合浦珠母贝26S蛋白酶体S4,S7亚基基因片段的分离与分析

26S蛋白酶体是真核生物中一种具有ATP依赖性的蛋白酶复合体,主要通过泛肽途径选择性降解细胞内与代谢调控、细胞周期有关的功能蛋白及异常蛋白,参与多种细胞活动的调控过程。26S蛋白酶体由具有催化活性的20S亚复合体和一个具有调节作用的19S亚复合体组成,其中19S亚复合体中的ATP酶亚基是调节26S蛋白酶体活性的重要组件。本通过简并引物PCR手段,从软体动物合浦珠母贝(Pinctada fucata)中扩增到参与构成19S亚复合体的S4和S7(MSS1)两个亚基的基因片段。这两个基因片段所编码的ATP酶组件包含有Gx4GKT,DEID,SAT和H／QRxGRxxR等26S蛋白酶体ATP酶亚基的共同功能基序。这是首次在软体动物中报道26S蛋白酶体的ATP酶亚基基因序列,为研究软体动物中26S蛋白酶体的结构与功能奠定了分子基础。

ISOLATING AND ANALYZING THE GENE FRAGEMENTS OF S4 AND S7 SUBUNIT OF 26S PROTEASOME FROM Pinctada fucata

The 26S proteasome is an eukaryotic ATP dependent protease complex that is responsible for selective degradation of abnomal proteins and naturally short lived proteins relate to metabolic regulation and cell cycle progression by ubiquitin pathway so as to regulate a variety of cellular activities. The complex is composed of two subcomplexes, the proteolytic core 20S particle and the 19S regulatory particle. Based on the sequences of amino acids and its corresponding DNA of 26S proteasomal ATPase subunits from human and rat, two degenerate primers were designed and synthesized, and two cDNA fragements, subunit S4 and subunit S7 gene of 26S proteasome, were amplified firstly from the mantle cDNA of Pinctada fucata by PCR. Each cDNA fragement encoded 190 amino acids contained four conserved motifs, Gx4GKT, DEID, SAT, and H/QRxGRxxR, which are characteristic of the ATPase subunits of 26S proteasome. The result will be the molecular bases for studying the function and structure of 26S proteasome of mollusc.