A specific genomic organization and a novel promoter sequence for both ZP2 and ZP3 gene expressions in the Pingxiang red transparent crucian carp, Carassius auratus var. pingxiangnensis

We cloned the full-length cDNA of ZP2 from Carassius auratus var. Pingxiangnensis (CaP_ZP2) and identified a cluster of three ZP genes through its DNA walker. These three genes, CaP_ZP3.1, CaP_ZP2 and CaP_ZP3.2 were located within a 10,855 bp region and each comprised of eight exons spanning 1348 bp, 1638 bp and 1348 bp, respectively. Protein bands of egg membrane between 40 and 70 kDa were in concordance with the deduced amino acid of these three CaP_ZP genes cDNA and with their molecular mass. This is the first report that two CaP_ZP3 genes were separated by CaP_ZP2 gene. A novel sequence of 1097 bp, located between CaP_ZP2 and CaP_ZP3.2, was inserted into the modified pAcGFP1-1 vector in the forward and reverse directions. Results showed that individual sequence served as promoters utilizing common regulatory elements in the forward and reverse directions for both CaP_ZP2 and CaP_ZP3.2 gene expressions. In situ hybridization against CaP_ZP2 confirmed that a strong positive signal was detected in the early development oocytes. Similarly, real-time PCR results also showed that CaP_ZP2 transcription increased mainly in a 4–8 month ovary, but was decreased dramatically in a 9–12 month ovary.