發(fā)布時間：2018-05-07 02:36

  本文選題：鯉魚(Cyprinus + carpio)�。� 參考：《東北農(nóng)業(yè)大學(xué)》2017年博士論文

【摘要】：鯉魚(Cyprinus carpio)作為世界性的經(jīng)濟(jì)魚類,其年產(chǎn)量超過445萬噸,約占全球淡水養(yǎng)殖年產(chǎn)量的10%,在全球水產(chǎn)養(yǎng)殖業(yè)中占據(jù)重要地位。我國是最大的鯉魚生產(chǎn)和消費(fèi)國,產(chǎn)量約占全球的75.3%。同時,我國也是培育出鯉魚優(yōu)良品種最多的國家,在過去的幾十年里大大促進(jìn)了我國水產(chǎn)養(yǎng)殖業(yè)的發(fā)展,滿足了市場“量”的需求,為人們提供了優(yōu)質(zhì)的動物蛋白。隨著消費(fèi)者生活水平的不斷提高,水產(chǎn)品品質(zhì)越來越受到人們重視。鯉魚肉質(zhì)、口感、營養(yǎng)價值等已遠(yuǎn)不能滿足市場日益增長的“質(zhì)”的需求。利用與肌肉品質(zhì)性狀緊密連鎖的基因,培育肉質(zhì)優(yōu)良、營養(yǎng)價值高的品種是解決此問題的有效技術(shù)途徑之一。肉質(zhì)性狀為復(fù)雜的經(jīng)濟(jì)性狀,受諸多因素影響。魚類中,肌肉脂肪酸組成及含量、脂肪含量等是影響肌肉品質(zhì)的重要因素。本研究以鏡鯉全同胞F2家系為材料,利用鯉魚250 K高密度SNP芯片對肌肉脂肪酸、脂肪含量等肌肉脂肪性狀進(jìn)行全基因組關(guān)聯(lián)分析(GWAS),獲得肌肉脂肪性狀顯著相關(guān)的SNP位點(diǎn);利用鯉魚全基因組注釋信息,獲得肌肉脂肪性狀候選基因。采用克隆、mRNA表達(dá)、基因多態(tài)性與性狀關(guān)聯(lián)分析等手段對部分候選基因進(jìn)行了驗(yàn)證,確定基因與性狀的關(guān)系。利用基因敲除手段,構(gòu)建了鯉魚和斑馬魚LPL基因突變體,探討了LPL基因敲除對脂肪沉積的影響及其作用機(jī)制。本研究結(jié)果將為鯉魚肌肉脂肪性狀的遺傳機(jī)制解析和品質(zhì)提升的分子選育奠定基礎(chǔ)。主要結(jié)果如下:1)采用全同胞家系設(shè)計,應(yīng)用混合線性模型,開展了肌肉脂肪酸、脂肪含量等性狀全基因組關(guān)聯(lián)研究。獲得了C22:0、C24:1n9、C18:3n6、C20:3n3、C22:2n6、EPA、DHA、EPADHA、HUFA和n-3PUFA等10個脂肪酸性狀,5%基因組水平顯著性(P5.56×10-6)SNP位點(diǎn)54個,注釋基因34個。獲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)和腹部脂肪占凈重比例(AbFP)等4個脂肪含量相關(guān)性狀,基因組潛在關(guān)聯(lián)顯著性水平(P1.11×10-4)SNP位點(diǎn)18個,注釋基因10個,其中,carp089419達(dá)到5%基因組水平顯著性(P5.56×10-6),且與AbFW和AbFP均顯著相關(guān)。利用熒光定量PCR(RT-qPCR)對8個已注釋基因(ANKRD10A、TANC2、FJX1、CHKA、ADAM8A、FASN、LPL-a和LPL-b)在脂肪含量極高、極低樣本中進(jìn)行了初步驗(yàn)證,結(jié)果表明:ANKRD10A和TANC2在脂肪含量高的肌肉樣本中表達(dá)量低于脂肪含量低的樣本,且差異極顯著(P0.01);而FJX1、CHKA、FASN、LPL-a和LPL-b在脂肪含量高的樣本中表達(dá)量顯著高于低脂肪含量樣本,其中CHKA、FASN、LPL-a和LPL-b在兩組樣本間表現(xiàn)出極顯著差異(P0.01)。因此,可將ANKRD10A、TANC2、FJX1、CHKA、FASN、LPL-a和LPL-b作為候選基因進(jìn)一步研究。2)克隆了FASN、LPL-a和LPL-b 3個候選基因cDNA全長,并對基因序列、生物學(xué)信息及基因表達(dá)規(guī)律進(jìn)行分析。FASN基因(KY378913)cDNA全長為8927bp,編碼2511個氨基酸,5’非編碼區(qū)(5’-UTR)為202bp,3’非編碼區(qū)(3’-UTR)為1192bp;LPL-a基因(KM213240.1)cDNA全長2631bp,編碼507個氨基酸,5’-UTR為162bp,3’-UTR為945bp;LPL-b基因(KM213241.1)cDNA序列全長2413bp,編碼507個氨基酸,5’-UTR為158bp,3’-UTR為732 bp。同源性及系統(tǒng)進(jìn)化分析發(fā)現(xiàn),FASN與其它魚類氨基酸同源性為68%-96%;LPL-a和LPL-b氨基酸同源性為95%,兩者與其它魚類氨基酸同源性為61%-93%;進(jìn)化樹顯示,鯉魚與金線渻FASN基因的進(jìn)化關(guān)系最近,鯉魚與鯽魚LPL基因的進(jìn)化關(guān)系最近。蛋白生物信息學(xué)分析顯示,FASN蛋白質(zhì)相對分子量274.1456 kD,理論P(yáng)I值為6.10,其不穩(wěn)定系數(shù)為41.14,說明該蛋白不穩(wěn)定。FASN蛋白以無規(guī)則卷曲結(jié)構(gòu)為主,占44.66%。LPL-a和LPL-b蛋白質(zhì)的相對分子量分別為57.6688kD和57.5877 kD。蛋白均以無規(guī)則卷曲為主,分別占50.10%和51.87%。兩者均含有脂肪酶和PLAT/LH2兩個結(jié)構(gòu)域。不同組織中RT-q PCR結(jié)果顯示,3個基因在多個組織中均廣泛表達(dá),FASN在腦組織中表達(dá)量最高,在背部肌肉與腹部肌肉中表達(dá)差異不顯著;LPL-a在腹部肌肉中表達(dá)量最高,LPL-b在脂肪組織中表達(dá)量最高,LPL-a和LPL-b在腹部肌肉和背部肌肉組織中表達(dá)量均具有極顯著性差異(P0.01)。不同發(fā)育時期RT-qPCR結(jié)果顯示,FASN在囊胚晚期表達(dá)量最高,囊胚時期相對受精卵期表達(dá)量顯著增加(P0.05);LPL-a和LPL-b均在在開口期的表達(dá)量達(dá)到最高,在7體節(jié)時期的表達(dá)量最低。3)開展了FASN、LPL-a和LPL-b基因多態(tài)性與肌肉脂肪性狀相關(guān)性研究。根據(jù)FASN、LPL-a和LPL-b基因在鯉魚基因組中的位置和鯉魚SNP數(shù)據(jù)庫,尋找到基因所含的SNP位點(diǎn)22個;采用測序法獲得22個SNP位點(diǎn)在鯉魚群體中基因型數(shù)據(jù),其中5個SNP位點(diǎn)(carp165086、carp165090、carp165099、carp076700和carp076701)為單態(tài),17個多態(tài)性SNP位點(diǎn)有效等位基因?yàn)?.0195-1.9998,平均值為1.8094;觀測雜合度為0.0145-0.7374,平均值為0.5070;期望雜合度為0.0192-0.5012,平均值為0.4216�；蛐团c性狀相關(guān)性分析顯示:8個SNP位點(diǎn)與脂肪性狀顯著相關(guān)(P0.05)。FASN基因上,carp165088(AT)與AbFW和SFA顯著相關(guān);carp165093(GA)與MFdo和n-3PUFA顯著相關(guān);carp165096(AT)和carp005641(GA)與EPA顯著相關(guān);carp005638(CT)與MFdo和n-6PUFA顯著相關(guān)。LPL-a基因上的carp076703(TC)與MFab顯著相關(guān);LPL-b基因上carp076738(TG)和carp076740(TC)與MFab顯著相關(guān);且carp 076740也與SFA顯著相關(guān)。綜上,FASN、LPL-a和LPL-b基因可作為鯉魚脂肪性狀的候選主效基因。4)利用CRISPR/Cas9基因編輯技術(shù),制備了鯉魚LPL基因突變F0代,獲得鯉魚LPL-a和LPL-b基因突變F0個體分別為203尾和197尾。并以斑馬魚為模型,探索了LPL基因敲除對斑馬魚肌肉脂肪沉積的影響。構(gòu)建了斑馬魚LPL基因突變純系2個,即1號突變系(-13 bp)和2號突變系(+11/-7 bp),形態(tài)結(jié)果顯示:LPL-/-斑馬魚在胚胎和出苗20天沒有明顯異常,2月齡檢測發(fā)現(xiàn)2個突變系LPL-/-斑馬魚生長速度顯著慢于野生型(P0.05);解剖發(fā)現(xiàn)LPL-/-斑馬魚腹腔腸系膜脂肪沉積減少,甚至無脂肪沉積;肌肉脂肪酸測定結(jié)果顯示:對照野生型,LPL-/-斑馬魚SFA、MUFA和PUFA含量降低,且SFA和PUFA差異顯著(P0.05);而三酰甘油(TG)含量增加,且差異極顯著(P0.01)。熒光定量PCR檢測顯示:LPL基因mRNA表達(dá)量在LPL-/-斑馬魚中顯著降低(P0.01)。同時,LPL基因突變導(dǎo)致其所在的糖代謝通路和PPAR通路中,其上下游基因mRNA表達(dá)量呈現(xiàn)不同程度下調(diào)。本結(jié)果將為LPL基因在鯉魚脂肪沉積中的作用機(jī)制研究提供參考。
[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.

【學(xué)位授予單位】：東北農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：S917.4

【參考文獻(xiàn)】

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本文編號：1855040