中华蜜蜂酚氧化酶和丝氨酸蛋白酶相关基因及其全长转录本发掘与分析  

Exploration and investigation of genes and full-length transcripts associated with phenoloxidases and serine proteases in Apis cerana cerana

作　　者：赵浩东 王思懿 李琪明 张天泽 张艺琼 高旭泽 陈大福[1,2,3] 郭睿 付中民[1,2,3] ZHAO Haodong;WANG Siyi;LI Qiming;ZHANG Tianze;ZHANG Yiqiong;GAO Xuze;CHEN Dafu;GUO Rui;FU Zhongmin(College of Bee Science and Biomedicine,Fujian Agriculture and Forestry University,Fuzhou 350002;National&Local United Engineering Laboratory of Natural Biotoxin,Fuzhou 350002;Apitherapy Research Institute of Fujian Province,Fuzhou 350002)

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]福建省蜂疗研究所,福州350002

出　　处：《安徽农业大学学报》2024年第1期82-87,共6页Journal of Anhui Agricultural University

基　　金：国家自然科学基金(32172792,31702190);国家现代农业产业技术体系专项资金项目(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项目(郭睿);福建农林大学动物科学学院(蜂学学院)科研扶持项目(郭睿,付中民)共同资助。

摘　　要：为深入开展中华蜜蜂相关研究提供参考信息和基础,利用已获得的纳米孔(Nanopore)测序数据对中华蜜蜂Apis cerana cerana的酚氧化酶(phenoloxidase,PO)和丝氨酸蛋白酶(serine protease,SP)相关基因和全长转录本进行发掘和分析。通过Blast工具将中华蜜蜂所有全长转录本比对KEGG和Nr数据库以筛选出PO和SP相关基因和全长转录本。采用gffcompare软件将PO和SP相关全长转录本与东方蜜蜂参考基因组(ACSNU-2.0)已注释基因进行比较以优化结构。使用Astalavista软件鉴定PO和SP相关基因的可变剪接(alternative splicing,AS)事件,再利用IGV浏览器进行结构可视化。通过PCR验证AS事件的真实性。利用TAPIS pipeline预测和分析可变多聚腺苷酸化(alternative polyadenylation,APA)位点,并通过TBtool软件鉴定APA位点上游的基序(motif)。鉴定到中华蜜蜂PO和SP相关的42个基因与146条转录本。优化了16个参考基因组已注释PO和SP相关基因的结构,其中5′端延长和3′端延长的基因均有7个,5′端和3′端同时延长的基因有2个。鉴定到PO和SP相关的10个基因的389次AS事件,其中最丰富的AS类型是5′端可变剪接。PCR结果证实了2次AS事件的真实性。共鉴定到34个PO和SP相关基因含有1个及以上的APA位点,其中含有3个APA位点的基因数量最多;在APA位点上游鉴定到多个motif,一致性序列为:GGHKSYWSHHTRATWTCNBHDMRRYWYRTNYTVACNGCKGCDCAYTGYR。鉴定到中华蜜蜂PO和SP相关的42个基因和146条全长转录本,优化了东方蜜蜂参考基因组已注释的PO和SP相关基因结构,并发掘出PO和SP相关基因的389次AS事件和237个APA位点。In order to further understand some information of Apis cerana and provide a basis for studying its function,the genes and full-length transcripts associated with phenoloxidases(PO)and serine proteases(SP)in Apis cerana cerana were explored and analyzed using previously gained Nanopore long-read sequencing data.All full-length transcripts in Apis cerana cerana were aligned to KEGG and Nr databases with Blast tool to screen genes and full-length transcripts relevant to PO and SP.Structures of annotated genes were optimized by comparing PO and SP-associated full-length transcripts with annotated transcripts in the Apis cerana reference genome(ACSNU-2.0)utilizing gffcompare software.Types of AS events in genes relative to PO and SP were identified by using Astalavista software,followed by visualization with IGV browser.The authenticity of AS events was confirmed by PCR.Predic-tion and investigation of APA sites were performed using TAPIS pipeline,and motifs at upstream of APA sites were then identified using TBtool software.Totally,42 genes and 146 full-length transcripts relative to PO and SP in Apis cerana cerana were identified.Structures of 16 annotated genes were optimized,among these 5′ends and 3′ends of seven genes were prolonged,and both 5′ends and 3′ends of two genes were prolonged.Additionally,389 AS events occurred in 10 genes were discovered,among these the most abundant type was alternative 3′splice site.PCR result validated the authenticity of randomly selected two AS events.Furthermore,34 genes associated with PO and SP were identified to include one or more APA sites,among which those genes containing three APA sites were in the largest group;multiple motifs at upstream of APA sites were detected,with the consistent sequence:GGHKSYW SHHTRATWTCNBHDMRRYWYRTNYTVACNGCKGCDCAYTGYR.This study identified 42 genes and 146 full-length transcripts related to Apis cerana cerana PO and SP,optimized the structures of 16 annotated PO and SPgenes in the reference genome of Apis cerana,and explored 389 AS e

关 键 词：中华蜜蜂 酚氧化酶 丝氨酸蛋白酶 纳米孔测序 全长转录本 可变剪切 可变多聚腺苷酸化 

分 类 号：S891[农业科学—特种经济动物饲养]