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

  • 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RACE-PCR技术首次在池蝶蚌中获得STAT的cDNA全长序列(GenBank ID: KY123702), 命名为HsSTAT, HsSTAT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%。HsSTAT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, HsSTAT与光滑双脐螺STAT5B和盘鲍STAT5聚在一支; 亚细胞定位结果显示HsSTAT定位于血细胞的细胞质中; 荧光原位杂交结果显示HsSTAT主要在细胞核。qPCR结果显示HsSTAT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HsSTAT的全长序列, HsSTAT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。

     

    Abstract: The cDNA full length sequence of STAT (HsSTAT, GenBank ID: KY123702) was obtained from Hyriopsis schlegelii first time using the TACE-PCR technique. The full length of HsSTAT is 2752 bp. The lengths of the 5′-untranslated region, 3′-untranslated region, and ORF region are 121, 264 and 2367 bp, respectively, and it encoded 788 amino 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 of 12.9% in the sequence, and lysine has the lowest percentage of 0.5%. The utilization frequency analysis of amino acid at the conservative locus showed that only tryptophan reached 100% utilization. The secondary structure of HsSTAT shows that the percentages of α-helix, β-folding, extension chain, random coil were 46.83%, 8.5%, 15.36% and 29.31%, respectively. Furthermore, the third structure analysis give a prediction of four classical domains. In addition, the hydrophobicity analysis show that the HsSTAT protein is hydrophilic with an index of –0.531. 1 palmitoylation modification site, 3 small ubiquitin-related modification sequences, and 22 phosphorylation sites were predicted. Phylogenetic tree analysis showed that HsSTAT is clustered with Biomphalaria glabrata STAT5B and Haliotis discus discus STAT5. Subcellular localization results indicate that HsSTAT is located in the cytoplasm of hemocytes. Fluorescence in situ hybridization shows that the expression of HsSTAT is mainly in nucleus. The results of qPCR show that HsSTAT is expressed in all tissues, among which it has the highest expression in the hepatopancreas and the lowest in hemocytes. The proliferation results show that HsSTAT helps promote the proliferation of SMCC-7721 cells.

     

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