Cloning And Expression Of The Egg Envelope Glycoprotein ZP3 In Pingxiang Red Transparent Crucian Carp, Carassius Auratus Var. Pingxiangnensis

Pingxiang red-transparent crucian carp, Carassius auratus var. Pingxiangnensis is a wildness natural triplod crucian carp mutant distributed in the area of Pingxiang in Jiangxi Province. It takes bi-sexual and gynogenetic reproduction mode which is similar to the silver crucian carp, Carassius auratus gibelio (Block). Zona pellucida (ZP) is the matrix glycoprotein extracellular around the peripheral eggs. ZP3 is a member of the ZP family. Experiments showed that, ZP3 is the receptor of primary sperm, and it could combine sperms and leads to acrosome reaction in mammals. Its O-linked glycosylated side chain plays a key role during sperm combination.The main content of this paper includes two parts. First: we cloned the cDNA sequence of ZP3 from Pingxiang red-transparent crucian carp by using SMART cDNA synthesis and RACE-PCR methods. The sequence we obtained is 1278bp. It has complete 3'end including terminal codon TAA, a tailing signal AATAAA and a tail with 30-bp long poly A. The partial open reading frame of ZP3 is from 16bp to 1230bp and it encodes 404 amino residues. Analysis of amino acid sequence homology indicated that Pingxiang red-transparent crucian carp showed the highest homolog with silver crucian carp. This results suggested they might have very close relationships. Second: The core fragment of cloned cDNA sequence was subcloned into pET-32a vector. The recombined vector was transformed into BL-21(DE3) E.coli competent cells. They were induced by isopropyl-β-thiogalactopyranoside (IPTG), and then expressed a fusion protien. This protien was purified with affinity resin-Ni-NTA, analysised through Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and quantitated by Brandford method. In result, we obtained the relatively purified fusion protien about 31KDa, with the concentration of 3.588mg/L. This research can be a foundation for preparation of ZP3 polyclonal antibody and further study about the expression characteristics of ZP3 in the process of oogenesis. At the same time it can be the foundation for the in-depth study of Pingxiang red-transparent crucian carp fertilization process through the early sperm and egg combination and identification, thus contributes to understand the sperm and egg identification mechanisms. Also this study provides a theoretical basis for guiding the propagation, breeding and production of Pingxiang red-transparent crucian carp.