like viral particles, which confirmed that BmBDV can be generated after transfection. Subsequently, a recombinant baculovirus BmBac-VD1 inserted with VD1 genome was constructed. Results of Western blot and immunofluorescence assay indicated that viral structural proteins of BmBDV were expressed in the BmBac-VD1-infected cells. Baculiform and spherical virions were also observed in infected cells by TEM, and two kinds of virions were separated. However, results of molecular biological detection revealed that infectious sequence from BmBac-VD1 was packaged within spherical virion. Therefore, we suggested that vector inserted with BmBDV genomic DNA showed infectivity, and BmBDV-like viral particles packaging recombinant DNA can be produced in the cultured BmN cells. Outcome of our current research provided not only a new method of infection to explore the gene function of BmBDV in vitro but also a protocol to facilitate development of more effective new-type pesticides.

Nosema bombycis, a microsporidium, is a pathogen of pebrine disease of silkworms, and its genomic DNA sequences had been determined. Thus far, the research of gene functions of microsporidium including N. bombycis cannot be performed with gain/loss of function. In the present study, we targeted to construct transgenic N. bombycis. Therefore, hemocytes of the infected silkworm were transfected with a nontransposon vector pIZT/V5-His vector in vivo, and the blood, in which the hemocyte with green fluorescence could be observed, was added to the cultured BmN cells. Furthermore, normal BmN cells were infected with germinated N. bombycis, and the infected cells were transfected with pIZT/V5-His. Continuous fluorescence observations exposed that there were N. bombycis with green fluorescence in some N. bombycis-infected cells, and the extracted genome from the purified N. bombycis spore was used as templates. PCR amplification was carried out with a pair of primers for specifically amplifying the green fluorescence protein (GFP) gene; a specific product representing the gfp gene could be amplified. Expression of the GFP protein through Western blotting also demonstrated that the gfp gene was perfectly inserted into the genome of N. bombysis. These results illustrated that exogenous gene can be integrated into N. bombycis genome by mediating with a non-transposon vector. Our research not only offers a strategy for research on gene function of N. bombycis but also provides an important reference for constructing genetically modified microsporidium utilized for biocontrol of pests.

Genes in the signal transducer and activator of transcription (STAT) family are vital for activities including gene expression and immune response. To investigate the functions of the silkworm Bombyx mori STAT (Bm-STAT) gene in antiviral immunity, two Bm-STAT gene isoforms, Bm-STAT-L for long form and Bm-STAT-S for short form, were cloned. Sequencing showed that the open reading frames were 2313 bp encoding 770 amino acid residues for Bm-STAT-L and 2202 bp encoding 734 amino acid residues for Bm-STAT-S. The C-terminal 42 amino acid residues of Bm-STAT-L were different from the last 7 amino acid residues of Bm-STAT-S. Immunofluorescence showed that Bm-STAT was primarily distributed in the nucleus. Transcription levels of Bm-STAT in different tissues were determined by quantitative PCR, and the results revealed Bm-STAT was mainly expressed in testes. Western blots showed two bands with molecular weights of 70 kDa and 130 kDa in testes, but no bands were detected in ovaries by using anti-Bm-STAT antibody as the primary antibody. Expression of Bm-STAT in hemolymph at 48 h post infection with B. mori macula-like virus (BmMLV) was slightly enhanced compared with controls, suggesting a weak response induced by infection with BmMLV. Hemocyte immunofluorescence showed that Bm-STAT expression was elevated in B. mori nucleopolyhedrovirus (BmNPV)-infected cells. Moreover, resistance of BmN cells to BmNPV was reduced by downregulation of Bm-STAT expression and increased by upregulation. Resistance of BmN cells to BmCPV was not significantly improved by upregulating Bm-STAT expression. Therefore, we concluded that Bm-STAT is a newly identified insect gene of the STAT family. The JAK-STAT pathway has a more specialized role in antiviral defense in silkworms, but JAK-STAT pathway is not triggered in response to all viruses.

High-throughput paired-end RNA sequencing (RNA-Seq) was performed to investigate the gene expression profile of a susceptible Bombyx mori strain, Lan5, and a resistant B. mori strain, Ou17, which were both orally infected with B. mori cypovirus (BmCPV) in the midgut. There were 330 and 218 up-regulated genes, while there were 147 and 260 down-regulated genes in the Lan5 and Ou17 strains, respectively. Gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment for differentially expressed genes (DEGs) were carried out. Moreover, gene interaction network (STRING) analyses were performed to analyze the relationships among the shared DEGs. Some of these genes were related and formed a large network, in which the genes for B. mori cuticular protein RR-2 motif 123 (BmCPR123) and the gene for B. mori DNA replication licensing factor Mcm2-like (BmMCM2) were key genes among the common up-regulated DEGs, whereas the gene for B. mori heat shock protein 20.1 (Bmhsp20.1) was the central gene among the shared down-regulated DEGs between Lan5 vs Lan5-CPV and Ou17 vs Ou17-CPV. These findings established a comprehensive database of genes that are differentially expressed in response to BmCPV infection between silkworm strains that differed in resistance to BmCPV and implied that these DEGs might be involved in B. mori immune responses against BmCPV infection.