大熊猫RPL35A基因的克隆及超表达研究  

Cloning and Overexpression of Ribosomal Protein L35A Gene(RPL35A) from Giant Panda

作　　者：杜玉杰 侯万儒[2] DU Yujie;HOU Wanru(Zhanjiang Preschool Education College,Zhanjiang Guangdong 524048,China;College of Life Sciences,China West Normal University,Nanchong Sichuan 637009,China)

机构地区：[1]湛江幼儿师范专科学校科学教育系,广东湛江524048 [2]西华师范大学生命科学院,四川南充637009

出　　处：《西华师范大学学报（自然科学版）》2018年第4期377-382,共6页Journal of China West Normal University(Natural Sciences)

基　　金：国家自然科学基金项目(31400016;31200012)

摘　　要：探究大熊猫核糖体蛋白基因RPL35A及其编码蛋白的特点,以进一步研究其编码蛋白的功能,为深入开展大熊猫分子生物学研究及大熊猫基因保护等积累科学资料;本研究以大熊猫骨骼肌为材料,采用RT-PCR和降落-PCR方法克隆RPL35A基因的cDNA基因组序列,IPTG法诱导基因在BL21细胞系中的表达;大熊猫RPL35A基因的cDNA序列为337 bp,编码110个氨基酸;该基因的基因组序列长1 275 bp,包含2个外显子和1个内含子;RPL35A基因在BL21细胞系中的表达产物与组氨酸标签蛋白融合形成18 k Da的融合蛋白;该基因的cDNA序列、基因组序列中的外显子片段及其编码的蛋白序列与已报道的人、牛、小家鼠、褐家鼠等物种的对应序列同源性很高,蛋白质理化性质和功能位点基本相同,这说明该基因及其编码蛋白在进化过程中非常保守,可用于分子进化方面的研究,在物种进化关系研究中具有一定的参考意义和研究价值;同时,该基因可在原核生物中表达出具有活性的产物,可进一步开展纯化和蛋白质功能的研究。To futher investigate the function of encoded protein,gather scientific data for molecular biology study of giant panda and protect gene resources of this endangered species,this paper explores the characteristics of ribosomal protein L35A gene(RPL35A)and its encoded protein.RT-PCR and touch-down PCR technology are used to clone the cDNA and the genomic sequence of RPL35A from giant panda and overexpressed in BL21 by IPTG.The results show that the length of cDNA is 333 bp encoding 110 amino acids and the genomic sequence is 1 275 bp in length possessing two exons and one introns.The expression protein of RPL35A in BL21 and N-terminally His-tagged protein give rise to the accumulation of an expected 18 kDa polypeptide fusion protein.The cDNA and its deduced amino acid sequence share a high homology with that of mammals reported,including Homo sapiens,Bos Taurus,Mus musculus and Rattus norvegicus.And their molecular weight as well as isoelectric point are basically the same.All of these illustrate that gene RPL35A and its encoded protein are very conservative in the process of evolution.These findings can be used in the study of molecular evolution.It is of certain reference significance and research value in phylogenic relationship.Meanwhile,its active overexpression in prokaryotic organism shows that further study on protein purification and its function can be conducted.

关 键 词：大熊猫 RT-PCR 降落-PCR RPL35A 克隆 表达 

分 类 号：Q786[生物学—分子生物学]