| Silver-lip pearl oyster Pinctada maxima is the best species of producing high quality of large-sized seawater pearls. Yet, since 1985, lots of P. maxima spats died in large scale in the course of culture in the sea, especially at the 5cm larval stage. In the mean time, the wild P. maxima was overfished significantly and thus is facing the danger of extinction. The pearl culture industry was in unprecedented difficulties. However, the causes of spat mortality were kept unknow until now; water pollution might be one of the reasons. Therefore, it is significant to investigate the impact of pollutants on the physiology of P. maxima spats in order to understand the reasons of death.Metallothioneins (MTs) are a family of proteins with low molecular weight, high cysteine content, and high affinity for divalent heavy metal ions. MT plays an important role in removal of free radicals, detoxification of heavy metal ions, metabolism of trace elements, and preventing the process of oncogenesis. In this study, P. maxima MT (PMMT) gene was isolated and cloned from cDNA library, recombinant protein was produced and its expression by cadmium-induced was studied. It is helpful to clarify its work mechanism and activation pathway, as well as to explore the mechanisms of stress response and immune defense. This can also laid a foundation for the comprehensive study on the immune defense system and for selective breeding of stress resistance of P. maxima.1. the full-length cDNA of PMMT was cloned from the full-length cDNA library from the mantle of P. maxima. The full length sequence is 599 bp, including a 5' untranslated region (UTR) of 75 bp, a 3' UTR of 296 bp and an open reading frame (ORF) of 228bp encoding a polypeptide of 75 amino acids. The contents of Cys, Lys and Gly in the PMMT amino acid sequence were 29.3%, 9.3%, 9.3%, respectively, while no Phe, His, Trp or Tyr amino acid was found. 2. The predicted protein from the PMMT sequence was rich in Cys-X(1-3)-Cys frames. It contained nine Cys-X-Cys frames, two Cys-X-X-Cys frames, four Cys-X-X-X-Cys frames and two Cys-Cys frames. The CXCXXXCXCX motif (X is the amino acid except Cys) was similar to that of invertebrates with just one amino acid difference. And the N-terminal sequence CgCgvgCTTlsdCnCt-dCsCK of PMMT was similar with the sequence CxCxxxCTGxxxCxCxxxCxCK of mollusks just with CTT being replaced by CTG. The result of sequence analysis indicated that PMMT was the member of MT family. It can be classified as Familiy 2(mollusc MTs) and subfamily mo (other mollusc MTs) according to the MT classification system. PMMT gene cloning provides a theoretical foundation for further study on evolution, diversity, relationship between structure and function of metallothionein family.3. Prokaryotic expression vector pET-32a-PMMT was constructed and recombinant PMMT was expressed in E. coli induced by IPTG. This lays a foundation for the study of the structure and functions of PMMT protein in vitro.4. A pair of RT-PCR primers was designed according to the cDNA sequence. Semi-quantitive RT-PCR method was used to analyze the quantitative expression of PMMT after exposing to different concentration of cadmium. PMMT gene expression can be detected in the control group without cadmium stimulus. At low concentrations of cadmium (0.005mg/L), PMMT expression increased by 1.70times compared to the control. For high concentration groups (0.05mg/L, 0.10mg/L), the ratios increased to1.79and 2.08 times, respectively. It showed that cadmium can induce the expression of PMMT gene and the gene expression level rises with the cadmium concentration increases. This indicated that PMMT gene can be used to inspect cadmium pollution levels in farming environment as a biomarker, and provides the basic reference for studying the death of P. maxima spats.
|
PDF Full Text Request