牦牛Cyclin D1基因的克隆及IGF-I对其在睾丸支持细胞表达的影响

doi:10.11843/j.issn.0366-6964.2018.02.010

摘要/Abstract

摘要：

本研究旨在克隆牦牛（Bos grunniens）Cyclin D1基因的CDS序列，分析其生物学特性，研究胰岛素样生长因子-I （Insulin-like growth factor-I，IGF-I）对Cyclin D1 mRNA和蛋白在睾丸支持细胞（Sertoli cell，SC）中表达的影响。采集5～8月龄健康牦牛的睾丸样品进行SC的分离培养，用RT-PCR技术克隆牦牛Cyclin D1基因，用ORF Finder、MEGA7.0和DNAMAN对其进行生物信息学分析，并采用免疫荧光染色技术对Cyclin D1蛋白在SC中的表达进行定位分析。向体外培养的SC中加入不同浓度（0（对照组）、25、50、100、150 ng·mL^-1）的IGF-I，采用实时定量PCR （qRT-PCR）和蛋白免疫印迹法检测Cyclin D1基因和蛋白的表达。结果表明：1）牦牛Cyclin D1基因（GenBank登录号：KY 420723）与其他物种的同源性均较高。2） Cyclin D1蛋白在SC胞核中高表达，为核蛋白。3）浓度为25和150 ng·mL^-1IGF-I，Cyclin D1 mRNA表达量与对照组差异不显著（P>0.05），浓度为50和100 ng·mL^-1IGF-I时，Cyclin D1 mRNA表达量显著高于其他各组（P<0.05）；IGF-I浓度为100 ng·mL^-1，Cyclin D1蛋白表达量与对照组差异不显著（P>0.05），25、50和150 ng·mL^-1 IGF-I组Cyclin D1蛋白表达量显著高于对照组（P<0.05）；IGF-I浓度为50 ng·mL^-1时，Cyclin D1 mRNA和蛋白表达量均为最高，分别为对照组的1.65和1.20倍。综上表明，Cyclin D1基因在进化过程中高度保守；IGF-I可调控SC中Cyclin D1的表达，其最佳作用浓度为50 ng·mL^-1。

关键词: 牦牛, 睾丸支持细胞, 细胞周期蛋白D1, 胰岛素样生长因子-I

Abstract:

The aim of this study was to clone the CDS of yak (Bos grunniens) Cyclin D1 gene, to analyse its biological characteristics, and to research the effects of insulin-like growth factor-I (IGF-I) on Cyclin D1 gene and protein expression in Sertoli cell (SC). The testicular samples were collected from 5-to 8-month-old healthy yaks for isolation and culture of SC. The Cyclin D1 gene was cloned by RT-PCR and its biological characteristics were analyzed by ORF Finder, MEGA7.0 and DNAMAN. The expression of Cyclin D1 protein in SC was localized by immunofluorescence staining. IGF-I was added into the culture medium of SC in the concentration of 0(control), 25, 50, 100, 150 ng·mL^-1, and then the relative expressions of Cyclin D1 mRNA and protein were detected by real-time quantitative PCR (qRT-PCR) and Western blotting, respectively. The results were as follows:1) Yak Cyclin D1 gene (GenBank accession number:KY 420723) had the high homology with other species. 2) Cyclin D1 protein belonged to nuclear protein because of high fluorescence intensity on cell nucleus. 3) The level of Cyclin D1 mRNA in 25 and 150 ng·mL^-1 groups were not significantly different with the control group (P>0.05), while 50 and 100 ng·mL^-1 groups were significantly higher than other groups (P<0.05); the difference of Cyclin D1 protein expression between 100 ng·mL^-1 group and the control group was not significantly different (P>0.05), however, a significant difference existed between 25, 50, 150 ng·mL^-1 groups and control group (P<0.05); the relative expression of Cyclin D1 mRNA and protein were all reached the highest levels when IGF-I was 50 ng·mL^-1, which were 1.65 and 1.20 times higher than that of the control group, respectively. The results suggested that Cyclin D1 gene was highly conserved during evolutionary process; IGF-I could regulate the expression of Cyclin D1 in SC and its optimal concentration was 50 ng·mL^-1.

Key words: yak, Sertoli cell, Cyclin D1, insulin-like growth factor-I

中图分类号:

S823.8⁺5.2

王亚营, 潘阳阳, 叶小琳, 李谷月, 何翃闳, 王萌, 樊江峰, 崔燕, 余四九. 牦牛Cyclin D1基因的克隆及IGF-I对其在睾丸支持细胞表达的影响[J]. 畜牧兽医学报, 2018, 49(2): 310-317.

WANG Ya-ying, PAN Yang-yang, YE Xiao-lin, LI Gu-yue, HE Hong-hong, WANG Meng, FAN Jiang-feng, CUI Yan, YU Si-jiu. Cloning of Yak Cyclin D1 Gene and the Effects of IGF-I on Its Expression in Sertoli Cells[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(2): 310-317.

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