Molecular Characterization,Expression Analysis And SNP Application Analysis Of SPI And LBP/BPI Genes From Hyriopsis Cumingii

Hyriopsis cumingii is the most important mussel species commercially exploited for freshwater pearl production in China. In recent years, H. cumingii cultivation has suffered seriously from high mortality. LPS-binding protein/Bactericidal Permeability Increasing protein(LBP/BPI), Kazal-type serines proteinase inhibitor(KSPI) and Kutiz-type serines proteinase inhibitor(Ku SPI) are very important in the immune reaction of biological body, but the study of KSPI in Hyriopsis cumingii has been rarely reported.. Based on the EST sequences of mantle of H. cumingii, the complete c DNA of LBP/BPI gene, KSPI gene, Ku SPI gene in H. cumingii were cloned by rapid amplification of c DNA ends(RACE), and obtained c DNA sequences and the expression were analysed with RT-PCR. Then, the full-length of KSPI gene was contained with PCR technique based on the c DNA sequences. Several SNPs were obtained through direct sequencing from two populations(resistant stock and susceptible stock). And we analyzed the association of these polymorphic locis with resistance trait by investigating the genotypes frequency between two groups. The major results were as follows:Full-length c DNA cloning and molecular characterization of three immune genes from H. Cumingii.The full length of c DNA sequence of LBP/BPI gene of H.cumingii was cloned. It was 1825 bp, containing 5’and 3’-UTRs apart of 97 bp and 219 bp, and an ORF of1509 bp encoding 502 amino acids, and the molecular weight was 56478.5u. The amino acid sequence contains two BPI domain structures, and the result of homology analysis showed that it has a few of similarity with other species, suggesting that this gene belongs to the typical LBP/BPI gene. Real-time quantitative PCR showed that LBP/BPIgene expressed in a wide range of tissues including the adductor; foot; liver; blood;mantle; gill; gonad; intestine; kidney and the highest expression was in blood, on the contrary, the lowest in foot. After being infected with Aeromonas hydrophila, the expression of LBP/BPI gene had significant increasing in blood and mantle compared with the control group. The results suggested that BPI gene is very important in the immune reaction of Hyriopsis cumingii.The full length of c DNA sequence of Kazal-type serines proteinase inhibitor(KSPI)gene of H.cumingii was cloned. It was 1029 bp, containing 5’and 3’-UTRs apart of 61 bp and 206 bp, and an ORF of 762 bp encoding 253 amino acids, and the molecular weight was 27397.5u. The amino acid sequence contains five Kazal domain structures, and the result of homology analysis showed that it has a few of similarity with other species,suggesting that this gene belongs to the typical KSPI gene. Real-time quantitative PCR showed that KSPI gene expressed in a wide range of tissues including the adductor; foot;liver; blood; mantle; gill; gonad, and the highest expression was in mantle,on the contrary,the lowest in liver. After being infected with Aeromonas hydrophila, the expression of KSPI gene had significant increasing in 7 tissues compared with the control group.The full length of c DNA sequence of Ku SPI gene of H.cumingii was cloned. It was624 bp, containing 5’and 3’-UTRs apart of 38 bp and 148 bp, and an ORF of 438 bp encoding 145 amino acids, and the molecular weight was 1637.5u. The amino acid sequence contains one Kuntiz domain structures, and the result of homology analysis showed that it has a few of similarity with other species, suggesting that this gene belongs to the typical Ku SPI gene. Real-time quantitative PCR showed that Ku SPI gene expressed in a wide range of tissues including the adductor; foot; liver; blood; mantle;gill; gonad; intestine; kidney and the highest expression was in mantle, on the contrary,the lowest in gonad. After being infected with Aeromonas hydrophila,the expression of Ku SPI gene had significant increasing in mantle compared with the control group. The results suggested that Ku SPI gene is very important in the immune reaction of Hyriopsiscumingii.The full length of DNA sequence of Kazal-type serines proteinase inhibitor(KSPI)gene of H.cumingii was cloned. It was 4749 bp, containing 5 exons and 4 introns. The length of 5 exons apart of 110 bp, 94 bp, 132 bp, 275 bp, 420 bp. The length of 4 introns apart of 521 bp, 1990 bp, 436 bp, 739 bp.Single nucleotide polymorphisms(SNPs) were obtained through direct sequencing genes of H. cumingii from resistant stock population and susceptible stock population.23 SNPs were obtained of KSPI gene and we investigated the association of these polymorphic locis with resistance trait. Among them,-1882G-A SNP、-3694A-C SNP、-3793A-C SNP,-4119T-A SNP、-4199C-T SNP、-4114G-C SNP、-1291A-G SNP in KSPI introns and-2812G-T SNP in KSPI exon were significant difference of the genotypes frequency and allelic frequency between two groups(P<0.05).