博士 副研究员 硕士生导师
郭睿，福建农林大学蜂学学院副研究员，研究方向为：蜜蜂对主要病原的胁迫应答机制，蜜蜂主要病原的增殖与侵染机制，非编码RNA介导蜜蜂及其病原互作的分子调控机制，蜜蜂及其病原互作中的表观调控机制，蜜蜂疾病的综合防控技术体系，现为动物科学学院（蜂学学院）蜜蜂保护课题组、蜜蜂-病原互作机制科研创新团队骨干。主持国家自然科学基金青年基金、福建省自然科学基金、福建省教育厅中青年教师教育科研项目、福建农林大学杰出青年学者科研人才项目、农业部授粉昆虫生物学重点实验室开放基金、福建农林大学科技发展专项基金项目等国家级、省部级、市厅级和校级科研项目；参与国家现代农业产业体系建设专项资金项目、国家重点实验室项目、国家自然科学基金面上项目、福建农林大学科技发展专项基金项目和福建农林大学发展基金项目。荣获2017和2018年度福建农林大学青年五四奖章、2013年度博士研究生国家奖学金以及苏州大学2012年度博士优秀奖学金；作为通讯作者或第一作者在《Applied Microbiology and Biotechnology》、《Journal of Invertebrate Pathology》、《Parasitology Research》、《Gene》、《Current Microbiology》、《Insects》、《Apidologie》、《Journal of Apicultural Research》、《微生物学报》、《昆虫学报》和《中国农业科学》等国内外学术刊物上发表论文60余篇；获授权的发明专利3项、新型实用专利2项；1篇学术论文获得第28届世界养蜂大会口头报告资格，4篇学术论文获得第29届世界养蜂大会Poster资格；受邀担任《RNA Biology》、《Current Bioinformatics》和《Journal of Agricultural Science and Technology》等期刊的审稿人。
杜宇， 童新宇， 周丁丁， 陈大福， 熊翠玲， 郑燕珍， 徐国钧， 王海朋， 陈华枝， 郭意龙， 隆琦， 郭睿
【目的】蜜蜂球囊菌（Ascosphaera apis，简称球囊菌）是一种能够侵染中华蜜蜂（Apis cerana cerana，简称中蜂）幼虫的致死性真菌病原。微小RNA（microRNA，miRNA）可通过在转录后水平靶向抑制或降解mRNA而参与宿主与病原互作过程。本研究旨在对球囊菌胁迫的中蜂6日龄幼虫肠道的差异表达miRNA（DEmiRNA）及其靶基因进行深入分析，进而揭示DEmiRNA在蜜蜂响应球囊菌胁迫应答过程中的作用。【方法】利用Illumina MiSeq平台对正常及球囊菌胁迫的中蜂6日龄幼虫肠道（AcCK和AcT）进行测序，通过相关生物信息学软件预测DEmiRNA及其靶基因。通过Blast将靶基因注释到GO和KEGG数据库。利用Cytoscape软件构建DEmiRNA与其靶mRNAs的调控网络。通过Stem-loop RT-PCR和qPCR验证测序数据的可靠性。【结果】本研究共预测出537个miRNA，其长度分布介于16 ~35 nt之间，且不同长度的miRNA首位碱基偏向性差异明显。通过Stem-loop RT-PCR证实了10个novel miRNA的表达。AcCK vs AcT比较组共有54个DEmiRNA，包含31个上调和23个下调miRNA，可分别靶向结合6170 和8199个靶基因。GO分类结果显示上调和下调miRNA的靶基因分别涉及47和47个条目，富集基因数最多的皆为结合、细胞进程和催化活性。KEGG代谢通路（pathway）富集分析结果表明上调和下调miRNA的靶基因分别富集在134和126条pathway，富集基因数最多的均为内吞作用和内质网中的蛋白质加工。调控网络分析结果表明，DEmiRNA及其靶mRNAs形成十分复杂的调控关系；31个DEmiRNA可靶向结合51个与泛素介导的蛋白水解相关的mRNAs，18个DEmiRNA可靶向结合14个与Jak-STAT信号通路相关的mRNAs；miR-1277-x、miR-26-x、miR-27-y、miR-30-x、miR-6052-x等16个miRNA共同参与了上述两条免疫通路的调控。最后，随机挑选3个DEmiRNA进行qPCR验证，结果证明了测序数据的可靠性。【结论】本研究提供了中蜂幼虫肠道在球囊菌胁迫后期的miRNA的表达谱和差异表达信息，揭示了球囊菌与宿主之间在miRNA组学水平存在复杂的互作。miR-6052-x和miR-1277-x作为调控网络的核心可能通过影响细胞凋亡参与宿主的免疫防御，miR-26-x和miR-30-x可能通过调控Jak-STAT信号通路参与宿主的胁迫应答。本研究筛选出的关键DEmiRNA有望作为治疗白垩病的分子靶标。
中华蜜蜂， 幼虫， 球囊菌， microRNA， 靶基因， 调控网络
郭睿， 陈华枝， 童新宇， 熊翠玲， 郑燕珍， 付中民， 解彦玲， 王海朋， 赵红霞， 陈大福
蜜蜂球囊菌Ascosphaera apis特异性侵染蜜蜂幼虫而导致白垩病。本研究利用RNA-seq技术对球囊菌菌丝和孢子进行深度测序，基于高质量的转录组数据对已注释基因进行结构优化，对未注释基因进行预测、鉴定和分析。本研究通过将测序得到的clean reads比对参考基因组和转录本重构，共对101个已注释基因的5'端或3'端进行了延长；利用Cuffcompare软件将重构转录本与参考基因组进行比对，共鉴定出373个新基因，随机挑选10个新基因进行RT-PCR验证，8个能扩增出符合预期的目的片段，表明预测出的多数新基因真实存在。进一步分析结果显示有147个球囊菌新基因可注释到Nr和eggNOG数据库中；85个新基因富集在29个GO条目；66个新基因富集在33条代谢通路；上述结果表明本研究预测出的新基因可能在球囊菌的新陈代谢和细胞生命活动中发挥重要作用。研究结果为球囊菌的基因结构和功能注释信息提供了有益补充，也为新基因的功能研究打下了初步基础。
蜜蜂球囊菌， 转录组， 已注释基因， 结构优化， 新基因
熊翠玲， 杜宇， 王鸿权， 郑燕珍 ， 付中民， 王海朋， 张璐， 陈大福， 郭睿
为探究中华蜜蜂（Apis cerana cerana）与意大利蜜蜂（Apis mellifera ligustica）幼虫的球囊菌抗性差异产生的原因，利用Venn分析、GO分类分析、KEGG代谢通路分析以及Ka/ks分析对二者的转录组进行比较研究。Venn分析结果显示，中蜂与意蜂的同源基因有17 656 个，归属于8 111 个基因家族，二者的特有基因分别有1 158和241 个，分别归属于458和86 个基因家族。GO分类结果显示，中蜂和意蜂的特有基因分别富集在38和28 个GO条目。KEGG代谢通路富集分析结果显示，中蜂和意蜂的特有基因分别富集于96和21 个代谢通路；进一步分析发现中蜂的特有基因富集在内吞作用、溶酶体、泛素介导的蛋白水解和黑化作用等4 个细胞免疫通路，以及MAPK信号通路和Toll-like受体信号通路等2 个体液免疫通路，而意蜂仅有1 个特有基因富集在MAPK信号通路。Ka/Ks分析结果显示受到强烈正向选择、弱正向选择、中性选择和负向选择的单拷贝同源基因分别有37、281、2 630和3 269 个。对受到强烈正向选择的基因进行GO分类和KEGG代谢通路富集分析，GO结果显示中蜂和意蜂的单拷贝同源基因富集的GO 条目类型相同；KEGG结果显示中蜂的单拷贝基因仅富集在氧化磷酸化。上述结果表明免疫相关基因数量的差异是二者的球囊菌抗性差异产生的重要原因之一，中蜂在与球囊菌的协同进化过程中可能通过提高能量利用从而限制球囊菌的增殖。本研究结果可为阐明中蜂及意蜂幼虫的球囊菌抗性差异产生的分子机制提供参考。
中华蜜蜂， 意大利蜜蜂， 幼虫， 比较转录组学， 球囊菌
郭睿， 杜宇， 童新宇， 熊翠玲， 郑燕珍， 徐国钧， 王海朋， 耿四海， 周丁丁， 郭意龙， 吴素珍， 陈大福
【目的】微小RNA（microRNA，miRNA）是一类重要的基因表达调控因子，可影响宿主与病原间的互作过程。蜜蜂球囊菌（Ascosphaera apis）是一种特异性侵染蜜蜂幼虫的致死性真菌病原。本研究旨在对意大利蜜蜂（Apis mellifera ligustica，简称意蜂）幼虫肠道在球囊菌胁迫前期的差异表达miRNA（differentially expressed miRNA，DEmiRNA）及其靶基因进行深入分析，在miRNA组学水平探究意蜂幼虫在球囊菌侵染前期的胁迫应答，并通过构建显著DEmiRNA的调控网络筛选出与宿主应答相关的关键miRNA。【方法】利用small RNA-seq（sRNA-seq）技术对正常及球囊菌侵染的意蜂4日龄幼虫肠道（AmCK和AmT）进行高通量测序，首先对原始数据进行质控和评估，随后将过滤后的数据与西方蜜蜂（Apis mellifera）参考基因组进行比对；将比对上的序列标签（tags）注释到miRBase数据库，得出已知miRNA的表达量；通过TPM（tags per million）算法对各样本中miRNA的表达量进行归一化处理，以|log2 fold change|≥1且P≤0.05作为标准筛选得到显著DEmiRNA；利用TargetFinder 软件预测显著DEmiRNA的靶基因，并对其进行GO和KEGG代谢通路（pathway）富集分析。利用Cytoscape软件对miRNA-mRNA调控网络进行可视化。最后，利用茎环反转录PCR（Stem-loop RT-PCR）和荧光定量PCR（qPCR）验证测序数据的可靠性。【结果】AmCK和AmT样品的测序分别得到13 553 302和10 777 534条原始读段（raw reads），经严格过滤后得到的有效读段（clean reads）数分别为13 186 921和10 480 913条。各样品的生物学重复间的Pearson相关性系数分别在0.9822和0.9508以上。共有10个显著DEmiRNA，包括4个上调miRNA和6个下调miRNA。显著DEmiRNA在AmT的整体表达水平低于AmCK。10个显著DEmiRNA可靶向结合3 788个靶基因，其中上调miRNA的1 240个靶基因可注释到GO数据库中的39个GO条目，主要富集在结合、细胞进程、代谢进程和应激反应等；下调miRNA的749个靶基因可注释到34个GO条目，主要富集在细胞进程、结合、代谢进程和应激反应等。KEGG数据库注释结果显示，上调miRNA和下调miRNA的靶基因分别注释到95和66条代谢通路，富集基因数最多的分别是Wnt信号通路、Hippo信号通路、光传导以及内吞作用、磷脂酰肌醇信号系统、嘌呤代谢。对于上调和下调miRNA，分别有31和52个靶基因注释到内吞作用，15和7个靶基因注释到泛素介导的蛋白水解，11和5个靶基因注释到Jak-STAT信号通路，1和3个靶基因注释到MAPK信号通路。显著DEmiRNA与靶mRNA之间形成复杂的调控网络，7个显著DEmiRNA靶向结合96个与Wnt信号通路相关的mRNA，8个显著DEmiRNA靶向结合55个与内吞作用相关的mRNA。Stem-loop RT-PCR和qPCR结果验证了测序数据的可靠性。【结论】对意蜂幼虫肠道在球囊菌侵染前期的DEmiRNA及其靶基因进行预测和分析，并构建和分析了DEmiRNA-mRNA调控网络，研究结果提供了宿主miRNA的表达谱和差异表达信息，揭示了DEmiRNA通过调控细胞生命活动、新陈代谢以及部分细胞和体液免疫等生物学过程参与宿主的胁迫应答。miR-4331-y、miR-4968-y、miR-8440-y、novel-m0023-5p和novel-m0025-3p共同参与了宿主的Wnt信号通路和内吞作用的调控，可作为白垩病治疗的潜在分子靶标。
意大利蜜蜂， 幼虫肠道， 发育， 差异表达微小RNA， 调控网络
付中民， 陈华枝， 刘思亚， 祝智威， 范小雪， 范元婵， 万洁琦， 张璐， 熊翠玲， 徐国钧， 陈大福， 郭睿
【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。
意大利蜜蜂， 东方蜜蜂微孢子虫， 中肠， 免疫应答， 细胞免疫， 体液免疫
Rui Guo， Simei Wang， Renyu Xue， Guangli Cao， Xiaolong Hu， Moli Huang， Yangqi Zhang， Yahong Lu， Liyuan Zhu， Fei Chen， Zi Liang， Sulan Kuang， Chengliang Gong
Applied Microbiology and Biotechnology，2015，99（12）：5175-5187
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.
Bombyx mori， Cypovirus， RNA-Seq， Gene expression profile
Rui Guo， Guangli Cao， Yahong Lu， Renyu Xue， Dhiraj Kumar， Xiaolong Hu， Chengliang Gong
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.
Transgenic Nosema bombycis， Bombyx mori， pIZT/， V5-His vector， BmN cells， Transfection， Fluorescence
Rui Guo， Guangli Cao， Yuexiong Zhu， Dhiraj Kumar， Renyu Xue， Yahong Lu， Xiaolong Hu， Chengliang Gong
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.
Infection system， Recombinant virus， BmBDV， Bombyx mori
Xiaoli Zhang， Rui Guo， Dhiraj Kumar， Huanyan Ma， Jiabin Liu， Xiaolong Hu， Guangli Cao， Renyu Xue， Chengliang Gong
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.
Bombyx mori， Bm-STAT gene， Expression pattern， Antiviral defense
Rui Guo， Dafu Chen， Cuiling Xiong， Chunsheng Hou， Yanzhen Zheng， Zhongmin Fu， Qingyun Diao， Lu Zhang， Hongquan Wang， Zhixian Hou， Wendong Li， Dhiraj Kumar， Qin Liang
Journal of Invertebrate Pathology，2018，156（1）：1-5
Ascospheara apis is a widespread fungal pathogen that exclusively invades honeybee larvae. Thus far, non-coding RNA in A. apis has not yet been documented. In this study, we sequenced A. apis using strand specific cDNA library construction and Illumina RNA sequencing methods, and identified 379 lncRNAs, including antisense lncRNAs, lincRNAs, intronic lncRNAs and sense lncRNAs. Additionally, these lncRNAs were found to be shorter in length and have fewer exons and transcript isoforms than protein-coding genes, similar to those identified in mammals and plants. Furthermore, the existence of 15 predicted lncRNAs of A. apis was confirmed using RT-PCR and expression levels of 11 were lower than those of adjacent protein-coding genes. Our findings not only enlarge the lncRNA database for fungi, but also lay a foundation for further investigation of potential lncRNAmediated regulation of genes in A. apis.
Long non-coding RNA， Chalkbrood， Ascospheara apis， Honeybee larvae
Rui Guo， Guangli Cao， Renyu Xue， Dhiraj Kumar， Fei Chen， Wei Liu， Yue Jiang， Yahong Lu， Liyuan Zhu， Zi Liang， Sunlan Kuang， Xiaolong Hu， Chengliang Gong
Applied Microbiology and Biotechnology，2018，102（3）：1367-1379
Bombyx mori cypovirus (BmCPV) is one of the major viral pathogen for silkworm, and the genome of BmCPV is composed of 10 dsRNA segments. As construction system of recombinant BmCPV (rBmCPV) is scanty, researchers achieved little progress in studying gene function of BmCPV in recent decades. Here, 10 recombinant plasmids with a full-length cDNA of viral genome segments S1-S10 containing T7 promoter were constructed. After cotransfecting the BmN cells with the mixture of 10 in vitrotranscribed RNAs, pathological changes were observed. Real-time PCR and Western blot showed viral gene vp1 and structural proteins were expressed. It is found the genome of the rBmCPV is composed of 10 dsRNA segments similar to those of wild-type BmCPV. Moreover, viral particles and polyhedron with virions can be generated in the cotransfected cells and the injected silkworm midguts. These findings confirmed the formation of infective rBmCPV. Additionally, we found viable rBmCPV was generated by cotransfecting the mixture of in vitro-transcribed S1-S9 RNAs into the cultured cells, confirming polh was not essential for BmCPV replication. Moreover, an infectious rBmCPV expressing the DsRed protein was constructed based on this system. Further investigation showed S2 and S7 segments are indispensible for viral proliferation. Our findings demonstrated the construction system of rBmCPV can be utilized for exploring viral replication and pathogenesis, and investigated method for constructing rBmCPV will certainly facilitate developing novel biopesticides and expressing exogenous gene in the midgut of silkworm.
Bombyx mori， Cypovirus， Recombinant virus， In vitro-transcribed RNAs， Exogenous gene
Rui Guo， Dafu Chen， Cuiling Xiong， Chunsheng Hou， Yanzhen Zheng， Zhongmin Fu， Qin Liang， Qingyun Diao， Lu Zhang， Hongquan Wang， Zhixian Hou， Dhiraj Kumar
Nosema ceranae is a unicellular fungal parasite of honey bees and causes huge losses for apiculture. Until present, no study on N . ceranae long non-coding RNAs (lncRNAs) was documented. Here, we sequenced purified spores of N . ceranae using strand-specific library construction and high-throughput RNA sequencing technologies. In total, 83 novel lncRNAs were predicted from N . ceranae spore samples, including lncRNAs, long intergenic non-coding RNAs (lincRNAs), and sense lncRNAs. Moreover, these lncRNAs share similar characteristics with those identified in mammals and plants, such as shorter length and fewer exon number and transcript isoforms than protein-coding genes. Finally, the expression of 12 lncRNAs was confirmed with RT-PCR, confirming their true existence. To our knowledge, this is the first evidence of lncRNAs produced by a microsporidia species, offering novel insights into basic biology such as regulation of gene expression of this widespread taxonomic group.
RNA-seq， Long non-coding RNA， Nosema ceranae， Honey bee
Dafu Chen， Huazhi Chen， Yu Du， Zhiwei Zhu， Jie Wang， Sihai Geng， Cuiling Xiong， Yanzhen Zheng， Chunsheng Hou， Qingyun Diao， Rui Guo
Applied Microbiology and Biotechnology，2020，104（1）：257-276
Currently, knowledge of circular RNAs (circRNAs) in insects including honeybee is extremely limited. Here, differential expression profiles and regulatory networks of circRNAs in the midguts of Apis cerana cerana workers were comprehensively investigated using transcriptome sequencing and bioinformatics. In total, 9589 circRNAs (201–800 nt in length) were identified from 8-day-old and 11-day-old workers’ midguts (Ac1 and Ac2); among them, 5916 (61.70%) A. cerana cerana circRNAs showed conservation with our previously indentified circRNAs in Apis mellifera ligucstica workers’ midguts (Xiong et al., Acta Entomologica Sinica 61:1363–1375, 2018). Five circRNAs were confirmed by RT-PCR and Sanger sequencing. Interestingly, novel_circ_003723, novel_circ_002714, novel_circ_002451, and novel_circ_001980 were highly expressed in both Ac1 and Ac2. In addition, the source genes of circRNAs were involved in 34 GO terms including organelle and cellular process and 141 pathways such as endocytosis and Wnt signaling pathway. Moreover, 55 DEcircRNAs including 34 upregulated and 21 downregulated circRNAs were identified in Ac2 compared with Ac1. circRNA-miRNA regulatory networks indicated that 1060 circRNAs can target 74 miRNAs; additionally, the DEcircRNA-miRNA-mRNA networks suggested that 13 downregulated circRNAs can bind to eight miRNAs and 29 miRNA-targeted mRNAs, while 16 upregulated circRNAs can link to 9 miRNAs and 29 miRNA-targeted mRNAs. These results indicated that DEcircRNAs as ceRNAs may play a comprehensive role in the growth, development, and metabolism of the worker’s midgut via regulating source genes and interacting with miRNAs. Notably, eight DEcircRNAs targeting miR-6001-y were likely to be key participants in the midgut development. Our findings not only offer a valuable resource for further studies on A. cerana cerana circRNA and novel insights into understanding the molecular mechanisms underlying the midgut development of eastern honeybee but also provide put
Apis cerana cerana， Honeybee， Midgut， Circular RNA， Competitive endogenous RNA， Regulatory network， Metabolism， Immunity
Rui Guo， Dafu Chen， Huazhi Chen， Zhongmin Fu， Cuiling Xiong， Chunsheng Hou， Yanzhen Zheng， Yilong Guo， Haipeng Wang， Yu Du， Qingyun Diao
Ascosphaera apis is a widespread fungal pathogen of honeybee larvae, which causes heavy losses in apiculture. To date, knowledge about non-coding RNA (ncRNA) including circular RNA (circRNA) in A. apis is lacking. In this study, A. apis mycelia and spores were sequenced using RNA-seq technology. A total of 551 circRNAs were predicted on the basis of bioinformatic analyses, and most of the circRNAs were 200–600 bp in length, which were different from animal and plant circRNAs. In addition, the expression of six circRNAs in A. apis were confirmed using divergent and convergent PCR. Moreover, circRNA-microRNA regulation networks in A. apis were constructed, and further investigation showed that A. apis circRNAs could regulate gene expression by competitively binding miRNAs. GO and KEGG pathway enrichment analyses of the miRNAs target genes of circRNAs demonstrated that these A. apis circRNAs are likely to play key roles in metabolism, environmental response and gene expression.
CircRNA， Ascosphaera apis， Fuangal pathogen， Honeybee larvae
Rui Guo， Dafu Chen， Huazhi Chen， Cuiling Xiong， Yanzhen Zheng， Chunsheng Hou， Yu Du， Sihai Geng， Haipeng Wang， Dingding Zhou， Yilong Guo
Circular RNAs (circRNAs) are newly discovered endogenous non-coding RNAs (ncRNAs) that play key roles in microRNA function and transcriptional regulation. Though a large number of circRNAs had been identifed in animals and plants, however, little is known regarding circRNAs in Nosema ceranae, a widespread fungal parasite of honeybee. In this study, using deep sequencing technology and bioinformatic analysis, we predicted 204 circRNAs from N. ceranae spore samples, including 174 exonic circRNAs and 30 intergenic circRNAs. In addition, the expression of seven N. ceranae circRNAs was confrmed by RT-PCR assay. Furthermore, regulation networks of circRNAs were constructed, and 15 circRNAs were found to act as sponges of the corresponding three miRNAs. GO categorization and pathway enrichment analysis suggested that the circRNAs are likely to play signifcant roles in N. ceranae spore. This is the frst report of circRNAs generated by a microsporidia species. Our results provide novel insights into understanding the basic biology of N. ceranae.
Circular RNA， Fungal parasite， Nosema ceranae， Honeybee
Dafu Chen， Rui Guo， Xijian Xu， Cuiling Xiong， Qin Liang， Yanzhen Zheng， Qun Luo， Zhaonan Zhang， Zhijian Huang， Dhiraj Kumar， Weijun Xi， Xuan Zou， Min Liu
Honeybees are susceptible to a variety of diseases, including chalkbrood, which is capable of causing huge losses of both the number of bees and colony productivity. This research is designed to characterize the transcriptome profiles of Ascosphaera apis-treated and un-treated larval guts of Apis mellifera ligustica in an attempt to unravel the molecular mechanism underlying the immune responses of western honeybee larval guts to mycosis. In this study, 24, 296 and 2157 genes were observed to be differentially expressed in A. apis-treated Apis mellifera (4-, 5- and 6-day-old) compared with un-treated larval guts. Moreover, the expression patterns of differentially expressed genes (DEGs) were examined via trend analysis, and subsequently, gene ontology analysis and KEGG pathway enrichment analysis were conducted for DEGs involved in up- and down-regulated profiles. Immunityrelated pathways were selected for further analysis, and our results demonstrated that a total of 13 and 50 DEGs were annotated in the humoral immune-related and cellular immune-related pathways, respectively. Additionally, we observed that many DEGs up-regulated in treated guts were part of cellular immune pathways, such as the lysosome, ubiquitin mediated proteolysis, and insect hormone biosynthesis pathways and were induced by A. apis invasion. However, more down-regulated DEGs were restrained. Surprisingly, a majority of DEGs within the Toll-like receptor signaling pathway, and the MAPK signaling pathway were up-regulated in treated guts, while all but two genes involved in the NF-κB signaling pathway were down-regulated, which suggested that most genes involved in humoral immune-related pathways were activated in response to the invasive fungal pathogen. This study's findings provide valuable information regarding the investigation of the molecular mechanism of immunity defenses of A. m. ligustica larval guts to infection with A. apis.
Immune response， Apis mellifera ligustica， Larval gut， Ascosphaera apis， RNA-seq， Transcriptome
Dafu Chen， Huazhi Chen， Yu Du， Dingding Zhou， Sihai Geng， Haipeng Wang， Jieqi Wan， Cuiling Xiong， Yanzhen Zheng， Rui Guo
Long non-coding RNAs (lncRNAs) have been proven to play pivotal roles in a wide range of biological processes in animals and plants. Here, we performed whole transcriptome strand-specific RNA sequencing of normal midguts of Apis mellifera ligustica workers (Am7CK, Am10CK) and Nosema ceranae-inoculated midguts (Am7T, Am10T), and 4749 conserved lncRNAs and 1604 novel lncRNAs were identified. These lncRNAs had minimal sequence similarities with other known lncRNAs in other species; however, their structural features were similar to counterparts in mammals and plants, including shorter exon and intron length, lower exon number, and lower expression level, compared with protein-coding transcripts. Further, 111 and 146 N. ceranae-responsive lncRNAs were identified from midguts at 7-days post-inoculation (dpi) and 10 dpi compared with control midguts. Twelve differentially expressed lncRNAs (DElncRNAs) were shared by Am7CK vs. Am7T and Am10CK vs. Am10T comparison groups, while the numbers of unique DElncRNAs were 99 and 134, respectively. Functional annotation and pathway analysis showed that the DElncRNAs may regulate the expression of neighboring genes by acting in cis and trans fashion. Moreover, we discovered 27 lncRNAs harboring eight known miRNA precursors and 513 lncRNAs harboring 2257 novel miRNA precursors. Additionally, hundreds of DElncRNAs and their target miRNAs were found to form complex competitive endogenous RNA (ceRNA) networks, suggesting that these DElncRNAs may act as miRNA sponges. Furthermore, DElncRNA-miRNA-mRNA networks were constructed and investigated, the results demonstrated that a portion of the DElncRNAs were likely to participate in regulating the host material and energy metabolism as well as cellular and humoral immune host responses to N. ceranae invasion. Our findings offer a rich genetic resource for further investigation of the functional roles of lncRNAs involved in the A. m. ligustica response to N. ceranae infection.
Long non-coding RNA， Competitive endogenous RNA， Regulatory network， Honeybee， Nosema ceranae， Stress response， Immune defense
Dafu Chen， Du Yu， Huazhi Chen， Yuanchan Fan， Xiaoxue Fan， Zhiwei Zhu， Jie Wang， Cuiling Xiong， Yanzhen Zheng， Chunsheng Hou， Qingyun Diao， Rui Guo
Here, the expression profiles and differentially expressed miRNAs (DEmiRNAs) in the midguts of Apis cerana cerana workers at 7 d and 10 d post-inoculation (dpi) with N. ceranae were investigated via small RNA sequencing and bioinformatics. Five hundred and twenty nine (529) known miRNAs and 25 novel miRNAs were identified in this study, and the expression of 16 predicted miRNAs was confirmed by Stem-loop RT-PCR. A total of 14 DEmiRNAs were detected in the midgut at 7 dpi, including eight up-regulated and six down-regulated miRNAs, while 12 DEmiRNAs were observed in the midgut at 10 dpi, including nine up-regulated and three downregulated ones. Additionally, five DEmiRNAs were shared, while nine and seven DEmiRNAs were specifically expressed in midguts at 7 dpi and 10 dpi. Gene ontology analysis suggested some DEmiRNAs and corresponding target mRNAs were involved in various functions including immune system processes and response to stimulus. KEGG pathway analysis shed light on the potential functions of some DEmiRNAs in regulating target mRNAs engaged in material and energy metabolisms, cellular immunity and the humoral immune system. Further investigation demonstrated a complex regulation network between DEmiRNAs and their target mRNAs, with miR-598-y, miR-252-y, miR-92-x and miR-3654-y at the center. Our results can facilitate future exploration of the regulatory roles of miRNAs in host responses to N. ceranae, and provide potential candidates for further investigation of the molecular mechanisms underlying eastern honeybeemicrosporidian interactions.
Apis cerana cerana， Midgut， Immune defense， Nosema ceranae， MicroRNA， Target mRNA， Regulatory network
Rui Guo， Dafu Chen， Qingyun Diao， Cuiling Xiong， Yanzhen Zheng， Chunsheng Hou
Journal of Invertebrate Pathology，2019，166（1）：170210-170
Chalkbrood is the most common fungal disease in honeybees. The objective of this study was to reveal immune responses in the Apis cerana cerana larval gut following Ascosphaera apis invasion. Combining a previously assembled transcriptome of A. c. cerana larval gut and the high-throughput sequencing data obtained in this study, 6152 differentially expressed genes (DEGs) were clustered into eight profiles. Trend analysis showed three significant up-regulated profiles (p ≤ 0.05) and three down-regulated profiles. Gene Ontology (GO) term analysis suggested that DEGs within significant up-regulated and down-regulated clusters were enriched in 46 and 38 functional groups, respectively. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis indicated a majority of DEGs were involved in ribosome structure or function, carbon metabolism, biosynthesis of amino acids, and oxidative phosphorylation. In addition, 142 and 14 DEGs were annotated in the cellular immune- and humoral immune-related pathways, respectively. Further investigation indicated that DEGs up-regulated in cellular immune and humoral immune pathways outnumbered those that were downregulated. Moreover, immune responses of A. c. cerana and Apis mellifera ligustica larvae were compared and studied to decipher resistance of eastern honeybee larvae to A. apis. These results demonstrated that a large number of genes involved in immunity-related pathways were activated by A. apis. Our findings provided valuable information for elucidating the molecular mechanisms underlying immune responses of A. c. cerana larvae to A. apis infection and pathogen-host interactions during chalkbrood infection.
Chalkbrood， Apis cerana cerana， Larval gut， Ascosphaera apis， Immune response， Transcriptome
Dafu Chen， Rui Guo， Cuiling Xiong， Yanzhen Zheng， Chunsheng Hou， Zhongmin Fu
Journal Apicultural Research，2018，57（4）：516-521
Chalkbrood is a fungal disease of honey bees and leads to serious apicultural loss worldwide. Ascosphaera apis, the pathogen of chalkbrood, has been found in Apis mellifera, Apis cerana, Xylocopa californica arizonensis larvae as well as adult bumble bees. Here, the fungal pathogen of Apis cerana cerana drone mummies was isolated and identified using morphological and molecular methods. Morphological observation indicated that the sizes of the fruiting bodies, spore balls and ascospores of the fungus isolated from A. c. cerana drone mummies were similar to those of A. apis. Phylogenetic analysis suggested that the fungus is identical to A. apis. Furthermore, the results of a cross infection assay demonstrated that the isolated fungus is capable of infecting eastern and western honey bee larvae and results in chalkbrood. These results confirmed that the isolated fungal pathogen is A. apis. This is the first documentation of morphological and molecular identification of A. apis in A. c. cerana. Our results not only provide novel insight to better understand the pathology of A. apis, but also lay a solid foundation for further investigations of host responses and pathogen-host interactions during chalkbrood of eastern honey bee larvae.
Chalkbrood， Ascosphaera apis， Apis cerana cerana， Drone， Molecular identification