西方蜜蜂几丁质合成酶基因的生物信息学分析及功能研究

doi:10.16380/j.kcxb.2023.04.004

昆虫学报 ›› 2023, Vol. 66 ›› Issue (4): 469-477.doi: 10.16380/j.kcxb.2023.04.004

西方蜜蜂几丁质合成酶基因的生物信息学分析及功能研究

郭意龙^1,#, 赵萧^1,#, 张天泽¹, 宋宇轩¹, 赵浩东¹, 范小雪¹, 任中民¹, 张婕¹, 赵红霞², 徐细建³, 陈大福^1,4,*, 郭睿^1,4,*

(1. 福建农林大学动物科学学院(蜂学学院), 福州 350002; 2. 广东省科学院动物研究所, 广州 510260; 3. 江西省养蜂研究所, 南昌 330000; 4. 福建省蜂疗研究所, 福州 350002)

出版日期:2023-04-20 发布日期:2023-06-01

Bioinformatic analysis and functional study on chitin synthase gene in Apis mellifera

GUO Yi-Long^1,#, ZHAO Xiao^1,#, ZHANG Tian-Ze¹, SONG Yu-Xuan¹, ZHAO Hao-Dong¹, FAN Xiao-Xue¹, REN Zhong-Min¹, ZHANG Jie¹, ZHAO Hong-Xia², XU Xi-Jian³, CHEN Da-Fu^1,4,*, GUO Rui^1,4,*

(1. College of Animal Sciences (College of Bee Science), Fujian Agriculture and Forestry University, Fuzhou 350002, China; 2. Institute of Zoology, Guangdong Academy Sciences, Guangzhou 510260, China; 3. Apicultural Research Institute of Jiangxi Province, Nanchang 330000, China; 4. Apitherapy Research Institute of Fujian Province, Fuzhou 350002, China)

Online:2023-04-20 Published:2023-06-01

摘要/Abstract

摘要： 【目的】本研究旨在明确西方蜜蜂Apis mellifera几丁质合成酶(chitin synthase, CHS)基因的分子特性，并揭示CHS在西方蜜蜂工蜂幼虫响应蜜蜂球囊菌Ascosphaera apis胁迫的免疫应答中的功能。【方法】利用相关生物信息学软件预测和分析西方蜜蜂CHS蛋白的分子特性、保守基序和结构域。使用MEGA X软件对西方蜜蜂和其他昆虫CHSs的氨基酸序列进行系统进化分析。采用体外转录法合成CHS和egfp的dsRNA，饲喂蜜蜂球囊菌胁迫的西方蜜蜂工蜂3日龄幼虫以进行RNAi，并利用RT-qPCR检测西方蜜蜂工蜂5日龄幼虫肠道中CHS及宿主响应蜜蜂球囊菌侵染的免疫相关基因abaecin, apidaecin, birc5, defensin-1和PGRP-S2的表达量。【结果】西方蜜蜂CHS蛋白含有1 572个氨基酸，属于20种氨基酸，其中正电荷氨基酸与负电荷氨基酸分别为177和169个，分子量约为178.77 kD，等电点为6.65；含纤维素合酶CESA3催化结构域。在西方蜜蜂和东方蜜蜂A. cerana等共13种昆虫的CHSs中均鉴定到3个Motif (Motif 1, Motif 2和Motif 3)和2个结构域(Chitin_synth_2和Glyco_trans_2_3)。系统进化树表明，膜翅目的西方蜜蜂与东方蜜蜂的CHSs的氨基酸序列一致性最高，聚为一支且自举值为100。相较于dsegfp饲喂对照组，dsCHS饲喂组5日龄幼虫肠道中CHS的表达量显著下调，干扰效率为29.40%，abaecin， birc5和
defensin-1的表达量均极显著上调，PGRP-S2的表达量显著上调，apidaecin的表达量极显著下调。【结论】西方蜜蜂CHS可能是胞内亲水性跨膜蛋白，西方蜜蜂与其他昆虫CHSs的氨基酸序列具有较高的保守性，其中与东方蜜蜂的CHS的氨基酸序列之间保守性最高，CHS影响西方蜜蜂工蜂幼虫响应蜜蜂球囊菌胁迫的免疫应答。

关键词: 西方蜜蜂, 蜜蜂球囊菌, RNA干扰, 几丁质合成酶, 免疫应答

Abstract: 【Aim】 This study aims to elucidate the molecular characters of chitin synthase (CHS) gene in Apis mellifera and uncover the function of CHS in immune response of A. mellifera worker larvae to Ascosphaera apis stress. 【Methods】 The molecular characters,
conserved motifs and structural domains of the A. mellifera CHS protein were predicted and analyzed using relevant bioinformatics software. Phylogenetic analysis of amino acid sequences of CHS proteins in A. mellifera and other insects was performed with MEGA X software. The dsRNAs of CHS and egfp were synthesized via in vitro transcription method, and fed to the A. apis-stressed 3-day-old larvae of A. mellifera workers to perform RNAi. RT-qPCR was used to determine the expression levels of CHS and immune-related genes abaecin, apidaecin, birc5, defensin-1 and PGRP-S2 in the 5-day-old larval gut of A. mellifera workers in response to A. apis stress. 【Results】 The CHS protein of A. mellifera contains 1 572 amino acids belonging to 20 types of amino acids, among which the numbers of amino acids with positive and negative charge are 177 and 169, respectively. CHS has the molecular weight of 178.77 kD, isoelectric point of 6.65 and cellulose synthase CESA3 catalytic structural domain. Three motifs (Motif 1, Motif 2 and Motif 3) and two structural domains (Chitin_synth_2 and Glyco_trans_2_3) were identified in CHSs in 13 insect species such as A. mellifera and A. cerana. Phylogenetic tree showed that the CHSs in A. mellifera and A. cerana of Hymenoptera showed the highest amino acid sequence identities, and clustered into one branch with the bootstrap value of 100. The expression level of CHS in the 5-day-old larval gut of A. mellifera in the dsCHS-fed group was significantly down-regulated as compared to that in the dsegfp-fed control group, with the interference efficiency of 29.40%. In the dsCHS-fed group, the expression levels of abaecin, birc5 and defensin-1 in the 5-day-old larval gut of A. mellifera were extremely significantly up-regulated, that of PGRP-S2 was significantly up-regulated, while that of apidaecin was extremely significantly down-regulated, as compared with those in the dsegfp-fed control groups. 【Conclusion】 A. mellifera CHS may be an intracellular hydrophilic transmembrane protein. The amino acid sequences of CHSs in A. mellifera and other insects are highly conservative, and those in A. mellifera and A. cerana are the most highly conservative. CHS affects A. mellifera worker larval immune response to A. apis stress.

Key words: Apis mellifera, Ascosphaera apis, RNA interference, chitin synthase, immune response

郭意龙, 赵萧, 张天泽, 宋宇轩, 赵浩东, 范小雪, 任中民, 张婕, 赵红霞, 徐细建, 陈大福, 郭睿. 西方蜜蜂几丁质合成酶基因的生物信息学分析及功能研究[J]. 昆虫学报, 2023, 66(4): 469-477.

GUO Yi-Long, ZHAO Xiao, ZHANG Tian-Ze, SONG Yu-Xuan, ZHAO Hao-Dong, FAN Xiao-Xue, REN Zhong-Min, ZHANG Jie, ZHAO Hong-Xia, XU Xi-Jian, CHEN Da-Fu, GUO Rui. Bioinformatic analysis and functional study on chitin synthase gene in Apis mellifera [J]. Acta Entomologica Sinica, 2023, 66(4): 469-477.

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西方蜜蜂几丁质合成酶基因的生物信息学分析及功能研究

Bioinformatic analysis and functional study on chitin synthase gene in Apis mellifera

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