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本实验采用RT-PCR方法克隆关岭牛MyoD基因的CDS区,利用Eco RⅠ、XhoⅠ酶对目的片段与pET32a(+)原核表达载体进行酶切,T4连接酶连接过夜,通过转化,测序,构建重组表达载体,并对该基因进行相关生物信息学分析。实验结果获得了关岭牛Myo D基因CDS区,成功构建了p ET-32a(+)-MyoD重组表达载体。MyoD基因编码区为954 bp,编码318个氨基酸,共有31个稀有密码子,表达蛋白大小为34.2 kD。该蛋白为水溶性蛋白,等电点p I=5.8,在体内带负电,二级结构中α-螺旋、无规则卷曲较多。蛋白没有信号肽和明显的跨膜区,为胞内蛋白,主要存在于细胞核中。重组载体表达的Myo D蛋白带有His等标签,蛋白大小为53.6 kD,共497个氨基酸。实验对关岭牛Myo D蛋白的理化性质、结构特点进行了初步分析,以期为研究牛MyoD蛋白特性及其作用机制奠定理论基础。
Abstract：The experiment used the method of RT-PCR to clone CDS region of Guanling Myo D gene and digested the fragment and prokaryotic expression vector p ET32a( +) by Eco R Ⅰ and Xho Ⅰ. Then it connected the two fragments to construct the recombinant expression vector by T4 ligase. Finally, it analysised the Myo D gene by bioinformatics software. The result showed that the experiment had succeeded in cloning CDS region of Guanling cattle Myo D gene and the recombinant prokaryotic expression vector of p ET-32a(+)-Myo D had been successfully constructed. Myo D gene coding region was 954 bp with a total of 31 rare codons and encoded 318 amino acids. The formula weight of Myo D protein was 34.2 k D. The protein was a kind of hydrophilic proteins and its isoelectric point was 5.8. It was the α-helix and random coil that mainly consisted of its secondary protein structure. Myo D protein was an intracellular protein, mainly in the nucleus, without transmembrane region and signal peptide. The protein prodced by recombinant Prokaryotic expression vector was a total of 497 amino acids and about 53.6 k D with tags, such as histidine tag. It is the preliminary analysis of the properties of Myo D protein that lays a theoretical foundation for further research on the characteristics and the regulation mechanism of Myo D protein.
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基本信息:
DOI：10.13417/j.gab.034.001413
中图分类号:Q78;Q811.4
引用信息:
[1]夏丹,许厚强,陈伟,等.关岭牛MyoD基因原核表达载体的构建及生物信息学分析[J].基因组学与应用生物学,2015,34(07):1413-1420.DOI:10.13417/j.gab.034.001413.
基金信息:
国家转基因生物新品种培育重大专项子课题[2013ZX08009-004];; 黔科合重大专项字[2013]6008号;; 贵州省科技厅农业攻关项目(黔科合NY字[2012]3008号);; 贵州大学研究生创新基金[研农2015031]共同资助
2015-07-25
2015-07-25