猪精氨酸-丝氨酸蛋白激酸3(SRPK3)基因的克隆、表达及多态性分析(英文)

Cloning,Expression and Polymorphism Analysis of Porcine SRPK3 Gene

通过对猪SRPK3基因初步的研究,为猪分子遗传育种提供基础分子生物学信息,为猪的遗传育种提供分子标记。以大白猪为实验材料,采用RT-PCR方法克隆了精氨酸-丝氨酸蛋白激酶3(serine/arginine-rich specific kinase 3,SRPK3)的全长基因CDS区;采用生物信息学方法分析了SRPK3基因核酸序列并对其所编码的的蛋白序列进行了预测与分析编码蛋白序列的结构特点;采用PCR-SSCP方法对大白猪,野猪,民猪及野家杂交猪的SRPK3基因的多态性进行了检验;采用实时荧光定量PCR(Real-time)方法检测了SRPK3在1日龄和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;采用皮下注射的方式构建猪骨骼肌损伤模型用于研究在骨骼肌修复过程中SRPK3基因表达特性。经拼接所得到的1 708bp核苷酸片段,涵盖了SRPK3基因的全长CDS(1 701bp),该基因编码含567个氨基酸片段;蛋白存在两个S_TKc结构域,猪SRPK3蛋白序列与人和牛的相似性较高。PCR-SSCP检测发现第6外显子上A629→G629,T653→T653的突变,氨基酸变化为Pro→His,Ile→Thr;第9外显子处的G1059→A1059,氨基酸无突变。利用荧光定量PCR研究发现,表达结果显示该基因表达具有组织和种间特异性。SRPK3基因的表达在整个骨骼肌细胞损伤修复过程中逐渐升高。SRPK3基因主要在肌肉和心肌内表达,骨骼肌损伤修复过程中伴随骨骼肌细胞分化SRPK3的表达持续升高,推测其可能与骨骼肌细胞发育相关。

Protein SRPK3 acted as a crucial element of transcription pre-initiation complex,which play an important role in regulation procession of gene expression.In order to explore the genetic characteristic of SRPK3 in pigs.SRPK3 gene came from Yorkshire a pig was cloned by RT-PCR yet coding sequence was completed.The distribution determination of mRNA came from ten Yorkshire pigs and Duroc pigs in heart,muscle,liver,kidney,lung stomach,small and large intestine,spleen,brain was finished by Real-time PCR by age one day and 30 days.Expression test of gene SRPK3 was implemented in a skeletal damage model during the period of skeletal muscles development.Sequence analysis of an mRNA fragment with a length of 1 708bp in gene SRPK3 of a Yorkshire pig revealed a full coding region,1 701bp,which coded 656 AAs including two S_TKc domains.The sequence of the porcine protein SRPK3 shared high similarity with its homolog from human and bovine,and they were closed in the Phylogenetic tree.PCR-SSCP test shows： On the Sixth exon（CDS： 629,CDS： 653） get A→G,T→C,which change the amino as Pro → His,Ile→Thr,on the ninth exon（CDS： 1059） get a G → A,but no change of amino.Both breeds specific expression and tissues specific expression were detected by RT-PCR,however high expression was mainly detected in skeletal muscle and heart.The quantity of mRNA of gene SPRK3 in the period of skeletal muscle destruction and repair,which showed gradually to be increased in different stage.Because of it＇s expressed in skeletal muscle and heart mainly,and gradually be increased in different stage of injury and repair process.SRPK3 may be related to skeletal muscle cell development.