MIF及其下游基因多态性与文蛤弧菌抗性机制的遗传解析

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	MIF及其下游基因多态性与文蛤弧菌抗性机制的遗传解析
	邹林虎
学位类型	硕士
导师	刘保忠
	2015-05-15
学位授予单位	中国科学院大学
学位授予地点	北京
学位专业	海洋生物学
关键词	文蛤弧菌抗性 Snps 关联分析 Mmmif Mmsaa Mmvegfa
摘要	文蛤是一种重要的经济养殖贝类。近年来，由细菌和病毒引发的病害问题使得文蛤养殖业损失惨重。因此，通过遗传育种的方式培育出具有病原抗性的文蛤品系对于文蛤养殖业来说显得尤为迫切重要。分子标记辅助选育(MAS)是一种有效的育种方法，借助MAS技术，育种家们能够选择出带有理想性状相关基因的生物个体。而运用MAS技术进行遗传育种，就必须先选择出与生物特定性状相关的分子标记。在众多的分子标记中，单核苷酸多态性(SNP) 具有共显性、二等位性、分布广泛以及高密度等优点，因而成为许多学者研究的焦点。候选基因集法(Candidate gene set approach) 是在比较和整合已有方法而提出的适于研究非模式动物SNP的新方法。本研究中，我们选取了Meretrix meretrix MIF(MmMIF)，Meretrix meretrix SAA(MmSAA) 和Meretrix meretrix VEGFA(MmVEGFA) 3个相关的免疫相关基因构成一个候选基因集，分析这3个基因及其序列多态性与文蛤弧菌抗性的相关性，希望获得与文蛤弧菌抗性相关的SNP标记，以指导文蛤的抗性育种工作。主要结果如下： 1、本研究从文蛤中克隆获得了一个与MIF同源的基因(MmMIF)。MmMIF基因包括4个外显子和3个内含子，编码114个氨基酸。蛋白序列比对结果表明MmMIF与紫贻贝的相似性最高(78%)。在MmMIF基因中，通过分型获得10个SNPs (g.727，g.737，g.807，g.810，g.820，g.821，g.829，g.875，g.886 和 g.889)与文蛤的弧菌抗性性状显著相关(P<0.05)。通过定点突变与原核重组表达试验验证了其中的错义突变SNP (g.737, T→C)对于MmMIF蛋白的氧化还原酶活性和互变异构酶活性没有显著影响(P>0.05)。对这10个抗性相关SNPs进行单体型分析共获得4个单体型。其中单体型Hap1(GAGATGTATG) 和Hap2(TGATCAATCA)与文蛤的弧菌抗性性状呈极显著相关(P<0.05)，同时Hap1为危险性单体型，Hap2为保护性单体型。文蛤的组织表达实验表明，MmMIF在所有检测的组织中广泛表达。分别用副溶血弧菌(Vibrio Parahaemolyticus)和PBS刺激文蛤后，不同单体型(Hap1, Hap1/Hap2, Hap2)文蛤中MmMIF基因的表达结果表明：在0 h control group中，不同单体型的文蛤中MmMIF基因的表达量之间没有显著性差异(P>0.05)；在24 h PBS-injected group中，单体型为Hap1和Hap1/Hap2的文蛤的MmMIF基因的表达量显著低于单体型为Hap2的文蛤的表达水平(P < 0.05)；在24 h vibrio-injected group中，单体型为Hap2 的文蛤MmMIF基因的表达水平显著低于单体型为Hap1和Hap1/Hap2的文蛤个体(P < 0.05)。这些研究结果不仅为文蛤的抗性选育提供了可能的分子标记，同时也为进一步探索MmMIF基因中抗性相关SNP发挥功能的机制奠定了基础。 2、从文蛤中克隆获得了一个SAA基因(MmSAA)。MmSAA基因的DNA序列为1407 bp，由3个外显子和2个内含子组成。MmSAA基因的组织表达分析表明，MmSAA在文蛤各个组织中广泛表达，在肝胰脏中的表达量最高。用副溶血弧菌注射文蛤后24 h，MmSAA基因在肝胰脏中的表达量相对于对照组显著上调(P < 0.05)。在MmSAA基因中通过分型获得5个SNP(g.42，g.72，g.82，g.147 和 g.165)与文蛤对于弧菌的抗性性状显著相关(P < 0.05)。通过定点突变与原核重组表达试验对其中的错义突变SNP(g.82, A / G)进一步研究，结果表明，野生型重组表达载体pET30a-MmSAA对于宿主菌生长速率的抑制效果显著高于突变型重组表达载体。此外，对09G3SPSB文蛤群体4个生长相关性状进行测量并初步统计，通过关联分析筛选与文蛤生长性状相关的SNP位点，结果表明，SNP g.176与文蛤的生长性状显著相关(P = 0.015)。本研究的结果表明，MmSAA基因的多态性与文蛤的弧菌抗性性状和文蛤的生长性状相关，且抗性与生长相关的SNP之间是没有重合和关联的。本实验的结果有望对选择弧菌抗性强、生长快的文蛤品题提供参考标记。 3、从文蛤中克隆获得了一个VEGFA基因(MmVEGFA)。MmVEGFA基因共编码308个氨基酸。蛋白序列比对分析表明MmVEGFA与长牡蛎的VEGFA蛋白的相似性最高(54%)。在MmVEGFA基因中，共获得8个SNPs(g.124，g.165，g.167，g.197，g.276，g.324，g.326 和 g.356)与文蛤弧菌抗性性状显著相关(P < 0.05)。单体型分析结果表明，Hap1(TGATCAATCA) 和Hap2(CGCTTACG) 与文蛤弧菌抗性显著相关(P<0.05)，同时Hap1为保护性单体型，Hap2为危险性单体型。以保护性单体型Hap1为参照，单体型Hap2显著地增加了文蛤感染弧菌的危险度(OR = 2.23, 95%, CI:1.21-4.12, P = 0.01)。此外，以MmVEGFA和MmMIF基因中与文蛤抗性性状相关的SNPs为对象进行tagSNPs的筛选，共获得3个tagSNPs (g.165，g.356 和 g.829)。为了探索不同基因中的SNPs对于文蛤弧菌抗性性状的贡献是否存在交互作用，本研究采用了两种方法(一般Logistic回归模型结合对数线性模型 & 多因子降维法)进行了分析。结果表明，这3个tagSNPs独立对文蛤的弧菌抗性性状有显著影响，但它们之间并不存在显著的交互作用。本研究获得的与文蛤弧菌抗性性状相关SNPs和单体型可作为文蛤抗性品种选育的分子标记。对于免疫相关基因和SNP的作用机制的初步探索，将为进一步解析文蛤在应对弧菌感染时机体的免疫应答机制提供有益的启示。
其他摘要	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. MmMIF, MmSAA and MmVEGFA were three immune-related genes and there are certain connect among them. In this study, MmMIF, MmSAA and MmVEGFA 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 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 (MmMIF) contained 4 exons and 3 introns, encoding a 114-amino acid protein. Sequence comparison indicated that the MmMIF 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, T→C) was detected by site-directed mutagenesis with fusion expression of protein assay, and no variation was found of the MmMIF’s redox enzyme activity and tautomerism enzyme activity between the wild-type MmMIF and mutant MmMIF. 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, MmMIF was expressed in all detected tissues. Distinct expression patterns of MmMIF 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 MmMIF at 0 h in control group; MmMIF 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 MmMIF 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 (MmSAA) was identified in the clam M. meretrix. The full length DNA of MmSAA was 1407 bp, consisting of 3 exons and 2 introns. The distribution of MmSAA in clam tissues was examined with the highest expression in hepatopancreas. In response to the Vibrio parahaemolyticus challenge, MmSAA mRNA 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 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 pET30a-MmSAA 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 09G3SPSB 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 MmSAA 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 (MmVEGFA) cDNA. It encodes a precursor protein of 308 amino acids. Sequence comparison indicated that the MmVEGFA 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 tagSNPs (g.165, g.356 and g.356) were abtained through analysis of the SNPs in MmVEGFA and MmMIF. 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 tagSNPs. 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.
学科领域	海洋生物学
语种	中文
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/22776
专题	实验海洋生物学重点实验室
作者单位	中国科学院海洋研究所
第一作者单位	中国科学院海洋研究所
推荐引用方式 GB/T 7714	邹林虎. MIF及其下游基因多态性与文蛤弧菌抗性机制的遗传解析[D]. 北京. 中国科学院大学,2015.

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