【目的】微小RNA（microRNA，miRNA）是一类在转录后水平对mRNA进行负调控的关键调控因子。本研究旨在通过分析意大利蜜蜂（Apis mellifera ligustica，简称意蜂）幼虫肠道发育过程中差异表达miRNA（differentially expressed miRNA，DEmiRNA）及其调控网络，提供miRNA的表达谱和差异表达信息，揭示DEmiRNA在幼虫肠道发育中的作用。【方法】利用small RNA-seq（sRNA-seq）技术对意蜂4、5和6日龄幼虫肠道样品（Am4、Am5和Am6）进行测序，将质控后的数据与西方蜜蜂（Apis mellifera）参考基因组进行比对，然后将比对上的序列标签（tags）注释到miRBase数据库，并利用TPM（tags per million）算法归一化处理所得miRNA的表达量，再通过相关生物信息学软件对miRNA进行表达量聚类、前体二级结构预测及差异表达分析。利用TargetFinder软件预测DEmiRNA的靶基因，使用Blast软件对靶基因进行GO和KEGG数据库注释，进而通过Cytoscape软件构建miRNA-mRNA的调控网络。采用茎环实时荧光定量PCR（Stem-loop RT-qPCR）验证测序数据的可靠性。【结果】意蜂幼虫肠道样品的测序分别得到10 841 644、14 655 839和9 230 496条有效序列标签；Am4 vs Am5比较组包含16个上调和10个下调miRNA，Am5 vs Am6比较组包含5个上调和7个下调miRNA。其中，novel-m0031-3p为两个比较组所共有，并结合5个与蜕皮激素诱导蛋白相关的靶基因;二者的特有DEmiRNA数分别为25和11个。Am4 vs Am5的26个DEmiRNA结合5 742个靶基因，其中2 725个靶基因可注释到GO数据库中的46个GO term，并主要富集在结合、细胞进程、代谢进程和单组织进程等；Am5 vs Am6的12个DEmiRNA结合3 733个靶基因，且其中2 725个靶基因富集在结合、细胞进程、单组织进程和代谢进程等41个GO term；此外，两个比较组中的DEmiRNA分别有1 046和676个靶基因可注释到116和92条KEGG代谢通路，且Am4 vs Am5比较组的DEmiRNA的靶基因富集在Wnt信号通路、Hippo信号通路、嘌呤代谢和内吞作用等通路上的数量均高于Am5 vs Am6比较组。进一步分析结果显示Am4 vs Am5中的上调和下调miRNA可分别结合611和85个靶基因，其中ame-miR-6052结合的靶基因数最多，可通过结合5个靶基因,参与对细胞色素P450的调控；miR-281-x结合49个靶基因,并间接调控组氨酸代谢、TGF-β信号通路以及Hippo信号通路；Am5 vs Am6中的上调和下调miRNA可分别结合43和431个靶基因，其中miR-iab-4-x结合靶基因数量最多，并广泛参与调控背腹轴的形成、Hippo信号通路、Wnt信号通路、FoxO信号通路、Notch信号通路以及mTOR信号通路等与生长发育相关的通路。调控网络分析结果表明，DEmiRNA与靶基因间形成较为复杂的调控网络，DEmiRNA居于调控网络的中心位置，而mRNA位于调控网络的外周。最后，通过茎环实时荧光定量PCR（Stem-loop RT-qPCR）对随机选取的3个DEmiRNA进行验证，结果证实了测序数据的可靠性。【结论】在全基因组水平对意蜂幼虫肠道的DEmiRNA及其靶基因进行预测和分析，并对DEmiRNA的调控网络进行构建及分析，发现意蜂幼虫通过调节包括ame-miR-6052、miR-iab-4-x、miR-281-x和novel-m0031-3p在内的多个miRNA的表达水平对肠道生长和发育进行调控，研究结果不仅提供了意蜂肠道发育过程的miRNA表达谱及差异表达信息，也为阐明意蜂幼虫肠道的发育机理打下了基础。

【目的】白垩病是困扰养蜂生产的顽疾。目前，尚无利用二代测序技术研究中华蜜蜂（Apis cerana cerana,简称中蜂）幼虫白垩病的报道。本研究利用RNA-seq技术对健康（AcCK）及球囊菌(Ascosphaera apis)胁迫的中蜂4日龄幼虫肠道（AcT）进行深度测序，在转录组水平研究中蜂幼虫在球囊菌胁迫早期的胁迫应答。【方法】通过Illumina HiSeq 2500平台对AcCK和AcT进行双端（PE125）测序，首先对测序数据进行质控和评估，利用edgeR软件进行差异表达基因（DEG）分析，进而对DEGs进行GO富集分析及KEGG代谢通路富集分析，最后，利用实时荧光定量PCR（qRT-PCR）验证测序数据的可靠性。【结果】 AcCK和AcT 的转录组测序共得到188 457 338条原始读段（raw reads）, 经过滤得到182 088 448条有效读段（clean reads），两端Q20与Q30均在97.96%和94.97%以上，说明测序数据质量良好；主成分分析（PCA）结果显示第一与第二主成分可分别解释基因表达总体方差的75.8%和10.7%；DEG分析结果显示上调基因与下调基因的数量分别为344和239个；GO富集分析结果显示DEGs共富集在36个GO分类（term）上，其中基因富集数最多的细胞（106 unigenes）、细胞组件（106 unigenes）和代谢进程（104 unigenes）；KEGG代谢通路富集分析结果显示上调与下调基因分别富集在72个和45个代谢通路上，其中上调基因富集数最多的是核糖体（72 unigenes）、碳代谢（16 unigenes）和糖酵解（14 unigenes），而下调基因富集数最多的是碳代谢（9 unigenes）、二羧酸代谢（8 unigenes）和氨基酸生物合成（7 unigenes），进一步分析表明中蜂幼虫肠道的部分细胞免疫对球囊菌的胁迫产生应答，而体液免疫不产生应答，宿主的代谢相关基因受到球囊菌的显著抑制。【结论】揭示了中蜂幼虫在球囊菌入侵早期的胁迫应答，为深入解析中蜂幼虫的胁迫应答机制提供了重要信息，也为在分子水平研究关键应答基因打下了基础。

【目的】通过对意大利蜜蜂（Apis mellifera ligustica，简称意蜂）工蜂中肠响应东方蜜蜂微孢子虫（Nosema ceranae）胁迫的差异表达基因（differentially expressed gene，DEG）及免疫通路进行深入细致的分析，揭示宿主的细胞和体液免疫应答，为关键免疫应答基因的筛选及功能研究打下基础。【方法】基于前期获得的正常及东方蜜蜂微孢子虫胁迫的意蜂7日龄和10日龄工蜂中肠（Am7CK、Am7T、Am10CK、Am10T）转录组数据，按照FDR≤1，P≤0.05和|log2 fold change|≥1的标准筛选出各组的DEG，利用相关生物信息学软件对DEG进行Pearson相关性分析、Venn分析、GO分类和KEGG代谢通路富集分析，进而对免疫通路富集基因进行统计和分析，利用实时荧光定量PCR（real-time quantitative PCR，RT-qPCR）验证转录组数据的可靠性。【结果】 差异分析结果显示，Am7CK vs Am7T比较组包含472个上调基因和385个下调基因；Am10CK vs Am10T比较组包含611个上调基因和360个下调基因。Venn分析结果显示，两个比较组特有的DEG分别为739和853个，共有的DEG为118个。GO分类结果显示，Am7CK vs Am7T比较组中上调和下调基因分别涉及23和29个功能条目，其中富集上调基因数最多的前5位分别为结合、催化活性、代谢进程、细胞进程和单组织进程；富集下调基因数最多的前5位分别为代谢进程、单组织进程、催化活性、细胞进程和结合。Am10CK vs Am10T比较组中上调和下调基因分别涉及36和26个功能条目，其中富集上调基因数最多的前5位分别为单组织进程、结合、细胞进程、催化活性和代谢活性；富集下调基因数最多的前5位分别为结合、细胞进程、催化活性、代谢进程和单组织进程。KEGG代谢通路富集分析结果显示，Am7CK vs Am7T比较组中上调和下调基因分别富集在38和33条代谢通路，富集上调基因数最多的前5条分别为胆汁分泌、内质网蛋白加工、泛素介导的蛋白水解、PI3K-Akt信号通路和神经营养因子信号通路；富集下调基因数最多的前5条分别为胞质DNA传感通路、嘌呤代谢、嘧啶代谢、RNA聚合酶和核糖体；涉及泛素介导的蛋白水解等3条细胞免疫通路，以及PI3K-Akt信号通路等7条体液免疫通路。Am10CK vs Am10T比较组中上调和下调基因分别富集在54和43条代谢通路，富集上调基因数最多的前5条分别为Hippo信号通路、药物代谢-细胞色素P450、P450对外源物质的代谢、泛素介导的蛋白水解和鞘脂类代谢；富集下调基因数最多的前5条分别为mRNA监测、鞘脂类信号通路、果糖和甘露糖代谢、半乳糖代谢和鞘脂类代谢；涉及泛素介导的蛋白水解等7条细胞免疫通路，以及NF-κB信号通路等2条体液免疫通路。RT-qPCR验证结果显示6个随机挑选的DEG的表达量变化趋势与测序结果一致，证实了本研究中测序数据的可靠性。进一步分析发现意蜂7日龄和10日龄工蜂中肠的NF-κB信号通路均被东方蜜蜂微孢子虫激活，随即启动了3种抗菌肽apidaecin、defensin-1和hymenoptaecin的合成，体现了它们在宿主抵御东方蜜蜂微孢子虫入侵中的重要性。【结论】在转录组水平解析了意蜂工蜂中肠对东方蜜蜂微孢子虫的免疫应答，揭示宿主在胁迫前期同时作出细胞和体液免疫应答，前者可能在抵御病原入侵方面发挥主要作用；宿主的细胞免疫在胁迫后期持续增强，但体液免疫较大程度地减弱；泛素介导的蛋白水解通路及富集DEG，NF-κB信号通路及富集DEG，以及apidaecin、defensin-1和hymenoptaecin编码基因可能在意蜂工蜂对东方蜜蜂微孢子虫的免疫应答中起到关键作用。

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.

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.