多浪羊PROKR 2基因克隆、生物信息学及组织差异表达分析  

Cloning,Bioinformatics and Tissue Differential Expression Analysis of PROKR 2 Gene in Dolang Sheep

作　　者：黄巧艳 李伟[1,2] 王鑫昆[1,2] 邢凤 HUANG Qiaoyan;LI Wei;WANG Xinkun;XING Feng(College of Animal Science and Technology,Tarim University,Alar 843300,China;Key Laboratory Tarim Animal Husbandry Science and Technology Xinjiang Production&Construction Corps,Alar 843300,China)

机构地区：[1]塔里木大学动物科学学院,阿拉尔843300 [2]新疆生产建设兵团塔里木畜牧科技重点实验室,阿拉尔843300

出　　处：《中国畜牧兽医》2024年第4期1339-1348,共10页China Animal Husbandry & Veterinary Medicine

基　　金：国家自然科学基金(31960655);塔里木大学研究生科研创新项目(TDGRI202243)。

摘　　要：【目的】克隆多浪羊促动力素受体2(prokineticin receptor 2,PROKR2)基因,并检测初情期启动过程中PROKR 2基因在多浪羊不同组织中的表达水平,为探究PROKR 2基因在绵羊初情期启动过程中的作用提供依据。【方法】以初情期后多浪羊下丘脑cDNA为模板,PCR扩增PROKR 2基因并克隆测序。利用DNAMAN软件对测序结果进行拼接,采用MegAlign软件进行物种间相似性比对并构建系统进化树,并利用生物信息学软件预测多浪羊PROKR2蛋白理化性质和结构功能。使用实时荧光定量PCR技术检测PROKR 2基因在多浪羊下丘脑、垂体、卵巢、输卵管及子宫中初情期前、初情期及初情期后的表达水平。【结果】克隆获得PROKR 2基因序列大小为2641 bp,包括5′-UTR 143 bp、3′-UTR 1343 bp和CDS区1155 bp,编码384个氨基酸,与GenBank中绵羊预测mRNA序列(登录号:XM_004014342.5)相似性为99.83%。系统进化树表明,多浪羊PROKR 2基因的遗传距离与山羊最近,与鸡最远。生物信息学分析表明,PROKR2蛋白为疏水稳定碱性蛋白,有7个跨膜结构,属于跨膜蛋白,存在31个磷酸化位点,主要在质膜上发挥作用。多浪羊PROKR2蛋白与GnRH1、PROK1、ANOS1等蛋白存在相互作用关系。实时荧光定量PCR结果显示,在多浪羊下丘脑各时期中PROKR 2基因表达量均显著高于其他组织(P<0.05),且在初情期后的表达量最高;垂体中PROKR 2基因表达量无显著变化(P>0.05);子宫中PROKR 2基因在初情期前的表达量显著高于初情期和初情期后(P<0.05);卵巢和输卵管中PROKR 2基因在初情期的表达量显著高于初情期前和初情期后(P<0.05)。【结论】试验成功克隆了多浪羊PROKR 2基因CDS区序列,PROKR2蛋白属于疏水稳定碱性蛋白,含有7个跨膜结构,主要在下丘脑中表达,且在卵巢和输卵管中的总趋势为先升后降。研究结果可为进一步探究PROKR 2基因在绵羊初情期启动过程中的调控作用提供参考。【Objective】This study was aimed to clone prokineticin receptor 2(PROKR2)gene,and detect the expression of PROKR 2 gene in different tissues during the initiation of puberty in Dolang sheep,so as to provide a basis for exploring the role of PROKR 2 gene in the initiation of puberty in sheep.【Method】Using the hypothalamic cDNA in Dolang sheep after first puberty as a template,PROKR 2 gene was amplified by PCR,cloned and sequenced.DNAMAN software was used to splice the sequencing results,MegAlign software was used to compare the similarity among different species and construct a phylogenetic tree,and the physicochemical property and structural function of PROKR2 protein in Dolang sheep were predicted by bioinformatics software.Real-time quantitative PCR was used to detect the expression of PROKR 2 gene in hypothalamus,hypophysis,ovary,oviduct and uterus of Dolang sheep in prepuberty,puberty and postpuberty.【Result】The cloned sequence size of PROKR 2 gene was 2641 bp,including 5′-UTR 143 bp,3′-UTR 1343 bp and CDS region 1155 bp,encoding 384 amino acids,and the similarity was 99.83%with the predicted mRNA sequence in sheep on GenBank(accession No.:XM_004014342.5).The phylogenetic tree showed that the genetic distance of the PROKR 2 gene in Dolang sheep was the closest to Capra hircus,and the farthest to Gallus gallus.Bioinformatics analysis showed that PROKR2 protein was a hydrophobic and stable basic protein with 7 transmembrane structures,which belonged to the transmembrane protein,and there were 31 phosphorylation sites,which mainly played a role in the plasma membrane.There was an interaction relationship between proteins such as GnRH1,PROK1,ANOS1 and PROKR2 protein in Dolang sheep.The results of Real-time quantitative PCR showed that the expression of PROKR 2 gene in each stage of hypothalamus was significantly higher than that of other tissues(P<0.05),and the expression of PROKR 2 gene was the highest in the postpuberty stage,while there was no significant change in hypophysis(P>0.05).The express

关 键 词：多浪羊 PROKR 2基因 克隆 初情期 表达 

分 类 号：S826[农业科学—畜牧学] Q78[农业科学—畜牧兽医]