Expression Of Different Tissues And Immunjofluorescence Localization In Oocytes Of ZP3 Protein In The Pingxiang Red-Transparent Crucian Caip, Carassius Auratus Var. Pingxiangnenesis

Pingxiang red-transparent crucian carp, Carassius auratus var. Pingxiangnensis, mainly distributed in Pingxiang city of Jiang Xi Province, is a triploid wildness crucian carp mutant. For its whole transparent body, it was named Carassius auratus var. Pingxiangnensis. The fish is a good crucian carp specie for edible value and ornamental value, and it is approved by national aquatic original improved variety committee as a new cultural fish specie in 2008. Carassius auratus var. Pingxiangnensis has two modes of reproduction,which includes bi-sexual and gynogenetic reproductive mode.The content of this research includes three parts:First:The ZP3 cDNA sequence from Carassius auratus var. Pingxiangnensis is cloned by the method of RACE-PCR. Its full length is 1385bp, and an ORF is 1308bp for encoding a peptide of 435 aa. By blasting of amino acid sequence, amino acid residue from 121 to 376 aa belongs to ZP family domains.Second:The ZP3 mRNA expression levels of different tissues in Carassius auratus var. Pingxiangnensis are detected by the technology of real-time PCR. The tissues include heart, liver, spleen, kidney, brain and ovary. The results of relative quantitative analysis show that mRNA expression in the liver was the lowest, and the ovary posses more higher expression level than other tissues. The ZP3 mRNA expression in the ovary is about 26,656 times as much as in the liver. Except for the ovary, there is a slight expression in all the other tissues.Third:The purified ZP3 protein is used as antigen to immunize the rabbits. The polyclonal antibody of rabbit-anti-fish serum is prepared. The potency of antiserum is detected by Elisa. The antiserum is used for oocytes frozen section immunofluorescence localization experiments. The result of Elisa shows that the potency of antiserum is about 25,600 and the specificity of antiserum is good. Immunofluorescence results show that:ZP3 protein existed in the early developmental oocytes, and with the development of oocytes, ZP3 protein synthesis accumulation gradually increases.In the V growth period of oocyte,the cumulant of ZP3 protein on the cell membrane and in the cytoplasm reachs maximum.ZP3 protein plays a great important role in the period of sperm-egg recognition. In order to investigate the function of ZP3 protein in the period of fertilization, this research provide a few bio-molecular dates.