Cloning and Expression of KIF2A Gene During Follicular and Oocyte Development in Yak
Manzhen YANG, Xingyu MIN, Luyu YANG, Hailing YU, Yulei HU, Yanjin ZHU, Bangting PAN, Jian LI, Xianrong XIONG
Cloning and Expression of KIF2A Gene During Follicular and Oocyte Development in Yak
The aim of this study was to clone the kinesin family member 2A gene(KIF2A),and explore the expression pattern of KIF2A gene in different tissues of yak,as well as the spatially and temporally expressed during the follicular development and oocyte meiotic maturation.The heart,liver,spleen,lung,kidney stomach,uterus and ovary tissues(n=5)were collected from 3—4 years old healthy female yak after slaughter.Yak KIF2A gene was amplified by RT-PCR with yak ovarian cDNA as template,and its protein were analyzed by bioinformatics software such as MAGA 7.0 and SWISS-MODEL etc.Quantitative Real-time PCR(RT-qPCR)was used to detect the expression level of KIF2A in yak various tissues and immunohistochemistry was used to analyze the expression and localization of KIF2A in follicles with different diameters.According to the diameter,the follicle were divided into three groups: small(diameter 1.0—2.9 mm),medium(diameter 3.0—5.9 mm),and large(diameter 6.0—9.0 mm)follicle.The expression characteristics of KIF2A gene in granulosa cells and oocyte meiosis in follicles were analyzed by RT-qPCR.The results showed that sequence length of KIF2A gene was 1 964 bp,and the CDS was 1 530 bp,which encoding 509 amino acids.KIF2A protein was negatively charged and belonged to hydrophobic protein.KIF2A gene was relatively conservative and widely expressed in various tissues of yaks, and KIF2A protein was mainly located in granulosa cells, its mRNA expression in follicular granulosa cells with different diameter and size increased with the growth and development of follicles. During the meiotic maturation of yak oocytes,the expression of KIF2A showed a sequential feature,and the G Ⅴ phase was significantly higher than that of M Ⅰ and M Ⅱ phase.The results showed that KIF2A might be involved in the regulation of follicular and oocyte maturation development in yaks,and the results of this study provided basic data for the functional study of KIF2A gene in yak follicular development.
Yak / KIF2A genes / Sequence characteristics / Tissue expression / Oocyte {{custom_keyword}} /
Tab.1 PCR primer sequences and products information表1 PCR引物序列及产物信息 |
| 基因 Gene | 引物(5'—3') Sequence of primer(5'—3') | 产物长度/bp Product size | 退火温度/℃ Tm | 用途 Utilization |
|---|---|---|---|---|
| KIF2A | F:TTTGCTTATGGGCAGACT | 1 964 | 59 | 克隆 |
| R:ACATTTGACCTCCCGTTC | ||||
| KIF2A | F:TCCCACCACTTGAACCTG | 151 | 60 | RT-qPCR |
| R:AATGCTAAAGAAGCCGAACT | ||||
| GAPDH | F:TGCGTGCTGAGTAGTTGGTG | 290 | 59 | RT-qPCR |
| R:TCTTCTGGGTGGCAGTGATGG |
| 注:F.正向引物;R.反向引物。 | |
| Note:F.Forword primer;R.Reverse primer. |
Tab.2 Bioinformatics analysis contentsand tools表2 生物信息学分析内容和工具 |
| 分析内容 Analysis contents | 分析工具 Analysis tools |
|---|---|
| 开放阅读框的预测及氨基酸序列的翻译 | |
| Prediction of ORF finder and translation of amino acid sequence | |
| 序列比对Sequence alignment | |
| 蛋白质理化性质分析Analysis of physicochemical properties of proteins | |
| 蛋白质亲疏水性分析Hydrophilic and hydrophobic analysis of proteins | |
| 相互作用蛋白预测Interaction protein prediction | |
| 蛋白质功能结构域预测Prediction of protein functional domain | |
| 蛋白质二级结构的预测Prediction of protein secondary structure | |
| 蛋白质三级结构的预测Prediction of tertiary structure of proteins | |
| 物种间氨基酸序列比对Amino acid sequence alignment between species | DNAMAN |
| 构建进化树Construction of evolutionary trees | MEGA 6.0 |
Fig.1 Amplification,and sequence comparison of yak KIF2A geneA: M.DL2000 DNA Marker,1.KIF2A gene;B. Predicted sequence of KIF2A gene and predicted amino acid sequence of yak;C. The difference between the determination sequence and the KIF2A coding region sequence of cattle.图1 牦牛KIF2A基因扩增、序列比对 A:M.DL2000 DNA Marker,1.KIF2A基因;B.牦牛KIF2A基因预测序列及推测的氨基酸序列;C.测定序列与黄牛KIF2A编码区序列差异。 |
Fig.3 Functional domain,secondary structure and tertiary structure of yak KIF2A proteinA.Functional domain analysis of yak KIF2A protein;B.The secondary structure of yak KIF2A protein;C.The tertiary structure of yak KIF2A protein.图3 牦牛KIF2A蛋白功能结构域、二级结构和三级结构 A.牦牛KIF2A蛋白功能结构域分析;B.牦牛KIF2A二级结构;C.牦牛KIF2A蛋白三级结构。 |
Fig.7 Relative expression levels of KIF2A in follicular granulosa cells and oocytes of yaks at different developmental stagesA.Relative expression levels of KIF2A in small,medium and large follicular granulosa cells; B.Relative expression levels of KIF2A in oocytes at different stages.图7 牦牛不同发育阶段卵泡颗粒细胞、不同时期卵母细胞KIF2A的相对表达水平 A.小、中、大卵泡颗粒细胞KIF2A的相对表达水平;B.不同时期卵母细胞KIF2A的相对表达水平。 |
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Tab.1 PCR primer sequences and products informationTab.2 Bioinformatics analysis contentsand tools
Fig.1 Amplification,and sequence comparison of yak KIF2A geneFig.2 Bioinformatics of yak KIF2A geneFig.3 Functional domain,secondary structure and tertiary structure of yak KIF2A proteinFig.4 Homology analysis and phylogenetic tree of yak KIF2A geneFig.5 Relative expression levels of KIF2A gene in different tissuesFig.6 Expression and location of KIF2A in yak ovaryFig.7 Relative expression levels of KIF2A in follicular granulosa cells and oocytes of yaks at different developmental stages/
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