西方蜜蜂肽聚糖识别蛋白相关基因的剪接异构体鉴定及分析  

Identification and analysis of splice variants of peptidoglycan recognition protein-associated genes in Apis mellifera

作　　者：冯佩林 朱乐冉 臧贺 刘小玉 康婧 邱剑丰 刘锋 徐细建 骆群 陈大福[1,2,3] 郭睿 徐国钧[1,2,3] FENG Pei-Lin;ZHU Le-Ran;ZANG He;LIU Xiao-Yu;KANG Jing;QIU Jian-Feng;LIU Feng;XU Xi-Jian;LUO Qun;CHEN Da-Fu;GUO Rui;XU Guo-Jun(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;Apiculture Research Institute of Jiangxi Province,Nanchang 330000,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]江西省养蜂研究所,南昌330000

出　　处：《昆虫学报》2025年第2期133-143,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项目(CARS-44-KXJ7);福建省自然科学基金面上项目(2022J01131334);宁夏回族自治区重点研发项目(2022BBF02037);江西省重点研发计划项目(20212BBF63008);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)。

摘　　要：【目的】本研究旨在系统鉴定和分析西方蜜蜂Apis mellifera肽聚糖识别蛋白(peptidoglycan recognition protein,PGRP)相关基因及其剪接异构体,探析PGRP基因剪接异构体编码蛋白的理化性质和分子特征,并测定PGRP基因剪接异构体在西方蜜蜂工蜂成虫应答东方蜜蜂微孢子虫Nosema ceranae侵染中的表达模式,以期为西方蜜蜂PGRP基因剪接异构体的功能研究提供参考和依据。【方法】基于已获得的西方蜜蜂8和11日龄工蜂成虫中肠纳米孔测序数据,利用Blast工具将前期鉴定到的所有全长转录本分别比对到Nr和KEGG数据库,筛选出西方蜜蜂PGRP基因及其剪接异构体,并随机选择PGRP基因的5条剪接异构体通过RT-PCR验证序列的真实性。使用Gffcompare软件将鉴定到的PGRP基因序列比对至西方蜜蜂参考基因组以优化基因结构。采用Astalavista软件鉴定PGRP基因的可变剪接(alternative splicing,AS)事件类型,再通过RT-PCR和Sanger测序验证AS事件。利用相关软件预测和分析PGRP基因剪接异构体编码蛋白的理化性质和分子特征。采用RT-qPCR检测东方蜜蜂微孢子虫接种后西方蜜蜂工蜂成虫中肠中PGRP基因剪接异构体的相对表达量。【结果】共鉴定到西方蜜蜂的4个PGRP相关基因(PGRP-3,PGRP-S2,PGRP-LC和PGRP-LB)及其19条剪接异构体。通过RT-PCR与Sanger测序证实了PGRP基因5条剪接异构体(rna14029,ONT.6350.8,ONT.6350.10,rna24089和ONT.6350.7)的表达和序列真实性。PGRP-3(gene10434)的5′和3′端分别延长了8和1055 bp,PGRP-S2(gene10435)的5′和3′端分别延长了27和234 bp。通过RT-PCR证实了PGRP-S2的AS事件的真实性。剪接异构体ONT.6350.2编码蛋白的分子式为C_(966)H_(1502)N_(272)O_(275)S_(7),分子量约为21527.63 D,理论等电点为8.94,平均亲水性为-0.119,含有信号肽和PGRP结构域,不含跨膜结构域;剪接异构体ONT.6350.6编码蛋白分子式为C_(841)H_(1301)N_(243)O_(244)S_(5),分子量约为18860.46 D,�【Aim】This study aims to systematically identify and analyze the genes related to peptidoglycan recognition proteins(PGRPs)and their splice variants in Apis mellifera,to explore the physicochemical properties and molecular characteristics of PGRP gene splice variants-encoding proteins,and to determine the expression profile of PGRP gene splice variants in response of adult workers of A.mellifera to Nosema ceranae infection,so as to offer the reference and basis for functional study on splice variants of PGRP genes of A.mellifera.【Methods】Based on the obtained nanopore sequencing data from the midguts of the 8-and 11-day-old adult workers of A.mellifera,the previously identified all full-length transcripts were aligned to the Nr and KEGG databases by using the Blast tool to screen PGRP genes of A.mellifera and their splice variants,and RT-PCR was used to validate the authenticity of the sequences of five splice variants of PGRP genes randomly selected.Gffcompare software was utilized to align the sequences of the identified PGRP genes to the reference genome of A.mellifera,thus optimizing the gene structures.Astalavista software was employed to identify the types of alternative splicing(AS)events in PGRP genes,and AS events were then verified by RT-PCR and Sanger sequencing.Related software was used to predict and analyze the physicochemical properties and molecular characteristics of the proteins encoded by the splice variants of PGRP genes.RT-qPCR was conducted to determine the relative expression levels of PGRP gene splice variants in the midguts of A.mellifera adult workers after inoculation with N.ceranae.【Results】A total of four PGRP-associated genes(PGRP-3,PGRP-S2,PGRP-LC and PGRP-LB)and their 19 splice variants of A.mellifera were identified.RT-PCR and Sanger sequencing results confirmed the authenticity of expression and sequences of five splice variants(rna14029,ONT.6350.8,ONT.6350.10,rna24089,ONT.6350.7)of PGRP genes.The 5′and 3′ends of PGRP-3(gene10434)were elongated by 8 and 1055 bp,res

关 键 词：西方蜜蜂 东方蜜蜂微孢子虫 肽聚糖识别蛋白 纳米孔测序 剪接异构体 

分 类 号：S891[农业科学—特种经济动物饲养]