蟾蜍TNFRSF11b、TNFRSF9基因克隆及生物信息学分析  

Cloning and Bioinformatics Analysis of TNFRSF11b and TNFRSF9 in Bufo gargarizans Cantor

作　　者：苏岩[1] 褚涵 余坤 潘瑞[1,3] 叶陈娟 李军德[4] 黄晓[2,5] 张恬 SUN Yan;CHU Han;YU Kun;PAN Rui;YE Chen-juan;LI Jun-de;HUANG Xiao;ZHANG Tian(Chongqing Key Laboratory of Traditional Chinese Medicine Resource,Chongqing Academy of Chinese Materia Medica,Chongqing 400065,China;College of Pharmacy,Hubei University of Chinese Medicine,Wuhan 430065,China;Chongqing Branch of National Resource Center for Chinese Materia Medica,China Academy of Chinese Medical Sciences,Chongqing 400065,China;State Key Laboratory of Dao-di Herbs,National Resource Center for Chinese Materia Medica,China Academy of Chinese Medical Sciences,Beijing 100700,China;Hubei Key Laboratory of Traditional Chinese Medicine Resource and Chemistry,Hubei University of Chinese Medicine,Wuhan 430060,China)

机构地区：[1]重庆市中药研究院重庆市中药资源学重点实验室,重庆400065 [2]湖北中医药大学药学院,湖北武汉430065 [3]中国中医科学院中药资源中心重庆分中心,重庆400065 [4]中国中医科学院中药资源中心,北京100700 [5]中药资源与中药化学湖北省重点实验室,湖北武汉430060

出　　处：《中国现代中药》2023年第8期1655-1667,共13页Modern Chinese Medicine

基　　金：重庆市科技局绩效激励引导专项(cqac20190001);湖北中医药大学2020年青苗计划资助项目(2020ZZX002)。

摘　　要：目的:克隆中华蟾蜍肿瘤坏死因子受体超家族(TNFRSF)BbgTNFRSF11b、BbgTNFRSF9基因的全长序列,并分析其序列特征。方法:根据转录组测序所得的TNFRSF11b、TNFRSF9基因片段设计特异性引物,以中华蟾蜍蟾皮为材料,利用逆转录聚合酶链式反应(PCR)技术获得BbgTNFRSF11b、BbgTNFRSF9基因全长互补脱氧核糖核酸(cDNA)序列,并采用生物信息学手段分析其序列特征,通过实时荧光定量PCR方法检测BbgTNFRSF11b、BbgTNFRSF9基因在中华蟾蜍7种组织/器官中的表达情况。结果:克隆获得蟾蜍BbgTNFRSF11b基因的全长cDNA序列为1233bp,编码410个氨基酸,理论相对分子质量为46.97kDa,等电点为8.64,不存在跨膜区及信号肽,为亲水性蛋白,具有多个磷酸化位点。BbgTNFRSF9基因的全长cDNA序列为829bp,编码247个氨基酸,理论相对分子质量为67.874kDa,理论等电点为5.12,存在信号肽,为疏水性蛋白,具有多个磷酸化位点,进化树分析发现BbgTNFRSF11b、BbgTNFRSF9基因与其他动物TNFRSF11b、TNFRSF9基因相似度不高,表明该基因虽有特殊结构域但不同物种间差异较大。组织表达分析显示,BbgTNFRSF11b、BbgTNFRSF9基因在耳后腺中表达显著高于其他部位。结论:成功获得蟾蜍BbgTNFRSF11b、BbgTNFRSF9基因序列,掌握其序列特征,为后续深入研究该蛋白的功能提供参考。Objective:To characterize the full-length sequences of BbgTNFRSF11b and BbgTNFRSF9(members of tumor necrosis factor receptor superfamily)cloned from Bufo gargarizans,so as to provide an experimental basis for deciphering the anti-tumor mechanism of B.gargarizans skin in the future.Methods:The full-length cDNA sequences of BbgTNFRSF11b and BbgTNFRSF9 genes were obtained from the skin of B.gargarizans with the specific primers designed based on the TNFRSF11b and TNFRSF9 sequences obtained by transcriptome sequencing,and their sequence characteristics were analyzed by bioinformatics tools.The expression levels of BbgTNFRSF11b and BbgTNFRSF9 in 7 tissues of B.gargarizans were measured by real-time fluorescence quantitative PCR.Results:The full-length cDNA sequence of BbgTNFRSF11b was 1233 bp,encoding 410 amino acid residues.The deduced protein had a theoretical relative molecular weight of 46.97 kDa and an isoelectric point of 8.64.It was a hydrophilic protein with multiple phosphorylation sites and no transmembrane domain or signal peptide.The full-length cDNA sequence of BbgTNFRSF9 was 829 bp,encoding 247 amino acid residues.The deduced protein had a theoretical relative molecular weight of 67.874 kDa and a theoretical isoelectric point of 5.12.It was a hydrophobic protein with a signal peptide and multiple phosphorylation sites.The phylogenetic tree showed that BbgTNFRSF11b and BbgTNFRSF9 were not similar to those in other animals,indicating that the gene had special domains different among species.The expression of BbgTNFRSF11b and BbgTNFRSF9 in the retroauricular gland was significantly higher than that in other tissues.Conclusion:We obtained and characterized the sequences of BbgTNFRSF11b and BbgTNFRSF9,which provided an experimental basis and a theoretical basis for further research on the function of the protein.

关 键 词：蟾蜍 基因克隆 序列分析 肿瘤坏死因子受体超家族 

分 类 号：R282.7[医药卫生—中药学]