Cloning, Molecular Characterization And SNP Application Analysis Of Three Immune Genes From Hyriopsis Cumingii

Triangle sail mussel (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. Understanding the functional genes especially genes related to immunity and their mechanism of H. cumingii is crucial for managing diseases and developing sustainable freshwater pearl mussel culture. Based on the EST sequences of mantle cDNA library of H. cumingii, the complete cDNA of GPX gene, SOD gene, IRF-2 gene in H. cumingii were cloned by rapid amplification of cDNA ends (RACE) in this paper, and obtained cDNA sequences and the amino acids sequences were analysed. Then, partial DNA sequences of these genes and their 5′-flanking regions were amplified from genome DNA isolated from H. cumingii with PCR and genome walking technique. Several SNPs were obtained through direct sequencing these genes of H. cumingii 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 and allelic frequency distribution between two groups. The study provided new available molecular markers for research of genetics and genetic breeding in H. cumingii. The major results were as follows:1. Full-length cDNA cloning and molecular characterization of three immune genes from H. cumingiiThe GPX full-length cDNA sequence was 1286 bp including a 39 bp 5′-untranslated region, a 659 bp 3′-untranslated region and an open reading frame (ORF) 588 bp in length, which encodes for 195 amino acids with the predicted molecular weight of 22.2 KDa and the isoelectric point of 8.44. Molecular structure analysis revealed that GPX was Se-GPX. The amino acid sequences have three highly conserved loop structures that have a stabilizing effect on the tertiary structure of the enzyme. Homologous analysis of amino acid sequences indicated that the GPX of H. cumingii shared a high similarity with GPX-2 and GPX-1 of vertebrates (73.1%-80.8%).The SOD full-length cDNA sequence was 763 bp including a 62 bp 5′-untranslated region, a 74 bp 3′-untranslated region and an open reading frame (ORF) 627 bp in length, which encodes for 208 amino acids with the predicted molecular weight of 22.8 KDa and the isoelectric point of 8.25. The N-terminus hydrophobic region predicted containing of 22 amino acid residues within the SOD displayed the characteristic features of a signal peptide. The amino acid sequences have two CuZnSOD signature sequences, and the amino acids required for the Cu (His-93, -95, -110 and -169) and Zn (His-110, -118, -129 and Asp-132) were conserved in CuZnSOD. Phylogenetic trees showed that CuZnSOD of H. cumingii was clustered together with extracellular CuZnSODs and far away from cytoplasmic CuZnSOD of Cristaria plicata. These results indicated CuZnSOD gene of H. cumingii cloned in this study was extracellular CuZnSOD.The IRF-2 full-length cDNA sequence was 2688 bp including a 110 bp 5′-untranslated region, a 1588 bp 3′-untranslated region and an open reading frame (ORF) 990 bp in length, which encodes for 329 amino acids with the predicted molecular weight of 37.6 KDa and the isoelectric point of 5.1. The amino acid sequences have highly conserved DBD structure in N-terminal that contains characteristic repeats of six tryptophan residues. Homologous analysis of amino acid sequences indicated that HcIRF-2 is overall 24.3-32.6% identical to homologous proteins from mammals, avians, amphibians, and fishs, with 56.6–62.8% identity at the level of the N-terminal DBD and only 9.4–23.1% identity in the C-terminus. 2. Gene sequences and 5′-flanking regions of three immune genes from H. cumingiiThe complete/partial DNA sequence of these genes and their 5′-flanking regions were amplified from genome DNA isolated from H. cumingii with PCR and genome walking technique. The whole sequence of GPX gene with the length of 6708 bp included 5′-flanking region(992 bp), 2 exons and 1 intron. The sequence length of exonⅠ, exonⅡ, and intronⅠwas 273 bp, 991 bp, and 4491 bp, respectively. Sequence analysis of the 5′-flanking region revealed that it contained core promoter element (TATA-box) and other transcription regulation elements such as AP1, C/EBP and CdxA. The partial DNA sequence of SOD gene included three introns, and the length of intronⅡ, intronⅢ, and intronⅣwas 1533 bp, 3973 bp, and 2912 bp respectively.The sequence length of intronⅠand 5′-flanking region of IRF-2 was 465bp and 1669 bp respectively. Sequence analysis of the 5′-flanking region revealed that it contained several transcription regulation elements such as AP1、CdxA、HSF、NIT2 and HNF-3b.3. SNP analysis of resistant stock and susceptible stock from H. cumingii Single nucleotide polymorphisms were obtained through direct sequencing genes of H. cumingii from Poyang Lake wild population (resistant stock, YS) and Jinhua cultured population (susceptible stock, YZ). Twenty-one SNPs were obtained of GPX gene and we investigated the association of these polymorphic locis with resistance trait. Among them, -894G-A SNP、-907A-C SNP、-944A-C SNP in GPX promter, -3833T-A SNP、-3839C-T SNP、-4138G-C SNP、-4142A-G SNP and -5637G-T SNP in GPX intron were significant difference of the genotypes frequency and allelic frequency between two groups (P<0.05).18 SNPs were obtained from SOD gene. Among them, -7994C-G SNP、-8087G-C SNP were significant difference of the genotypes frequency and allelic frequency between two groups (P<0.05). C/G genotype present specifically in resistant stock at -7994C-G site, and G/C genotype present specifically in susceptible stock at -8087G-C site. -8001A-G SNP、-8035G-A SNP、-8191T-A SNP were significant difference of the genotypes frequency and allelic frequency between two groups (P<0.05).12 SNPs were obtained from IRF-2 gene. Among them, -1918C-T SNP、-3786G-T SNP were significant difference of the allelic frequency between two groups (P<0.05); -3917T-C SNP were significant difference of the genotypes frequency between two groups (P<0.05); -4034C-T SNP were significant difference of the genotypes frequency and allelic frequency between two groups (P<0.05). C/C genotype present specifically in resistant stock at -3917T-C site, and T/T genotype present specifically in resistant stock at -4034C-T site.