猪精氨酸-丝氨酸蛋白激酶1(SRPK1)克隆表达及功能初步分析  

Cloning and Functional Analysis of Serine/arginine-rich Specific Kinase 1 in Pig

作　　者：俄广鑫[1] 刘娣 张冬杰[2] 杨秀琴[1] 崔羽[3] 祝继原[1] 杨少成[1] 王嘉博[1] 谢宇彤[1] 

机构地区：[1]东北农业大学动物科学技术学院,哈尔滨150030 [2]黑龙江省农业科学院畜牧中心,哈尔滨150086 [3]海南大学农学院,海口570228

出　　处：《畜牧兽医学报》2010年第11期1379-1386,共8页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金："十一五"国家科技支撑计划重点项目"优质瘦肉型猪新品种选育与产业化开发"(2008BADB2B02)

摘　　要：本研究旨在阐明猪SRPK1基因的分子特点和表达谱。以大白猪为研究对象,利用RT-PCR技术克隆SRPK1基因CDS区域,同时利用反向PCR(inverse PCR,I-PCR)技术得到猪部分SRPK1的5′UTR序列;利用生物信息学方法分析SRPK1基因核苷酸序列及编码蛋白序列的结构特点;利用荧光定量PCR(real-time PCR)检测SRPK1在1和30日龄大白猪及杜洛克的心脏、肌肉、脾脏、肝脏、肾脏、肺脏、胃、小肠、大肠、脑的表达情况;构建猪骨骼肌损伤模型,研究在骨骼肌发育过程中SRPK1基因的表达特性。结果,将克隆片段拼接得到长2499bp的大白猪SRPK1基因序列,包含了1968bp完整CDS区域,分析表明其编码含656个氨基酸的蛋白质;包括2个P_Kc结构域,猪SRPK1蛋白序列与人和黑猩猩的相似性较高。通过生物信息学方法预测所得5′UTR含有多个转录结合位点:HSF1、HSF2、Ik-1、IK-2、SRY、SP1MyoD、USF、E47、p300、CP2、RREB-1、E2F和AP-1。利用荧光定量PCR研究发现,表达结果显示猪该基因表达具有组织和种间特异性,主要都是在胃、大肠和小肠中表达。SR-PK1基因在整个损伤修复过程中的表达反复出现升高和降低。5′UTR处有多个和肌肉生长相关蛋白的转录结合位点,主要在消化系统组织表达,在骨骼肌修复中表达上调,推测其可能与骨骼肌细胞发育或其他肌肉生长相关因子的表达有关。The objective of this study was to illuminate the molecular features and the expression profile of the porcine SRPK1 gene.The SRPK1 gene from the Yorkshire pig was cloned and sequenced,and then its molecular features were analyzed.Use inverse PCR to clone the partial 5′UTR sequence of SRPK1 in pig.The mRNA distribution profile of the porcine SRPK1 gene in ten tissues（heart,muscles,liver,kidney,lung,stomach,small and large intestine,spleen,brain） of Yorkshire and Duroc（one-day,one-month） were examined by real-time PCR.Construct pig skeletal muscle injury model by subcutaneous injection to study the expression characteristics of SRPK1 gene in repair process of skeletal muscle.A sequence with a length of 2 499 bp was cloned,it contained the CDS（coding sequence） region of 1 968 bp of the porcine SRPK1 gene,encoded a protein composed of 656 amino acids,including two P_Kc domains.The sequence of the porcine SRPK1 protein shared high similarity with human and chimpanzee,and they were closed in the Phylogenetic tree.The result of bioinformatics analysis showed that 5′UTR obtained in the study contained multiple transcription binding sites：HSF1,HSF2,Ik-1,IK-2,SRY,SP1 MyoD,USF,E47,p300,CP2,RREB-1,E2F and AP-1.The study results showed that,by real-time PCR,pigs had the organization-and species-specific in expression,but highly in stomach,small intestine and large intestine.The expression of SRPK1 gene fluctuated in the injury and repair process.There are several transcription binding sites in 5′UTR related to muscle development,it mainly expressed in digestive system and the expression level increased in injury and repair process of skeletal muscle.It was inferred that SRPK1 gene may be related to skeletal muscle cell development,or other growth-related factor expression.

关 键 词：精氨酸/丝氨酸蛋白激酶1 猪 实时荧光定量PCR 反向PCR 骨骼肌损伤模型 生物信息学分析 

分 类 号：S828[农业科学—畜牧学] Q527[农业科学—畜牧兽医]