【作者】 谢斯思；

【导师】 李万程；

【作者基本信息】 湖南师范大学 ， 细胞生物学， 2010， 硕士

【摘要】 在许多种不同的细胞进程中,如细胞分裂分化、生长繁殖、信号转导、DNA复制、基因调控和细胞衰老凋亡等生命进程中蛋白质Ser/Thr磷酸酶2A(PP2A)起着重要的调节作用。结构亚基A,调节亚基B和催化亚基C组装构成一个PP2A的异源三聚体。其中各有两种异构体存在在结构亚基A和催化亚基C中,而调节亚基B则有十多种异构体按其特异性分为至少四个不同的家族,分别为B,B′,B″和B′″。多种不同的基因分别编码这些不同的调节亚基,而调节亚基B的功能除了能介导催化特异性的底物外还能介导PP2A全酶的在细胞内的亚定位。调节亚基B中的striatin的在低等脊椎动物中的功能和作用及其独特的表达定位引起了我们的研究兴趣,为此在本实验中我们采用RACE克隆方法克隆了斑马鱼PP2A调节亚基B’’’-striatin基因的全长序列。所得序列结果为斑马鱼striatin基因包含2349bp的CDS其编码的氨基酸序列长782个aa。较高的同源性存在于斑马鱼striatin氨基酸序列与其他物种的striatin氨基酸序列之间。关于斑马鱼striatin基因的表达模式分析,RT-PCR分析显示在9个组织中striatin mRNA有不同程度的表达,而在斑马鱼胚胎发育各时期中在两细胞期、多细胞期、囊胚期和原肠期中表达量很高,而在其他发育时期表达量较低。Western Blot结果分析,在脑组织中检测到striatin有最高的表达。然而在心脏、鳃和鳍中striatin蛋白没有被检测到。在发育过程中,striatin蛋白在原肠、神经胚和视原基期有非常强烈的表达,随后表达量在多细胞和心跳期下调。这些结果揭示出striatin在成体组织中和斑马鱼发育过程中有很重要的作用。更多还原

【Abstract】 It is well established that the protein serine/threonine phosphatase 2A (PP2A) plays very important roles in many different cellular processes, including cell proliferation and differentiation, gene expression, neurotransmission, apoptosis, aging, and so on. PP2A consists of three heterogenic subunits:the scaffold subunit A, the catalytic subunit C, and the regulatory subunit B. While both the scaffold and the catalytic subunits contain only two isoforms, at least four subfamilies of the regulatory subunits, B, B’, B’’, and B’’’ have been identified. These regulatory subunits from different families are encoded by different genes and bear other functions besides directing the specificity of PP2A.To explore the possible functions of the regulatory subunits of PP2A in lower vertebrates, we have cloned the full-length cDNA sequence of the gene encoding the regulatory subunit striatin of PP2A from zebrafish, Danio rerio using RACE cloning method. The result of sequence showed that the stiatin cDNA of zebrafish contains an ORF of 2349 bp encoding a protein of 782 amino acids. The striatin protein displays a very high level of sequence identity with the striatin regulatory subunit from other species of vertebrates. Regarding its expression pattern, RT-PCR analysis reveals that the striatin gene is differentially expressed in 9 tissues examined. During twelve different developmental stages of the zebrafish, the highest level of mRNA expression was detected at the stages of two-cell, multiple-cell, blastula and gastrula, and a decreased level of the striatin mRNA was detected in other developmental stages.At the protein level, the highest expression level of the striatin protein was found in brain. In contrast, the striatin protein was hardly detectable in kidney, heart, gill and fin. Developmentally, the striatin protein was initially expressed at low level in two-cell, multiple-cell and blastula stage, then significantly upregulated at the stages of gastrula, neurula, and optic vesicle, and then decreased at heart beating and later stages. These results suggest that striatin appears to play a very important role during zebrafish development and also in adult tissue homeostasis.更多还原