关岭牛MyoD基因CDS区的克隆、序列分析及其真核表达载体构建

Cloning and sequence analysising of the CDS region of Guanling cattle MyoD gene and construction of its eukaryotic expression vector

[目的]通过关岭牛MyoD基因CDS区的克隆、序列分析及pcDNA3.1(+)-MyoD真核表达载体的构建,为后续研究MyoD基因的调控机制奠定基础。[方法]通过设计特异性引物克隆关岭牛MyoD基因的CDS区,并用DNAStar、Ex PASy等软件对该基因CDS区进行序列分析;同时利用双酶切的方法构建真核表达载体pcDNA3.1(+)-MyoD。[结果]关岭牛MyoD基因的CDS区全长957bp,编码318个氨基酸;其编码的蛋白属于亲水性蛋白,蛋白二级结构主要以α-螺旋及无规则卷曲为主,含有b HLH结构域,无明显的跨膜区域。同源性分析显示,关岭牛MyoD基因的CDS区序列与海福特牛和山羊的同源性最高,核苷酸同源性为98.96%和96.9%,氨基酸同源性为100%和89.3%。MyoD蛋白的氨基酸肽链长度由低等动物到高等动物有逐渐加长的趋势。[结论]成功克隆了关岭牛MyoD基因的CDS区,并构建了其真核表达载体pcDNA3.1(+)-MyoD,为进一步研究MyoD基因在成肌分化过程中的作用及其调控机制奠定基础。

[Objective] To clone the CDS region of Guanling cattle MyoD gene and analysis its sequence and construct its eukaryotic expression vector,which maked preparations for follow-up study the regulation mechanism of Guanling cattle MyoD gene. [Methods]The primers were designed to amplify the CDS region of Guanling cattle MyoD gene. DNAStar and Ex PASy softwares are used to analyse the sequenceing of Guanling cattle MyoD gene. At the same time,the Guanling cattle MyoD gene was inserted in plasmid vector pcDNA3. 1（＋）after double enzymed to construct eukaryotic expression vector pcDNA3. 1（＋）-MyoD. [Results]The results indicated that the total length of CDS region of Guanling cattle MyoD gene is 957 bp,which encoded 318 amino acids. The formula weight of Guanling cattle MyoD protein is about 34. 22 k Da,and its theory isoelectric point is 5. 67. It is a hydrophilic protein. Its secondary structure mainly consists of α-helix and random curl,contain basic helix-loop-helix structure domain. There is no obvious transmembrane domain in its amino acid sequence. Homology analysis showed that it has the highest homology with Hereford and goat,nucleic acid sequence with Hereford and goat homology,are 98. 96% and 96. 9%,respectively,the amino acid homology are 100% and 89. 3%. The result indicated that the amino acid peptides length of MyoD protein is a tendency of gradually increasing from lower to higher animals. [Conlusion]This experiment cloned the CDS area of Guanling cattle MyoD gene and constructed its eukaryotic expression vector pcDNA3. 1（＋） – MyoD successfully,which lay a experiment foundation for further research on the mechanism of regulation and the effect of MyoD protein in myogenic differentiation process.