大口黑鲈摄食调控相关基因SNPs的筛选及与生长关联分析

发布时间：2018-05-19 03:41

本文选题：大口黑鲈(Micropterus + salmoides)　；参考：《上海海洋大学》2016年硕士论文

【摘要】：大口黑鲈(Micropterus salmoides)是鲈形目(Perciformes)中的一种,在我国的养殖品种里,也是养殖面积较大的鱼类之一。大口黑鲈是典型的强肉食性鱼类,其传统的养殖方式主要是投喂冰鲜下杂鱼,这不仅使其养殖业受海洋渔业资源不足的限制,还存在冰鲜鱼体引入病原体污染水体的风险,导致难以开展工厂化养殖和无公害水产品的生产。近年来,使用人工配合饲料投喂大口黑鲈取得了一定的成效,但人工配合饲料养殖大口黑鲈在中后期生长速度迟缓的现象普遍发生,这也严重限制到了我国大口黑鲈养殖业的进一步发展。培育出能较好摄食并利用人工配合饲料、具有优良生长性状的大口黑鲈新品系,是现阶段大口黑鲈选育工作的重要目标。分子标记辅助育种技术是鱼类遗传改良的重要手段,借助分子标记对候选基因和目标性状进行关联和选择分析,能很大程度提高相关经济性状选择的效率,并可有效缩短育种时间。根据已有研究报道,鱼类的摄食受多种内在和外在因素的影响,其中食欲状况是鱼类主要的内在影响因素,当养殖鱼类处于食欲兴奋状态时,它们的饲料摄入量以及利用率都会相应的得到改善;鱼类摄食的调控主要通过大脑和神经外周信号的相互作用完成,食欲相关基因是参与鱼类摄食调控的主效基因,该类基因的突变会对生长性状产生影响;本研究选取Ghrelin、PACAP和NPY等摄食相关基因作为大口黑鲈生长性状相关的候选基因,分析其单核苷酸多态性(single nucleotide polymorphism,SNP)与生长性状的关联性,以期为大口黑鲈的分子标记辅助育种提供有效的标记,促进食性改良大口黑鲈新品系的培育。主要研究内容如下:1.大口黑鲈Ghrelin基因SNPs的筛选及与生长性状关联性分析Ghrelin是脊椎动物的一种脑肠肽,有促进摄食功能,并能促进生长激素(GH)释放,参与能量平衡调控和糖类代谢。为探索ghrelin基因多态性与大口黑鲈生长性状的相关性,实验针对大口黑鲈ghrelin基因的启动子序列,采用直接测序法获得了2个SNPs位点:S1(A-642C)和S2(A-639C)。选取327尾同批繁殖、同塘养殖的大口黑鲈,然后采用Sna Pshot方法进行SNPs位点检测和分型。结果显示实验群体在ghrelin基因两个SNPs位点上均处于哈温平衡,S1和S2共组成了5种双倍型(D1、D2、D3、D4和D5)。关联分析的结果表明,S1位点上的AC基因型和S2位点上的AA基因型分别是两个突变位点的优势基因型,双倍型分析表明,双倍型D1在各生长性状上具有显著优势。2.大口黑鲈PACAP基因SNPs的筛选及与生长性状关联分析垂体腺苷酸环化激酶多肽(pituitary adenylate cyclase-activating polypeptide,PACAP)是一种能够促进生长激素、促性腺激素和催乳素分泌的多效激素,属于胰高血糖素/分泌肽家族的成员。为探索PACAP基因多态性对大口黑鲈生长性状的影响,使用直接测序法在PACAP基因上筛选到一个SNP位点:A-2282C,存在3种基因型:AA、AC和CC,其基因型频率分别为0.034,0.645和0.321,A等位基因频率为0.3563,C等位基因频率为0.6437。选取同批繁殖、同塘养殖的327尾大口黑鲈用Sna Pshot方法进行SNPs位点检测和分型,构建最小二乘分析模型,分析突变位点基因型与生长性状的相关性。卡方检测结果显示,哈温-平衡常数为0.0615,即该位点在所检测大口黑鲈群体中基本处于Hardy-Weinbery平衡状态,有效等位基因数为1.8465,期望杂合度和观测杂合度分别为0.5409和0.6442。方差分析结果显示:AC基因型群体在各生长性状上的平均表型值比AA和CC基因型都要高出很多;且AC基因型群体在体质量、体高和全长上显著高于AA和CC基因型群体(P0.05)。AA基因型群体的平均体质量、体高和全长相对于CC基因型群体分别高出了18.1%、5.8%和5.4%,但AA型群体和CC群体在各生长性状上没有显著差异(P0.05)。推测该位点可能是影响大口黑鲈生长性状的的主效数量性状核苷酸(quantitative trait nucleotides,QTN)或与之紧密连锁。3.大口黑鲈北方亚种和佛罗里达亚种NPY基因的DNA和c DNA克隆及序列分析神经肽Y(Neuropeptide Y,NPY)在机体的摄食活动中发挥重要作用,是哺乳动物最重要的一种内源性促食欲因子。为了解大口黑鲈NPY基因的结构及进一步研究该基因在大口黑鲈中的功能作用,采用RT-PCR和RACE技术,克隆了大口黑鲈北方亚种(M.salmoides salmoides)和佛罗里达亚种(M.salmoides floridanus)NPY基因c DNA序列,两亚种NPY c DNA均包括一个编码99个氨基酸的ORF框和长度为52bp的5′非编码区(5′-UTR);结合PCR和基因组步移技术获得了长度分别为3561bp和3565bp的大口黑鲈北方亚种和佛罗里达亚种NPY基因DNA序列。序列分析结果表明,大口黑鲈北方亚种和佛罗里达亚种NPY基因均由4个外显子和3个内含子组成。经MATINSPECTOR软件预测,在北方亚种和佛罗里达亚种启动子序列分布有TATA框、CAAT框、CCAAT-Box、GATA-Box等基本转录调控元件。实验在大口黑鲈两亚种NPY DNA序列间发现了6个单个位点碱基差异,与大口黑鲈北方亚种相比佛罗里达亚种启动子区域出现一个4个碱基的插入。不同物种间NPY基因的序列同源性分析表明大口黑鲈与鳜鱼和石斑鱼的NPY基因核苷酸同源性最高,达90%和88%,氨基酸同源性分别为93%和95%。研究结果旨在为进一步研究大口黑鲈神经肽Y基因功能作用奠定基础。4.大口黑鲈NPY基因的SNPs与生长性状相关性的初步研究在获得大口黑鲈NPY基因DNA序列的基础之上,选取该基因作为研究大口黑鲈生长性状的候选基因,鉴于神经肽Y是鱼类摄食调控网络中最强有力的促进食欲的摄食因子,推测该基因上的单核苷酸多态性会影响鱼类的摄食以及相关激素的分泌,进而对大口黑鲈的生长性状产生影响。实验根据克隆得到的大口黑鲈NPY基因的DNA序列,在NPY基因5′非编码区筛选获得了1个SNP位点:S1(A-85C)。根据实验所选取的分型群体,采样Sna Pshot方法对实验群体的NPY基因进行SNPs位点分型。对分型结果进一步处理分析后结果表明,实验群体在突变位点处于哈温平衡,该位点位点共有3种基因型:AA、AC和CC。AC基因型群体的平均体质量、体宽、全长和体高分别高于AA基因型群体6.51%、2.56%、2.18%和3.34%;相对于CC基因型分别高出了15.37%、7.97%、4.09%和5.12%,但在统计上不同基因型在各项生长性状间均没有达到显著相关性(P0.05)。但实验发现的SNPs位点对以后进一步研究大口黑鲈NPY基因与其他性状的联系提供了参考。5.三个基因多态性及合并基因型对大口黑鲈生长性状的遗传效应为进一步探讨将Ghrelin、PACAP和NPY基因作为影响大口黑鲈生长性状的候选基因的可能性,在采用直接测序法检测了这三个基因的单核苷酸多态性并分别分析了这3个基因SNPs与大口黑鲈生长性状的关联性的基础之上,分析这三个基因间合并基因型对大口黑鲈生长性状的影响。结果表明:Ghrelin基因的两个突变位点与PACAP基因上的SNP位点分别有4种和5种合并基因型,与NPY基因也分别形成了4种和5种合并基因型,PACAP和NPY基因的合并基因型有4种,三个基因一起组成的合并基因型分别有9种和6种。关联分析表明:Ghrelin-PACAP基因优势基因型聚合体ACAC和AAAC个体在各生长性状上基本显著优于其它合并基因型个体(P0.05);以Ghrelin基因的S1位点与PACAP基因进行合并基因型分析时,ACAC基因型在体质量、体宽和全长上比劣势基因型CCCC分别高出了14.1%,4.3%和3.6%,以Ghrelin基因的S2位点与PACAP基因进行合并基因型分析时,AAAC基因型在体质量、体宽和全长上比劣势基因型ACAC分别高出了16.2%,4.5%和3.0%。Ghrelin-NPY基因优势基因型聚合体的生长性状均值也优于其它合并基因型,但在统计上没有达到显著相关性(P0.05)。PACAP-NPY基因的合并基因型中,ACAC基因型个体在体高和全长上显著优于CCAA基因型个体,在体质量、体宽和全长上分别高出了12.4%,4.3%和3.3%。三个基因组成的合并优势基因型中,以S2位点参与合并而组成的AAACAC为最优基因型,且大于任意两基因间的最优合并基因型在各生长性状上的均值,以S1位点参与合并而组成的CCCCAA为劣势基因型,AAACAC合并基因型个体在体质量、体宽和全长上比CCCCAA基因型个体分别高出了26.8%,8.5%和4.2%。结果显示:两基因合并后对大口黑鲈生长性状的影响效应优于任意单个基因优势基因型的影响效应,三个基因合并后的优势基因型在各生长性状上也优于任意两个的合并优势基因型;据此我们做出以下判断:在符合遗传机理的前提下,可能合并的生长相关基因的优势单基因型越多,对大口黑鲈生长性状的影响效应就越大。
[Abstract]:Micropterus salmoides, a kind of bass (Perciformes), is one of the large aquaculture fishes in our country. The big mouth bass is a typical strong carnivorous fish. The traditional way of breeding is to feed the icy fresh fish, which not only makes the aquaculture limited by the shortage of marine fishery resources. There are also the risk of the ice fish body introducing the pathogen to the water body, which is difficult to carry out the production of the plant culture and the harmless aquatic products. In recent years, the use of artificial feed to feed the big mouth bass has achieved certain results. It is seriously restricted to the further development of Chinese big mouth bass breeding industry. It is an important goal for the breeding of big mouth bass that can be better feeding and using artificial mix feed and good growth traits. The molecular marker assisted breeding is an important means of genetic improvement of fish, with the help of molecular markers. The correlation and selection analysis of the candidate genes and target traits can greatly improve the efficiency of the selection of the related economic traits and shorten the breeding time effectively. According to the previous study, the feeding of fish is influenced by many internal and external factors, in which the appetite condition is the main internal influence factor of fish, and it is used as a fish fish. When they are in the state of appetite, their feed intake and utilization are improved correspondingly. The regulation of fish feeding is done mainly through the interaction of brain and nerve peripheral signals, and appetite related genes are the main genes involved in the regulation of fish feeding. Select Ghrelin, PACAP and NPY as the candidate genes related to the growth traits of the large mouth bass, analyze the association between the single nucleotide polymorphism (single nucleotide polymorphism, SNP) and the growth traits, in order to provide effective marker for the molecular marker assisted breeding of the big mouth bass, and promote the food to improve the new product of the big mouth bass. The main research contents are as follows: 1. the screening of the Ghrelin gene SNPs of the big mouth bass and the correlation analysis with the growth traits, Ghrelin is a kind of brain gut peptide of vertebrates, which can promote the feeding function, promote the release of growth hormone (GH), participate in the regulation of energy balance and carbohydrate metabolism. In order to explore the polymorphism of ghrelin gene and the large mouth bass The correlation of long characters, 2 SNPs loci, S1 (A-642C) and S2 (A-639C) were obtained by direct sequencing by direct sequencing of the promoter sequence of the ghrelin gene of large mouth bass. The SNPs loci were detected by the same batch of 327 tails, and the Sna Pshot method was used to detect and classify the SNPs loci. The results showed that the experimental population was in ghrelin gene. The two SNPs loci were in the HW equilibrium, and S1 and S2 were composed of 5 double types (D1, D2, D3, D4 and D5). The results of correlation analysis showed that the AC genotypes and AA genotypes on the S1 loci were the dominant genotypes of two mutation sites respectively. Screening of the PACAP gene SNPs of the oral bass and its association with the growth traits, the pituitary adenylate cycled kinase polypeptide (pituitary adenylate cyclase-activating polypeptide, PACAP) is a multi effect hormone that promotes growth hormone, gonadotropin and prolactin secretion. It is a member of the glucagon / secretory peptide family. It is to explore PACAP. The effect of genetic polymorphism on the growth traits of big mouth bass was found by direct sequencing. The PACAP gene was screened by direct sequencing: A-2282C, with 3 genotypes: AA, AC and CC, the genotype frequencies were 0.034,0.645 and 0.321, the A allele frequency was 0.3563, C allele frequency was 0.6437. selected for the same batch reproduction and 327 tail in the same pond culture. The Sna Pshot method was used to detect and classify the SNPs loci, and the least square analysis model was constructed to analyze the correlation between the genotypes of the mutant loci and the growth traits. The ksquare test results showed that the hash equilibrium constant was 0.0615, that is, the site was basically in the equilibrium state of Hardy-Weinbery in the detected large mouth bass population, and the effective allele was effective. The number of genes was 1.8465, and the expected heterozygosity and observed heterozygosity were 0.5409 and 0.6442. variance analysis showed that the average phenotypic values of AC genotypes were much higher than those of AA and CC genotypes, and the AC genotypes were significantly higher in body mass, in body height and in full length than in AA and CC genotypes (P0.05).AA genes. The average body mass of the type population, body height and full length were 18.1%, 5.8% and 5.4% higher for CC genotypes, but there was no significant difference in the growth traits between AA and CC populations (P0.05). It is presumed that this locus may be the main quantitative trait nucleotide (quantitative trait nucleotides, QTN) affecting the growth traits of large mouth bass. The cloning and sequence analysis of the DNA and C DNA of the northern subspecies and subspecies of Florida subspecies of Florida subspecies NPY, neuropeptide Y (Neuropeptide Y, NPY) plays an important role in the feeding activities of the organism. It is the most important endogenous appetite promoting factor of mammalian. To understand the structure and further of the NPY gene of the large mouth bass. To study the function of the gene in the big mouth bass, RT-PCR and RACE techniques were used to clone the DNA sequence of the northern subspecies (M.salmoides salmoides) and the NPY gene C of Florida subspecies (M.salmoides floridanus) of the large mouth bass. The two subspecies NPY C DNA included a 99 amino acid coded frame and the 5 'non coding region (5'). -UTR); PCR and genomic step technique were used to obtain the DNA sequence of NPY gene of the northern subspecies and Florida subspecies of Florida subspecies, large mouth bass and 3565bp, respectively. The sequence analysis showed that the NPY genes of the northern subspecies and the subspecies of Florida subspecies were composed of 4 exons and 3 introns. The subspecies of northern subspecies and subspecies of Florida subspecies are distributed with TATA frame, CAAT frame, CCAAT-Box, GATA-Box and other basic transcriptional regulators. The experiment found 6 single site base differences between the two subspecies NPY DNA sequences of the large mouth bass, and a 4 base insertion in the subspecies of the subspecies of the northern subspecies of the big mouth bass. The sequence homology of NPY gene among different species showed that the nucleotide homology of the NPY gene of the large mouth bass and the mandarin fish and the grouper was the highest, reaching 90% and 88%. The amino acid homology was 93% and 95%., respectively, to lay the foundation for the further study of the Y gene function of the large mouth perch neuropeptide Y gene, which lay the foundation for the SNPs of the.4. large mouth bass NPY gene. A preliminary study on the correlation of growth traits on the basis of obtaining the DNA sequence of the NPY gene of the large mouth bass, selected the gene as a candidate gene for the study of the growth traits of large mouth bass. In view of the fact that neuropeptide Y is the most powerful feeding factor promoting appetite in the fish feeding regulation network, it is speculated that the single nucleotide polymorphisms on this gene will be affected. The feeding of fishes and the secretion of related hormones had an effect on the growth traits of the large mouth bass. According to the DNA sequence of the NPY gene of the cloned bass bass, 1 SNP loci were obtained in the 5 'non coding region of the NPY gene: S1 (A-85C). According to the selected group of the experiment, the Sna Pshot method was sampled for the experimental population. The NPY gene was typed at the SNPs locus. The results of further analysis showed that the experimental population was at the equilibrium at the mutation site, and there were 3 genotypes at the loci: the average body mass of the AA, AC and CC.AC genotypes, the body width, the total length and the body height were 6.51%, 2.56%, 2.18% and 3.34%, respectively, compared with the AA genotypes; relative to C. The C genotypes were 15.37%, 7.97%, 4.09% and 5.12% higher respectively, but there was no significant correlation between the different genotypes of the genotypes (P0.05). However, the SNPs loci found in the experiment further studied the relationship between the NPY gene and other traits of the big mouth bass, which provided the reference to three gene polymorphisms and the combination genotype of.5.. The genetic effect on the growth traits of large mouth bass is to further explore the possibility of Ghrelin, PACAP and NPY genes as candidate genes affecting the growth traits of large mouth bass. The single nucleotide polymorphisms of these three genes were detected by direct sequencing and the correlation between the 3 gene SNPs and the growth traits of large mouth bass was analyzed. On the basis of the analysis, the effects of the combined genotypes of the three genes on the growth traits of the big mouth bass were analyzed. The results showed that the two mutation sites of the Ghrelin gene and the SNP loci on the PACAP gene were 4 and 5 genotypes respectively. 4 and 5 genotypes were formed respectively with the NPY gene, and the combined genotype of the PACAP and NPY genes had the same genotypes. There were 9 and 6 types of combined genotypes composed of three genes. The correlation analysis showed that the Ghrelin-PACAP gene dominant genopolymer ACAC and AAAC were significantly better than other combined genotype individuals (P0.05) in the growth traits, and ACAC was combined with the S1 site of the Ghrelin gene and the PACAP gene, ACAC Genotypes were 14.1%, 4.3% and 3.6% higher in body mass, body width and length than inferior genotypes, respectively. When the S2 locus of Ghrelin gene and PACAP gene were combined with genotype analysis, the AAAC genotype was 16.2%, 4.5%, and 3.0%.Ghrelin-NPY gene dominant genotype in body mass, body width and full length than inferior genotype ACAC, respectively. The average growth traits of the polymers were also better than those of other combined genotypes, but in the statistics of P0.05.PACAP-NPY gene combination, the ACAC genotype was significantly better than the CCAA genotype in the body height and length, and the body mass, body width and the whole length were 12.4%, 4.3% and 3.3%. three genomes respectively. In the combined dominant genotypes, AAACAC is the best genotype with the S2 site involved in the combination, and it is larger than the average of the optimal combination genotype between any two genes. The CCCCAA is a inferior genotype with the S1 locus involved in the combination, and the AAACAC combined with the genotypes is in body mass, body width and total length than CCCCA The A genotype individuals were 26.8%, 8.5% and 4.2%. respectively. The results showed that the effect of the two gene combination on the growth traits of the big mouth bass was better than that of any single gene dominant genotype, and the dominant genotypes of the three genes were also superior to the two combined dominant genotypes in the growth traits. In accordance with the genetic mechanism, the more dominant single genotypes of the associated growth related genes, the greater the effect on the growth traits of the big mouth bass.
【学位授予单位】：上海海洋大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S917.4

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