光裸星虫CyclinB基因克隆及其在卵母细胞中的表达分析  

Cloning of CyclinB gene from Sipunculus nudus and its expression analysis in oocytes

作　　者：邬婧 苏泳霖 周丹 钟如卓 郭志诚 王庆恒[1,2] WU Jing;SU Yong-lin;ZHOU Dan;ZHONG Ru-zhuo;GUO Zhi-cheng;WANG Qing-heng(Fisheries College,Guangdong Ocean University,Zhanjiang,Guangdong 524025,China;Guangdong Provincial Engineering Laboratory for Mariculture Organism Breeding,Zhanjiang,Guangdong 524088,China)

机构地区：[1]广东海洋大学水产学院,广东湛江524088 [2]广东省海水养殖生物育种工程实验室,广东湛江524088

出　　处：《南方农业学报》2021年第7期1980-1990,共11页Journal of Southern Agriculture

基　　金：广东省科技计划项目(163-2019-XMZC-0009-02-0059,2016A020209010);广东省大学生创新创业训练计划项目(201810566049)。

摘　　要：【目的】探究细胞周期蛋白B(CyclinB)基因在光裸星虫(Sipunculus nudus)卵母细胞发育成熟过程中的作用,为揭示光裸星虫卵母细胞发育的分子机制提供理论依据。【方法】利用RACE克隆光裸星虫CyclinB(Sn-CyclinB)基因cDNA序列,通过ProtParam、DNAMAN 9.0、COBALT、SOPMA、Phyer2、Pfam及BLAST等在线软件进行生物信息学分析,并以实时荧光定量PCR检测分析Sn-CyclinB基因在光裸星虫卵母细胞不同发育时期的表达情况。【结果】Sn-CyclinB基因cDNA序列全长2468 bp,包括141 bp的5'端非翻码区(5'-UTR)、1097 bp的3'端非翻码区(3'-UTR)及1230 bp的开放阅读框(ORF),编码409个氨基酸残基,在3'-UTR中存在3个胞质多聚腺苷酸化元件(CPE)、1个翻译调控元件(TCE)和1个K-box翻译调控元件。Sn-CyclinB蛋白分子量为46.304 kD,理论等电点(pI)为8.68,具有CyclinB特征序列(FLRRxSK)和Cyclin降解框(RxALGxIxN),属于亲水性蛋白,含有周期蛋白框(Cyclin box);其二级结构中α-螺旋、延伸链、β-转角和无规则卷曲分别占56.72%、3.91%、0.73%和38.63%;CyclinB蛋白C端的保守性较N端高;与其他物种相比,光裸星虫Cyclin降解框(RALGTISN)的位置前移了13个氨基酸;光裸星虫与小头虫和欧洲帽贝的CyclinB蛋白三级结构高度相似。基于CyclinB氨基酸序列相似性构建的系统发育进化树分为无脊椎动物和脊椎动物两大支,其中光裸星虫与小头虫及水蛭等无脊椎动物聚为一支。Sn-CyclinB基因在光裸星虫卵母细胞不同发育时期均有表达,且整体上呈双峰型变化趋势,其相对表达量的峰值分别出现在卵黄旺盛合成后期(O3)和肾管发育时期(O5)。【结论】Sn-CyclinB蛋白具有CyclinB特征序列,其3'-UTR含有多种与翻译调控相关的调控元件,参与光裸星虫卵母细胞减数分裂G_(1)/S期和G_(2)/M期的转换,对促进卵母细胞减数分裂有序进行起重要作用。【Objective】Exploring the role of CyclinB gene in the development and maturation of Sipunculus nudus oo-cytes to provide a theoretical basis for further understanding of the molecular mechanism of S.nudus oocyte development.【Method】RACE technology was used to clone the full length of S.nudus CyclinB(Sn-CyclinB)cDNA and the bioinfor-matics analysis was performed by some online softwares like ProtParam,DNAMAN 9.0,COBALT,SOPMA,Phyer2,Pfam,and BLAST.The real-time fluorescence quantitative PCR technology was used to analyze the expression pattern of Sn-CyclinB in different developmental stages of oocytes.【Result】The full-length cDNA of Sn-CyclinB was 2468 bp with 141 bp 5'-untranslated region(5'-UTR),1097 bp 3'-untranslated region(3'-UTR),and 1230 bp open reading frame(ORF).It encoded 409 amino acids and contained three cytoplasmic polyadenylation elements(CPE),a translation con-trol element(TCE)and a K-box translation regulation element in the 3'-UTR.The theoretical molecular weight and iso-electric point(pI)of Sn-CyclinB protein were 46.304 kD and 8.68 respectively and it had a signature sequence(FLR-RxSK)and Cyclin degradation box(RxALGxIxN)which was a hydrophilic protein.Sn-CyclinB protein also contained a cyclin box and the secondary structure of protein,alpha-helix,extension chain,beta-corner,and random curl accounted for 56.72%,3.91%,0.73%,and 38.63%respectively.It suggested that the C-terminus of CyclinB homologous protein was more conservative than N-terminus.Compared with other species,the position of the Cyclin degradation box(RAL-GTISN)had moved forward by 13 amino acids and the tertiary structure of Sn-CyclinB protein was highly similar to those of Capitella teleta and Patella vulgata.Phylogenetic tree analysis,based on the similarity of amino acid sequences of Cy-clinB,showed that CyclinB clustered into invertebrates and vertebrates branches among which S.nudus,C.teleta and He-lobdella triserialis were clustered into the invertebrate branch.Sn-CyclinB was expressed in different development sta

关 键 词：光裸星虫 细胞周期蛋白B(CyclinB) Cyclin降解框 表达分析 卵母细胞 减数分裂 

分 类 号：S963.219[农业科学—水产养殖]