金鱼PP2A调节亚基PR55基因的克隆及表达分析
MOLECULAR CLONING AND DIFFERENTIAL EXPRESSION PATTERNS OF THE GENE ENCODING THE PR55/B? OF PP-2A IN GOLDFISH, CARASSIUS AURATUS
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摘要: 蛋白磷酸酶2A是一种重要的丝氨酸/苏氨酸蛋白磷酸酶,对于调控多细胞的生命活动起重要作用。以金鱼大脑为材料,运用RT-PCR技术克隆得到PP2A调节亚基B55家族中PR55基因编码区部分序列。结果显示PR55基因cDNA长1218 bp,编码的多肽共含405个氨基酸。序列分析表明,该基因编码的蛋白与已知其他物种对应的PR55蛋白质均有着很高的同源性。用RT-PCR的方法检测了PR55基因在金鱼不同组织和胚胎发育不同时期的mRNA表达水平。结果表明,PR55基因表达呈现明显的组织和胚胎发育阶段差异性。在成体组织中,仅在大脑和鳍中有表达。在胚胎发育过程中,PR55从神经胚开始出现,整体呈现上升趋势,在出膜期达到最高水平。据此推测,PR55基因可能在金鱼胚胎发育中具有多种重要作用。Abstract: The reversible phosphorylation of proteins is an important posttranslational modification in eukaryotes that modulates the functional status of more than thirty percent of total cellular proteins. In the present study, we reported the molecular cloning of a partial cDNA coding for the PR55/B of PP-2A from the brain of goldfish through 5 RACE PCR strategy. The partial PR55 cDNA contained 1218 nucleotides which encoded a deduced partial protein of 405 amino acids. Sequence homology analysis showed that the PR55/B of PP-2A displayed a high level of amino acid identity with the counterpart from other species including human and rat, indicating the conservation of PR55/B. RT-PCR analysis revealed that PR55/B mRNA was specifically expressed in the brain and fin of goldfish. Our demonstration that PR55/B was expressed in the fish fin was a novel finding for the first time. This result suggested that the PP-2A with PR55/B as the regulatory subunit in fish likely played an important role in swimming, balancing and sensitivity to the water environment. Moreover, during the development of goldfish, PR55/B mRNA was initially detected at neurula stage, suggesting that the PP-2A with PR55/B as the regulatory subunit was likely implicated in control of the brain development differentiation in fish. Furthermore, we found that PR55/B became gradually increased from the optic vesicle stage and reached a peak level at the muscle movement stage, and thereafter, PR55/B mRNA maintained at this level with slight fluctuation from heart beat to hatching larvae. These results indicated that PP-2A with PR55/B as the regulatory subunit was actively regulating the development processes of these different stages. The consistent develop-mental expression patterns of PR55/B (Fig. 2) and the catalytic subunits (PP-2Ac)[28] also supported that PR55/B may regulate fish development in holoenzyme, which was different from that of certain regulatory subunits of PP-2A[20―22]. Thus, our present study demonstrated that the specific PP-2A with PR55/B as regulatory subunit may play an important role in regulating fish development. In addition, PP-2A containing PR55/B as the regulatory subunit may play impor-tant roles of homeostasis in brain and fin.
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Keywords:
- Signal Transduction /
- Protein Dephosphorylation /
- PP-2A /
- PR55 /
- Development /
- Morphogenesis
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