【作者】 张依裕；

【导师】 陈国宏；

【作者基本信息】 扬州大学 ， 动物遗传育种与繁殖， 2010， 博士

【摘要】 脂肪沉积过多是现今肉鸭育种中面临的一个重要难题,脂肪过度沉积导致饲料利用率降低和胴体品质下降,更为重要的是人体摄入过多脂肪导致的相关疾病引起了广泛关注,这在一定程度上制约了养鸭业的发展。脂肪沉积作为一个数量性状,受多基因调控。但是,目前对鸭脂肪沉积相关候选基因的研究报道较少,急切需要进一步开展相关研究工作。本研究采用气质联用(GC-MS)技术和血液自动生化分析仪对10周龄樱桃谷鸭、金定鸭、苏牧麻鸭和白羽番鸭4个群体的胸肌脂肪酸组成和9项血清生化指标甘油三酯(TG)、总胆固醇(TC)、白蛋白(Alb)、总蛋白(TP)、胆碱酯酶(ChE)、碱性磷酸酶(ALP)、免疫球蛋白A(IgA)、球蛋白(GLO)和白/球比值(Alb/GLO)进行测定和分析;采用RT-PCR法对樱桃谷鸭和白羽番鸭LXRα基因cDNA进行克隆测序和生物信息学分析;采用PCR-SSCP和DNA测序相结合研究了4个群体3个脂肪沉积相关候选基因LXRα、Adiponectin和ApoVLDL-II的遗传变异及其与肉质性状的相关性;采用实时荧光定量PCR法研究了3个基因在10周龄金定鸭12个组织(心、肝、胸肌、小肠、大肠、小脑、大脑、下丘脑、肾、肺、脾和腺胃)的表达差异以及白羽番鸭和金定鸭肝脏组织在不同发育时期(0d、2w、4w、6w、8w和10w)的表达规律,旨在对鸭的育种或遗传改良工作提供科学参考依据。研究主要取得如下成果:1.鸭胸肌脂肪酸组成分析4个群体中均检测到16种脂肪酸,以油酸(C18:1)、棕榈酸(C16:0)、硬脂酸(C18:0)和亚油酸(C18:2)为主要成分,占脂肪酸质量分数的95%左右。脂肪酸组成在群体间存在一定的差异,除C14:1不存在群体效应外(P>0.05),其它脂肪酸均存在群体效应,其中C15:0存在显著的群体效应(P<0.05),其它脂肪酸均存在极显著的群体效应(P<0.01),所有脂肪酸均不存在性别效应和群体×性别的互作效应(P>0.05)。樱桃谷鸭的UFA最高,而PUFA和EFA最低,白羽番鸭恰好相反。2.鸭血清生化指标测定结果4个群体的9项血清生化指标均存在极显著的群体效应(P<0.01),TG存在显著的性别效应(P<0.05),TC和ALP存在极显著的性别效应(P<0.01),公鸭TG、TC和ALP显著高于母鸭(P<0.05);TG和TC存在极显著的群体×性别的互作效应(P<0.01)。3.鸭LXRα基因的克隆和生物信息学分析首次克隆了樱桃谷鸭和白羽番鸭LXRα基因cDNA序列1626 bp,GenBank登录号分别为FJ966078和GU132847,包括部分5′-UTR序列73 bp、CDS全序列1230 bp和3′-UTR序列323 bp,编码409个氨基酸,樱桃谷鸭和白羽番鸭LXRα基因共存在14个核苷酸(CDS区:9个;UTR区5个)和3个氨基酸(Ser163Gly、Gln171Glu和Asn361Lys)的差异。鸭LXRα蛋白与哺乳动物和鱼类的同源性在74%-78%,与鸡的同源性高达97%。聚类分析显示:哺乳动物、禽类和鱼类各为一类。生物信息学分析表明:鸭LXRα蛋白含有17个磷酸化位点、2个低组分复杂性区域、1个ZnF-C4和1个HOLI结构域,无信号肽,无跨膜螺旋;2条LXRα基因CDS区碱基差异和氨基酸差异导致了RNA折叠结构、蛋白二级结构和糖基化位点发生了改变。4.鸭LXRα基因遗传变异及其与肉质性状的关联分析在鸭LXRα基因LXR-E5位点检测到C277G同义突变,LXR-E12位点检测到G1396C突变和LXR-I6位点检测到C44T突变,其它6个位点LXR-E4、LXR-E6、LXR-E7、LXR-E8、LXR-E10和LXR-E11均没有检测到多态;关联分析表明:LXR-E5位点与嫩度显著相关(P<0.05),LXR-E12和LXR-I6位点与pH、失水率、IMF、TC、TG、UFA、PUFA和EFA显著相关(P<0.05);LXR-E5×LXR-I6互作与UFA显著相关(P<0.05),BBCC组合基因型最高;LXR-E12×LXR-I6互作与pH、嫩度和TC显著相关(P<0.05),分别是BBDD、ABCC和BBDD组合基因型最高。5.白羽番鸭LXRα基因遗传变异及其与肉质性状的关联分析在白羽番鸭LXRα基因LXR-E4和LXR-E12位点分别检测到G53A和-1483/T突变,其它7个位点均没有检测到多态;关联分析表明:LXR-E4位点与IMF、UFA和肉色显著相关(P<0.05),LXR-E4×LXR-E12互作与UFA显著相关(P<0.05),BBCC组合基因型最高。6.鸭Adiponectin基因遗传变异及其与肉质性状的关联分析在鸭Adiponectin基因4个位点ADP1、ADP2、ADP3和ADP4中发现了15个SNPs,其中3′-UTR区1个:G887A,CDS区12个:C86T、C104T、C146T、C155T、C456T、A574G、C651T、C684T、T768C、G784A、A801C和C807T,内含子2个:C273T和C295T。A574G、G784A和A801C为有义突变,分别导致氨基酸序列中144位的Thr(T)变成Ala(A)、214位的Ile(I)变成Val(V)和219位的Asp(D)变成Glu(E);相关分析结果表明:ADP1位点与IMF、UFA、PUFA和EFA显著相关(P<0.05);ADP2位点与失水率、IMF、TC和UFA显著相关(P<0.05);ADP4位点与失水率、TC、UFA和PUFA显著相关(P<0.05);ADP1×ADP3和ADP2×ADP3互作与UFA显著相关(P<0.05),分别是组合基因型CDBC和AACC最高;ADP1×ADP4和ADP3×ADP4互作与失水率和IMF显著相关(P<0.05),失水率分别是组合基因型CDAC和CCBB最高,IMF分别是组合基因型DDAA和CCAA最高。7.白羽番鸭Adiponectin基因遗传变异及其与肉质性状的关联分析在白羽番鸭Adiponectin基因3个位点ADP1、ADP2和ADP4中发现了3个SNPs,其中CDS区2个:A167G和G711A,均为同义突变;内含子1个:C290T;ADP3位点没有检测到多态。关联分析表明:ADP1和ADP2位点与IMF和失水率显著相关(P<0.05);ADP1×ADP4和ADP2×ADP4互作与UFA显著相关(P<0.05),分别是组合基因型BBFF和TTFF最高。8.鸭ApoVLDL-II基因遗传变异及其与肉质性状的关联分析本研究获得鸭ApoVLDL-II基因组DNA序列(GQ 180104),并对该基因的5个位点Exon1、Exon2、Exon3、Exon4和Intron1进行SSCP检测,结果发现:Exon1和Exon2没有检测到多态,在另外3个位点中检测到了12个SNPs:T667C、C669G、T673C、G674A、G683A、G688A、C708G、T715G、G2106A、T2723C、C2743T和A2944C,2个插入/缺失:764位后插入/缺失TG,1910位碱基后插入/缺失CC,除A2944C突变发生在外显子4非编码区外,其它突变均在内含子内,整个编码区没有检测到突变;相关分析表明:Exon3和Exon4位点与失水率、嫩度、IMF、UFA、PUFA和EFA显著相关(P<0.05),Intron1位点与pH、失水率、IMF、TC、TG和UFA显著相关(P<0.05),Exon3×Exon4互作与TC显著相关(P<0.05),组合基因型CCBB最高;Exon3×Intron1互作与UFA显著相关(P<0.05),组合基因型DDBB最高。9.白羽番鸭ApoVLDL-II基因遗传变异及其与肉质性状的关联分析本研究获得白羽番鸭ApoVLDL-II基因组DNA序列(GQ 180103),5个位点中仅Exon3、Exon4和Intron1检测到多态,共发现了3个SNPs和1个插入/缺失:外显子3发生T1986C突变,为沉默突变;外显子4的UTR区检测到C2901T突变;内含子1检测到A720G突变和在687 bp碱基之后插入/缺失1个长度为13 bp的序列AAAATCTTGTTTA;相关分析表明:Intron1位点与IMF和TG显著相关(P<0.05),Exon3/Exon4×Intron1互作没有对肉质性状产生显著性影响(P>0.05)。10. LXRα、Adiponectin和ApoVLDL-II基因的组织表达规律分析实时荧光定量PCR法检测结果表明:LXRα基因在金定鸭的肝脏中表现为高度表达,肺、脾、肾、心和下丘脑表现为中度表达,胸肌、小脑、大脑、腺胃、小肠和大肠表现为低度表达;金定鸭和白羽番鸭肝脏组织LXRα基因的发育性表达规律相似,均表现为0日龄下降到2周龄,随后逐渐增加,且公鸭的表达量均低于母鸭的表达量,白羽番鸭公母鸭各个时期的表达量均低于金定鸭。Adiponectin基因在鸭的胸肌、大肠和心表现为高度表达,肺、肝、小肠、脾和肾表现为中度表达,腺胃、下丘脑、小脑和大脑表现为低度表达,公母鸭Adiponectin基因表达量均随日龄的增加而降低,公鸭Adiponectin基因在不同时期的表达量均高于母鸭,0-4周龄公、母金定鸭均高于白羽番鸭,6-10周龄则低于白羽番鸭;金定鸭在4-6周龄表达量下降最快,而白羽番鸭则为6-8周龄。肝脏ApoVLDL-II基因在公鸭的表达量一直呈缓慢下降趋势,而母鸭呈缓慢上升趋势,说明ApoVLDL-II基因的表达存在性别差异,并且在不同性别中可能发挥不同的生物学作用。11. LXRα、Adiponectin和ApoVLDL-II基因表达调控关系基因的表达调控分析结果表明:3个基因在金定鸭和白羽番鸭中的表达调控关系一致,0-2周龄,公鸭肝脏组织的3个基因彼此间呈正调控关系,母鸭LXRα和Adiponectin基因与ApoVLDL-II基因呈负调控关系,而LXRα和AMP1基因呈正调控关系。4-10周龄,公鸭的LXRα基因与ApoVLDL-II和Adiponectin基因呈负调控关系,ApoVLDL-II与Adiponectin基因呈正调控关系;母鸭的Adiponectin基因与ApoVLDL-II和LXRα则为负调控关系,ApoVLDL-II与LXRα基因呈正调控关系。协同表达分析结果说明:3个基因的表达调控存在性别差异。更多还原

【Abstract】 At present, excessive fat deposition is one of the main problems encountered by the duck industry, which makes feed utilization decrease and carcass quality decline. More importantly, that the diseases caused by excessive intake of fat-related has aroused widespread interest, and to some extent, it restricted the duck industry. Fat deposition, as a quantitative trait, is regulated by multi-gene. However, the current reported studies are still lacking in duck fat deposition related to candidate genes, and further research is very necessary. In this study, 4 populations, Cherry Valley duck, Jinding duck, White Muscovy, and Sumu Sheldrake (10w) were used. The fatty acid content of breast muscle and 9 Serum biochemical parameters, including triglyceride (TG), total cholesterol (TC), albumin (Alb), total protein (TP), cholinesterase (ChE), alkaline phosphatase (ALP), immunoglobulin A (IgA), globulin (GLO) and Alb/GLO ratio were determined and evaluated by GC-MS and Blood Automatic Biochemical Analyzer. LXRαgene was cloned from Cherry Valley and White Muscovy by using RT-PCR method, and it’s structure and function were further predicted by bioinformatics. Genetic variation of LXRα, Adiponectin, and ApoVLDL-II genes and its relationship with meat quality traits in 4 populations were studied by using PCR-SSCP and DNA sequencing. Real fluorescent quantitative PCR was conducted to investigate expression pattern of LXRα, Adiponectin, and ApoVLDL-II genes in 12 tissues (heart, liver, chest muscle, small intestine, large intestine, cerebellum, brain, hypothalamus, kidney, lung, spleen and proventriculus) of 10-week-old Jinding duck and the developmental expression pattern in liver of White Muscovy and Jinding duck at different developmental stages (0d, 2w, 4w, 6w, 8w and 10w). Probably, the achievement of this study will contribute to duck breeding or genetic improvement and provide reasonable scientific ground. The main results were showed as following:1. Analysis of fatty acid content in breast muscle of duck 16 fatty acids were detected in each duck population, among which oleic acid (C18: 1), palmitic acid (C16:0), stearic acid (C18:0) and linoleic acid (C18:2) were the main composition, accounted for 95% or so. Fatty acids content were distinct from each other among populations, and all fatty acids except C14:1 (P>0.05) exhibited population effect, and C15: 0 was significant (P<0.05) and the rest showed extremely significant population effect (P<0.01). No fatty acid existed sex effect, and the population and sex interaction effect existed (P>0.05). UFA of Cherry Valley duck was the highest, whereas PUFA and EFA were the lowest, however, White Muscovy just the opposite.2. Analysis of serum biochemical parameters The 9 serum biochemical parameters of all populations exhibited a very significant population effect (P<0.01), among which TG showed a significant sex effect (P<0.05), and TC and ALP presented significant sex effect (P<0.01), and TG, TC, and ALP of the male was significantly higher than the female (P<0.05); The interaction effect for TG and TC between population and sex was significant (P<0.01).3. LXRαgene cloning and bioinformatics LXRαgene cDNA was cloned, whose size was 1626 bp, in Cherry Valley Duck and White Muscovy for the first time, and GenBank accession number were FJ966078 and GU132847 respectively. It embraces 5’-UTR sequence of 73 bp, CDS all sequence of 1230 bp and 3’-UTR sequence of 323 bp, and encoded 409 amino acids. There were 14 nucleotides (9 CDS and 5 UTR) and 3 amino acids (Ser163Gly, Gln171Glu and Asn361Lys), which were different between these 2 populations. LXRαprotein in duck had 74-78% homology with mammals and fish, and up to 97% with chicken. Cluster analysis revealed that probably mammals, birds and fishes, each of these belonged to different categories separately. Bioinformatics analysis indicated that duck LXRαprotein contained 17 phosphorylation sites, two low compositional complexity region, a ZnF-C4 and a HOLI domain, without signal peptide and transmembrane helix; The diversity of CDS and the structure of amino acids in 2 LXRαgenes resulted in the RNA folding, protein secondary structure and O-glycosylation sites differences.4. Associations of genetic variations of LXRαgene with meat quality traits in duck It was the first time that silent mutation 277(C/G) was identified in LXR-E5 locus, and 1396(G/C) mutation and 44(C/T) mutation were found in LXR-E12 locus and LXR-I6 locus of duck LXRαgene respectively. Other 6 loci (LXR-E4, LXR-E6, LXR-E7, LXR-E8, LXR-E10 and LXR-E11) had no polymorphism. Correlation analysis showed that LXR-E5 locus of LXRαgene significantly associated with tenderness (P<0.05), and LXR-E12 and LXR-I6 loci were significantly related to pH, water loss rate, IMF, TC, TG, UFA , PUFA and EFA (P<0.05). Interaction between LXR-E5 and LXR-I6 loci had a significant impact on UFA (P<0.05), and that BBCC genotype was the highest. Interaction between LXR-E12 and LXR-I6 loci had an extremely significant effect on tenderness, pH, and TC (P<0.05), and that ABCC, BBDD, and BBDD were the highest respectively.5. Associations of genetic variations of LXRαgene with meat quality traits in White Muscovy There were 53(G/A) and 1483(-/T) mutations first found in LXR-E4 and LXR-E12 loci of White Muscovy LXRαgene respecticely. There were no polymorphism in other 7 loci(LXR-E5、LXR-E6、LXR-E7、LXR-E8、LXR-E10, LXR-E11 and LXR-I6). Correlation analysis suggested that LXR-E4 locus had a significant genetic effect on IMF, UFA and meat color (P<0.05). Interaction between LXR-E4 and LXR-E12 loci had a significant effect on UFA (P <0.05), and that BBCC was the highest.6. Associations of genetic variations of adiponectin gene with meat quality traits in duck 15 SNPs were discovered in 4 loci of ADP1, ADP2, ADP3 and ADP4 of duck adiponectin gene, of which G887A of 3’-UTR, 12 SNPs (C86T, C104T, C146T, C155T, C456T, A574G, C651T, C684T, T768C, G784A, A801C and C807T) of CDS, C273T and C295T of intron 2. A574G, G784A, and A801C were missense mutations, resulting in amino acid sequence altered, that were 144 of Thr(T) into Ala(A), 214 of Ile(I) into a Val(V), and 219 of Asp(D) into Glu(E). Association results showed that ADP1 locus of duck adiponectin gene presented significant genetic effects on IMF, UFA, PUFA and EFA (P<0.05). ADP2 locus showed significant genetic effects on water loss rate, IMF, TC, and UFA (P<0.05). ADP4 locus conducted significant genetic effect on water loss rate, TC, UFA and PUFA (P<0.05). Interactions between ADP1 and ADP3, and ADP2 and ADP3 had a significant influence on UFA (P<0.05), and genotypes of AACC and CDBC were the highest respectively. Interactions between ADP1 and ADP4, and ADP3 and ADP4 on water loss rate and IMF appeared a significant effect (P<0.05), and genotypes of CDAC and CCBB for water loss rate, and DDAA and CCAA for IMF were the highest, respectively.7. Associations of genetic variations of adiponectin gene with meat quality traits in White Muscovy 3 SNPs were first found in White Muscovy adiponectin gene, including A167G and G711A of CDS, and C290T of intron, which were nonsense mutations. ADP1 and ADP2 loci of adiponectin gene conducted significant effect on water loss rate and IMF (P<0.05). Interactions between ADP1 and ADP4, and ADP2 and ADP4 on UFA appeared a significant influence (P<0.05), and genotypes of BBFF and TTFF showed the highest respectively. 8. Associations of genetic variation of ApoVLDL-II gene with meat quality traits in duck Genomic DNA sequence(GQ 180104) of duck ApoVLDL-II were first cloned, and 5 loci of Exon1, Exon2, Exon3, Exon4 and Intron1 were detected by PCR-SSCP. There were no polymorphism in Exon1 and Exon2, whereas 12 SNPs (T667C, C669G, T673C, G674A, G683A, G688A, C708G, T715G, G2106A, T2723C, C2743T, and A2944C), and insertion/deletion TG and CC after the 764 bp and 1910 bp respectively were discovered in another 3 loci. In addition to A2944C mutation located in exon 4 UTR, others were in introns, and the complete coding region mutation was not detected. Correlation analysis showed that Exon3 and Exon4 loci had significant genetic effects on water loss rate, tenderness, IMF, UFA, PUFA and EFA (P<0.05), and Intron1 locus had a significant genotype effect on the pH, water loss rate, IMF, TC, TG and UFA (P<0.05). Interactions between Exon3 and Exon4 had a significant impact on TC (P<0.05), and the genotype of CCBB presented the highest. Interactions between Exon3 and Intron1 had a significant influence on UFA (P<0.05), and the genotype of DDBB was the highest.9. Associations of genetic variation of ApoVLDL-II gene with meat quality traits in White Muscovy Genomic DNA sequence (GQ 180103) of White Muscovy ApoVLDL-II gene was first discovered. Only Exon3, Exon4 and Intron1 of 5 loci exhibited polymorphism, while exon 3 occured T1986C was silent mutation, C2901T mutation was detected in UTR of exon 4, and A720G mutation and insertion/deletion 13bp sequence AAAATCTTGTTTA after the 687bp was discovered in intron1. Association analysis suggested that Intron1 had a significant genetic effect on IMF and the TG in White Muscovy (P<0.05). Interactions between Exon3/Exon4 and Intron1 exhibited no significant impact on all traits detected (P>0.05).10. Tissue expression pattern analysis of LXRα, Adiponectin, and ApoVLDL-II genes By real-time fluorescent quantitative PCR, the results revealed that LXRαgene in Jinding duck given the performance of highly specific expression of liver, and then lung, spleen, kidney, heart and hypothalamus showed moderate expression, and last chest muscle, cerebellum, brain, proventriculus, small intestine and large intestine presented low expression. Developmental expression pattern of LXRαgene in liver of Jinding duck was in agreement with White Muscovy, performing that the level from 0-day-old dropped to 2 weeks, and then gradually increased, and male was lower than female. Regardless of male or female, levels of expression of White Muscovy were lower than Jinding duck during various periods. Adiponectin gene performed highly specific expression in Jinding duck breast muscle, intestine and heart, and showed moderate expression in lung, liver, small intestine, spleen and kidneys, presented low expression in proventriculus, hypothalamus, cerebellum and brain. With age increasing, level of Adiponectin gene expression decreased in male and female. However, male showed higher than female at different phases. 0-day-old to 4-week-old male and female Jinding duck was higher than White Muscovy, whereas 6-10 weeks lower. The sharpest decline for Jinding duck was the 4-6 weeks, while the 6-8 weeks for White Muscovy. ApoVLDL-II gene expression level in liver of the male had shown a slow decline, whereas a slowly rising for the female, indicating that sex affects ApoVLDL-II gene expression, and may play a special biological role in diferent genders.11. Associations of LXRα, Adiponectin and ApoVLDL-II genes expression and regulation Analysis of gene expression and regulation of 3 genes in Jinding duck and White Muscovy reveled that they positively regulated with each other in liver tissue of male during 0 day to 2 weeks, but in female, ApoVLDL-II negatively regulated by LXRαand Adiponectin, and LXRαand Adiponectin presented a positive control. During 4-10 weeks of age, LXRαwas negatively regulated by ApoVLDL-II and Adiponectin, while ApoVLDL-II and Adiponectin gene was up-regulated relationship in male; Adiponectin gene was negatively controlled by ApoVLDL-II and LXRα, while there was the up-regulated relationship between ApoVLDL-II and LXRαgene in female. Results of synergistic expression analysis showed that: There were sex differences in gene expression and regulation of 3 genes.更多还原