鸭A-FABP基因cDNA克隆与基因多态性的研究
摘要
沉积在肌肉内的脂肪是影响肉质和风味的一项重要经济性状。肌内脂肪(Intramuscular fat,IMF)被认为与肉质及口感呈正相关,影响肉质的多汁性、嫩度及风味。脂肪酸结合蛋白(Fatty acid binding protein,FABP)基因在动物体内脂肪代谢过程中的作用,与肌内脂肪含量等肉质性状显著相关,其中A-FABP (Adipocyte fatty acid bingding protein,A-FABP)和H-FABP (Muscle and heart fatty acid binding protein,H-FABP)已被证实是这些数量性状的候选基因,进行了重点研究。目前家禽育种工作已经取得了很大进展,然而高强度选择又带来了许多负面影响。其中控制脂肪在鸭体内的过多蓄积,提高鸭胴体质量和适口性是中国养禽业急需解决的问题。本研究把A-FABP基因作为鸭肌内脂肪含量的候选基因加以研究,旨在通过cDNA末端快速扩增技术(Rapid amplification of cDNA ends,RACE)克隆并分析鸭A-FABP基因的结构与功能,利用聚合酶链式反应-单链构象多态性(Polymerase chain reaction-Single strand conformation polymorphism,PCR-SSCP)技术分析鸭A-FABP基因的单核苷酸多态性,以阐明鸭A-FABP基因的遗传变异,继而以此为分子遗传标记进行辅助选择应用于鸭的育种,对降低鸭体脂、提高鸭肌内脂肪含量和改善鸭肉品质具有重要意义。
为揭示鸭A-FABP基因的结构和功能,应用RACE技术首次克隆并鉴定了鸭A-FABP基因cDNA序列。首先用一对含高度保守的DNA片段的兼并引物,从鸭腹部脂肪组织总RNA扩增部分A-FABP片段,经验证与已知序列一致；接着测序并设计新引物分别从5’和3'RACE扩增延长该片段。最后根据全基因的序列设计上、下游引物,经SEQMAN软件拼接5'RACE产物和3'RACE产物以及已知序列获得片段大小为652bp的cDNA序列。与鸡A-FABP基因进行BLAST比对分析,结果表明该652bp的cDNA序列由5’-UTR区、CDS区和3’-UTR区组成,其中CDS区长度为399bp,5’非编码区长度为64bp,3’非编码区长度为189bp。包含完整的1、2、3、4外显子序列,与鸡A-FABP基因序列4个外显子一样也分别编码24,58,34和16个氨基酸。鸭A-FABP基因399bp的开放阅读框编码132个氨基酸,推导和分析氨基酸序列表明,分子结构与鸡A-FABP相似,其中有些氨基酸残基表现出较高的保守性。利用DNAMAN软件比较核苷酸序列,与猪、鸡和鹅的A-FABP基因分别有78%、95%和98%的同源性。
根据RACE获得鸭A-FABP基因的cDNA序列,设计引物P0,扩增出该基因的第二内含子序列,在此基础上又设计3对引物利用PCR-SSCP方法对获得的序列进行多态性研究。结果引物P1在北京鸭、缙云麻鸭等品种中发现4处单碱基突变,分别为：386处“A-T”突变：388处的“T-C”突变；403处的“T-C”突变；444处的“C-T”突变。这些点突变共产生了3种基因型,且基因型在各鸭品种间的分布有显著差异。
Intramuscular Fat (IMF) content which is the crucial parameter for meat quality has important effect on meat sapor, succulence, tenderness and delicate flavor. IMF is deemed to be positive correlation with meat palatability, and the fatty acid binding proteins (FABPs) are regarded as relevant to the intramuscular fat content and other meat parameters in the lipid metabolic process of animal body. Adipocyte fatty acid binding protein (A-FABP) and Muscle and heart fatty acid binding protein (H-FABP) are selected as candidate genes for these quantitative traits and studied intensively. Presently, the improvement in poultry breeding has been made. However, negative effects have appeared accompanied with high intensity selection. Problems need resolved by poultry industry in china, exist in controlling excessive fatness accumulation that affects duck carcass quality. This experiment deals with the research of A-FABP gene effect on duck meat quality. This study was designed to investigate the genetic variation and the genotype diversity, which will be served as genetic maker to select fat and flavor traits using marker-assisted selection.
In order to study the structure and function of duck A-FABP gene, cDNA of the A-FABP gene was cloned using a method of Rapid amplification of cDNA ends (RACE). Firstly, according to high conservative nucleotide sequence of homologous fragment in A-FABP gene, a pair of degenerated primer was designed, and a special DNA fragment was gained from duck total RNA by Reverse transcription-Polymerase chain reaction (RT-PCR). Then based on the sequence of gained DNA fragment, some new primers were designed, and the 3'terminal and the 5'terminal of A-FABP gene were cloned by RACE respectively. Finally a complete cDNA of A-FABP was to extend with newly designed primer by PCR. The product was identified by electrophresis and sequence analysis. The results of sequencing indicate that A-FABP gene cDNA is 652bp in length, which includes an open reading frame of 399bp encoding a protein of 132 amino acid residues. In comparison with the nucleotide sequences of other species A-FABP genes, duck A-FABP gene is similar to chicken's and goose's, showing an overall identity of 95% with chicken and 98% with goose,71% with human and 78% with pig. In addition, some amino acid residues in A-FABP molecule manifest extremely conservative among animals, which suggests that they could have an important biological function.
A pairs of primers were designed according to the sequence of duck adipocyte fatty acid of binding protein gene, and then the A-FABP gene was amplified by RACE from duck genome. The product of 652bp length of PCR was cloned and sequenced, it includes complete exonl,2,3 and 4. Homologous comparison was done between duck and chicken, goose and porcine A-FABP gene and the homology are 95%,98% and 78% respectively. The intron 2 was amplified and then 3 pairs of primers were designed on the base of the cloned gene, the SNPs was detected by the technique of single strand conformation polymorphism (SSCP) and then confirmed by sequencing. Four nucleotides mutation were found, A—T at-386bp, T—C at-388bp, T—C at-403bp and C—T at-444bp respectively. Three genotypes were detected based on these mutation and genotypes frequencies among duck breeds were different significantly.
为揭示鸭A-FABP基因的结构和功能,应用RACE技术首次克隆并鉴定了鸭A-FABP基因cDNA序列。首先用一对含高度保守的DNA片段的兼并引物,从鸭腹部脂肪组织总RNA扩增部分A-FABP片段,经验证与已知序列一致；接着测序并设计新引物分别从5’和3'RACE扩增延长该片段。最后根据全基因的序列设计上、下游引物,经SEQMAN软件拼接5'RACE产物和3'RACE产物以及已知序列获得片段大小为652bp的cDNA序列。与鸡A-FABP基因进行BLAST比对分析,结果表明该652bp的cDNA序列由5’-UTR区、CDS区和3’-UTR区组成,其中CDS区长度为399bp,5’非编码区长度为64bp,3’非编码区长度为189bp。包含完整的1、2、3、4外显子序列,与鸡A-FABP基因序列4个外显子一样也分别编码24,58,34和16个氨基酸。鸭A-FABP基因399bp的开放阅读框编码132个氨基酸,推导和分析氨基酸序列表明,分子结构与鸡A-FABP相似,其中有些氨基酸残基表现出较高的保守性。利用DNAMAN软件比较核苷酸序列,与猪、鸡和鹅的A-FABP基因分别有78%、95%和98%的同源性。
根据RACE获得鸭A-FABP基因的cDNA序列,设计引物P0,扩增出该基因的第二内含子序列,在此基础上又设计3对引物利用PCR-SSCP方法对获得的序列进行多态性研究。结果引物P1在北京鸭、缙云麻鸭等品种中发现4处单碱基突变,分别为：386处“A-T”突变：388处的“T-C”突变；403处的“T-C”突变；444处的“C-T”突变。这些点突变共产生了3种基因型,且基因型在各鸭品种间的分布有显著差异。
Intramuscular Fat (IMF) content which is the crucial parameter for meat quality has important effect on meat sapor, succulence, tenderness and delicate flavor. IMF is deemed to be positive correlation with meat palatability, and the fatty acid binding proteins (FABPs) are regarded as relevant to the intramuscular fat content and other meat parameters in the lipid metabolic process of animal body. Adipocyte fatty acid binding protein (A-FABP) and Muscle and heart fatty acid binding protein (H-FABP) are selected as candidate genes for these quantitative traits and studied intensively. Presently, the improvement in poultry breeding has been made. However, negative effects have appeared accompanied with high intensity selection. Problems need resolved by poultry industry in china, exist in controlling excessive fatness accumulation that affects duck carcass quality. This experiment deals with the research of A-FABP gene effect on duck meat quality. This study was designed to investigate the genetic variation and the genotype diversity, which will be served as genetic maker to select fat and flavor traits using marker-assisted selection.
In order to study the structure and function of duck A-FABP gene, cDNA of the A-FABP gene was cloned using a method of Rapid amplification of cDNA ends (RACE). Firstly, according to high conservative nucleotide sequence of homologous fragment in A-FABP gene, a pair of degenerated primer was designed, and a special DNA fragment was gained from duck total RNA by Reverse transcription-Polymerase chain reaction (RT-PCR). Then based on the sequence of gained DNA fragment, some new primers were designed, and the 3'terminal and the 5'terminal of A-FABP gene were cloned by RACE respectively. Finally a complete cDNA of A-FABP was to extend with newly designed primer by PCR. The product was identified by electrophresis and sequence analysis. The results of sequencing indicate that A-FABP gene cDNA is 652bp in length, which includes an open reading frame of 399bp encoding a protein of 132 amino acid residues. In comparison with the nucleotide sequences of other species A-FABP genes, duck A-FABP gene is similar to chicken's and goose's, showing an overall identity of 95% with chicken and 98% with goose,71% with human and 78% with pig. In addition, some amino acid residues in A-FABP molecule manifest extremely conservative among animals, which suggests that they could have an important biological function.
A pairs of primers were designed according to the sequence of duck adipocyte fatty acid of binding protein gene, and then the A-FABP gene was amplified by RACE from duck genome. The product of 652bp length of PCR was cloned and sequenced, it includes complete exonl,2,3 and 4. Homologous comparison was done between duck and chicken, goose and porcine A-FABP gene and the homology are 95%,98% and 78% respectively. The intron 2 was amplified and then 3 pairs of primers were designed on the base of the cloned gene, the SNPs was detected by the technique of single strand conformation polymorphism (SSCP) and then confirmed by sequencing. Four nucleotides mutation were found, A—T at-386bp, T—C at-388bp, T—C at-403bp and C—T at-444bp respectively. Three genotypes were detected based on these mutation and genotypes frequencies among duck breeds were different significantly.
引文
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