Single Nucleotide Polymorphism Of Hemocyanin Ig-like Domains In Litopenaeus Vannamei

Although it was long believed that the immune system of invertebrate didn't possess molecular diversity because of restrict germline genes. However, recent evidence from numerous researches suggest that some highly variable genes which are possibly immune-related have been identified in invertebrates. Hemocyanin is a multifunctional protein in the shrimp and other invertebrates, such as antiviral, antibacterial, phenoloxidase activity and other immunological activities,and our early results showed that it's multifunction may be correlated to the molecular polymorphism. Combined with bioinformatics, SSCP(single-strand conformation polymorphism), plasmid library construction, DNA sequencing, gene cloning , prokaryotic expression and other molecular biological techniques, we studied the polymorphism characteristics of Litopenaeus vannamei hemocyanin, the preliminary mechanism and the significance in immunological function. The results were as following:1. Hemocyanin shows polymorphisms with SNPsFirst, we cloned two subunits sequences of hemocyanin from Litopenaeus vannamei (named HcS and HcL). Bioinformatics analysis revealed that either subunit possesses an Ig-like domain which has a highly homologous with IgSF (immunoglobulin superfamily).To invest it's molecular diversity ,from Litopenaeus vannamei EST database, we found 14 and 35 significant mutations sites in HcS and HcL cDNA respectively, concentrated in Ig-like domains through, suggesting that there maybe exist molecular diversity of SNPs (single nucleotide polymorphism) in hemocyanin.Then, through PCR-SSCP, we found that there are 4 and 5 bands each in the SSCP images of HcSC3 and HcLC1, and they have different sequences respctively, there were 2318, 2370, 2413, 2460, 2466 and 1315, 1410, 1432 SNPs respectively. illustrated the SNPs polymorphism in hemocyanin. Further, we constructed 24 plasmid library and sequenced hemocyanin Ig-like domains up to 2428 sequences. The results showed that:1)there are 78 sites of SNPs in HcS Ig-like domain ,and the SNPs polymorphisms in DNA exon, intron and mRNA were 0.36-8.00%, 0.36-31.27% and 0.27-3.48% respectively, the consistency between DNA and mRNA level was about 11.1%. At the individual level, the distribution of SNPs polymorphism was similar. In tissue level, the polymorphisms in four tissues were different. 2)The polymorphism in HcL was obviously higher than HcS, the polymorphisms in DNA exon, intron and mRNA were 0.34-14.36%,0.34-14.63% and 0.27%-25.60% respectively. The rate of five important polymorphic sites, 1272,1315,1410,1450,1938 SNPs polymorphism were over 10%.and we found the similar polymorphisms phenomenon of important SNPs sites in different tissues and individuals except gill, and the consistency between DNA and mRNA level was about 43.9%.2. Hemocyanin SNPs maybe mainly related to inheritance and somatic mutationsIn order to investigate the the relationship between polymorphisms and genetic or environmental in hemocyanin Ig-like domains, we used SSCP and sequencing methods to expore the SSCP difference in different development stages, and constucted plasmid library of nauplius phase I. The result found that, HcSC3 SSCP pattern different in four development stages, and compared with adult shrimp, the SNPs consistency in DNA and mRNA level were 35.71% and 21.43% respectively. HcLC1 SSCP pattern almost same in four development stages, and compared with adult shrimp, the SNPs consistency in DNA and mRNA level were 66.67% and 63.64% respectively. So, we suppose the polymorphism of HcS Ig-like domain maybe associated with genetic and environmental, and HcL Ig-like domain polymorphism should be caused by genetic.3. Hemocyanin SNPs maybe one of the molecule basis against pathogenic microorganismsTo research the relationship of hemocyanin diversity and it's immunological mutifunction, through PCR-SSCP technique, we studied the HcSC3 and HcLC1 SSCP after bacteria and poly(I:C) stimulation. The results showed that HcSC3 polymorphism was significant change at 9 h, 12 h, 24 h after Vibrio parahaemolyticus stimulation, and poly(I:C) stimulation changed SSCP pattern at 3 h. However, the change of HcLC1 is not as obvious as HcSC3, only at 24h after poly(I:C) stimulation, the SSCP graphics have a little change.Furthermore, we explore HcS SNPs related to its resistance to Vibrio parahaemolyticus. Compared with susceptible group, we found a extra band in resistance group, there was 4 SNPs in 2334,2342,2354 and 2374 sites. It proved that the existence of hemocyanin SNPs associated with shrimp resistance.In conclusion, our present results showed that hemocyanin Ig-like domains exhibited SNPs polymorphisms, the possible mechanism of HcS Ig-like domain polymorphism maybe associated with genetic and environmental, and HcL Ig-like domain polymorphism should be caused by genetic. We speculate that hemocyanin SNPs maybe one of the molecule basis against pathogenic microorganisms. Our results are helpful to interpret the molecular foundation of hemocyanin diversity, make up for the deficiencies in the natural immune system.