Molecular Cloning And Differential Expression Patterns Of PP2A-PR55α Gene In Carassius Auratus And Danio Rerio

Reversible protein phosphorylation is an essential regulatory mechanism in many cellular processes. Protein kinases and Protein phosphatases are responsible for protein phosphorylation and dephosphorylation respectively. Protein phosphatases2A (PP2A) represents a conserved family of important protein serine/threonine phosphatases in species ranging from yeast to human. The PP2A core enzyme comprises a scaffold subunit and a catalytic subunit. The heterotrimeric PP2A holoenzyme consists of the core enzyme and a variable regulatory subunit. PR55/B family is one of identified regulatory B subunits of PP2A and is encoded by four genes:PR55a, PR55β, PR55y and PR55δ.In the present thesis study, We have cloned cDNA sequence of PR55a of PR55/B family of PP2A from goldfish and zebrafish using RT-PCR and RACE cloning strategies. Our results revealed that the full length of PR55a cDNA of goldfish is2179bp, which contains an open reading frame (ORF) of1338bp encoding a deduced protein of446amino acids. And the partial PR55a cDNA of zebrafish consists of1784nucleotides, which contains an ORF of1338bp encoding a deduced protein of446amino acids. Amino acids sequence analysis showed that the PR55a protein displays a very high level of sequence identity with their counterparts in other vertebrate species, thus suggesting the conservation of PR55a gene in its evolution and fuction. Amino acid sequence analysis through SMART and other programs revealed that the WD40repeats were conserved in PR55a protein. This structure is thought to mediate protein-protein interactions. And it is also found in PR55/B family as a highly conserved structure, suggesting that this domain provides a binding site for the PR55/B family with the PP2A-A subunits to form the PP2A holoenzyme.Subsequently, to further explore the possible functions of the regulatory subunits of PP2A in tissue differentiation and embryonic development in lower vertebrates, we analysed the differential expression patterns of PR55a in different tissues and different developmental stages in goldfish and in zebrafish using RT-PCR and Western Blot. At the expression level of mRNA, PR55a was widely expressed in goldfish and zebrafish tissues, but showed a significant tissues differences and species diversity. At the expression level of protein, PR55a was widely expressed in goldfish tissues whereas is expressed in a tissue-specific manner in zebrafish tissues. The PR55a protein was hardly detectable in kidney, muscle and heart. In addition, the PR55a protein was highly expressed in liver, spermary, ovary and brain in goldfish and zebrafish, which suggests that PR55a may be important for regulating the development of gonad and maintaining the function of liver and brain.In the process of the embryonic development of goldfish and zebrafish, the expression of PR55a also showed species diversity. At the expression level of mRNA at the12different developmental stages of goldfish, the lowest expression level was detected at two-cell, multiple-cell and blastula stage. And the mRNA expression increased abruptly at gastrula stage, then maintain constant and high level at the following development stages. And the highest level of mRNA was detected at brain vesicle stage. In the process of embryonic development of zebrafish, the PR55a mRNA was highly expressed at two cell stage, then dropped continuously at the early developmental stages. And the expression of PR55a mRNA maintain constant and low from optic vesicle stage to hatch stage. This suggests that the highest expression level of PR55a mRNA at two cell stage maybe from the maternal expression, and then decline gradually in the process of developmental stage.At the expression level of protein, the PR55a expression maintain substantially constant and high level at the different developmental stages of goldfish, which suggests that PR55a has an important role in the regulation of embryonic development of goldfish.In the process of the embryonic development of zebrafish, the expression level of the PR55a protein rise quickly at multiple cell stage and body pigment stage, suggesting that the PR55a begin expresses highly at the cleavage stage of development and has an important role in the formation of body pigment.