牦牛CCND2基因的克隆及其在不同发情时期卵巢中的表达

发布时间：2018-05-10 10:25

本文选题：牦牛 + CCND　；参考：《中国农业科学》2017年13期

【摘要】：【目的】研究牦牛细胞周期蛋白D2(Cyclin D2,CCND2)基因序列特征及其在牦牛不同发情时期卵巢中的表达。【方法】以牦牛为研究对象,提取卵巢的总RNA进行反转录为第一链c DNA,根据Gen Bank中黄牛CCND2的m RNA序列(Gen Bank登录号:NM_001076372.1),使用Primer 5.0软件设计引物。应用PCR方法对CCND2基因扩增克隆,并测序获得CCND2基因完整的CDS区序列及部分5′端和3′端UTR区。使用Protparam、SOPMA、SWISS-MODEL及SMART分别对牦牛CCND2蛋白的理化性质、二级结构、三级结构及结构域进行预测分析,并进行同源性分析和系统进化树构建,利用半定量PCR方法对CCND2基因在牦牛各组织中的表达进行检测;同时采用实时荧光定量PCR技术分析CCND2基因在牦牛不同发情时期(卵泡期、红体期、黄体期)卵巢中的相对表达量。【结果】克隆获得牦牛CCND2基因序列为909 bp,其中包含可编码289个氨基酸残基的长为870 bp的CDS区。与黄牛相比,牦牛CCND2的CDS区存在5个碱基突变,导致其中一个氨基酸改变。牦牛CCND2基因CDs序列与野牦牛、黄牛、印度水牛、绵羊、野猪、马、家犬、猫、人类的进行比对同源性分别是99.54%、99.31%、99.31%、98.16%、94.81%、90.46%、90.80%、91.49%、88.62%,物种之间同源性较高,与进化树分析显示的亲缘关系远近一致,表明CCND2基因编码区在进化中较为保守。对CCND2蛋白预测分析知牦牛CCND2基因编码蛋白的分子式为C1445H2315N377O440S18,相对分子质量约为32.59k D,理论等电点为4.75,总平均亲水性为-0.107,不稳定指数为44.56,属亲水不稳定的酸性蛋白,该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,且与三级结构分析结果一致。蛋白含位于第26—152位氨基酸处的Cyclin_N和位于第154—257位氨基酸处的Cyclin_C两个结构域。CCND2基因在牦牛各组织中均有表达,其中在胃、卵巢和脑中表达相对较高;q RT-PCR结果显示在牦牛不同发情时期卵巢中CCND2 m RNA均有表达,且卵泡期卵巢中的表达水平最高(P0.01),在红体期和黄体期卵巢中表达水平无差异(P0.05)。【结论】成功获得了牦牛CCND2基因的CDS区全长序列和组织表达谱,对其进行生物信息分析及蛋白功能结构分析为该基因在牦牛繁殖调控中的进一步研究提供理论依据;在牦牛不同发情时期卵巢中CCND2m RNA表达水平存在差异,可能与卵泡发育过程中颗粒细胞增殖分化、卵泡发生波及卵巢内分泌有关,为进一步研究牦牛卵巢发育机制及改善牦牛繁殖效率提供了基础资料。
[Abstract]:[objective] to study the sequence characteristics of yak cell cycle protein D2(Cyclin D2 (CCND2) gene and its expression in the ovaries of yak at different estrous stages. [methods] Yak was studied. The total RNA extracted from the ovary was reverse transcribed into the first strand of c-DNA. According to the m RNA sequence of Gen Bank, the Bank accession number of Gen Bank was: NM00776372.1, and the primers were designed with Primer 5.0 software. The CCND2 gene was amplified by PCR and sequenced to obtain the complete CDS region of the CCND2 gene and part of the 5 'end and 3' end UTR region of the CCND2 gene. The physicochemical properties, secondary structure, tertiary structure and domain of CCND2 protein in yak were predicted and analyzed by using the SWISS-MODEL and SMART, and the homology analysis and phylogenetic tree construction were carried out. The expression of CCND2 gene in yak tissues was detected by semi-quantitative PCR, and real-time fluorescence quantitative PCR was used to analyze the expression of CCND2 gene in different estrous stages (follicle stage, red body stage) of yak. [results] the sequence of yak CCND2 gene was 909 BP, which contained 870 BP CDS region encoding 289 amino acid residues. Compared with yellow cattle, there were 5 base mutations in the CDS region of yak CCND2, which resulted in one amino acid change. The homology of CDs sequence of yak CCND2 gene with wild yak, yellow cattle, Indian buffalo, sheep, wild boar, horse, dog, cat and human were 99.5454, 99.31, 99.31, 98.16, 90.46, 90.80 and 91.4988.62, respectively. The results showed that the coding region of CCND2 gene was conserved in evolution. The prediction and analysis of CCND2 protein showed that the molecular formula of yak CCND2 gene encoding protein was C1445H2315N377O440S18, the relative molecular weight was about 32.59kD, the theoretical isoelectric point was 4.75, the total mean hydrophilicity was -0.107, and the instability index was 44.56.It was a hydrophilic acid protein. The protein has no transmembrane region and signal peptide, and the secondary structure mainly contains 伪 -helix and random crimp, which is consistent with the results of tertiary structure analysis. The protein contains two domains, Cyclin_N located at the 26-152 amino acid and Cyclin_C at the 154-257 amino acid. CCND2 gene is expressed in all yak tissues, including the stomach. The expression of CCND2 m RNA in ovary and brain was higher than that in brain. The results showed that the expression of CCND2 m RNA was found in the ovary of yak at different estrous stages. The highest expression level of P0.01 was found in follicular ovary, but there was no difference in expression level between erythroid and luteal stage. [conclusion] the full-length CDS sequence and tissue expression profile of CCND2 gene in yak were successfully obtained. The biological information analysis and protein functional structure analysis provide theoretical basis for further study of the gene in yak reproductive regulation, and the expression level of CCND2m RNA in the ovaries of yak at different estrus stage is different. It may be related to the proliferation and differentiation of granulosa cells during follicular development, follicle development and ovarian endocrine, which provides basic data for further study on the mechanism of ovarian development and improvement of reproductive efficiency of yak.
【作者单位】：西南民族大学生命科学与技术学院;西南民族大学青藏高原研究院;
【基金】：四川省科技支撑计划(2014NZ0114) 西南民族大学中央高校基本科研业务费专项资金(2016NZYQN38)
【分类号】：S823.85

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