关键词: 牦牛 , 胞红蛋白 , 基因克隆 , 生物信息学

Abstract: 【Objective】In order to enrich basic data in yak CYGB gene, CDS region of yak CYGB gene was cloned and analyzed by bioinformatics method. 【Method】 Total RNA of yak hippocampus tissue was extracted and reverse transcribed into cDNA by RT-PCR technology. Specific primers were designed according to cDNA sequence of cattle CYGB gene in the GenBank (GenBank accession No.：DV874786.1) by online software Primer 3.0. The CDS region and part of 5′ UTR and 3′ UTR in yak CYGB gene were cloned from yak hippocampus total RNA by PCR amplification, TA cloning and nucleic acid sequencing technology. The primary structure, secondary structure, tertiary structure, physicochemical properties, homology were analyzed and phylogenetic tree of CYGB was constructed by online software like ProtParam, PredictProtein, SWISS-MODEL and Lasergene7.1 software package. The three-dimensional structure was modified and output by PyMol software. The protein subcellular localization was predicted by online subcellular localization tool PSORT II Prediction, and the protein function was predicted by Protfun software.【Result】The 650 bp length CYGB gene in yak was got by cloning, including the 573 bp length CDS region (GenBank accession No.：KF669898), and the bases composition were 20.59%A, 16.40%T, 33.33%G, 29.67%C, encoding 190 amino acids. Alignment with CDS and amino acid sequence of cattle CYGB gene, four base mutations were found and amino acid was not mutated, four mutations are synonymous mutations. The formula of protein encoded by CYGB gene in yak was C964H1513N263O278S7, and the molecular weight was 21.5 kD, the theory isoelectric point was 6.32, the extinction coefficient was 24075, the instability index was 48.43, the aliphatic index was 83.63, and the grand average of hydropathicity was -0.301. It was an unstable and soluble protein. Its estimated half-life is 30 hours in mammal reticulocyte. The secondary structure of CYGB was mainly α-helices and random coil, α-helices was 64.21% and random coil was 35.79%, CYGB was an α class protein. The tertiary structure of CYGB was a “three-over-three” α-helical sandwich structure. Subcellular localization of CYGB was in the cytoplasm (65.2%), nucleus (17.4%), mitochondria (13.0%) and vesicles of secretory system (4.3%), mainly in the cytoplasm, which CYGB may play a role of signal transducer or transcription regulation in the energy metabolism and cofactor biosynthesis. The amino acid homology of CYGB compared yak to cattle, sheep, dog, mouse, rat, chicken, monkey, chimpanzee and human were 100%，98.9%，97.8%，95.3%，93.7%，78.8%，98.4%，95.8% and 96.8%, respectively. There was a high homology among different species, and phyletic evolution was the same as their genetic relationship. The research indicated that the CYGB gene coding region was conservative in the course of evolution.【Conclusion】The 573 bp length CDS region of yak CYGB gene was got by RT-PCR, TA cloning and nucleic acid sequencing technology for the first time, and the nucleotide sequence, the encoded amino acid sequence and protein structure and function were analyzed. CYGB in yak was a soluble and acidic protein that 190 amino acid residues composed, which plays an important role in the energy metabolism and cofactor biosynthesis. CYGB gene coding region was conservative in the course of long-term biological evolution. The gene was cloned and analyzed, thus providing a theoretical basis for revealing the genetic characteristics of yak CYGB gene.

Key words: yak , cytoglobin , gene cloning , bioinformatics