Functional Verification And SNP Analysis Of Serine Proteinase Genes Of Portunus Trituberculatus

The swimming crab Portunus trituberculatus supports a large crab fishery and aquaculture in China. In recent years, however, with the development of intensive culture, various diseases caused by bacteria and viruses had frequently occurred in cultured P. trituberculatus stocks and then causing enormous economic losses in P. trituberculatus culture. The emulsification disease, a serious disease causing high mortality in P. trituberculatus, is mainly induced by the infection of Vibrio alginolyticus. Therefore, the study of immune defense mechanism and resistance strains for crabs breeding and sustainable development is very important.Traditional selective breeding techniques are always expensive, time-consuming, and easily influenced by the environment and could not fulfill the urgent need for resistant strains. One of the methods that could be used to improve breeding strategies is marker assisted selection(MAS), which is already successfully used in the improvement of agricultural population. Among all kinds of DNA markers, single nucleotide polymorphism(SNP) has been the most widely used due to its just a single base change in a DNA sequence, genetic stability, representative and its high density in genomes.In this study, the genetic polymorphisms of PtcSP and PtSPH in P. trituberculatus were identified and analized on the basis of the serine protease genes of P. trituberculatus, which were reported about their sequences cloning and expression in our laboratory. And the association between the polymorphisms and susceptibility/resistance of P. trituberculatus to V. alginolyticus was investigated. This research will be hopefully to select some molecular markers for selective breeding of crab. Meanwhile, the experiments of protein reconstruction and antibacterial test were carried out to verify the function of the PtcSP gene.The main results are as follows:(1) A 2355 bp fragment of PtcSP gene was obtained. By direct sequencing from 22 susceptible specimens and 22 resistant specimens of P. trituberculatus, a total of 109 SNPs including 77 transitions, 22 transversions and 10 ins-dels were detected. Of these SNPs, 66 SNPs were observed in introns, 42 in coding exons and one in noncoding exons. In the coding region, a total of 16 synonymous mutations and 26 non-synonymous mutations were found in the PtcSP. The ratio of non-synonymous/synonymous(dN/dS) was 1.6, which is widely used as an indicator of selective pressure, suggesting that PtcSP gene had evolved under positive selection(dN/dS > 1). The deletion of C799, G1480 and CG(1750-1751) led to the early termination of open reading frame(ORF).Genotype and allele frequencies of SNPs in PtcSP were numbered and analyzed between susceptible and resistant stocks. Totally eleven polymorphic sites were found to be significantly different in the two stocks by examining the distributions of polymorphisms with ?2 test, suggesting that they might be associated with susceptibility/resistance to V. alginolyticus. One SNP(I1-(170-175) AACAGT-Ins/del) exhibited significant difference in genotype frequencies(P < 0.05) and extremely significant difference in allele frequencies(P < 0.01) in the two stocks, of which AACAGT-Ins allele was more prevalent in resistant stock(59.1%) than in susceptible stock(22.7%). Besides, other 10 SNPs were significantly different in allele frequencies(P < 0.05) between susceptible and resistant stocks.Five SSRs,(AGG)n,(AGG)nAA(GAG)m,(AGG)nTA(GAG)m,(AGG)nTG(GAG)m and(AGG)nTGGTG(GAG)m, were detected in intron 3 of PtcSP gene. No statistically significant difference in polymorphisms of these SSRs was observed between susceptible and resistant stocks. However, some differences were found by analysis of the statistics. Among the five alleles of the unit based on the difference of repeat number of each unit,(AGG)10 demonstrated the highest frequency(66.7% in susceptible crabs and 83.3% in resistant crabs, respectively), followed by(AGG)6-9(13.3% and 16.7%, respectively). Similarly, for the polymorphism of(AGG)4TGGTG(GAG)5, only two crabs were detected in resistant crabs and the others were not found.(2) Genomic sequence of PtSPH was amplified from 21 susceptible samples and 21 resistant samples. In the 1965 bp fragment, totally 77 SNP polymorphisms were detected, including 53 transitions, 18 transversions and 6 ins-dels. Of these, 20 polymorphic sites were detected in introns, 19 in coding exons and 38 in noncoding exons. Among 19 SNPs in the coding region, 12 synonymous mutations and 7 non-synonymous mutations were observed in the PtSPH. The non-synonymous/synonymous ratio(dN/dS = 0.58) was lower than the expected ratio(ratio = 1) under neutral evolution, which may result from that some sites of PtSPH gene were under negative(purifying) selection for functional constrains.Genotype and allele frequencies of SNPs in PtSPH were analyzed and confirmed between susceptible and resistant stocks with the software spss 16.0. According to the data of ?2 test, three SNP loci(I1-326, I1-524 and I2-607) showed significant differences only in allele frequencies(P < 0.05).(3) To further characterize the function of PtcSP2 protein, the expiement was conducted to assay various biologicalfunctions of the clip domain and Tryp_SPc domain of PtcSP by the recombinant of the proteins. The expirment of the proteinase activity exhibited that clip domain of PtcSP2 had antibacterial activity and the Tryp_SPc domain had trypsin-like protease activity. The expirment of the antimicrobial activity was showed that clip domain of PtcSP2 exhibits the same minimum inhibitory concentration, while it showed no yeast bacteriostatic action.