Studies On Genetic Breeding And Two Immune Genes Of Pinctada Fucate

Pearl oyster Pinctada fucata is distributed over coastal area of South China and isthe most popular farming shellfish for seawater pearl production. However, since themid-1990s, pearl oyster begins to die largely in China and leads to a dramatic decline inSouth China Seawater pearl production. Although it is still unclear about the causes forpearl oyster high mortality, it is believed that the high mortality is related to theseawater pollutions, the disease outbreaks and the stock degeneration. In order tocontrol disease and enhance the yields and quality of seawater pearl, it is necessary toresearch the genetic breeding and the innate immune defense mechanisms of pearloyster, which lacks the adaptive immune system.This studies using the pedigree selection and family breeding method to foster thenew strain of P. fucata with fast growth, strong resistance and better economy benefit. In thecolor selective breeding, four different shell color selective breeding with no radiationgray (white arrises), no radiation belt golden (golden edges), radiation belt edgesectors-connecting black (black arrises), radiation belt red (red arrises) are establishedrespectively. Twenty color families of pearl oyster P. fucata were established. The growthtraits and shell width index of these P. fucata families were compared, and also the growthat each month of Pinctada fucata families were observed; In order to provide the theorybasis and the data support for the genetic breeding of P. fucata , the heritability and geneticcorrelation of fifteen full sib-families larvae of P. fucata were estimated using themethod of REML. Meanwhile,Catalase (Catalase, PoCAT) and antimicrobial peptidestheromacin (PoAP) related to the immune system in pearl oyster P. fucata have beenclone and analyzed. Futhur, The PoAP gene was linked into prokaryotic vector pET-32α,and the fusion proteins were successfully expressed in E.coli BL21. In addition, theantibacterial spectrum of PoAP has been studied . The main results were listed as below: 1. The foundation and comparation of the growth traits of P. fucata color familiesTwenty color families of pearl oyster P. fucata are established by using single pairmating method and their growth traits of six months are studied. The results showed thatBSF4-3, RSF4-1, RSF4-2 and RSF4-5 were more excellent than other ones in thegrowth traits as a whole. So they were considered as the materials for further slecetivebreeding for rapid growth. The family BSF4-3 was the biggest one in shell width indexunder the comparation of each familiy, WSF4-2,WSF4-3 and RSF4-5 took the secondplaces,RSF4-1 and RSF4-2 relatively low. Compared to the four color selecting lines ,the growth trait of the red and black color selecting lines significantly different from theother shell color lines. The red shell color line had the largest shell length ,shell height,shell width and the gross weight. while the white shell color line displayed the smallest.2.Estimates of heritability and genetic correlations for growth of P. fucata larvaeHeritability in the narrow sense (h2) expresses the proportion of the totalphenotypic variance in a trait that is attributable to additive genetic effects. In march2010, the black,gold,yellow and white shell color lines of pearl oyster P. fucata wereestablished by separately selecting black,red,yellow and white shell color breeders inXincun stock,Lingshui of Hainan. Fifteen Full sib-families were obtained by artificialfertilization based on each sire was mated to one dams, including four gold colorfamilies, five black color families, three white color families and three red colorfamilies. On days 5,15,25 and 40,50 individuals were randomly sampled from each offamilies and shell length and shell height of each individual were measured. Analysis ofvariances was conducted to evaluate growth differences among the four shell color lines.Genetic parameters of shell length, shell height and condition factor in P. fucata on 5,15,25 and 40 days were estimated byDF-REML．Significant differences were found ingrowth among the four shell color lines at each sampling time(P<0.05). During the fourtimes, the white shell color line had the largest shell length and the largest shell height,while the white shell color line displayed the smallest. The estimates of narrow-senseheritabilites were 0.17～0.23 for shell height and 0.12～0.22 for shell length. Positivephenotypic correlations were found among shell length and shell height (P<0.01)．Thecorrelations were significant. The trend of genetic correlation was the same as thephenotypic correlation. The estimates of genetic and phenotypic correlation coefficients are 0.825～0.989 and 0.832～0.965,respectively. Medium heritability estimates andpositive genetic correlations indicate that both shell height and shell length shouldrespond favorably to direct and indirect selection for growth．Further studies are neededto establish the precise contributions of these variation sources on adult stages indifferent environments to assess the real potential for the response to selection．3. The characteristics and expression analysis of catalase gene of P. fucataCatalase (EC 1.11.1.6) is an important antioxidant enzyme that protects aerobicorganisms against oxidative damage by degrading hydrogen peroxide to water andoxygen. In the present study, a catalase cDNA of peal oyster P. fucata (designated asPoCAT) is cloned and characterized by expressed sequence tag (EST) and rapidamplification of cDNA ends (RACE) approaches. PoCAT is 2428 bp long and consistsof a 5'-UTR of 140 bp, an unusually long 3'-UTR of 749 bp and an open readingframe (ORF) of 1539 bp. The ORF of PoCAT encodes a polypeptide of 512 aminoacids with molecular weight of 58.1 kDa and the theoretical isoelectric point of 8.4.PoCAT shares 62.3-82.2% identity and 73.0-92.0% similarity to other catalase aminoacid sequences. Sequence alignment indicates that PoCAT contains the proximalheme-ligand signature sequence (R351LFSYSDT358), the proximal active site signature(F61NRERIPERVVHAKGGGA78) and the three catalytic amino acid residues (His72,Asn145, and Tyr 355). Twelve amino acids (N145, H191,F195,S198,R200,N210,Y212,K234,I299,W300,H302,Y355) were identified as putative residues involved in NADPH binding.PoCAT has two potential glycosylation sites (N436YS438 and N478FS480). PoCAT mRNAis ubiquitously expressed in all detected tissues, and the expression level of PoCATmRNA is higher in intestine and mantle. The expression profile analysis shows that theexpression level of PoCAT mRNA in intestine is significantly up-regulated at 2, 4 and12 h after Vibrio alginolyticus stimulation. These results demonstrated that PoCAT is atypical member of catalase family and might be involved in innate immune responsesof pearl oyster.4.cDNA cloning and procaryotic expression of antimicrobial peptides theromacinof P. fucataAntimicrobial peptides (AMPs) have been proven to be one of the mostimportant humoral components that afford resistance to pathogen infection. The AMP gene to be identified was that encoding theromacin in the pearl oyster P. fucta (PoAPtheromacin); The full-length theromacin cDNA contains 522 bp and consists of a5'-UTR of 6 bp, an unusually long 3'-UTR of 749 bp and an open reading frame (ORF)of 375 bp that encodes a 124 amino acid peptide with molecular weight of 13.67 kDaand the theoretical isoelectric point of 9.25. Homology analysis of the deduced aminoacid sequence of the PoAP with other known theromacin sequences by MatGATsoftware revealed that the PoAP shared 29.0%-46.8% similarity to the other knowntheromacin sequences. Signal P-N program showed that Hc theromacin contains 33amino acid signal peptide and a mature peptide located at amino acids 34-124; themature peptidecontains 12 cysteine residues and 13 alcaline amino acid residues withmolecular weight of 13.67 kDa and the theoretical isoelectric point of 9.25. TMpredprogram suggests that PoAP theromacin is a membrane protein with 1 transmembranehelix between amino acids 13 and 34. The PoAP gene was linked into prokaryoticvector pET-32α, and the PoAP fusion protein with 31 kDa molecular mass wassuccessfully expressed in E.coli BL21. Using His- Bind Purification Kit Protocolpurify the antimicrobial peptides recombinant protein and compared to thebacteriolysis effect on gram-positive bacteria and gram-negative bacteria using thepurified protein. The results show that PoAP theromacin proteins had obviousbacteriolysis to the gram-negative bacteria,but no gram-positive organisms .Thisshows antibacterial peptides theromacin play a important antibacterial function in theimmune responses of pearl oyster.