CDNA Cloning And Expressive Characterization Of Vitellogenin In Neocaridina Denticulata Sinensis

Neocaridina denticulata sinensis is a good material in ecological, physiological and toxicological experiments. Because of the large quantity of production, it has a certain economic value. Vitellogenesis(Vg) is the central event of egg maturation in all oviparous species, it is the major yolk protein precursors in most animals including crustaceans. Crustacean’s Vg is a high-molecular-weight protein associated with lipidic, glucidic, and carotenoid prosthetic groups. Vg is transported to the oocyte via the hemolymph, and internalized into the oocyte through receptor-mediated endocytosis. In the oocyte, Vg undergoes further modifications to vitellin. Then, the vitellins are eventually used as a nutrient source for the developing embryos. Crustacean’s vitellogenesis and the structure of Vg have been studied by histology, immunohistochemistry and electrophoretic techniques. In recent years, people have a better understanding in the expression of Vg through the study of shrimp’s Vg cDNA synthesis. The study of Vg cDNA of N. denticulata sinensis has not been reported.First, the Vg cDNA sequences of Penaeus semisulcatus, Litopenaeus vannamei, Marsupenaeus japonicas, Fenneropenaeus chinensis, Metapenaeus ensis, Homarus americanus were searched in the system of the NCBI’s blastn, then those sequences were compared by the bioedit to find conservative regions. At last, a 474 bp cDNA sequence from the ovary of mature shrimp, N. denticulata sinensis, was cloned through homologous cloning method.The Vg cDNA sequences of Macrobrachium rosenbergii and Penaeus monodon were compared by DNAMAN. The results showed that the identity of this sequence and Vg mRNA sequence of M. rosenbergii, P. monodon was 54.8% and 51.5%. A phylogenetic tree was established based on the corresponding Vg mRNA of eleven species by MEGA 4.1. The results showed that N. denticulata sinensis, P. monodon and M. rosenbergii has a closer affinity. The farthest kinship with N. denticulata sinensis is P. trituberculatus. The results of phylogenetic tree showed that the Vg conservative and the evolution of shrimp has a strong correlation.According to the Vg cDNA sequence of N. denticulata sinensis, two primers were designed to prokaryotic expression. A recombinant plasmid(pET-VgE) was constructed. A protein was successfully expressed. The Western blot results showed that the Vg cDNA sequences was the correct result. Because the method of the protein expression and protein purification are relatively easy, so this protein will be used to establish a more mature, simple identification system of N. denticulata sinensis’Vn.The Semi-quantitative PCR results showed that N. denticulata sinensis’s Vg mRNA were expressed both in the ovary and hepatopancreas. The hepatopancreas is the main Vg synthesis site in the early and medium-term stage of ovarian development. Ovary is the main Vg synthesis site when it’s mature. There are two stages of the Vg formation in N.denticulata sinensis. The first stage is that Vg is synthesized in the hepatopancreas. The second stage is that Vg synthesized in the hepatopancreas is absorbed by oocytes. Vg is modified in the ovary to produce Vn for the embryo developing. The model regarding to the Vg creation in the hepatopancreas of N. denticulata sinensis needs further study.