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C_(1428)H_(2168)N_(372)O_(403)S_(13),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。
[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 C_(1428)H_(2168)N_(372)O_(403)S_(13).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.
作者
吴伯文
范小雪
刘彩珍
冯睿蓉
付志英
吴杨
郭睿
WU Bo-Wen;FAN Xiao-Xue;LIU Cai-Zhen;FENG Rui-Rong;FU Zhi-Ying;WU Yang;GUO Rui(Fujian Vocational College of Agriculture,Fuzhou 350000,China;National&Local United Engineering Laboratory of Natural Biotoxin,Fuzhou 350002,China;Apitherapy Research Institute of Fujian Agriculture and Forestry University,Fuzhou 350002,China;College of Bee Science and Biomedicine,Fujian Agriculture and Forestry University,Fuzhou 350002,China)
出处
《应用昆虫学报》
北大核心
2025年第2期371-380,共10页
Chinese Journal of Applied Entomology
基金
国家自然科学基金项目(32372943)
福建省自然科学基金面上项目(2022J01133)
福建省教育厅中青年教师教育科研项目(JAT2312)。
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
Apis mellifera
autophagy
autophagy related gene 5
molecular characteristics
phyletic evolution
expression pattern
