Cloning and Expression of KIF2A Gene During Follicular and Oocyte Development in Yak

doi:10.7668/hbnxb.20193224

Abstract

Abstract:

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.

Key words: Yak, KIF2A genes, Sequence characteristics, Tissue expression, Oocyte

YANG Manzhen, MIN Xingyu, YANG Luyu, YU Hailing, HU Yulei, ZHU Yanjin, PAN Bangting, LI Jian, XIONG Xianrong. Cloning and Expression of KIF2A Gene During Follicular and Oocyte Development in Yak[J]. Acta Agriculturae Boreali-Sinica, 2022, 37(6): 219-227. doi: 10.7668/hbnxb.20193224.

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Figures/Tables 9

Tab.1 PCR primer sequences and products information

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

Tab.2 Bioinformatics analysis contentsand tools

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	https://www.ncbi.nlm.nih.gov/orffinder/
Prediction of ORF finder and translation of amino acid sequence
序列比对Sequence alignment	https://blast.ncbi.nlm.nih.gov/Blast.cgi
蛋白质理化性质分析Analysis of physicochemical properties of proteins	https://web.expasy.org/protparam/
蛋白质亲疏水性分析Hydrophilic and hydrophobic analysis of proteins	https://web.expasy.org/protscale/
相互作用蛋白预测Interaction protein prediction	https://cn.string-db.org/cgi/networki
蛋白质功能结构域预测Prediction of protein functional domain	https://myhits.sib.swiss/cgi-bin/motif_scan
蛋白质二级结构的预测Prediction of protein secondary structure	https://npsa-prabi.ibcp.fr/cgi-bin/
蛋白质三级结构的预测Prediction of tertiary structure of proteins	https://swissmodel.expasy.org/interactive
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 gene A: 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.

Fig.2 Bioinformatics of yak KIF2A gene A.Hydrophilic and hydrophobic analysis of yak KIF2A protein;B.Phosphorylation site analysis of yak KIF2A protein;C.Prediction of yak KIF2A protein interaction protein.

Fig.3 Functional domain,secondary structure and tertiary structure of yak KIF2A protein A.Functional domain analysis of yak KIF2A protein;B.The secondary structure of yak KIF2A protein;C.The tertiary structure of yak KIF2A protein.

Fig.4 Homology analysis and phylogenetic tree of yak KIF2A gene A.Homology analysis of KIF2A among different species;B.Phylogenetic tree of yak KIF2A.

Fig.5 Relative expression levels of KIF2A gene in different tissues Different lowercase letters indicate significant difference (P<0.05).The same as Fig.7.

Fig.6 Expression and location of KIF2A in yak ovary A.General picture of yak ovary;B.Antral follicle,C.Local enlargement of antral follicle: GC.Granular cell,TC.Theca cells.

Fig.7 Relative expression levels of KIF2A in follicular granulosa cells and oocytes of yaks at different developmental stages A.Relative expression levels of KIF2A in small,medium and large follicular granulosa cells; B.Relative expression levels of KIF2A in oocytes at different stages.

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