Cloning Of ZPB Genes And Effects Of Endocrine Disrupting Chemicals On ZPB Of Rare Minnow

Zona pellucida (ZP) containing proteins are glycoproteins in teleost chorion whcih playsimportant role in the development of egg envelope, the process of fertilization and protectingfertilized egg et al. ZP proteins are encoded by several gene subfamilies, mainly includingZPA, ZPB, ZPC and ZPX genes. In teleost species, ZP genes are expressed exclusively in liverunder the regulation of estrogen or exclusively in ovary, or expressed in liver and ovary. Theregulation mechanism of ZP genes in ovary is unknown yet.To state the effects of endocrine disrupting chemicals (EDCs) in aquatic environment onZP genes and then provide the basic theories for detecting EDCs and risk prediction andevaluation on EDCs in aquatic environment, five ZPB genes were isolated and characterizedby using gene cloning and RACE method in rare minnow. These five G. rarus ZPB geneisoforms were named as grZPB.1, grZPB.2, grZPB.3, grZPB.4and grZPB.5. The lengths ofthese five grZPB isoforms are1857bp,1821bp,1713bp,1929bp and1965bp, respectively.And the putative amino acid sequences are602,590,554,626and638aa. These amino acidsequences are highly similar with carp and goldfish ZP2/ZPB proteins. The putative aminoacid sequences of five grZP gene isoforms contain typical conserved domains: trefoil motif,ZP domain, CFCS site, IHP and EHP site. The grZPB precursors contain eighteen cysteineresidues, six of which are present in the TFF domain and twelve residues in the ZP domainrespectively. The repetitive domains of grZPB putative amino acid sequences are the leastconserved, which are rich in glutamine and proline. The sequence of predominant amino acidresidues of the repeat unit is VQQQASQQFPQR/Q. And the numbers of repeat unit in fivegrZPBs are16,15,12,18and19respectively.Tissue distribution was analyzed with real-time quantitative reverse transcription PCR(RT-qPCR) after the toal RNAs were extracted from various tissus in female and male rareminnow. Results showed that grZPB mRNA isoforms were exclusively expressed in ovary. G.rarus juveniles at the age of21day postfertilization were exposed to17α-ethinylestradiol(EE2;0.01,0.1and1nM),4-nonylphenol (4-NP;10,100and1000nM) and bisphenol A (BPA;0.1,1and10nM) for3days. mRNA expressions of ZPB isoforms following thexenoestrogens' exposure were detected by RT-qPCR. And the data was analyzed by the2-△△Cqmethod. The results indicated that the induction of EE2at00.1-1nM on mRNAexpressions of the grZPB isoforms is weaker than that on vitellogenin. Low dose EE2(0.01,0.1nM) significantly induced the expression of grVTG and1nM EE2strongly significantlyinduced the expression of grVTG.4-NP exposures at three concentrations had differentialeffects on the grZPBs.10nM4-NP significantly depressed the mRNA expression of grZPB.5while100nM4-NP up-regulated the mRNA expression of grZPB.1. BPA at threeconcentrations weakly induced mRNA expressions of the grZPB isoforms.