西方蜜蜂ATG5基因的克隆、分析及时空表达谱测定
Cloning, analysis, and expression, of the Apis mellifera ATG5 gene
吴伯文1** 范小雪2, 3, 4** 刘彩珍1 冯睿蓉1 付志英1 吴 杨1 郭睿 2, 3, 4***
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DOI:10.7679/j.issn.2095-1353.2025.035
作者单位:1. 福建农业职业技术学院,福州 350000;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. 福建农林大学蜂学与生物医药学院,福州 350002
中文关键词:西方蜜蜂;自噬;自噬相关基因5;分子特征;系统进化;表达谱
英文关键词:Apis mellifera; autophagy; autophagy related gene 5; molecular characteristics; phyletic evolution; expression pattern
中文摘要:
【目的】 对西方蜜蜂Apis mellifera自噬相关基因5(Autophagy related gene 5,AmATG5)进行分子克隆和生物信息学分析,并检测AmATG5在工蜂7个组织和7个发育时间的表达谱,为进一步探究AmATG5的功能提供详实的参考依据。【方法】 通过PCR扩增AmATG5的编码序列(Coding sequence,CDS)并进行Sanger测序验证。使用相关软件预测和分析AmATG5蛋白的理化性质、分子特征、保守基序、高级结构和蛋白互作网络。通过核质分离与RT-qPCR验证AmATG5的亚细胞定位。采用RT-qPCR检测AmATG5的相对表达水平。【结果】 成功克隆到AmATG5的CDS;AmATG5含265个氨基酸,相对分子量约为31.41 kD,分子式为C1428H2168N372O403S13,含31个磷酸化位点和7个保守基序,不含跨膜结构域和信号肽;AmATG5 mRNA主要分布于细胞质;AmATG5与小蜜蜂Apis florea的ATG5在进化树上聚为一支;AmATG5包含110个无规则卷曲,93个α-螺旋,48条延伸链和14个β-转角;AmATG5与自噬相关蛋白1和自噬相关蛋白6等9个蛋白构成1个互作网络;AmATG5在工蜂的毒腺、中肠、咽下腺、脂肪体、脑、触角和表皮7个组织中差异表达,在脑中的表达量最高,而在表皮中的表达量最低;AmATG5的表达量在卵、1、3和5日龄幼虫阶段持续下降,在8日龄预蛹、11和16日龄蛹阶段持续上升,至16日龄蛹时达到最高。【结论】 AmATG5为疏水性蛋白和胞内蛋白,通过与ATG6等9个蛋白潜在互作发挥作用;AmATG5在西方蜜蜂工蜂的不同组织和不同发育阶段动态差异表达,在脑和16日龄蛹中特异性高表达。
英文摘要:
[Aim] To successfully clone, and then determine the
function and expression of the autophagy related gene 5 (AmATG5)
in different tissues and developmental
stages of Apis mellifera,
thereby providing a basis for further investigation of the function of this
gene. [Methods] The CDS of AmATG5 was amplified by PCR followed by
Sanger sequencing. Relevant software was used to predict and analyze its
physicochemical properties, molecular characteristics and protein interaction
network. RT-qPCR was used to ascertain the relative expression of AmATG5
in seven diverse tissue types and seven developmental stages. [Results] The CDS of AmATG5 was
successfully cloned. AmATG5 contains 265 amino acids, has a molecular weight of
approximately 31.41 kD, and a molecular formula of C1428H2168N372O403S13.
AmATG5 includes 31 phosphorylation modification sites and 7 conserved motifs,
but there is no transmembrane structure and signal peptide. AmATG5 mRNA was mainly distributed in
the cytoplasm. AmATG5 and ATG5 in Apis florea clustered into a single
phylogenetic clade. AmATG5 contains 110 random coils, 93 alpha helices, 48
extended strands, and 14 beta turns. AmATG5 has an interaction network with 9
other proteins, such as ATG1 and ATG6. AmATG5
was differentially expressed in 7 different tissues of A. mellifera workers, including the venom gland, midgut, hypopharyngeal gland, fat body,
brain, antenna and cuticle. Its expression level was highest in the brain and
lowest in the cuticle. AmATG5 was continuously down-regulated in
eggs, 1-day-old larvae, 3-day-old larvae and 5-day-old larvae, but continuously
up-regulated in 8-day-old prepupae, 11-day-old pupae and 16-day-old pupae. Its
expression peaked in 16-day-old pupae. [Conclusion] AmATG5
is a hydrophobic, intracellular protein that potentially functions by
interacting with nine other proteins, including ATG6. Its expression varies in
different tissues and developmental stages of A. mellifera workers, and
it appears to be most highly expressed in the brain and in 16-day-old pupae.