牦牛<i>PIK3CB</i>基因克隆及其在卵泡发育过程中的表达研究

doi:10.7668/hbnxb.20191376

摘要/Abstract

摘要： 旨在探讨磷脂酰肌醇-3激酶催化亚基β（Phosphoinositide-3 kinase，PI3K，catalytic subunit beta，PIK3CB/P110β）基因序列特征，比较分析其在牦牛不同发育阶段卵泡中的表达规律。通过RT-PCR技术从牦牛卵巢组织中克隆获得PIK3CB基因序列并对其进行生物信息学分析；采用RT-qPCR方法检测PIK3CB基因在牦牛不同组织中的表达水平；将采集的牦牛卵泡根据直径大小分为大（≥7 mm）、中（3.0~6.9 mm）、小（≤2.9 mm）3组，分别收集各组卵泡中的壁颗粒细胞及卵母细胞提取总RNA，采用RT-qPCR方法检测PIK3CB mRNA在各级别卵泡壁颗粒细胞及卵母细胞中的相对表达量。结果显示：克隆获得牦牛PIK3CB基因CDS区长3 213 bp，共编码1 070个氨基酸。蛋白质分析显示，PIK3CB蛋白为亲水酸性蛋白，无跨膜结构无信号肽，二级结构主要由α-螺旋和无规卷曲组成。PIK3CB核苷酸同源性及遗传进化树分析显示，牦牛与野牦牛和黄牛亲缘关系最近。PIK3CB基因在牦牛心脏、脾脏、肾脏、肌肉、肝脏、肺脏、子宫、胃、小肠、卵巢组织中均有表达，尤其在脾脏、子宫和卵巢组织中表达量显著高于其他组织（P<0.05）。RT-qPCR结果显示，PIK3CB基因在卵泡发育过程中均有表达，其中在各级别卵泡壁颗粒细胞中，mRNA表达量随着卵泡发育进程呈现上升趋势，且大、中级别卵泡中的表达量极显著高于小卵泡组（P<0.01）；但是卵母细胞中mRNA表达量差异不显著（P>0.05）。结果提示，mRNA基因参与了牦牛卵泡发育调控，是PI3K信号通路在调节颗粒细胞的功能中不可或缺的催化亚基之一，具体调控机制有待进一步研究。本研究为深入探讨PI3K-AKT信号通路在卵巢发育中的调控机理提供基础资料。

关键词: 牦牛, PIK3CB, 组织表达, 卵泡

Abstract: The aim of this study was to investigate the sequence characteristics of phosphatidylinositol-3 kinase catalytic subunit β(PIK3CB/P110β) gene and compare its expression profile in different developmental stages of yak follicles.The sequence of PIK3CB gene was cloned from yak ovary tissue by RT-qPCR and analyzed by bioinformatics. The expression level of PIK3CB gene in different tissues of yak was detected by RT-qPCR method. The collected yak follicles were divided into three groups according to their diameter:large(≥ 7 mm), medium(3.0-6.9 mm) and small(≤ 2.9 mm). The total RNA was extracted from the oocytes and parietal granulosa cells of each group. Then, the relative expression of PIK3CB gene mRNA was detected by RT-qPCR.The results showed that the full-length CDS region of yak PIK3CB gene was 3 213 bp, encoding 1 070 amino acids. Bioinformatics analysis showed that PIK3CB protein was a hydrophilic acidic protein with no transmembrane structure and signal peptide, and the secondary structure was mainly composed of α-helix and random coil. The nucleotide homology and genetic tree analysis showed that yak PIK3CB gene was closely related to that of Bos mutus and Bos taurus. PIK3CB gene was expressed in heart, lung, kidney, liver, small intestine, stomach and muscle of yak, especially in spleen, uterus and ovary, the expression level was significantly higher than that in other tissues(P<0.05). The results of RT-qPCR showed that PIK3CB gene was expressed during follicular development, and in different follicular wall granulosa cells, the expression of mRNA increased with the development of follicles, and the expression in large and intermediate follicles was significantly higher than that in small follicles (P<0.01), but there was no significant difference in mRNA expression in oocytes(P>0.05).The results suggested that PIK3CB gene was involved in the regulation of yak follicular development, and it was one of the indispensable catalytic subunits of PI3K signal in regulating the function of granulosa cells. This study provides basic data for further study on the mechanism of PI3K-AKT signaling pathway in ovarian development.

Key words: Yak, PIK3CB, Tissue expression, Follicle

中图分类号:

S823.8+5
Q78

马鸿程, 熊显荣, 王晗, 海卓, 闵星宇, 李键. 牦牛PIK3CB基因克隆及其在卵泡发育过程中的表达研究[J]. 华北农学报, 2021, 36(1): 210-218. doi: 10.7668/hbnxb.20191376.

MA Hongcheng, XIONG Xianrong, WANG Han, HAI Zhuo, MIN Xingyu, LI Jian. Cloning of Yak PIK3CB Gene and Its Expression During Follicular Development[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2021, 36(1): 210-218. doi: 10.7668/hbnxb.20191376.

http://www.hbnxb.net/CN/Y2021/V36/I1/210

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牦牛PIK3CB基因克隆及其在卵泡发育过程中的表达研究

Cloning of Yak PIK3CB Gene and Its Expression During Follicular Development

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牦牛PIK3CB基因克隆及其在卵泡发育过程中的表达研究

Cloning of Yak PIK3CB Gene and Its Expression During Follicular Development

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