池蝶蚌STAT基因的克隆及功能分析  

CLONING AND FUNCTION ANALYSIS ON SIGNAL TRANSDUCER AND ACTIVATOR OF TRANSCRIPTION IN HYRIOPSIS SCHLEGELII

作　　者：舒福兴 周伟 盛军庆 王军花 王小敏 高沁 洪一江 SHU Fu-Xing;ZHOU Wei;SHENG Jun-Qing;WANG Jun-Hua;WANG Xiao-Min;GAO Qin;HONG Yi-Jiang(Key Lab of Acqutic Resources and Utilization of Jiangxi, School of Life Sciences, Nanchang University, Nanchang 330031,China;Medical College, Nanchang University, Nanchang 330031, China)

机构地区：[1]南昌大学生命科学学院,江西省水产动物资源与利用重点实验室,南昌330031 [2]南昌大学医学院,南昌330031

出　　处：《水生生物学报》2018年第2期284-292,共9页Acta Hydrobiologica Sinica

基　　金：十三五国家贝类产业技术体系淡水珍珠贝养殖与育珠岗(CAR-49);国家自然科学基金(31660337);江西省科技落地计划项目(KJLD12001);江西省教育厅一般项目(GJJ150166)资助

摘　　要：为了探究池蝶蚌(Hyriopsis schlegelii)信号转导及转录激活因子STAT基因的初步功能,通过RACEPCR技术首次在池蝶蚌中获得STAT的c DNA全长序列(Gen Bank ID:KY123702),命名为Hs STAT,Hs STAT全长为2752 bp,5′-非编码区(5′UTR)为121bp,3′-非编码区3′UTR为264 bp,开放阅读框(ORF)为2367 bp,编码788个氨基酸;生物信息学分析发现该蛋白结构域主要由STAT_int、STAT_alpha、STAT_bind、SH2四个经典保守性功能域组成;缬氨酸最多占12.9%。色氨酸使用频率为100%。二级结构中α螺旋最多占46.83%、β折叠最少占8.5%。Hs STAT蛋白亲水性指数为–0.531,是亲水性蛋白。预测到棕榈酰化修饰位点1个,小泛素相关修饰序列3个,磷酸化修饰位点22个。系统进化树分析显示,Hs STAT与光滑双脐螺STAT5B和盘鲍STAT5聚在一支;亚细胞定位结果显示Hs STAT定位于血细胞的细胞质中;荧光原位杂交结果显示Hs STAT主要在细胞核。q PCR结果显示Hs STAT在所有的组织中均有表达,其在组织中的表达量在肝胰腺中最高,在血细胞中最低。细胞增殖实验显实验组增殖率为119%,高于对照组。实验已克隆到Hs STAT的全长序列,Hs STAT可能为STAT5亚型,具有促进SMCC-7721细胞增殖的功能,推测具有池蝶蚌细胞的信号转导功能参与池蝶蚌的先天免疫。The cDNA full length sequence of STAT(HsSTAT,GenBank ID:KY123702)was obtained from Hyriopsisschlegelii first time using the TACE-PCR technique.The full length of HsSTAT is2752bp.The lengths of the5′-untranslatedregion,3′-untranslated region,and ORF region are121,264and2367bp,respectively,and it encoded788amino acids.The protein domain consists of four classical conservative function domains,including STAT_int,STAT_alpha,STAT_bind and SH2.Valine possesses the highest percentage of12.9%in the sequence,and lysine hasthe lowest percentage of0.5%.The utilization frequency analysis of amino acid at the conservative locus showed thatonly tryptophan reached100%utilization.The secondary structure of HsSTAT shows that the percentages ofα-helix,β-folding,extension chain,random coil were46.83%,8.5%,15.36%and29.31%,respectively.Furthermore,the thirdstructure analysis give a prediction of four classical domains.In addition,the hydrophobicity analysis show that theHsSTAT protein is hydrophilic with an index of–0.531.1palmitoylation modification site,3small ubiquitin-relatedmodification sequences,and22phosphorylation sites were predicted.Phylogenetic tree analysis showed that HsSTATis clustered with Biomphalaria glabrata STAT5B and Haliotis discus discus STAT5.Subcellular localization results indicatethat HsSTAT is located in the cytoplasm of hemocytes.Fluorescence in situ hybridization shows that the expressionof HsSTAT is mainly in nucleus.The results of qPCR show that HsSTAT is expressed in all tissues,among which ithas the highest expression in the hepatopancreas and the lowest in hemocytes.The proliferation results show that HsSTAT helps promote the proliferation of SMCC-7721cells.

关 键 词：池蝶蚌 HsSTAT 生物信息学分析 亚细胞定位 荧光原位杂交 qPCR 细胞增殖 

分 类 号：Q718[生物学—分子生物学]