水貂PRLR基因的克隆及生物信息学分析

Cloning and Bioinformatics Analysis of Prolactin Receptor Gene of Mink

本研究旨在从水貂卵巢组织中克隆催乳素受体(prolactin receptor,PRLR)基因编码区全长序列,并进行生物学信息分析,为研究其结构和功能奠定基础。根据GenBank中登录的犬PRLR基因的mRNA预测序列(登录号:XM_025436332.1)设计1对引物,采集性成熟的健康水貂卵巢组织样本,提取RNA并反转录合成cDNA,以cDNA为模板PCR扩增PRLR基因,将其插入pEASY-Blunt Simple Vector中,筛选阳性克隆测序后进行生物信息学分析。结果显示,水貂PRLR基因编码区序列全长为1 878bp,编码了625个氨基酸,水貂PRLR基因核苷酸序列与海獭同源性最高,为97.7%。系统进化树分析也显示其和海獭亲缘关系最近。对水貂卵巢PRLR基因编码蛋白进行生物信息学分析表明,PRLR蛋白含有1个信号肽,1个细胞外区域,1个跨膜区和1个膜内区;水貂PRLR蛋白为单次跨膜蛋白,其N端的D1部分含有2对保守的二硫键(Cys36—Cys46和Cys75—Cys86),D2部分含有保守的WS-基序(WS-E-WS)。水貂PRLR蛋白具有和其他PRLR蛋白相似的膜外和膜内结构。本试验结果为进一步研究和揭示水貂PRLR蛋白的结构及功能奠定了基础。

The aim of this study was to clone the full-length coding sequence of prolactin receptor(PRLR)gene from mink ovary tissues and analyze its biological characteristics to lay a foundation for studying its structure and function.A pair of primers was designed based on the predicted mRNA sequence of the dog PRLRgene in GenBank(accession No.:XM025436332.1),and the ovary tissue samples of sexually mature healthy mink were collected,and RNA was extracted and then was reverse transcribed to synthesize cDNA,and PRLRgene was amplified by PCR using cDNA as template.The PCR products then were inserted into the pEASY-Blunt Simple Vector.Bioinformatics analysis was carried out after positive clone sequencing.The results showed that the coding region of PRLRgene of mink was 1 878 bp in length and encoded 625 amino acids.The homology of nucleotide sequence of PRLRgene between mink and Enhydra lutris kenyoni wasthe highest which reached 97.7%.The phylogenetic tree analysis also showed the closest relationship with Enhydra lutris kenyoni.The bioinformatics analysis of the PRLR protein of mink ovary showed that it contained a signal peptide,an extracellular region,a transmembrane region and an intramembranous region.It was a single transmembrane protein,and the D1 domain at the N-terminal contained two pairs of conserved disulfide bonds(Cys36-Cys46 and Cys75-Cys86),the D2 domain contained a conserved WS-motif(WS-E-WS).The PRLR protein of mink had a similar structure to other PRLR proteins.In conclusion,the study provided basic experimental data and theoretical basis for the further study of the structure and function of PRLR protein.