鹅AKR1D1基因编码区克隆、序列分析及在卵泡中的表达特性研究

Molecular Cloning,Bioinformatics of the Goose AKR1D1 Gene cDNA Coding Domain Sequence,and Its Differential Expression Profiles in Ovarian Follicles of Different Stages

【目的】克隆鹅AKR1D1基因编码区序列，预测其蛋白结构、功能，并研究其在鹅各等级卵泡中的表达特性。【方法】以天府肉鹅母系为材料，采用RT—PCR技术克隆鹅AKR1D1基因编码区序列，利用多种生物信息学分析软件预测其结构与功能，并应用荧光定量PCR技术检测其在各等级卵泡中的表达特性。【结果】结果表明：鹅AKR1D1基因编码区全长981bp，编码326个氨基酸，氨基酸水平上与鸡相似性高达95．71％；氨基酸序列分析表明鹅AKR1D1蛋白相对分子量为37263．7Da，主要定位在细胞质和线粒体内，属于水溶性蛋白；预测鹅AKR1D1氨基酸含有磷酸化位点18个、糖基化位点3个；其二级结构以无规则卷曲为主，三级结构呈弯曲螺旋结构；荧光定量PCR结果显示，AKR1D1在鹅2-4mm卵泡颗粒层和膜层表达最高，在膜层F5和颗粒层F1中表达最低。【结论]AKR1D1可能通过调控类固醇激素的动态平衡从而对卵泡募集、筛选、闭锁以及排卵起到重要作用。

[Objective]The study was to clone the AKR1D1 gene＇s cDNA coding domain sequence of geese and predict its structure, function and expression profiles.[Methods]Using Tianfu meat-type geese as materials, we cloned the goose＇s AKRID1 coding domain sequence （CDS） by RTPCR, and predicted its protein structure and functions through several bioinformational tools and analyzed its expression in ovarian follicles of different stages of goose by real-time quantitative PCR.[Results]The results showed that the goose＇s AKR1D1 CDS consisted of 981 nucleotides that encoded 326 amino acids residues, and the amino acid homology of goose AKR1D1 shared 95.71% identity with chicken. The analysis of amino acid sequence revealed that the goose AKRIDI encoded water-soluble protein and its relative molecular weight was 37 263.7 Da. Subcellular location of AKR1D1 was primarily in the cytoplasm and chondriosome,and it did not belong to the secreted protein. It is predicted that the AKR1D1 protein contained 18 phosphorylation sites, 3 glycosylation sites. The secondary structure of AKR1D1 was mainly composed of random coil,while the tertiary structure of domain area showed a formic form helix structure. The results of real-time quantitative PCR revealed that goose AKR1D1 expressed most in theca layer and granulosa layer with 2-4 ram, at the lowest in theca layer of F5 and granulosa layer of F1. [Conclusion] AKRID1 may play an important role in the recruitment, selection, screening, atresia and ovulation of the follicle by regulating the dynamic balance of steroid hormones.