摘要
【目的】本研究旨在丰富中华蜜蜂Apis cerana cerana 14-3-3ζ基因的基本信息,为其进一步的功能研究提供参考和依据。【方法】通过PCR扩增14-3-3ζ基因的编码序列(coding sequence,CDS),再进行TA克隆和Sanger测序;使用相关软件预测14-3-3ζ的理化性质和分子特征,并对14-3-3ζ进行系统进化分析;利用RT-qPCR检测14-3-3ζ基因在中华蜜蜂不同发育阶段(卵、幼虫、预蛹、蛹和成虫)、刚出房工蜂成虫不同组织(触角、中肠、脂肪体、咽下腺、脑、表皮和毒腺)及蜜蜂球囊菌Ascospaera apis接种中华蜜蜂工蜂3日龄幼虫后4,5和6日龄幼虫肠道中的表达量。【结果】成功克隆到中华蜜蜂14-3-3ζ基因的CDS,含744个核苷酸,编码247个氨基酸,中华蜜蜂14-3-3ζ的分子量约为28.0 kD,含26个磷酸化位点、4个结构域和1个保守基序,不含跨膜结构域与信号肽;中华蜜蜂、西方蜜蜂Ap.mellifera、黑大蜜蜂Ap.laboriosa、小蜜蜂Ap.florea、芦蜂Ceratina calcarata、火红熊蜂Bombus pyrosoma、地熊蜂B.terrestris、苜蓿切叶蜂Megachile rotundata、壁蜂Osmia lignaria和东南蓝莓蜂Habropoda laboriosa 14-3-3ζ均包含4个相同的保守基序和1个相同的结构域(14-3-3_1),中华蜜蜂和西方蜜蜂的14-3-3ζ在系统进化树上聚为一支。14-3-3ζ基因在中华蜜蜂卵中的表达量显著高于3日龄幼虫、1和2日龄预蛹及4日龄蛹中的表达量,在中华蜜蜂各日龄工蜂成虫体内的表达量无显著差异;14-3-3ζ基因的表达量在中华蜜蜂刚出房工蜂成虫毒腺中最高,且显著高于触角、中肠、咽下腺、脑、表皮和脂肪体中的表达量;蜜蜂球囊菌接种中华蜜蜂工蜂3日龄幼虫后,14-3-3ζ基因在中华蜜蜂工蜂4,5和6日龄幼虫肠道中的表达量与对照组比显著下调。【结论】中华蜜蜂14-3-3ζ基因在工蜂的毒腺和卵中特异性高量表达,幼虫肠道中14-3-3ζ基因的表达在蜜蜂球囊菌侵染过程中被激活,14-3-3ζ是一种潜在的亲水性、非跨膜和胞内蛋白,中华蜜蜂与上述其他10种蜂的14-3-3ζ较为保守,中华蜜蜂和西方蜜蜂的14-3-3ζ之间亲缘关系最近。
【Aim】This study aims to enrich the basic information of 14-3-3ζ gene of Apis cerana cerana,so as to provide a reference and basis for its further functional study.【Methods】The coding sequence(CDS)of 14-3-3ζ gene was amplified by RT-PCR,followed by TA cloning and Sanger sequencing.The physicochemical properties and molecular features of 14-3-3ζ were predicted using the relevant software,and the phylogenetic analysis of 14-3-3ζ was performed.RT-qPCR was used to detect the expression levels of 14-3-3ζ gene in different developmental stages(egg,larva,pre-pupa,pupa and adult),and different tissues(antennae,midgut,fat body,hypopharyngeal gland,brain,cuticle and venom gland)of the newly emerged adult workers of Ap.cerana cerana,as well as in the guts of the 4-,5-and 6-day-old larvae of Ap.cerana cerana after inoculating the 3-day-old larval workers with Ascosphaera apis.【Results】The CDS of 14-3-3ζ gene of Ap.cerana cerana was successfully cloned,including 744 nucleotides and encoding 247 amino acids.14-3-3ζ of Ap.cerana cerana had the molecular weight of about 28.0 kD,included 26 phosphorylation sites,four structural domains and one conserved motif,but had no transmembrane domains and signal peptides.The 14-3-3ζ proteins of Ap.cerana cerana,Ap.mellifera,Ap.laboriosa,Ap.florea,Ceratina calcarata,Bombus pyrosoma,B.terrestris,Megachile rotundata,Osmia lignaria and Habropoda laboriosa all contained four identical conserved motifs and one same structural domain(14-3-3_1).The 14-3-3ζ proteins of Ap.cerana cerana and Ap.mellifera clustered into a single clade on the phylogenetic tree.The expression level of 14-3-3ζ gene in Ap.cerana cerana eggs was significantly higher than those in the 3-day-old larvae,1-day-old prepupae,2-day-old prepupae and 4-day-old pupae.The differences in the expression level of 14-3-3ζ gene in various day-old adult workers of Ap.cerana cerana were non-significant.The expression level of 14-3-3ζ gene in the venom gland of the newly emerged adult workers of Ap.cerana cerana was the highest,significantly higher than those in the antennae,midgut,hypopharyngeal gland,brain,cuticle and fat body.Following inoculation of the 3-day-old larval workers of Ap.cerana cerana with As.apis,the expression levels of 14-3-3ζ gene in the 4-,5-and 6-day-old larval worker guts of Ap.cerana cerana were significantly down-regulated as compared with those in the control group.【Conclusion】Ap.cerana cerana 14-3-3ζ gene is specifically and highly expressed in the venom gland and egg of worker,and the expression of 14-3-3ζ gene in the larval guts is activated in the process of As.apis infection.14-3-3ζ is a putative hydrophilic,non-transmembrane and intracellular protein,and highly conserved in Ap.cerana cerana and the above other ten bee species.There is the closest genetic relationship between 14-3-3ζ of Ap.cerana cerana and Ap.mellifera.
作者
陈颖
康婧
臧贺
王勇杰
张凯遥
叶道有
冯睿蓉
陈大福
徐国钧
郭睿
邱剑丰
CHEN Ying;KANG Jing;ZANG He;WANG Yong-Jie;ZHANG Kai-Yao;YE Dao-You;FENG Rui-Rong;CHEN Da-Fu;XU Guo-Jun;GUO Rui;QIU Jian-Feng(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;Fujian Vocational College of Agriculture,Fuzhou 350000,China)
出处
《昆虫学报》
北大核心
2025年第8期1031-1039,共9页
Acta Entomologica Sinica
基金
国家自然科学基金项目(32172792,32372943)
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)
福建省教育厅中青年教师教育科研项目(JAT231226)
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)。
作者简介
陈颖,女,2001年3月生,江西宜春人,硕士研究生,研究方向为蜜蜂分子生物学,E-mail:13979562297@163.com;共同第一作者:康婧,女,2000年2月生,甘肃兰州人,硕士研究生,研究方向为蜜蜂分子生物学,E-mail:kj000213@163.com;通讯作者:郭睿,E-mail:ruiguo@fafu.edu.cn;通讯作者:邱剑丰,E-mail:jfqiu@fafu.edu.cn。
