中华蜜蜂工蜂幼虫肠道细胞自噬与凋亡相关基因及其剪接本鉴定  

Identification of genes related to intestinal cells'autophagy and apoptosis and their transcript variants in Apis cerana cerana worker larvae

作　　者：荆欣 胡颖 臧贺 宋宇轩 李坤泽 叶道有 康婧 陈大福[1,2,3] 郭睿 JING Xin;HU Ying;ZANG He;SONG Yuxuan;LI Kunze;YE Daoyou;KANG Jing;CHEN Dafu;GUO Rui(College of Bee Science and Biomedicine,Fujian Agriculture and Forestry University;National&Local United Engineering Laboratory of Natural Biotoxin,Fujian Agriculture and Forestry University;Apitherapy Research Institute of Fujian Province,Fuzhou,Fujian 350002,China)

机构地区：[1]福建农林大学蜂学与生物医药学院 [2]福建农林大学天然生物毒素国家地方联合工程实验室 [3]福建省蜂疗研究所,福建福州350002

出　　处：《福建农林大学学报（自然科学版）》2024年第6期786-791,共6页Journal of Fujian Agriculture and Forestry University:Natural Science Edition

基　　金：国家自然科学基金面上项目(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(X202310389074,X202310389077)。

摘　　要：【目的】通过鉴定和解析中华蜜蜂(Apis cerana cerana)工蜂幼虫肠道细胞自噬与凋亡相关基因及其剪接本,为进一步的功能研究提供依据。【方法】使用Blast工具将中华蜜蜂剪接本与Nr数据库进行比对,以鉴定自噬与凋亡相关基因及其剪接本。采用Astalavista软件挖掘相关基因的可变剪接(alternative splicing,AS)事件。通过RT-PCR验证AS事件的真实性。利用TAPIS pipeline软件预测相关基因的可变多聚腺苷酸化(alternative polyadenylation,APA)位点。使用TBtools软件预测APA位点上游的基序(motif)。【结果】鉴定出中华蜜蜂工蜂幼虫肠道中与自噬相关的49个基因及其170种剪接本,以及与凋亡相关的76个基因及其347种剪接本。鉴定出细胞自噬相关的8个基因的88次AS事件及凋亡相关的15个基因的339次AS事件。PCR扩增结果符合预期,证实了随机选择的3种类型AS事件(3'端可变剪接、外显子跳跃、内含子保留)的真实性。预测结果表明,自噬相关的36个基因含有1个或多个APA位点,但在这些APA位点的上游未鉴定到任何motif;而凋亡相关的55个基因含有1个或多个APA位点,并在其上游识别出多个motif,具有一致性序列:NNNDNDNNDNDNDNNDNDNDNNDNDNNNNDDHNNN,NNNNNVNNDNNNNNDHNNHNNNNVNNNH。【结论】中华蜜蜂工蜂幼虫肠道细胞自噬与凋亡相关基因可通过AS和APA形成数量丰富的剪接本,自噬和凋亡相关基因中最丰富的AS类型分别是3'端可变剪接和外显子跳跃,含1个APA位点的基因数目最多。【Objective】This study aims to provide reference and foundation for further functional investigation by identifying and analyzing autophagy-and apoptosis-relative genes and their transcript variants in the gut of Apis cerana cerana worker larvae.【Method】The transcript variants of A.cerana cerana were aligned to Nr database by Blast tool to identify genes associated with autophagy and apoptosis and their transcript variants.The alternative slicing(AS)events of these relevant genes were explored by Astalavista software.The authenticity of AS events was verified using RT-PCR.The alternative polyadenylation(APA)sites of related genes were predicted utilizing TAPIS pipeline.TBtools software was employed to predict motifs upstream these APA sites.【Result】In total,49 autophagy-related genes and corresponding 170 transcript variants as well as 76 apoptosis-associated genes and their 347 transcript variants were identified.Additionally,88 AS events of 8 autophagy-relative genes and 339 AS events of 15 apoptosis-relevant genes were identified.The sizes of PCR amplification fragments were in accordance with the expected sizes,confirming the authenticity of three randomly selected types of AS events including alternative 3'splice site,exon skipping,intron retention.Furthermore,36 autophagy-related genes were predicted to contain at least 1 APA site,without any motif upstream of these APA sites;55 apoptosis-relevant genes were predicted to include 1 or more APA sites,and severalmotifs were identified upstream of these APA sites,with a consensus sequence of NNNDNDNNDNDNDNNDNDNDNNDNDNNNNDDHNNN,NNNNNVNNDNNNNNDHNNHNNNNVNNNH.【Conclusion】The autophagy-and apoptosis-associated genes in the gut of A.cerana cerana worker larvae are able to generate abundant transcript variants via AS and APA,the most abundant AS types of autophagy-and apoptosis-related genes were alternative 3'splice site and exon skipping,respectively,which were dominated by genes with 1 APA site.

关 键 词：中华蜜蜂 自噬 凋亡 剪接本 可变剪接 可变多聚腺苷酸化 

分 类 号：S895.1[农业科学—特种经济动物饲养]