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

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。

Abstract:

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 II 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.