新西兰白兔CYP11A1基因的克隆、生物信息学分析及其对繁殖相关基因的影响

【目的】旨在通过分析细胞色素P450家族成员11A1（CYP11A1）的生物信息功能及其在新西兰白兔卵巢颗粒细胞中对繁殖性能相关基因的调控作用,为探究其在卵泡发育过程中调控功能奠定基础。【方法】根据GenBank上兔CYP11A1基因的序列,设计特异性引物,以新西兰白兔卵巢组织cDNA为模板,PCR扩增并克隆CYP11A1基因序列,构建pcDNA3.1-CYP11A1过表达重组载体;用Mega 5.1在线软件构建系统进化树,并通过生物信息学软件对CYP11A1蛋白的氨基酸组成、理化性质、磷酸化位点、保守结构域、二级结构、三级结构、亚细胞定位、蛋白互作进行预测分析。分离新西兰白兔卵巢颗粒细胞,并通过免疫荧光法鉴定颗粒细胞特异性抗体卵泡刺激素受体（FSHR）的表达。设计3条干扰CYP11A1基因表达的引物（siRNA-1、siRNA-2、siRNA-3）,筛选出干扰效率最高的1条,并将其与pcDNA3.1-CYP11A1过表达重组载体转染到卵巢颗粒细胞中,用实时荧光定量PCR检测颗粒细胞中羟基类固醇17-β脱氢酶1（HSD17B1）、骨形态发生蛋白15（BMP15）和促卵泡刺激素受体（FSHR）等繁殖相关基因mRNA表达水平。【结果】成功克隆新西兰白兔CYP11A1基因,其CDS全长序列为1 557 bp,编码518个氨基酸,其中亮氨酸含量（10.6%）最高,精氨酸（7.6%）、缬氨酸（7.4%）和丙氨酸（7.2%）次之。进化树分析显示,CYP11A1蛋白序列与家鼠、人和猩猩的距离最近。生物信息学分析显示,CYP11A1蛋白理论等电点为7.4,正负电荷残基数各占50个,脂肪族氨基酸指数为82.16,不稳定性指数39.54,其中氨基酸序列中亲水性残基数量多于疏水性残基;CYP11A1蛋白具有40个潜在的磷酸化位点,其中以苏氨酸、丝氨酸和酪氨酸最为丰富;CYP11A1蛋白二级结构由α-螺旋（48.31%）、无规则卷曲（40.22%）、延伸链（7.42%）和β-转角（4.04%）组成,三级结构为弯曲螺旋状。亚细胞定位预测结果显示,CYP11A1蛋白主要分布于线粒体（52.2%）、细胞质（17.4%）和细胞核（13.0%）中,还具有1个p450家族的结构域,并与多个繁殖性能相关的蛋白（STAR、CYP21A2、CYP17A1和FDX1）相互作用。分离的兔颗粒细胞表达FSHR,可以用于后续试验;CYP11A1基因在颗粒细胞中过表达后,HSD17B1和FSHR基因表达量极显著上调（P＜0.05）,BMP15基因表达量显著上调（P＜0.01）;干扰CYP11A1基因表达后,BMP15和FSHR基因的表达量极显著下调（P＜0.01）,HSD17B1基因表达量显著下调（P＜0.05）。【结论】CYP11A1是一个稳定、亲水、相对保守且具有抗氧化和类固醇激素合成功能的蛋白。CYP11A1基因可调控卵巢颗粒细胞中与繁殖性能相关的基因HSD17B1、BMP15、FSHR的表达,说明CYP11A1基因在母兔繁殖过程中发挥一定作用。

Cloning and Bioinformatics Analysis of CYP11A1 Gene in New Zealand White Rabbits and Its Effects on Reproduction-related Genes

【Objective】 To analyze the bioinformatics function of cytochrome P450 family 11 subfamily A member 1 (CYP11A1) and the regulation of genes related to reproductive performance in New Zealand White rabbit ovarian granulosa cells,in order to lay the foundation for exploring its regulatory function in follicular development.【Method】 According to the sequence of rabbit CYP11A1 gene in GenBank,specific primers were designed to clone CYP11A1 gene and construct pcDNA3.1-CYP11A1 overexpression recombinant vector from ovary tissue of New Zealand White rabbits.Phylogenetic tree was constructed by Mega 5.1 software. The amino acid composition,physicochemical properties,phosphorylation site,conserved domain,secondary structure,tertiary structure,subcellular location,protein cross-linking of CYP11A1 protein were predicted by bioinformatics softwares. Ovarian granulosa cells were further isolated from ovarian tissue of New Zealand White rabbits and identified by immunofluorescence using specific antibody follicle stimulating hormone receptor (FSHR).Three primers (siRNA-1,siRNA-2,siRNA-3) were designed to interfere the expression of CYP11A1 gene,and the one with the highest interference efficiency was screened,and the overexpressed recombinant vector pcDNA3.1-CYP11A1 and siRNA-CYP11A1 were transfected into ovarian granulosa cells.After RNA was extracted from granulosa cells,then Real-time quantitative PCR was used to analyze the effects of CYP11A1 overexpression and knockdown on the mRNA expression levels of reproductive genes such as HSD17B1,BMP15 and FSHR.【Result】 CYP11A1 gene was successfully cloned from New Zealand White rabbits.The full-length sequence of CDS of CYP11A1 gene of New Zealand White rabbits was 1 557 bp,encoding 518 amino acids,among which leucine (10.6%) was the highest,followed by arginine (7.6%),valine (7.4%) and alanine (7.2%).Evolutionary tree analysis showed that the protein sequence of rabbit CYP11A1 was closest to mice,humans and orangutans.Bioinformatics analysis showed that the theoretical isoelectric point of CYP11A1 protein was 7.4,the number of positive and negative charge residues was 50,the aliphatic amino acid index was 82.16,and the instability index was 39.54,among which the number of hydrophilic residues in amino acid sequence was more than that of hydrophobic ones.This protein had 40 potential phosphorylation sites,among which threonine,serine and tyrosine were the most abundant.The secondary structure of CYP11A1 protein consisted α-helix (48.31%),random coil (40.22%),extended chain (7.42%) and β-turn (4.04%),and the tertiary structure was predicted to be curved helix.Subcellular localization predicted that CYP11A1 protein was mainly distributed in mitochondria (52.2%),cytoplasm (17.4%) and nucleus (13.0%).It also had a domain of P450 family and interacted with multiple proteins related to reproductive performance (STAR,CYP21A2,CYP17A1 and FDX1).The expression of FSHR in isolated granulosa cells was positive,which could be used for subsequent experiments.After CYP11A1 gene was overexpressed in granulosa cells,the expression of HSD17B1 and FSHR genes were significantly upregulated (P＜0.05), the expression of BMP15 gene was extremely significantly upregulated (P＜0.01).After CYP11A1 gene was interfered,the expression of BMP15 and FSHR genes were extremely significantly down-regulated (P＜0.01),and the expression of HSD17B1 gene was significantly down-regulated (P＜0.05).【Conclusion】 CYP11A1 gene could regulate the expression of reproductive performance related genes HSD17B1,BMP15 and FSHR in ovarian granulosa cells,suggesting that CYP11A1 played a certain role in the reproductive process of female rabbits.