内蒙古白绒山羊erk2基因的克隆及表达模式分析  

Cloning and expression pattern of erk2 gene in Inner Mongolia Cashmere Goat

作　　者：王彦凤[1] 吴曼琳[1] 王晓晶[2] 王婧[1] 李洋[1] 连梦瑶 王志钢[1] 

机构地区：[1]内蒙古大学生命科学学院,内蒙古呼和浩特010021 [2]赤峰市医院,内蒙古赤峰024000

出　　处：《生物工程学报》2013年第12期1743-1752,共10页Chinese Journal of Biotechnology

基　　金：国家自然科学基金(No.31160469);内蒙古自然科学基金(No.2011MS0521)资助~~

摘　　要：旨在克隆内蒙古白绒山羊erk2基因cDNA并分析其基本表达模式。采用RT-PCR方法克隆白绒山羊erk2基因cDNA。通过在线软件Blast进行核酸序列分析,用SMART与Psite进行氨基酸序列分析。定量RT-PCR检测erk2基因在绒山羊组织中的表达特异性。免疫组化法检测绒山羊睾丸中erk2表达。克隆到的内蒙古白绒山羊erk2基因cDNA片段(GenBank Accession No.JX569765)长1 083 bp,包含了编码360个氨基酸残基的全长ORF,氨基酸序列与牛的ERK2(Bos Taurus BC133588.1)同源性为100%。SMART分析表明,ORF编码的蛋白包含了活化位点"TEY"及具有丝氨酸/苏氨酸激酶催化活性的S-TKc结构域。Psite分析表明,含2个N-糖基化位点、1个依赖于cAMP/cGMP的蛋白激酶磷酸化位点、3个蛋白激酶c磷酸化位点、5个酪蛋白激酶Ⅱ磷酸化位点、2个N-豆蔻酰化位点、2个异戊二烯基结合区(CAAX box)、7个微体羧基端靶向信号、2个蛋白激酶ATP结合区标记及一个丝/苏氨酸蛋白激酶活性区域标记。PSORT(k-NN prediction)程序预测其定位于细胞质中。定量RT-PCR分析显示erk2基因mRNA丰度在心脏、皮肤以及乳腺组织中mRNA丰度较高,脾、肾中的表达相对较低。在睾丸中检测到ERK2蛋白表达。The study aims at cloning the CDS fragment of erk2 gene cDNA in Inner Mongolia Cashmere Goat and analyzing its tissue-specific expression, erk2 gene cDNA was cloned by RT-PCR. The nucleotide sequence was analyzed by Blast and amino acid sequence was analyzed by online softwares SMART and Psite. The tissue-specific expression pattern of erk2 was analyzed by quantitative RT-PCR. The expression of erk2 in testis of goat was detected by Immunohistochemistry. The cloned erk2 gene cDNA （GenBank Accession No. JX569765） was 1 083 bp in length, including a complete ORF encoding 360 amino acids residues. The amino acid sequence shares 100% identity with the Bos Taurus ERK2 （Bos Taurus BC133588.1）. Analysis by SMART suggests that the encoded protein contained a ＂TEY＂ structure and an S-TKc domain possessing serine/threonine kinase catalytic activity. Analysis with Psite indicates one cAMP-/cGMP-dependent protein kinase phosphorylation site, 3 protein kinase C phosphorylation sites, 5 casein kinase lI phosphorylation sites, 2 protein kinases ATP-binding region signatures and one serine/threonine protein kinases active-site signature in this protein. Analysis by Psort （k-NN prediction） suggestes that this protein most probably is localized in cytoplasm. The results of quantitative RT-PCR show that the expression of erk2 mRNA was higher in heart, skin and breast, whereas lower in spleen and kidney. ERK2 protein was detected in testis by immunohistochemistry.

关 键 词：内蒙古白绒山羊 ERK2 基因克隆 表达模式分析 

分 类 号：S827[农业科学—畜牧学]