东方蜜蜂微孢子虫γ-tubulin基因的表达验证与分子特性分析  

Expression,validation and molecular characteristics of the Nosema ceranaeγ-tubulin gene

作　　者：叶亚萍 高旭泽 樊念 张佳欣 赵浩东 钱加珺 张文德 赵红霞 徐细建 陈大福 郭睿 YE Ya-Ping;GAO Xu-Ze;FAN Nian;ZHANG Jia-Xin;ZHAO Hao-Dong;QIAN Jia-Jun;ZHANG Wen-De;ZHAO Hong-Xia;XU Xi-Jian;CHEN Da-Fu;GUO Rui(College of Animal Sciences(College of Bee Science),Fujian Agriculture and Forestry University,Fuzhou 350002,China;Apitherapy Research Institute of Fujian Province,,Fuzhou 350002,China;Institute of Zoology,Guangdong Academy of Sciences,Guangzhou 510260,China;Apicultural Research Institute of Jiangxi Province,Nanchang 330000,China)

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

出　　处：《应用昆虫学报》2023年第3期808-815,共8页Chinese Journal of Applied Entomology

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(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);福建省大学生创新创业训练计划(202310389028,X202310389073)。

摘　　要：【目的】本研究对东方蜜蜂微孢子虫Nosema ceranae的γ-微管蛋白(γ-tubulin)基因γ-tubulin进行表达验证,明确γ-tubulin的分子特性,并对东方蜜蜂微孢子虫和其他物种的γ-tubulin蛋白进行保守基序和系统进化分析,以期丰富东方蜜蜂微孢子虫的γ-tubulin信息,并为深入开展γ-tubulin的功能研究提供基础。【方法】通过PCR验证γ-tubulin的表达。通过Expasy网站上的相关软件预测和分析γ-tubulin的理化性质、信号肽、磷酸化位点、二级结构和三级结构。使用MEME软件预测东方蜜蜂微孢子虫和其他物种γ-tubulin的保守基序。通过Mega 11.0软件对东方蜜蜂微孢子虫和其他物种的γ-tubulin进行氨基酸序列多重比对,并采用邻接法构建基于γ-tubulin的系统进化树。【结果】γ-tubulin在东方蜜蜂微孢子虫孢子中真实表达。γ-tubulin编码433个氨基酸,分子量约为49.11 kD,分子式为C_(2186)H_(3422)N_(582)O_(663)S_(20),脂溶系数为86.65,等电点为5.75;γ-tubulin包含29个丝氨酸酸化位点,5个酪氨酸酸化位点及14个苏氨酸磷酸化位点,包含191个α-螺旋,66条延长链,17个β-转角及159个无规则卷曲。另外,γ-tubulin同时定位于细胞核、细胞质和线粒体。东方蜜蜂微孢子虫与蜜蜂微孢子虫Nosema apis等9个物种的γ-tubulin均含有5个相同的保守基序。系统进化分析结果显示,东方蜜蜂微孢子虫与蜜蜂微孢子虫、家蚕微孢子虫Nosema bombycis、兔脑炎微孢子虫Encephalitozoon cuniculi、海伦脑炎微孢子虫Encephalitozoon hellem、角膜条孢虫Vittaforma corneae的γ-tubulin蛋白聚为一个大支,而小孢根霉Rhizopus microsporus与奇孢根霉Rhizopus azygosporus的γ-tubulin聚为另一个大支;东方蜜蜂微孢子虫及其姐妹种蜜蜂微孢子虫的γ-tubulin进化距离最近。【结论】证实了东方蜜蜂微孢子虫孢子中γ-tubulin的真实表达,明确了γ-tubulin的分子特性,并揭示东方蜜蜂微孢子虫与蜜蜂微�[Objectives]To validate the expression of the Nosema ceranaeγ-tubulin gene,clarify its molecular characteristics,and conduct a conserved motif and phylogenetic investigation ofγ-tubulin proteins in N.ceranae and other species.[Methods]The expression ofγ-tubulin was verified by PCR.Its physical and chemical properties,signal peptide,phosphorylation site and secondary and tertiary structures,were predicted and analyzed using related software on the Expasy website.MEME software was used to predict conserved motifs in theγ-tubulin of N.ceranae and other species.Mega 11.0 software was used to perform multiple alignment of amino acid sequences ofγ-tubulin from N.ceranae and other species,followed by construction of a phylogenetic tree based on the neighbor-joining method.[Results]The expression ofγ-tubulin was confirmed in N.ceranae spores.The molecular formula of the gene is C_(2186)H_(3422)N_(582)O_(663)S_(20).It contains 433 amino acids and has a molecular weight of about 49.11 kD.Its lipid solubility coefficient is 86.65,and its hydrophilic coefficient is 5.75.The N.ceranaeγ-tubulin gene contains 29 serine,5 tyrosine and 14 threonine phosphorylation sites,and also includes a 191α-helix,66 extension,17β-turn and 159 random coil.The gene is found in the nucleus,cytoplasm and mitochondria.The same five conserved motifs were identified in theγ-tubulin gene of N.ceranae and those of another nine species.Phylogenetic analysis indicates that theγ-tubulin gene of N.ceranae,Nosema apis,Nosema bombycis,Encephalitozoon cuniculi,Encephalitozoon hellem and Vittaforma corneae cluster within a single large clade,whereas that of Rhizopus microsporus and Rhizopus azygosporus cluster in another large clade.The closest evolutionary distance was that between theγ-tubulin protein of N.ceranae and that of its sister species N.apis.[Conclusion]Our results confirm the expression ofγ-tubulin in N.ceranae spores,clarify the molecular characteristics of this gene,reveal a close genetic relationship betweenγ-tubulin in N.ceranae

关 键 词：微孢子虫 东方蜜蜂微孢子虫 Γ-微管蛋白 蜜蜂 分子特性 系统进化 

分 类 号：S895.236[农业科学—特种经济动物饲养]