【作者】 赵小玲；

【导师】 朱庆；

【作者基本信息】 四川农业大学 ， 动物遗传育种与繁殖， 2008， 博士

【摘要】 脂肪性状是优质鸡肉品质评价的重要指标,此类性状的发育会直接影响其商品性能。因此,有必要开展调控脂肪性状发育的基因的研究。Adipocyte differentiation-related protein(ADFP)和Perilipin是覆盖在脂滴表面的磷蛋白,对人和鼠脂肪细胞的脂质代谢有重要的调控作用。由于两种蛋白参与了糖尿病、肥胖症和动脉粥样硬化的异常脂质代谢,在国外,ADFP和Perilipin在持续十七年的时间中一直是研究的热点。在人和小鼠上开展ADFP和Perilipin的研究对深入研究鸡脂肪性状遗传机制有很好的借鉴作用。本研究以地方品种四川山地乌骨鸡为试验材料,克隆获得了鸡PAT家族成员ADFP和基因PLIN的全编码区序列(1317bp和1554bp)(GenBank accession numbers:EU620700和EU620701),并翻译获得其氨基酸序列(438aa和515aa)。通过多种生物信息学分析方法,确定两条氨基酸序列具有PAT家族的结构特征,是鸡PAT家族成员ADFP和PLIN蛋白。本研究以地方品种四川山地乌骨鸡、藏鸡和培育优质鸡“大恒”S01系为试验材料,采用SYBR greenⅠ定量PCR分析ADFP基因和PLIN基因在1日龄和84日龄两个时间点品种间的表达差异,同时分析了四川山地乌骨鸡胚胎期(第9天和第15天)、1日龄和84日龄等4个时间点ADFP基因和PLIN基因表达的组织差异,以及胸肌、肝脏、腹脂和皮脂组织中ADFP基因和PLIN基因表达量的发育性变化同皮脂厚、肌内脂肪含量、活重和冠重等四个表型性状累积生长值之间的关系。结果表明,ADFP基因和PLIN基因在鸡不同生长发育阶段都有表达,并且在不同阶段各有优势表达组织,同一组织中也有表达的优势时间点;腹脂组织中ADFP基因的发育性变化与活重和冠重的累积生长值成显著正相关(P＜0.05),胸肌组织中ADFP基因的发育性变化与肌内脂肪含量的累积生长值成显著正相关(P＜0.05);腹脂组织中PLIN基因的发育性变化与皮脂厚的累积生长值成极显著地正相关(P＜0.01),胸肌组织中PLIN基因的发育性变化与肌内脂肪含量的累积生长值成极显著地正相关(P＜0.01)。这说明,ADFP和PLIN基因表达量与鸡脂肪组织,尤其是肌内脂肪含量的发育紧密相关。通过研究四川山地乌骨鸡各组织的ADFP和PLIN基因表达量倍比值的发育性变化,发现皮脂和腹脂组织中ADFP和PLIN表达量比值变化随机体的生长发育呈先升后降的趋势。由此推测,随着脂肪组织中脂肪细胞的发育成熟,ADFP和PLIN基因先后对鸡脂肪组织的代谢发挥主要调控作用。本研究以地方品种四川山地乌骨鸡、广东惠阳胡须鸡、封开杏花鸡、清远麻鸡、广西霞烟鸡、四川大恒优质鸡8个品系、岭南黄鸡矮小型麻羽专门化品系(E2)、岭南黄鸡Ⅱ号配套系(DC)和引进肉鸡品种爱拔益加等16个群体,共430个个体为试验材料,采用PCR-SSCP的方法设计引物,扫描ADFP和PLIN基因外显子序列和邻近内含子序列。结果,在ADFP基因中检测到3个单核苷酸突变位点,分别位于内含子4、内含子5和外显子8(第398氨基酸残基处)。具体为Z染色体33 394 913位(SNP1)A→T,33 395 676位(SNP2)C→T,33 397 903位(SNP3)A→G。与dbSNP数据库的SNP比对分析,发现三个突变位点为新的突变位点。在ADFP基因的三个突变位点中,等位基因A、C和G依次为SNP1(A/T)、SNP2(C/T)和SNP3(A/G)的优势等位基因。ADFP基因单突变位点与16个群体的屠宰性状关联分析结果显示,SNP3对肌内脂肪含量的遗传效应达到显著水平(P＜0.05)。同时,SNP1对全净膛重、腹脂重和腹脂率的遗传效应达到显著水平(P＜0.05),SNP2对胸肌重、腹脂重和皮脂厚的遗传效应达到显著水平(P＜0.05)。单倍型与16个群体的屠宰性状关联分析结果显示,单倍型组合对胸肌重、腹脂重和腹脂率的遗传效应达到显著水平(P＜0.05)。这说明ADFP基因突变位点与屠宰性状,尤其是脂肪性状显著相关,该基因可能是影响鸡的脂肪性状的主效基因或与控制这些性状的QTL连锁。在PLIN基因中检测到3个单核苷酸突变位点,分别位于外显子2(第7氨基酸残基)和外显子7(第285氨基酸残基和第289氨基酸残基)。具体为10染色体14 521351位(SNP1)T→C,14 523 391位(SNP2)C→T,14 523 402位(SNP3)G→A。与dbSNP数据库的SNP比对分析,发现三个突变位点为新的突变位点。在PLIN基因的三个突变位点中,SNP1(T/C)和SNP2(C/T)各基因型和等位基因在16个群体中随机分布,没有明显的优势基因型和等位基因;SNP3(G/A)基因型GG和等位基因G在16个群体中均为优势基因型和等位基因。突变位点SNP1和SNP2具有中等的多态信息含量。单突变位点与16个群体的屠宰性状关联分析结果显示,SNP1、SNP2和SNP3分别对肌内脂肪含量、胸肌重和屠体重有显著地遗传效应(P＜0.05)。单倍型与16个群体的屠宰性状关联分析结果显示,单倍型组合对皮脂厚有显著的遗传效应(P＜0.05)。这说明PLIN基因突变位点与屠宰性状,尤其是脂肪性状显著相关,该基因可能是影响鸡脂肪性状的主效基因或与控制这些性状的QTL连锁。更多还原

【Abstract】 Fatness traits were important factors of meat quality evaluation for high-quality chicken.The development of them would directly affect the economic value of high-quality chicken.Thus,it is necessary to study on the genes which control the development of fatness traits.Adipocyte differentiation-related protein and Perilipin were the phosphoprotein located on the surface of lipid droplet.The former researches reported ADFP and Perilipin controlled the lipid metabolism in human and mouse. Because both of them participated in the exceptional lipid metabolism of 2 type diabetes,adiposity and artery atherosclerosis,ADFP and Perilipin have been the study hotspot in medical field about two decades.The study results of ADFP and Perilipin in human and mouse were good references for study on the genetic mechanism of fatness traits in chickens.The current research adopted Sichuan Mountainous Land Black-Bone chicken as material.We successfully obtained the coding sequence of ADFP and PLIN gene, which were 1317bp and 1554bp.GenBank assigned them accession numbers EU620700 and EU620701,respectively.Based on these two cDNA sequences,we deduced two amino acid sequences.Their lengths were 438aa and 515aa.Via bioinformative analyses,we found both "the two amino acid sequences had the structure characters of PAT family and had high identity with ADFP and PLIN protein in other species,respectively.Thus,we concluded that they were the protein of chicken ADFP and PLIN.We inferred they had the function of controlling chicken lipid metabolism.The present study took Sichuan Mountainous Land Black-Bone chicken,Tibetan chicken and line S01 of Dahcng chicken as materials.We adopted the real-time PCR （SYBR greenⅠ） method to test the expression quantity of ADFP and PLIN genc in the breeds mentioned above at the first day and the 84^th day aider birth.And wc also tested the expression quantity of ADFP and PLIN genc in different growth points and had correlation analysis of these relative quantities and traits in Sichuan Mountainous Land Black-Bone chicken.The results indicated that ADFP and PLIN genc express in all tissues at all growth points,and there was advantageous tissue for gcnc expression at each growth point and there was also advantageous growth point for gene expression in a certain tissue.Furthermore,the developmental change of ADFP gene expression quantity in abdominal fat was positively related with the cumulative values of living weight and comb weight（P＜0.05）,and the developmental change of ADFP genc expression quantity in breast muscle was positively related with the cumulative values of intramuscular fat content（P＜0.05）.And the developmental change of PLIN gene expression quantity in abdominal fat was positively related with the cumulative values of subcutaneous fat thick（P＜0.05）,and the developmental change of PLIN genc expression quantity in breast muscle was positively related with the cumulative values of intramuscular fat content（P＜0.05）.The results indicated that the developmental change of ADFP and PLIN gene expression quantity were tightly correlated with the development change of fatness traits,especially with the development change of intramuscular fat content.Study on the ratioes of the expression quantity of ADFP gene and PLIN gene along with the growth points in Sichuan Mountainous Land Black-bone chicken discovered that the ratioes in subcutaneous fat and abdominal fat increased firstly and then deceased.So,we inferred that with adipocytes growing and being mature,ADFP and PLIN gcnc may have key role in turns in control chicken lipid metabolism.The current study adopted Mountainous Land Black-Bone chicken and Daheng high-quality chicken in Sichuan province,Huxu chicken,Xinghua chicken,Qingyuan ma chicken,and the cultivating line E2 and DC in Guangdong province,Xiayang chicken in Guangxi province,and one broiler breed,Arbor Acres chicken,totally 430 samples.We screened all the extrons and part ofintron sequences of ADFP gene and got three single nucleotide polymorphisms（SNP）.They were located in the 4^th intron, the 5^th intron and the 8^th extron(at the 398^th amino acid position).The SNP1 was a transition of A to T at 33 394 913 in chromosome Z;the SNP2 was a transversion of C to T at 33 395 676 in chromosome Z;the SNP3 was a transversion of Ato G at 33 397 903 in chromosome Z.All the SNPs detected in the current study were novel and synonymous SNPs.The allele A,C and G were the predominant allele in SNP 1,SNP2 and SNP3,respectively.The results of single locus analysis showed that SNP3 had significant genetic effect on intramuscular fat content（P＜0.05）;SNP1 had significant genetic effect on eviscerated weight,abdominal fat weight and percentage of abdominal fat（P＜0.05）;and SNP2 had significant genetic effect on breast muscle weight,abdominal fat weight and subcutaneous fat thick（P＜0.05）.The haplotype analysis indicated that the main haplotype combinations had significant genetic effect on breast muscle weight,abdominal fat weight and percentage of abdominal fat（P＜0.05）.In a word,the polymorphisms of ADFP gene were significantly associated with carcass traits,especially the fatness traits.We inferred that ADFP gene probably was the major gene affecting fatness traits or was in linkage disequilibrium with the QTL controlling the fatness traits in chickens.We also found three SNPs in PLIN gene.They were located in extron 2(at the 7^th amino acid position) and extron 7(at the 285^th and the 289^th amino acid position).In chromosome 10,their positions were 14 521 351（T→C）,14 523 391（C→T）,14 523 402（G→A）.All of them were synonymous but novel SNPs.There were no predominant allele at SNP1 and SNP2.However,allele G was the predominant allele at SNP3.And the SNP1 and SNP2 had middle polymorphic informative content（PIC）. The single locus analysis indicated that SNP1（T/C）,SNP2（C/T） and SNP3（G/A） had significant genetic effect on intramuscular fat content,breast muscle weight and carcass weight（P＜0.05）.The haplotype analysis had significant genetic effect on subcutaneous fat thick（P＜0.05）.Generally,the results of marker-trait linkage analyses indicated the polymorphisms of PLIN gene were significantly associated with carcass traits,especially the fatness traits.We inferred that PLIN gene probably was the major gene affecting fatness traits or was in linkage disequilibrium with the QTL controlling the fatness traits in chickens.更多还原