Abstract: 【Objective】The aim of the study was to investigate the sequence characteristics and expression level of cyclin D2(CCND2) in yak ovary during different periods of estrus. 【Method】 Total RNA of yak ovary were extracted and reverse transcribed into cDNA. Specific primers were designed according to mRNA sequence of cattle CCND2 gene in the GenBank(GenBank accession No: NM_001076372.1) by software Primer 5.0. The CDs region and part of 5′UTR and 3′UTR in yak CCND2 gene were cloned from yak ovary by polymerase chain reaction(PCR) amplification and sequencing technology. The physicochemical properties, secondary structure, tertiary structure, domain, homology were analyzed and phylogenetic tree of CCND2 was constructed by online software like Protparam, SOPMA, SWISS-MODEL and SMART. Semi-quantitative PCR was used to examine the expression profiles of CCND2 gene in different tissues. Quantitative real-time PCR (qRT-PCR) was used to detect the expression level of CCND2 gene in the ovary during different periods of estrus (ovarian follicular phase, red body phase and luteal phase).【Result】The 909 bp length sequences of CCND2 gene in yak, including 870 bp length CDS region encoding 289 amino acids, were obtained. Alignment with cattle CCND2 gene CDS and amino acid, five bases mutations and one amino acid mutation were found. The CDs homology of CCND2 compared yak to wild yak, cattle, Indian buffalo, sheep, wild boar, horse, dog, cat and human was 99.54%, 99.31%, 99.31%, 98.16%, 94.81%, 90.46%, 90.80%, 91.49% and 88.62%, respectively. There was a high homology among different species, and phyletic evolution was the same as their genetic relationship. The research indicated that the CCND2 gene CDs was conservative in the course of evolution. The formula of protein encoded by CCND2 gene in yak was C₁₄₄₅H₂₃₁₅N₃₇₇O₄₄₀S₁₈, and the molecular weight was 32.59 kD, the theory isoelectric point was 4.75, the grand average of hydroparthicity was -0.107, and the instability index was 44.56. It was an unstable, soluble and acidic protein without transmembrane regions and signal peptide. Random coil and α-helix were mainly in the secondary structure of CCND2, which were the same as the tertiary structure of CCND2 protein. The amino acids sequence of CCND2 contains Cyclin_N domain in the 26-152 amino acids and Cyclin_C domain in the 154-257 amino acids. The CCND2 gene was expressed in various tissues of yak, and relatively higher in stomach, ovary and brain. qRT-PCR results showed that the mRNA expression level of CCND2 in ovary of follicular phase was the highest (P＜0.01), and no difference between the red body phase and luteal phase (P＞0.05).【Conclusion】The complete CDs of CCND2 gene was successfully cloned and the tissues expression profiles were clarified, and its bioinformatics and encoded protein function and structure were investigated. These provide a theoretical basis for further study of CCND2 gene in regulation of yak reproduction. The different expressions of CCND2, in different periods of estrus in yak, might be related to proliferation and differentiation of granulose cells, development process of follicular wave, and ovarian endocrine. These indicated that CCND2 plays an important role during the reproductive cycle, and these results of study will provide basic information for further study on the ovarian development mechanism and improving reproductive competence in yak.

Key words: yak, CCND2, cloning, estrous cycle, ovary, expression