蛋鸡INHBA的克隆分析及其对卵泡发育调控的研究

Cloning and Analysis of INHBA in Layers and Its Regulation for Follicular Development

试验旨在探讨INHBA(Inhibin subunit beta A)基因在鸡卵泡发育过程中的作用,为提高蛋鸡产蛋性能提供理论依据。首先从海兰褐蛋鸡各级卵泡组织中提取总RNA,经反转录后对INHBA CDS编码区进行克隆且进行生物信息学分析,并采用实时荧光定量PCR法检测鸡INHBA在不同卵泡以及卵泡膜细胞和颗粒细胞中的表达规律。结果表明:扩增片段为1547 bp,包含1275 bp的编码区,编码424个氨基酸;海兰褐蛋鸡与日本鹌鹑、黄牛、绵羊、兔、猪、人、小鼠和非洲爪蛙的INHBA同源性分别为97.7%、78.2%、78.0%、77.4%、77.0%、72.8%、79.8%和72.8%,进化分析表明海兰褐蛋鸡的INHBA在进化过程中保守性较高,与日本鹌鹑关系最近,与哺乳动物相差也不远,与非洲爪蛙等两栖类动物相差最远。INHBA蛋白质理化性质分析表明,其属于弱酸性蛋白,不稳定,流动性较好。蛋白结构分析显示,其无跨膜域,N端有信号肽,无规则卷曲为整体的主要结构。不同卵泡INHBA表达显示,INHBA随着卵泡的发育表达量逐渐升高,并且在等级卵泡颗粒细胞中表达量最高,在等级卵泡颗粒细胞中添加低浓度促卵泡素(Follicle-stimulating hormone,FSH)处理后,INHBA表达显著升高,表明其在卵泡发育过程中可能受FSH的调控。研究结果可为后期INHBA功能以及其对鸡卵泡发育调控机制的研究提供一定的参考依据。

The purpose of this study was to investigate the role of INHBA in the development of chicken follicles,and to provide theoretical basis for improving egg laying performance of layers.Firstly,total RNA was extracted from follicular tissues of different hierarchical of Hy-Line Brown Layers and the first strand of cDNA was synthesized with reverse transcription.Finally,the CDS coding region of INHBA was cloned and bioinformatics analysis was conducted.Then,the expression of chicken INHBA in different follicles,follicular theca cells and granulosa cells was detected by real-time fluorescence quantitative PCH.The results showed that the amplified fragment was 1547 bp,containing 1275 np coding region and 424 amino acids.The homology of Hv-I,ine Brown layer anti Coturnix japonica.Bos taurus^Ovis aries,Oryctolagus cuniculust.Sus scrofa,Xenopus tropicalis^Mus musculus^and Xenopus tropicalis INHBA gene was respectively 97.7%,78.2%,78.0%,77.4%,77.0%,72.8%,79.8%,and 72.8%.The evolutionary analysis indicated that the INHBA of Hy-Line Brown Layers was highly conserved during the evolution process,and it was closely related to Coturnix japonica,not far from mammals,and the furthest from amphibians such as Xenopus tropicalis.The physicochemical properties of INHBA protein showed that it was weak acid protein,unstable and had good fluidity.The protein structure analysis showed that there was no transmemhrane domain,and there was signal peptide at the N terminal,and the random coil was the main structure.The expression of INHBA in different follicles showed that the expression level of INHBA gene gradually increased with the development of follicles,and mainly expressed in follicular granulosa cells.When low-concentration follicle-stimulating hormone(FSH)was added lo hierarchical follicular granulosa cells,the expression of INHBA was significantly increased,which indicated that it may be regulated by FSH during follicular development.The results of this study could provide some references for further study of the function of INHBA gene and its effect on chicken follicular development.