Molecular cloning, tissue expression and polymorphism analysis of the Caveolin-3 gene in ducks

1. Duck meat is considered a delicacy, but choosing the best meat is problematic. Caveolin-3 (CAV-3) is a muscle-specific protein marker in animals. The goal of the current study was to detect the characteristics of CAV-3 gene in ducks. 2. Full-length CAV-3 was acquired from ducks (Anas platyrhynchos) using reverse transcriptase PCR and rapid amplification of cDNA ends. DNAMAN software was used for homology comparisons. Quantitative reverse transcription-PCR, PCR-single-strand conformation polymorphism, and sequencing were used to determine CAV-3 expression and polymorphism of a single nucleotide, respectively. The study examined four types of ducks, including Jinding, Chaohu, Cherry Valley, and Gaoyou ducks. 3. The study acquired 1066 bp of CAV-3 cDNA sequences, including a 456 bp complete open reading frame encoding 151 amino acids. Both coding sequences (CDSs) and translated amino acids exhibited highest homology with Gallus gallus (CDS homology 91.67%, amino acids 94.04%), followed by mammalian species (CDS homology 79.0%, amino acids 78.0%). Single nucleotide polymorphism analysis revealed five mutations in exons (A489G, G501A, A557G, T563A, and A602G), and a C805T mutation in an intron. Among amplified polymorphic loci detected using primer 2, allele frequency was higher for A (489A501G507A563T602A) than B (489G501G507G563T602C) or C (489G501A507G563A602C). The highest occurred in Cherry Valley ducks (0.7587). Using primer 4, the M allele frequency was higher than that of the N allele. CAV-3 was most highly expressed in the heart, followed by skeletal muscles. Additionally, CAV-3 had higher expression in heart and breast muscle of overfed Muscovy ducks than control ducks, but no difference was seen in thigh muscle. 4. CAV-3 in ducks had the highest homology with Gallus gallus CAV-3, and it could be used as a marker for muscle quality in ducks.

1. 鸭肉常被视为珍馐佳肴，但优质鸭肉的甄选仍存在难题。小窝蛋白3（Caveolin-3, CAV-3）是动物体内的肌肉特异性蛋白标记物。本研究旨在探究鸭源CAV-3基因的特征。
2. 本研究通过逆转录聚合酶链式反应（reverse transcriptase PCR）与cDNA末端快速扩增技术（rapid amplification of cDNA ends, RACE），从绿头鸭（Anas platyrhynchos）中克隆获得了鸭源CAV-3的全长序列；采用DNAMAN软件进行同源性比对分析。分别采用实时定量逆转录聚合酶链式反应（quantitative reverse transcription-PCR）、PCR-单链构象多态性（PCR-single-strand conformation polymorphism）与测序技术，检测CAV-3的表达水平与单核苷酸多态性。本研究共纳入4个鸭品种：金定鸭、巢湖鸭、樱桃谷鸭与高邮鸭。
3. 本研究获得的鸭源CAV-3 cDNA序列全长为1066 bp，其中包含一个长度为456 bp的完整开放阅读框，可编码151个氨基酸。该基因的编码序列（coding sequences, CDS）与翻译所得氨基酸序列均与家鸡（Gallus gallus）的同源性最高，其中编码序列同源性达91.67%，氨基酸序列同源性达94.04%；其次为哺乳动物类群，编码序列同源性为79.0%，氨基酸序列同源性为78.0%。单核苷酸多态性分析显示，外显子区域存在5处突变位点：A489G、G501A、A557G、T563A及A602G；内含子区域存在1处C805T突变。在引物2扩增的多态位点中，等位基因A（单倍型：489A501G507A563T602A）的频率高于等位基因B（489G501G507G563T602C）与C（489G501A507G563A602C），其中樱桃谷鸭中的等位基因A频率最高，达0.7587。采用引物4扩增时，M等位基因的频率高于N等位基因。CAV-3在心脏中的表达量最高，其次为骨骼肌。此外，填饲番鸭的心脏与胸肌中CAV-3的表达水平显著高于对照组，但其腿肌中的表达水平无组间差异。
4. 鸭源CAV-3与家鸡CAV-3的同源性最高，该基因可作为鸭肌肉品质评定的分子标记物。