Isolation of AccGalectin1 from Apis cerana cerana and its functions in development and adverse stress response

The galectins are a family of lectins that play important roles in development, immunity, and the regulation of cellular responses. Much research has focused on the functions of galectins in mammals, though less in insects. Here, we identified the AccGalectin1 gene in Apis cerana cerana for the first time and explored its functions. The open reading frame of AccGalectin1 is 1449 base pairs and encodes a 482-amino-acid protein. AccGalectin1 expression was high during the transition between developmental stages and was high in the head, thorax, and epidermis compared with its levels in other tissues. In addition, the expression of AccGalectin1 was induced by several adverse stresses, including both abiotic and biotic stresses. A disk diffusion assay of recombinant AccGalectin1 protein revealed possible roles in protecting cells from oxidative stress. Furthermore, the expression levels of multiple oxidative genes (AccCAT, AccTpx1, AccTrx2, etc) were increased after AccGalectin1 was knocked down in Apis cerana cerana using RNA interference. We also observed that the malondialdehyde content in the AccGalectin1-silenced bees was higher than that in the control bees, while the antioxidant enzymatic activities of superoxide dismutase and peroxidase were lower. Considering these results, we suggest that AccGalectin1 may be indispensable for protecting honeybees from biotic and abiotic damage by participating in the oxidative resistance response and the immune response. These results may provide insight into the precise functions of galectins in mammals and other insects.     

Role of c-Jun NH2-terminal kinase-mediated mitogen-activated protein kinase pathway in response to pesticides in Apis cerana cerana

The mitogen-activated protein kinase (MAPK) cascade pathway plays an important role in regulating stress responses. The function of the c-Jun NH₂-terminal kinase (JNK), a component of the MAPK cascade pathway, in Apis cerana cerana (Acc) remains unclear. Here, JNK was isolated and identified from Acc. Bioinformatics analyses revealed there is a typical serine/threonine protein kinase catalytic domain in the AccJNK protein. An expression profile analysis showed that AccJNK was significantly induced by pesticide treatments. To further explore the functional mechanisms of AccJNK, a yeast 2-hybrid screen was performed, activator protein-1 (AP-1) was screened as the interaction partner of AccJNK, and the interaction relationship was further verified by pull-down assay. Quantitative real-time polymerase chain reaction showed the expression pattern of AccAP-1 was similar to that of AccJNK. After a knockdown of AccJNK or AccAP-1 by RNA interference, the survival rate of Acc after pesticide treatments increased. Additionally, the expression levels of antioxidant-related genes and the activities of antioxidant enzymes increased, suggesting that the knockdown of AccJNK or AccAP-1 increased the antioxidant capacity of bees. Our study revealed that the JNK-mediated MAPK pathway responds to pesticide stress by altering the antioxidant capacity of Acc.     

中华蜜蜂的欧洲幼虫腐臭病病原研究

采用中华蜜蜂Apis cerana cerana F.临床自然发病的欧洲幼虫腐臭病(European Foulbrood, EFB)幼虫,对其病原体进行了分离鉴定。结果表明：中华蜜蜂EFB的病原系蜂房蜜蜂球菌Melissococcus pluton。该菌为革兰氏阳性的兼性厌氧菌,形态学及染色特性、致病性试验、血清学试验和细菌DNA G+C mol%试验均证明中华蜜蜂和西方蜜蜂的EFB病原属于同属的蜂房蜜蜂球菌。生理生化特性结果与国外的早期研究结果相近似。该试验为不同地理位置、不同种寄主间蜂房蜜蜂球菌的遗传差异的研究,以及中华蜜蜂EFB病的防治学研究打下基础。     

澜沧江流域北部中华蜜蜂食源和营养生态位随海拔梯度的变化特征

为了解澜沧江流域北部中华蜜蜂(Apis cerana cerana)的分布,探究中华蜜蜂的食源和营养生态位沿海拔梯度的变化特征,调查了澜沧江流域北部各海拔区域中华蜜蜂的种群分布,运用蜂蜜孢粉学(melissopalynology)分析了各海拔区域中华蜜蜂蜂蜜中花粉的组成特征和变化规律,并综合分析了海拔、中华蜜蜂营养生态位和蜂蜜中花粉的相关性,探讨了自然环境与中华蜜蜂的分布,海拔梯度与蜜粉源植物、中华蜜蜂的食源和营养生态位的关系。结果表明:澜沧江流域北部中华蜜蜂的食源种类丰富,中华蜜蜂种群主要分布于2200—2800 m海拔区域。不同海拔区域中华蜜蜂采食花粉的种类和数量不同,中华蜜蜂种群分布多的海拔区域,蜂蜜中花粉的种类较多,但花粉数量相对少。随着海拔梯度升高,中华蜜蜂蜂蜜中花粉的数量表现为先降后增,而花粉的种类则表现为先增后降。不同海拔区域的中华蜜蜂营养生态位存在差异,推测各海拔区域蜜粉源植物分布、中华蜜蜂种内和种间授粉昆虫及食草动物等竞争因素不同,但各海拔梯度间的变化差异不显著。海拔与中华蜜蜂营养生态位呈正相关(r=0.051),相关性不显著;海拔与蜂蜜中花粉数量呈正相关(r=0.047),与蜂蜜中花粉种类呈正相关(r=0.144),相关性都不显著;中华蜜蜂营养生态位与蜂蜜中花粉的种类呈负相关(r=-0.305),相关性显著(P!0.05);与花粉的数量呈负相关(r=-0.064),相关性不显著。蜂蜜中花粉的数量与种类呈正相关(r=-0.303),且相关性显著(P!0.05)。     

A novel 1-Cys thioredoxin peroxidase gene in Apis cerana cerana: characterization of AccTpx4 and its role in oxidative stresses

Thioredoxin peroxidase (Tpx), also named peroxiredoxin (Prx), is an important peroxidase that can protect organisms against stressful environments. AccTpx4, a 1-Cys thioredoxin peroxidase gene from the Chinese honey bee Apis cerana cerana, was cloned and characterized. The AccTpx4 gene encodes a protein that is predicted to contain the conserved PVCTTE motif from 1-Cys peroxiredoxin. Quantitative real-time PCR (Q-PCR) and Western blotting revealed that AccTpx4 was induced by various oxidative stresses, such as cold, heat, insecticides, H₂O₂, and HgCl₂. The in vivo peroxidase activity assay showed that recombinant AccTpx4 protein could efficiently degrade H₂O₂ in the presence of DL-dithiothreitol (DTT). In addition, disc fusion assays revealed that AccTpx4 could function to protect cells against oxidative stresses. These results indicate that AccTpx4 plays an important role in oxidative stress responses and may contribute to the conservation of honeybees.     

中华蜜蜂6日龄幼虫响应蜜蜂球囊菌侵染的长链非编码RNA应答研究

【目的】本研究旨在探究长链非编码RNA (long non-coding RNA, lncRNA)在中华蜜蜂(Apis cerana cerana,简称中蜂)6日龄幼虫应答蜜蜂球囊菌(Ascosphaera apis,简称球囊菌)侵染过程中的差异表达谱及调控作用。【方法】利用链特异性cDNA建库的RNA-seq技术对未被侵染及球囊菌侵染的中蜂6日龄幼虫肠道(AcCK和AcT)进行深度测序。通过相关生物信息学软件分析lncRNA的结构特征和表达谱。筛选并分析差异表达lncRNA (differentially expressed lncRNA,DElncRNA)的顺式(cis)作用及竞争性内源RNA (competing endogenous RNA, ceRNA)调控网络。采用RT-qPCR验证测序数据及DElncRNA差异变化趋势的可靠性。【结果】AcCK和AcT共预测出642个已知lncRNA和487个新lncRNA。与蛋白编码基因相比,上述中蜂lncRNA外显子数更少、长度更短且表达量更低。43个antisenselncRNA与40个正义链mRNA之间互补配对。AcCK和AcT比...     

Characterization of the CDK5 gene in <Emphasis Type="Italic">Apis cerana cerana</Emphasis> (<Emphasis Type="Italic">AccCDK5</Emphasis>) and a preliminary identification of its activator gene, <Emphasis Type="Italic">AccCDK5r1</Emphasis>

Cyclin-dependent kinase 5 (CDK5) is an unusual CDK whose function has been implicated in protecting the central nervous system (CNS) from oxidative damage. However, there have been few studies of CDK5 in insects. In this study, we identified the AccCDK5 gene from Apis cerana cerana and investigated its role in oxidation resistance. We found that AccCDK5 is highly conserved across species and contains conserved features of the CDK5 family. The results of qPCR analysis indicated that AccCDK5 is highly expressed during the larval and pupal stages and in the adult head and muscle. We further observed that AccCDK5 is induced by several environmental oxidative stresses. Moreover, the overexpression of the AccCDK5 protein in E. coli enhances the resistance of the bacteria to oxidative stress. The activation of CDK5 requires binding to its activator. Therefore, we also identified and cloned cyclin-dependent kinase 5 regulatory subunit 1, which we named AccCDK5r1, from Apis cerana cerana. AccCDK5r1 contains a conserved cell localization targeting domain as well as binding and activation sites for CDK5. Yeast two-hybrid analysis demonstrated the interaction between AccCDK5 and AccCDK5r1. The expression patterns of the two genes were similar after stress treatment. Collectively, these results suggest that AccCDK5 plays a pivotal role in the response to oxidative stresses and that AccCDK5r1 is a potential activator of AccCDK5.     

中华蜜蜂和意大利蜜蜂蜂毒前溶血肽原蛋白cDNA的克隆与序列分析

分别从中华蜜蜂Apis cerana cerana和意大利蜜蜂Apis mellifera工蜂毒腺中抽提总RNA,通过RT-PCR方法扩增,各得到了蜂毒前溶血肽原蛋白的cDNA,再将扩增产物克隆到pGEM-Teasy载体上,进行测序和序列分析。结果表明,所扩增到的这两个片段长度均为213 bp,均为编码蜂毒前溶血肽原的cDNA,并分别推导出两者所编码的氨基酸序列。经序列比较,中华蜜蜂前溶血肽原与意大利蜜蜂、印度蜜蜂Apis cerana indica前溶血肽原的同源性都为97%。所报道的中华蜜蜂蜂毒前溶血肽原的核苷酸序列的GenBank登录号为AF487907。     

华东地区中华蜜蜂六地理种群的遗传多样性及遗传分化

目的】利用23对微卫星标记对来自于南昌、黄山、桐庐、费县、宜兴、武夷山6个华东地区的中华蜜蜂Apis cerana cerana种群进行遗传多样性及遗传分化分析。【方法】通过计算多态信息含量、平均杂合度、等位基因数、遗传距离、基因流、F 统计量等参数, 评估各中蜂种群遗传多样性和各种群间遗传分化。【结果】各座位的等位基因数为5（A014）至30（AP043）。所有种群均显示较高水平的期望杂合度, 其中, 武夷山中蜂最低, 为0.4280; 南昌中蜂最高, 为0.6329。各中蜂种群间存在极显著的遗传分化, 平均分化系数(Fst)为0.344。基于Nei氏遗传距离运用NJ聚类法将6个中蜂种群划分为3类。【结论】华东6个中蜂种群的遗传多样性较高, 遗传分化显著; 分析遗传分化与地理距离的关系发现, 华东6个中蜂种群间的遗传分化与地理距离不存在显著相关。     

中华蜜蜂化学感受蛋白基因Acer-CSP1克隆与表达特征分析

化学感受蛋白(chemosensory proteins, CSPs)是昆虫化学感受系统中重要的组成部分之一。本研究克隆了中华蜜蜂Apis cerana cerana化学感受蛋白基因Acer-CSP1, 其核苷酸全长351 bp （GenBank登录号为FJ157352）, 编码116个氨基酸残基, 预测蛋白分子量为13.85 kD, 等电点为4.89, 且含有4个保守的半胱氨酸残基, 均符合昆虫CSPs的一般特征, 且与意蜂CSP1基因具有99.1%的相似性, 与其他昆虫也有45.3%~68.0%的相似性。利用2^-ΔΔCt法及绝对定量法的real-time PCR技术对Acer-CSP1在中蜂不同器官表达特征进行了研究, 得出的一致结论为Acer-CSP1显著水平地高丰度表达于中华蜜蜂触角, 其次大量表达于头部。由于触角为中华蜜蜂最主要的嗅觉器官, 而头部则具有发达的感觉神经系统和味觉系统, 这也提示Acer-CSP1极有可能参与中华蜜蜂的嗅觉以及其他化学感受功能。     

Molecular characterization and tissue localization of sensory neuron membrane protein from Chinese honey bee, Apis cerana cerana (Hymenoptera: Apidae)

Sensory neuron membrane protein (SNMP) is an olfactory receptor with photoaffinity analogs, capable of binding the pheromone membrane protein receptor deduced from receptor membrane protein with the pheromone–pheromone binding protein complex. However, this hypothesis has not yet been experimentally verified. In this experiment, the cDNA sequence encoding an open reading frame (ORF) of the SNMP gene AccSNMP1 (GenBank, KC012595) was cloned from Chinese honey bee, Apis cerana cerana Fabricius. Results from sequence analysis showed that this gene is 1,563 bp long, and that the ORF encodes 520 amino acids with a predicted molecular weight of 58.02 kDa, and has a theoretical isoelectric point of 5.83. Furthermore, there are two putative transmembrane domains. Multiple sequence alignment indicated that the AccSNMP1 gene from A. cerana cerana had different degrees of identity with the corresponding genes in nineteen other insects at the amino acid level. Phylogenetic analysis of the aligned sequences showed that A. cerana cerana is closely related to Apis mellifera Linnaeus and Bombus impatiens Cresson. Its distribution in tissues, as quantified using real-time RT-PCR, indicated that AccSNMP1 is highly expressed in the antennae and legs of A. cerana cerana, and there was a significant difference (p < 0.05) in gene expression between those tissues and tissues in the thorax, abdomen, snout, and head (not including antennae). Western blotting also confirmed the existence in the antennae of AccSNMP1 with an M _W of 58.0 kDa, which is the same as the expected value of 58.02 kDa. An immunohistochemistry study showed that AccSNMP1 is expressed in the trichoid sensilla of A. cerana cerana antenna. Therefore, the results of this study provide the basis for further studies of the function of SNMP from A. cerana cerana.