牦牛hnRNP K基因的克隆及序列分析

Cloning and Sequence Analysis of hnRNP K Gene in Bos grunniens

为研究hnRNP K基因的生物学功能及其在牦牛中的特异性,利用RT-PCR和粘性末端连接法,分两段克隆了牦牛hnRNP K基因cDNA序列。序列分析结果表明,牦牛hnRNP K基因cDNA序列长11706bp,开放阅读框(ORF)长1389bp,编码463个氨基酸。序列比对结果表明,牦牛与黄牛hnRNP K cDNA序列的同源性达99.1%,编码的氨基酸同源性达到97.0%;在牦牛氨基酸序列中有15个突变。通过同源建模的方法成功构建了牦牛hnRNP K蛋白质三级结构,结果表明牦牛hnRNP K属于A型结构,而黄牛hnRNP K蛋白属于B型结构,其差异是由第459-463位氨基酸序列由"ADVEG"突变为"SGKFF"所致。乙酰化分析结果显示,牦牛hnRNP K对基因转录的影响水平跟黄牛是一致,表明不同物种hnRNP K功能的差异可能跟其氨基酸序列的差异有关。成功克隆的牦牛hnRNP K基因的cDNA序列为进一步分析该基因的功能提供参考。

In order to study the biological function of hnRNP K gene and its specificity in Bos grunniens,the eDNA sequence of hnRNP K gene in Bos grunniens was cloned by RT-PCR and cohesive end connecting method. Results showed that the size of hnRNP K eDNA was 11 706 bp, and the ORF was 1 389 bp, encoding 463 amino acids, which performed similarity to Bos taurus （nueleotide： 99.1% ;amino acids：97.0% ）. There were 15 mutations in deduced amino acids of hnRNP K protein sequence in Bos grunniens, The tertiary structure of Bos grunniens hnRNP K protein belongs to type A according to the results of homology modeling,and Bos taurus hnRNP K protein belongs to type B. The tertiary structure difference between two kinds of structure model was due to the mutations from ADVEG（type B）to SGKFF（ type A）at position 459 -463 in hnRNP K amino acids sequence. Acetylizing analysis showed that hnRNP K of Bos grunniens performs the same functions as Bos taurus＇ during the process of transcription. Therefore,it can be deduced the different functions of hnRNP K among different species were relate to the discrepancy of its amino acid sequence. The resuits of this study suggests that hnRNP K of Bos grunniens was cloned successfully and could provide reference for future analysis.