意大利蜜蜂piR-ame-1186994的靶基因与功能分析  

Target gene and functional analysis of piR-ame-1186994 in Apis mellifera ligustica(Hymenoptera:Apidae)

作　　者：张艺琼 那志豪 李琪明 王梦怡 李婧娴 代梦远 邱剑丰 张荣华 卢兆辉 陈大福[1,2,3] 严提珍 郭睿 ZHANG Yi-Qiong;NA Zhi-Hao;LI Qi-Ming;WANG Meng-Yi;LI Jing-Xian;DAI Meng-Yuan;QIU Jian-Feng;ZHANG Rong-Hua;LU Zhao-Hui;CHEN Da-Fu;YAN Ti-Zhen;GUO Rui(College of Bee Science and Biomedicine,Fujian Agriculture and Forestry University,Fuzhou 350002,China;National&Local United Engineering Laboratory of Natural Biotoxin,Fuzhou 350002,China;Apitherapy Research Institute of Fujian Agriculture and Forestry University,Fuzhou 350002,China;Dongguan Maternal and Children Health Hospital,Dongguan 523000,China)

机构地区：[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

出　　处：《昆虫学报》2025年第3期260-270,共11页Acta Entomologica Sinica

基　　金：国家自然科学基金面上项目(32372943,32172792);国家现代农业产业技术体系专项资金(CARS-44-KXJ7);福建省自然科学基金面上项目(2022J01131334);福建农林大学硕士生导师团队项目(郭睿);福建农林大学科技创新专项基金(KFb22060XA);福建省大学生创新创业训练计划项目(S202410389057,S202410389061)。

摘　　要：【目的】本研究旨在明确意大利蜜蜂Apis mellifera ligustica piR-ame-1186994的调控功能,为进一步探究piR-ame-1186994的调控作用机制提供科学依据。【方法】通过Stem-loop RT-PCR和Sanger测序分别验证piR-ame-1186994在意大利蜜蜂6日龄工蜂成虫中肠、12日龄雄蜂成虫精巢和7日龄蜂王成虫卵巢中的表达和序列真实性。使用相关软件预测piR-ame-1186994的靶mRNA并进行GO和KEGG数据库注释,进而构建发育信号通路、能量代谢通路及细胞和体液免疫通路相关调控网络。饲喂刚出房工蜂成虫piR-ame-1186994的模拟物(mimic)和mimic-NC(阴性对照组),利用RT-qPCR检测工蜂成虫中肠中的piR-ame-1186994及其关键2个关键靶基因(YAP 1和PLD 2)的相对表达量。【结果】在意大利蜜蜂6日龄工蜂成虫中肠、12日龄雄蜂成虫精巢和7日龄蜂王成虫卵巢中均扩增出piR-ame-1186994的特异性片段。piR-ame-1186994共靶向1097条mRNA,可注释到新陈代谢过程、结合和细胞等30条GO条目以及Wnt信号通路、内吞作用和催产素信号通路等182条KEGG通路。其中,分别有36和16条靶mRNA涉及5条发育相关信号通路(mTOR,Wnt,Hippo,AMPK和Notch信号通路)和4条能量代谢相关通路(氨基糖和核苷酸糖代谢、果糖和甘露糖代谢、糖酵解/糖异生及磷酸戊糖通路)。此外,分别有29和8条靶mRNA涉及4条细胞免疫通路(溶酶体、内吞作用、吞噬体和泛素介导的蛋白水解)和3条体液免疫通路(PI3K-Akt,MAPK和FoxO信号通路)。相较于阴性对照组mimic-NC,mimic-piR组中piR-ame-1186994的表达量在1,3和5日龄工蜂中肠中显著上调,在2和4日龄工蜂中肠中极显著上调;靶基因YAP 1的表达量在1,3,4和5日龄工蜂中肠中极显著下调,在2日龄工蜂中肠中显著下调;靶基因PLD 2的表达量在2,3和5日龄工蜂中肠中显著下调,在4日龄的工蜂中肠中极显著下调。【结论】piR-ame-1186994在意大利蜜蜂工蜂中肠、雄蜂精巢和蜂王卵巢�【Aim】The objective of this study is to elucidate the regulatory function of piR-ame-1186994 of Apis mellifera ligustica,so as to offer a scientific basis for further investigation of the underlying regulatory mechanism of piR-ame-1186994.【Methods】The expression and sequence authenticity of piR-ame-1186994 in the 6-day-old adult worker’s midgut,12-day-old adult drone’s testis and 7-day-old adult queen’s ovary of A.m.ligustica were verified by Stem-loop RT-PCR and Sanger sequencing,respectively.Relevant software was utilized to predict the target mRNAs of piR-ame-1186994 followed by GO and KEGG database annotation.Regulatory networks related to developmental signaling pathways,energy metabolism pathways and cellular and humoral immune pathways were further constructed.Newly emerged adult workers were fed with mimic and mimic-NC(negative control)of piR-ame-1186994,followed by the detection of the relative expression levels of piR-ame-1186994 and its key target genes(YAP 1 and PLD 2)in the midguts of adult workers using RT-qPCR.【Results】The specific fragment of piR-ame-1186994 was amplified from the 6-day-old adult worker’s midgut,12-day-old adult drone’s testis and 7-day-old adult queen’s ovary of A.m.ligustica.piR-ame-1186994 targeted 1097 mRNAs,which could be annotated to 30 GO terms involved in metabolic process,binding,cell,etc.,and 182 KEGG pathways including Wnt signaling pathway,endocytosis and oxytocin signaling pathway.Thirty-six and 16 target mRNAs were respectively involved in five developmental-related signaling pathways(mTOR,Wnt,Hippo,AMPK and Notch signaling pathways)and four pathways related to energy metabolism(amino sugar and nucleotide sugar metabolism,fructose and mannose metabolism,glycolysis/gluconeogenesis,and pentose phosphate pathway),respectively.Additionally,29 and eight target mRNAs were engaged in four cellular immune pathways(lysosome,endocytosis,phagosome and ubiquitin-mediated protein degradation)and three humoral immune pathways(PI3K-Akt,MAPK and FoxO signaling

关 键 词：意大利蜜蜂 中肠 卵巢 精巢 PIRNA 

分 类 号：Q965.9[生物学—昆虫学]