牦牛KDM4A基因克隆、组织表达谱及其在卵母细胞和颗粒细胞中的表达

doi:10.11843/j.issn.0366-6964.2018.02.008

摘要/Abstract

摘要：

本研究旨在阐明牦牛KDM4A基因的表达特性及其在牦牛卵母细胞和颗粒细胞中的作用。采集牦牛心、肝、脾、卵巢、肺、肾、睾丸、小肠、子宫、胃、大脑组织，以GenBank上已发布的黄牛KDM4A基因序列设计引物，利用RT-PCR方法扩增克隆牦牛KDM4A基因及检测其在牦牛各组织中的表达谱，并使用在线软件ExPASY分析KDM4A基因的结构和功能。采用实时荧光定量PCR法检测牦牛不同时期卵母细胞和颗粒细胞中KDM4A基因的表达水平。结果表明，本研究克隆得到牦牛KDM4A基因3 289 bp的cDNA序列。牦牛KDM4A核苷酸序列与其它哺乳动物的遗传距离较近，表明KDM4A基因在进化中较为保守。牦牛KDM4A基因CDs区为3 201 bp，编码1 066个氨基酸残基，相对分子质量122.48 ku。KDM4A蛋白为亲水不稳定的酸性蛋白，无跨膜区和信号肽，二级结构主要包含α-螺旋和无规卷曲，与三级结构分析结果一致。KDM4A基因在所选牦牛组织样本中均有表达，其中在卵巢、脾和睾丸中表达量最高。牦牛KDM4A mRNA在MⅡ期颗粒细胞中表达量显著高于在MI期和GV期颗粒细胞中的表达量（P<0.05），GV期卵母细胞中，KDM4A mRNA的表达水平显著高于在MI期和MⅡ期卵母细胞中的表达量（P<0.05）。综上表明，本研究成功克隆了KDM4A基因及其在牦牛卵母细胞和颗粒细胞成熟过程中mRNA表达水平的差异，表明KDM4A基因在卵母细胞及颗粒细胞成熟过程中发挥一定作用。

Abstract:

The aim of this study was to clarify the expression characteristics of yak KDM4A gene and its role in yak oocytes and granulosa cells. The primer was designed according to the published bovine (Bos taurus) KDM4A gene sequence on the GenBank, and the expression pattern of KDM4A was detected by RT-PCR in yak various tissues which included heart, liver, spleen, lung, kidney, small intestine, ovary, uterus, stomach, testicle and cerebrum.While the coding sequence of KDM4A gene was cloned by RT-PCR.The structure and function of KDM4A were analyzed by ExPASY software on line. The mRNA expression level of KDM4A in different periods of yak oocytes and granulosa cells were detected by qRT-PCR. Results showed that KDM4A gene of yak contained a 3 289 bp cDNA fragment, and had high homology with other mammals in nucleotide sequence through sequence alignment and phylogenetic tree, indicating that KDM4A was relatively conservative in the evolutionary process. The complete CDs of yak KDM4A gene was 3 201 bp, encoding 1 066 amino acids, and the molecular weight was 122.48 ku. The protein encoded by KDM4A gene in yak 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 KDM4A, consistent with the results of the tertiary structure analysis. KDM4A gene was expressed in various tissues of yak, and high abundance in ovary, spleen and testicle.The qRT-PCR results showed that the mRNA expression level in MⅡ phase granulosa cell was significantly higher than that in GV and MI phases (P<0.05). Meanwhile, the mRNA expression level of KDM4A was significantly higher in GV phase oocyte than that in MI and MⅡ phases (P<0.05). The complete CDs of KDM4A gene was successfully cloned and the KDM4A had a significant difference expression among the 3 periods of yak oocytes and granulosa cells, which indicated that KDM4A played role in the maturing process of yak oocytes and granulosa cells.

中图分类号:

S823.8⁺5.2

韩杰, 熊显荣, 蔡雯祎, 杨显英, 阿果约达, 张燕红, 李键. 牦牛KDM4A基因克隆、组织表达谱及其在卵母细胞和颗粒细胞中的表达[J]. 畜牧兽医学报, 2018, 49(2): 291-299.

HAN Jie, XIONG Xian-rong, CAI Wen-yi, YANG Xian-ying, A GUO Yue-da, ZHANG Yan-hong, LI Jian. Cloning of KDM4A Gene and Its Expression in Different Tissues, Oocyte and Granulosa Cell of Yak[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(2): 291-299.

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https://www.xmsyxb.com/CN/Y2018/V49/I2/291

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