The Genetic Analysis Of Vibrio Resistant Mechanism Of The Clam Meretrix Meretrix Based On The Polymorphism Of MIF And Its Downstream Genes

The clam(Meretrix meretrix) is an important economic species of marine bivalve. In recent years, clam diseases caused by bacteria and virus are becoming more and more common, which inflicted serious financial losses. Hence, it is urgent and crucial to carry out genetic breeding for the selection of high Vibrio-resistance strains of the clam M. meretrix. Marker-assisted selection(MAS) is a powerful method with which breeders can select animals with desirable combination of genes. While screening of molecular markers associated with certain traits is the first step for MAS. Among the molecular markers, single nucleotide polymorphisms(SNPs) are co-dominant, biallelic and distributed widely with high density. The candidate gene set approach is a new method for research on animal traits related SNPs, which was derived through comparing and integrating the existing methods. Mm MIF, Mm SAA and Mm VEGFA were three immune-related genes and there are certain connect among them. In this study, Mm MIF, Mm SAA and Mm VEGFA were chosed as the candidates and formed a gene set which have potential association with vibrio-resistance. Identification of these genes and analysis of their polymorphism, and screening SNPs 5 associated with vibrio-resistance, will provide useful markers for selective breeding of this clam. The research results are as follows:1. A homolog of MIF was identified from clam M. meretrix. The M. meretrix MIF(Mm MIF) contained 4 exons and 3 introns, encoding a 114-amino acid protein. Sequence comparison indicated that the Mm MIF corresponds to the Mytilus galloprovincialis MIF with 78% similarity. Genotyping result showed 10 single SNPs(g.727, g.737, g.807, g.810, g.820, g.821, g.829, g.875, g.886 and g.889) were significantly associated with vibrio-resistance(P<0.05). The effect of a missense mutation(g.737, Tâ†'C) was detected by site-directed mutagenesis with fusion expression of protein assay, and no variation was found of the Mm MIF’s redox enzyme activity and tautomerism enzyme activity between the wild-type Mm MIF and mutant Mm MIF. Four haplotypes were developed based on the 10 SNPs associated with vibrio-resistance. Hap1(GAGATGTATG) and Hap2(TGATCAATCA) were significantly associated with vibrio-resistance and the Hap1 was considered to be the dangerous haplotype and Hap2 was the protective one. In healthy clam, Mm MIF was expressed in all detected tissues. Distinct expression patterns of Mm MIF were observed among different Haplotypes(Hap1, Hap1/Hap2, Hap2) after challenge with Vibrio Parahaemolyticus and PBS. The result indicated that, haplotypes did not affect the expression of Mm MIF at 0 h in control group; Mm MIF expression in clams with Hap1 and Hap1/Hap2 was significantly lower than that with Hap2 at 24 h in PBS-injected group(P < 0.05); the expression of Mm MIF in clams with Hap2 was significantly lower than that with Hap1 and Hap1/Hap2 at 24 h in vibrio-injected group(P < 0.05). These studies provide potential markers as well as theoretical basis for resistance breeding of this clam.2. A SAA gene(Mm SAA) was identified in the clam M. meretrix. The full length DNA of Mm SAA was 1407 bp, consisting of 3 exons and 2 introns. The distribution of Mm SAA in clam tissues was examined with the highest expression in hepatopancreas. In response to the Vibrio parahaemolyticus challenge, Mm SAA m RNA showed significantly higher expression at 24 h post-challenge in experimental clams(P < 0.05). Five single SNPs(g.42, g.72, g.82, g.147 and g.165) were significantly 6 associated with Vibrio-resistance(P < 0.05). The effect of a missense mutation(g.82, A/G) was detected by site-directed mutagenesis with fusion expression of protein assay, and the result showed that the recombinant plasmids containing wild-type p ET30a-Mm SAA had more inhibition effect than the mutant one on the growth rate of the host bacteria. In addition, four growth traits of the clams in 09G3 SPSB population were recorded and the SNP g.176 was found to be significantly associated with the growth traits with the Global score value 0.790(P = 0.015). Our findings suggested that gene polymorphisms in Mm SAA might contribute to the risk of susceptibility to Vibrio infection and might be associated with the growth traits in the clam M. meretrix.3. In this study, we cloned and sequenced a clam M. meretrix vascular endothelial growth factor A(Mm VEGFA) c DNA. It encodes a precursor protein of 308 amino acids. Sequence comparison indicated that the Mm VEGFA corresponds to the Crassostrea gigas VEGFA isoform with 54% similarity. Eight SNPs(g.124, g.165, g.167, g.197, g.276, g.324, g.326 and g.356) were found to be significantly associated with Vibrio-resistance(P<0.05). Haplotype analysis indicated that Hap1(TGATCAATCA) and Hap2(CGCTTACG) were significantly associated with vibrio-resistance. Regarding Hap1 as the baseline, Hap2 was found to significantly increase the risk of susceptibility to vibrio infection with the adjusted odds ratio(OR = 2.23, 95%, CI:1.21-4.12, P = 0.01). Besides, three tag SNPs(g.165, g.356 and g.356) were abtained through analysis of the SNPs in Mm VEGFA and Mm MIF. To investigate the interactions between different SNPs which were significantly associated with vibrio-resistance, two methods(Logistic regression with log-linear model & Multifactor dimensionality reduction) were used respectively in this study. The result indicated that these three SNPs showed their effects on the resistanct of clam respectively and there was no significant interactive efficiency between each two tag SNPs.SNPs which were associated with clam vibrio-resistance obtained in this study may be used as genetic markers for clam breeding.The preliminary exploration for the function of immune related genes/SNPs, will provide the important basis for further study about immune response mechanism of M. meretrix in response to Vibrio infection.