Analysis Of Function Of Protein Phosphatase-1cγ In Fish Embryos Development

The reversible phosphorylation of protein plays an vital regulatory role in the growth of many creatures. Protein kinase and protein phosphatase play an important role in phosphorylation and dephosphorylation, respectively. PP-1is a kind of ser/threonine protein. In this paper, we will focus on elucidating the function of the gene of the catalytic subunit isomer y(PP-1cy) in the growth and development of animal embryos. First, we use RACE PCR to clone the full length cDNA of PP-1cy in the goldfish. Then, by using RT-PCR, we study the mRNA expression level of PP-1cy in different development period of embryos and different tissues. Finally, we injected the Morpholino Oligomers (MO) of PP-1cy or the overexpression vector of PP-1cy into the goldfish embryos to analyze the role of the catalytic subunit of the PP-lc isomer gamma gene (PP-lcy) in the development of goldfish. The results as follows:(1) The cDNA full length of PP-1cy is1078bp, and its coding region is972bp, which encodes a protein containing323amino acids. The molecular weight of PP-lcy is36.8kDa. The homology analysis of amino acid sequence indicated that goldfish PP-1cy shares a high level of identity to that from zebrafish (97.5%), mouse (96.5%) and human (96.5%). The result shows that PP-lcy shares the high homology between different species, which suggests that PP-1cy has higher conservative in the function and evolution, and at the same time PP-1cy may play a important role in the development of vertebrate.(2) Semi-quantitative PCR showed that PP-1cy expressed differently in9different tissues of goldfish and12different development period of goldfish embryos. In12different stages, the expression level of PP-1cy in eye pigment stage is highest, then blastula stage and hatching larvae. The mRNA expression of PP-1cy in5stages (optic vesicle, heart beat, brain differentiation) is relative high, while that in other stages(multiple cells, neurula, gastrula, muscle differentiation and body pigmentation) is relative low. The mRNA expression of PP-1cy in two cells stage is lowest. In9different tissues, the expression level of PP-lcy in ovary is highest, then liver and gill. The mRNA expression of PP-1cy in4tissues (spermary, muscle, fin, brain) is relative high, while that in2tissues(kidney, liver) is relative low.(3) The result of microinjection showed the goldfish embryos injected with MO of PP1-cy and overexpression vectors appear severe deformity or death, such as curved spine, eyes dysplasia and brain malformations. It explains that PP1-cy plays a vital role in the development of goldfish embryos. According the result of paraffin section, the eye development of goldfish appeared abnormal, large or small eye, absent or deformity of the lens, and phenotypic abnormality of retinal development. This indicates that PPl-cy is very important for the development of eyes. The results will lay a solid foundation for our further study on the relevant function of PP1-cγ in other vertebrates.