西方蜜蜂caspase-3基因克隆、分析与表达模式
Cloning, analysis and expression of the Apis mellifera caspase-3 gene
张天泽1** 李婧娴1** 王梦怡1 范小雪1, 2, 3 邱剑丰1, 2, 3 骆庆明2, 4 卢兆辉4 陈大福1, 2, 3 严提珍2, 4*** 郭 睿1, 2, 3***
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DOI：10.7679/j.issn.2095-1353.2025.088
作者单位：1. 福建农林大学蜂学与生物医药学院，福州 350002；2. 天然生物毒素国家地方联合工程实验室, 福州 350002； 3. 福建农林大学蜂疗研究所，福州 350002；4. 东莞市妇幼保健院, 东莞 523000
中文关键词：西方蜜蜂；caspase-3基因；细胞凋亡；东方蜜蜂微孢子虫；蜜蜂球囊菌
英文关键词：Apis mellifera; caspase-3 gene; apoptosis; Nosema ceranae; Ascosphaera apis
中文摘要：【目的】 本研究旨在为深入开展西方蜜蜂Apis mellifera caspase-3基因（Am-caspase-3）的功能研究提供参考和依据。【方法】 通过PCR扩增Am-caspase-3的编码序列（Coding sequences，CDS）后进行Sanger测序验证，并利用生物信息学工具预测Am-caspase-3蛋白的理化性质、分子特征及蛋白互作网络。利用MEGA软件进行西方蜜蜂和其他物种caspase-3系统进化分析。采用RT-qPCR检测Am-caspase-3在不同组织和发育阶段，工蜂成虫应答东方蜜蜂微孢子虫Nosema ceranae侵染过程以及工蜂幼虫应答蜜蜂球囊菌Ascosphaera apis侵染过程中的相对表达量。【结果】 Am-caspase-3的CDS长度为1 023 bp，编码340个氨基酸。Am-caspase-3的分子式为C1760H2703N459O539S23，相对分子量为39 kD，不含跨膜结构域和信号肽区，但包含44个磷酸化位点，主要分布于细胞质。Am-caspase-3的二级结构包括184条无规则卷曲，90个α-螺旋，46条延伸链和20个β-转角。在Am-caspase-3中鉴定到1个结构域和3个保守基序；Am-caspase-3与其模版1m72.1.A之间的同源性达73.00%。Am-caspase-3与CytC等12个蛋白构成1个互作网络。西方蜜蜂与东方蜜蜂Apis cerana的caspase-3之间同源性达81.23%，在进化树上聚为一支。Am-caspase-3在西方蜜蜂工蜂不同组织中差异表达，在中肠中的表达量最高且极显著高于其他6个组织（P < 0.01）。Am-caspase-3在3日龄幼虫中的表达量达峰值且极显著高于卵、7-8日龄预蛹及12日龄蛹（P < 0.01）。Am-caspase-3在2、6、15和18日龄成虫体内的表达量显著高于1日龄成虫体内的表达量（P < 0.05）。Am-caspase-3在工蜂成虫应答东方蜜蜂微孢子虫侵染的1、4、7、10和13 d皆显著下调（P < 0.05），Am-caspase-3在工蜂幼虫应答蜜蜂球囊菌侵染的1、2和3 d均显著上调（P < 0.05）。【结论】 成功克隆到Am-caspase-3的CDS；Am-caspase-3是潜在的酸性蛋白、亲水性蛋白和胞内蛋白，与CytC等11个蛋白之间存在互作关系；西方蜜蜂与东方蜜蜂的caspase-3之间同源性最高；Am-caspase-3潜在参与西方蜜蜂工蜂不同组织和虫态的发育过程及宿主应答病原真菌侵染的过程。
英文摘要：
[Aim] To provide a reference and foundation for
functional research on the Apis mellifera caspase-3 gene (Am-caspase-3). [Methods] The CDS of Am-caspase-3 was amplified with RT-PCR and verified
using Sanger sequencing. The physicochemical properties, protein structure and
protein interaction network of the Am-caspase-3 protein were then analyzed with bioinformatics software. A
phylogenetic analysis of caspase-3
was conducted by using MEGA software. RT-qPCR was used to detect the expression
of Am-caspase-3 in different
tissues of adult workers infected with Nosema
ceranae, and in worker larvae infected with Ascosphaera apis. [Results] The CDS of Am-caspase-3 is 1 023 bp in length and encodes 340 amino acids. The molecular formula
of the Am-caspase-3 protein is C1760H2703N459O539S23,
and its relative molecular weight is 39 kD. There are 44 potential
phosphorylation modification sites and no transmembrane structure or signal
peptide region. Am-caspase-3 is mainly distributed in the
cytoplasm. Its secondary structure is
comprised of 184 random coils, 90 α-helices, 46 extended strands and 20
β-turns. One structural domain and ten conserved motifs were identified. There is 73.00% homology between Am-caspase-3 and its template 1m72.1.A. There is an interaction network
among Am-caspase-3 and 12 other proteins such as CytC.
There is 81.23% homology between the caspase-3 of A. mellifera and A. cerana, with the homologous genes of
each species clustering within a single clade on the phylogenetic tree.
Am-caspase-3 was differentially expressed in
different worker tissues. Expression was significantly higher (P <
0.01) in the midgut than in the other 6 tissues tested, and significantly
higher (P < 0.01) in 3-day-old larvae than in eggs, 7-day-old
prepupae, 8-day-old prepupae or 12-day-old pupae. Expression of Am-caspase-3 in 2, 6, 15 and 18-day-old workers was significantly higher (P <
0.05) than in 1-day-old workers. Expression was significantly downregulated (P < 0.05) 1, 4, 7, 10
and 13 days, after infection with Nosema
ceranae. Conversely, expression was significantly upregulated
(P < 0.05) 1, 2, and 3 days, after infection with Ascosphaera apis. [Conclusion] The CDS of Am-caspase-3 was successfully cloned. Am-caspase-3 is a putative acidic,
hydrophilic, intracellular protein that interacts with 11 other proteins
including CytC, Am-caspase-3 is most homologous to Apis
cerana caspase-3. Am-caspase-3 potentially participates in the
developmental processes of different tissues and developmental stages of A.
mellifera workers, as well as in the immune response to infection by
pathogenic fungi.