鲤鱼肌肉脂肪性状相关基因筛选及功能研究

发布时间：2018-05-07 02:36

本文选题：鲤鱼(Cyprinus + carpio)　；参考：《东北农业大学》2017年博士论文

【摘要】：鲤鱼(Cyprinus carpio)作为世界性的经济鱼类,其年产量超过445万吨,约占全球淡水养殖年产量的10%,在全球水产养殖业中占据重要地位。我国是最大的鲤鱼生产和消费国,产量约占全球的75.3%。同时,我国也是培育出鲤鱼优良品种最多的国家,在过去的几十年里大大促进了我国水产养殖业的发展,满足了市场“量”的需求,为人们提供了优质的动物蛋白。随着消费者生活水平的不断提高,水产品品质越来越受到人们重视。鲤鱼肉质、口感、营养价值等已远不能满足市场日益增长的“质”的需求。利用与肌肉品质性状紧密连锁的基因,培育肉质优良、营养价值高的品种是解决此问题的有效技术途径之一。肉质性状为复杂的经济性状,受诸多因素影响。鱼类中,肌肉脂肪酸组成及含量、脂肪含量等是影响肌肉品质的重要因素。本研究以镜鲤全同胞F2家系为材料,利用鲤鱼250 K高密度SNP芯片对肌肉脂肪酸、脂肪含量等肌肉脂肪性状进行全基因组关联分析(GWAS),获得肌肉脂肪性状显著相关的SNP位点;利用鲤鱼全基因组注释信息,获得肌肉脂肪性状候选基因。采用克隆、mRNA表达、基因多态性与性状关联分析等手段对部分候选基因进行了验证,确定基因与性状的关系。利用基因敲除手段,构建了鲤鱼和斑马鱼LPL基因突变体,探讨了LPL基因敲除对脂肪沉积的影响及其作用机制。本研究结果将为鲤鱼肌肉脂肪性状的遗传机制解析和品质提升的分子选育奠定基础。主要结果如下:1)采用全同胞家系设计,应用混合线性模型,开展了肌肉脂肪酸、脂肪含量等性状全基因组关联研究。获得了C22:0、C24:1n9、C18:3n6、C20:3n3、C22:2n6、EPA、DHA、EPADHA、HUFA和n-3PUFA等10个脂肪酸性状,5%基因组水平显著性(P5.56×10-6)SNP位点54个,注释基因34个。获得背部肌肉脂肪含量(MFdo)、腹部肌肉脂肪含量(MFab)、腹部脂肪重(Ab FW)和腹部脂肪占净重比例(AbFP)等4个脂肪含量相关性状,基因组潜在关联显著性水平(P1.11×10-4)SNP位点18个,注释基因10个,其中,carp089419达到5%基因组水平显著性(P5.56×10-6),且与AbFW和AbFP均显著相关。利用荧光定量PCR(RT-qPCR)对8个已注释基因(ANKRD10A、TANC2、FJX1、CHKA、ADAM8A、FASN、LPL-a和LPL-b)在脂肪含量极高、极低样本中进行了初步验证,结果表明:ANKRD10A和TANC2在脂肪含量高的肌肉样本中表达量低于脂肪含量低的样本,且差异极显著(P0.01);而FJX1、CHKA、FASN、LPL-a和LPL-b在脂肪含量高的样本中表达量显著高于低脂肪含量样本,其中CHKA、FASN、LPL-a和LPL-b在两组样本间表现出极显著差异(P0.01)。因此,可将ANKRD10A、TANC2、FJX1、CHKA、FASN、LPL-a和LPL-b作为候选基因进一步研究。2)克隆了FASN、LPL-a和LPL-b 3个候选基因cDNA全长,并对基因序列、生物学信息及基因表达规律进行分析。FASN基因(KY378913)cDNA全长为8927bp,编码2511个氨基酸,5’非编码区(5’-UTR)为202bp,3’非编码区(3’-UTR)为1192bp;LPL-a基因(KM213240.1)cDNA全长2631bp,编码507个氨基酸,5’-UTR为162bp,3’-UTR为945bp;LPL-b基因(KM213241.1)cDNA序列全长2413bp,编码507个氨基酸,5’-UTR为158bp,3’-UTR为732 bp。同源性及系统进化分析发现,FASN与其它鱼类氨基酸同源性为68%-96%;LPL-a和LPL-b氨基酸同源性为95%,两者与其它鱼类氨基酸同源性为61%-93%;进化树显示,鲤鱼与金线渻FASN基因的进化关系最近,鲤鱼与鲫鱼LPL基因的进化关系最近。蛋白生物信息学分析显示,FASN蛋白质相对分子量274.1456 kD,理论PI值为6.10,其不稳定系数为41.14,说明该蛋白不稳定。FASN蛋白以无规则卷曲结构为主,占44.66%。LPL-a和LPL-b蛋白质的相对分子量分别为57.6688kD和57.5877 kD。蛋白均以无规则卷曲为主,分别占50.10%和51.87%。两者均含有脂肪酶和PLAT/LH2两个结构域。不同组织中RT-q PCR结果显示,3个基因在多个组织中均广泛表达,FASN在脑组织中表达量最高,在背部肌肉与腹部肌肉中表达差异不显著;LPL-a在腹部肌肉中表达量最高,LPL-b在脂肪组织中表达量最高,LPL-a和LPL-b在腹部肌肉和背部肌肉组织中表达量均具有极显著性差异(P0.01)。不同发育时期RT-qPCR结果显示,FASN在囊胚晚期表达量最高,囊胚时期相对受精卵期表达量显著增加(P0.05);LPL-a和LPL-b均在在开口期的表达量达到最高,在7体节时期的表达量最低。3)开展了FASN、LPL-a和LPL-b基因多态性与肌肉脂肪性状相关性研究。根据FASN、LPL-a和LPL-b基因在鲤鱼基因组中的位置和鲤鱼SNP数据库,寻找到基因所含的SNP位点22个;采用测序法获得22个SNP位点在鲤鱼群体中基因型数据,其中5个SNP位点(carp165086、carp165090、carp165099、carp076700和carp076701)为单态,17个多态性SNP位点有效等位基因为1.0195-1.9998,平均值为1.8094;观测杂合度为0.0145-0.7374,平均值为0.5070;期望杂合度为0.0192-0.5012,平均值为0.4216。基因型与性状相关性分析显示:8个SNP位点与脂肪性状显著相关(P0.05)。FASN基因上,carp165088(AT)与AbFW和SFA显著相关;carp165093(GA)与MFdo和n-3PUFA显著相关;carp165096(AT)和carp005641(GA)与EPA显著相关;carp005638(CT)与MFdo和n-6PUFA显著相关。LPL-a基因上的carp076703(TC)与MFab显著相关;LPL-b基因上carp076738(TG)和carp076740(TC)与MFab显著相关;且carp 076740也与SFA显著相关。综上,FASN、LPL-a和LPL-b基因可作为鲤鱼脂肪性状的候选主效基因。4)利用CRISPR/Cas9基因编辑技术,制备了鲤鱼LPL基因突变F0代,获得鲤鱼LPL-a和LPL-b基因突变F0个体分别为203尾和197尾。并以斑马鱼为模型,探索了LPL基因敲除对斑马鱼肌肉脂肪沉积的影响。构建了斑马鱼LPL基因突变纯系2个,即1号突变系(-13 bp)和2号突变系(+11/-7 bp),形态结果显示:LPL-/-斑马鱼在胚胎和出苗20天没有明显异常,2月龄检测发现2个突变系LPL-/-斑马鱼生长速度显著慢于野生型(P0.05);解剖发现LPL-/-斑马鱼腹腔肠系膜脂肪沉积减少,甚至无脂肪沉积;肌肉脂肪酸测定结果显示:对照野生型,LPL-/-斑马鱼SFA、MUFA和PUFA含量降低,且SFA和PUFA差异显著(P0.05);而三酰甘油(TG)含量增加,且差异极显著(P0.01)。荧光定量PCR检测显示:LPL基因mRNA表达量在LPL-/-斑马鱼中显著降低(P0.01)。同时,LPL基因突变导致其所在的糖代谢通路和PPAR通路中,其上下游基因mRNA表达量呈现不同程度下调。本结果将为LPL基因在鲤鱼脂肪沉积中的作用机制研究提供参考。
[Abstract]:Cyprinus carpio, as a world economic fish, has an annual output of more than 4 million 450 thousand tons, accounting for about 10% of the annual output of global freshwater aquaculture, and occupies an important position in the global aquaculture industry. China is the largest producer and consumer of carp, and the output is about 75.3%. in the world, and China is also the country that produces the most excellent varieties of carp. In the past few decades, it has greatly promoted the development of aquaculture industry in China, met the demand of the "quantity" of the market and provided people with high quality animal protein. With the continuous improvement of the living standard of consumers, the quality of aquatic products has been paid more and more attention. The meat quality, taste and nutritional value of carp are far from increasing the market. Long "quality" demand. It is one of the effective ways to solve this problem by using genes closely linked with muscle quality traits to cultivate good meat quality and high nutritive value. Meat quality is complex economic character and is influenced by many factors. In fish, muscle fatty acid composition and content, fat content and so on are the influence of muscle quality. In this study, the total genomic association analysis (GWAS) was performed on the muscle fatty acids and fat content of the carp 250 K high density SNP chip, using the 250 K high density SNP chip of carp, and the muscle fat traits were obtained by using the whole genome annotation information of carp. Some candidate genes were verified by cloning, mRNA expression, gene polymorphism and trait association analysis. The relationship between genes and characters was determined. The LPL gene mutation of carp and zebrafish was constructed by gene knockout. The effect of LPL knockout on fat deposition and its mechanism were discussed. The main results are as follows: 1) the main results are as follows: 1) using a full sibling family design and using a mixed linear model, a complete genome association study of fatty acids and fat content was carried out. C22:0, C24:1n9, C18:3n6, C20:3n3, C22:2n6, EPA, DHA, EP were obtained. 10 fatty acid traits such as ADHA, HUFA and n-3PUFA, 5% genomic level significant (P5.56 x 10-6) SNP site 54, and 34 annotated genes, and 4 fat content related traits, such as back muscle fat content (MFdo), abdominal muscle fat content (MFab), abdominal fat weight (Ab FW) and abdominal fat percentage (AbFP), were found to be associated with the potential association of the genome. There were 18 loci (P1.11 x 10-4) SNP and 10 annotated genes, of which, carp089419 reached the 5% genome level (P5.56 x 10-6), and was significantly related to AbFW and AbFP. Using fluorescent quantitative PCR (RT-qPCR), the 8 annotated genes (ANKRD10A, TANC2, FJX1, CHKA, CHKA, CHKA, P5.56, etc.) were in high fat and very low samples. The results showed that the expression of ANKRD10A and TANC2 in the muscle samples with high fat content was lower than those with low fat content, and the difference was very significant (P0.01), while the expression of FJX1, CHKA, FASN, LPL-a and LPL-b in the samples with high fat content was significantly higher than that of the low fat content samples, of which CHKA, FASN, LPL-a and LPL-b were in two groups of samples. P0.01, so ANKRD10A, TANC2, FJX1, CHKA, FASN, LPL-a and LPL-b were used as candidate genes to further study.2) and the 3 candidate genes of FASN, LPL-a and LPL-b were cloned, and the whole length of gene sequence, biological information and gene expression was analyzed, encoding 251 1 amino acids, 5 'non coding region (5' -UTR) are 202bp, 3 'non coding region (3' -UTR) is 1192bp, LPL-a gene (KM213240.1) cDNA full length 2631bp, encoding 507 amino acids, 5 '-UTR for 162bp, 3' -UTR as 945bp, 507 amino acids, 5 ', 3' as 732 homology and system The homology of FASN and other fish amino acids is 68%-96%, the homology of LPL-a and LPL-b amino acids is 95%, and the homology of both of them and other fish amino acids is 61%-93%. The evolutionary relationship between the carp and the gold line FASN gene is the closest. The protein bioinformatics analysis of the carp and the carp LPL gene is the closest. The relative molecular weight of FASN protein was 274.1456 kD, the theoretical PI value was 6.10, the instability coefficient was 41.14, indicating that the protein unstable.FASN protein was dominated by irregular curl structure, and the relative molecular weights of 44.66%.LPL-a and LPL-b proteins were 57.6688kD and 57.5877 kD. eggs, respectively, with irregular curls, accounting for 50.10% and 51.87%, respectively. Both of which contain two domains of lipase and PLAT/LH2. The results of RT-q PCR in different tissues show that 3 genes are widely expressed in multiple tissues. The expression of FASN is the highest in the brain tissue, and there is no significant difference in the expression between the back muscles and the abdominal muscles; LPL-a is the highest in the abdominal muscles, and the highest expression of LPL-b in the adipose tissue. The expression of LPL-a and LPL-b in the abdominal muscles and back muscles had a very significant difference (P0.01). The expression of FASN at the late stage of blastocyst was the highest and the expression of FASN in the blastocyst period increased significantly (P0.05) at the blastocyst period (P0.05) at different developmental stages, and the expression of LPL-a and LPL-b at the opening period was highest, at the time of 7 body joints. The correlation of FASN, LPL-a, and LPL-b gene polymorphisms with the muscle fat traits was studied. According to the location of FASN, LPL-a and LPL-b genes in the carp genome and the carp SNP database, 22 SNP loci contained in the gene were found, and the genetic data of 22 SNP loci in the carp population were obtained by sequencing. 5 SNP loci (carp165086, carp165090, carp165099, carp076700 and carp076701) were single state, and the effective allele of 17 polymorphic SNP loci was 1.0195-1.9998, the average value was 1.8094, the observed heterozygosity was 0.0145-0.7374, the average value was 0.5070, and the expected heterozygosity was 0.0192-0.5012, the average value was the analysis of the 0.4216. genotype and the trait correlation analysis. The 8 SNP loci were significantly related to the fat traits (P0.05).FASN gene, carp165088 (AT) was significantly related to AbFW and SFA; carp165093 (GA) was significantly related to MFdo and n-3PUFA; Carp076738 (TG) and carp076740 (TC) were significantly related to MFab, and carp 076740 was also significantly related to SFA. To sum up, FASN, LPL-a and LPL-b genes could be used as the main candidate genes for the fat traits of carp. The effect of LPL gene knockout on the fat deposition of zebrafish muscle was explored with zebrafish as a model, and the effect of LPL gene knockout on zebrafish muscle fat deposition was explored. 2 mutant lines of zebrafish LPL gene mutation, the 1 mutation line (-13 BP) and 2 mutant line (+11/-7 BP), were constructed. The morphological results showed that the LPL-/- zebra fish had no obvious abnormalities in the embryo and emergence 20 days, and 2 month old detected hair. The growth rate of the present 2 mutant lines LPL-/- zebrafish was significantly slower than that in the wild type (P0.05); the abdominal mesentery fat deposition of the LPL-/- zebrafish decreased and even no fat deposits were dissected; the muscle fatty acid determination showed that the control of the wild type, the LPL-/- zebra fish SFA, the MUFA and PUFA content decreased, and the SFA and PUFA were significantly different (P0.05), while the three acyl glycerol (TG) The content increased and the difference was very significant (P0.01). The fluorescence quantitative PCR detection showed that the mRNA expression of LPL gene decreased significantly in LPL-/- zebra fish (P0.01). At the same time, the LPL gene mutation resulted in the decrease of the mRNA expression of the upstream and downstream genes in the sugar metabolism pathway and PPAR pathway. The result will be the LPL gene in carp fat. The study of the mechanism of action in deposition provides a reference.

【学位授予单位】：东北农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S917.4

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