The Histochemistry Of Oocyte、the Molecular Characterization And Expression Of Zona Pellucida Genes (ZP) In Pingxiang Red-transparent Crucian Carp, Carassius Auratus Var. Pingxiangnensis

The Pingxiang red-transparent crucian carp, Carassius auratus var. Pingxiangnensis(designated as CaP), distributed in the Pingxiang region of Jiangxi province of China,was a natural wild triploid crucian carp mutant and had two different reproductivedevelopment modes including gynogenesis and bisexual reproduction. There was littleknown about the expression and regulation of ZP (designated as CaP_ZP) genes inspecial triploid CaP. The content of this research included three parts:1. Cytochemical method was used to demonstrate the changes of main biochemicalmaterials during oogenesis of CaP. A great deal of protein was produced in the earlydevelopment of oocytes from phase1and phase2. During this early period, PAS andSudan black reactions were nearly negative. As the further development of oocytes, alot of these two positive materials appeared in cytoplasm of oocytes. Results showedconsiderable glycogen, protein and lipid were produced in the maturation oocytes.These materials are prepared for embryonic development.2. The proteins of egg membrane were isolated and purified by sucrose-urea. Proteinbands of egg membrane between40and70kDa were in concordance with thededuced amino acid of CaP_ZP2and CaP_ZP3genes cDNA with their molecularmass. The full-length cDNA of CaP_ZP2was cloned using rapid amplification ofcDNA ends (RACE) from the ovary of CaP. The CaP_ZP2was1682bp in length andcontained an open reading frame (ORF) of1584bp encoding for527amino acidresidues. A cluster of three genes coding for CaP_ZP proteins were isolated. Thesethree genes, CaP_ZP3.1, CaP_ZP2and CaP_ZP3.2were located within a10,855bpregion and were each comprised of eight exons spanning1348bp,1638bp and1348bp; respectively. The exon-intron structures of three genes were nearly identical,suggesting that they arose from a common ancestral gene.3. The CaP_ZP2mRNA expression levels of different tissues were detected by thetechnology of real-time PCR. These tissues included heart, liver, spleen, kidney, brainand ovary. The results of relative quantitative analysis showed that mRNA expression in the liver was the lowest, and the ovary had more higher expression level than othertissues. The CaP_ZP2mRNA expression in the ovary was about85,932times asmuch as in the liver. Except for the ovary, there was a slight expression in all the othertissues. Real-time PCR was also applied to determine CaP_ZP2and CaP_ZP3mRNAexpression levels in the ovary at different stages of maturation. Real-time PCRanalysis confirmed that the high expression was observed in a4-8month ovary andthe expression level decreased dramatically in a9-12month ovary. In situhybridization against CaP_ZP2also confirmed that a strong positive signal wasdetected in the early development oocytes. And these two genes had a sameexpression pattern during the ovarian development.4. A novel sequence of1097bp, which was the common promoter region of CaP_ZP2and CaP_ZP3.2genes, was found. To investigate the activities of CaP_ZP2andCaP_ZP3.2promoters, the entire length of the1097bp sequence was inserted into themodified pAcGFP1-1vector in the forward and reverse directions. This was then usedto generate CaP_ZP2and CaP_ZP3.2: GFP (green fluorescence protein) transgeniccos-7lines. We demonstrated an intense GFP expression in the transgenic lines andthus that we found an individual ZP gene promoter region, which utilized differentregulatory elements in the forward and reverse directions for both CaP_ZP2andCaP_ZP3.2gene expressions.