西农萨能羊乳腺脂肪酸合酶基因exon 9-15的克隆与序列分析

Cloning and Sequence Analysis on exon 9-15 of FAS Gene in Mammary Gland of Xinong Saanen Goat

【目的】山羊FAS基因exon 9-15编码的乙酰/丙二酸单酰基转移酶(acetyl-CoA and malonyl-CoA transacylases,AT/MT)区域对山羊乳短、中链脂肪酸的合成起重要调控作用。本研究针对西农萨能羊乳腺FAS基因exon 9-15进行克隆和序列分析。【方法】以处于泌乳期28d的西农萨能羊乳腺组织mRNA反转录的cDNA为模板,通过RT-PCR方法首次扩增出西农萨能羊乳腺FAS基因exon 9-15的cDNA序列全长及exon 8的3′端和exon 16的5′端部分cDNA序列(GenBank收录号为DQ915966),并对其进行了同源性分析和功能预测。【结果】克隆片段全长1449bp,其中包括exon 9-15共1388bp、exon 8的3′端9bp和exon 16的5′端52bp,编码483个氨基酸,包含编码的AT/MT区域951bp(454～1404nt);西农萨能羊乳腺FAS基因exon 9-15与牛(NM_001012669),人(NM_004104),大鼠(NM_017332)和鸡(NM_205155)核苷酸序列相似度分别为95%、85.7%、82.7%、73.2%,氨基酸相似度分别为92.9%、77.7%、82.3%、64.7%;exon 10较其它物种缺失1个氨基酸。【结论】克隆获得西农萨能羊FAS基因片段全长1449bp,外显子9-15大小分别为463、185、190、95、135、204和116bp;核苷酸及氨基酸序列与牛相应序列的同源性均高于其它物种;AT/MT区域的活性位点在各物种间均为高度保守的丝氨酸,但西农萨能羊exon 10较其它物种缺失1个氨基酸,这可能对AT/MT区域的空间构象及其生理功能产生重要影响。本研究为西农萨能羊FAS基因cDNA全长克隆以及基因功能研究奠定了重要基础。

【Objective】The acetyl-CoA and malonyl-CoA transacylases region encoded by exon 9-15 of goat FAS gene plays a central role in synthesis of short- and medium-chain fatty acids in goat milk. In this work, exon 9-15 of Xinong Saanen goat mammary gland FAS gene was cloned and sequenced. 【Method】 The mRNA from Xinong Saanen goat mammary gland on 28th day of lactation was isolated, and exon 9-15 of FAS gene in Xinong Saanen goat mammary gland and partial cDNA of 3′ end of exon 8 and 5′ end of exon 16 were amplified with RT-PCR using the mammary gland cDNA as the template by reverse transcription （GenBank accession no. DQ 915966）, after sequencing the homology of sequence among species was analyzed and its function was speculated by bioinformatics. 【Result】Sequencing of the fragments showed that the cDNA was of 1 449 bp in length and it contained 1 338 bp exon 9-15 and 9 bp 3′ end of exon 8 and 52 bp 5′ end of exon 16, which encoded 483 amino acids including the 951 bp （454-1404 nt） AT/MT domain;The homologies of nucleotide and amino acid sequence of exon 9-15 of Xinong Saanen goat mammary gland FAS gene with those of bovine （NM_001012669）, human （NM_004104）, rattus （NM_017332） and chicken （NM_205155） were 95%, 85.7%, 82.7%, 73.2% and 92.9%, 77.7%, 82.3%, 64.7%, respectively. Compared with the other four species, an amino acid in exon 10 of goat FAS gene was absent.【Conclusion】The length of the obtained cDNA fragments of Xinong Saanen goat FAS gene was 1 449 bp, and the size of exon 9-15 was 463, 185, 190, 95, 135, 204 and 116 bp, respectively. Xinong Saanen goat and cow had higher homology in nucleotide sequence and amino acid sequence than other species. The active sites of the AT/MT region was the highly conserved serine among species, but an amino acid in exon 10 of goat FAS gene was deleted, which may bring about significant influence on conformation and physiological functions. This work laid an important foundation for cloning the full-length cDNA of Xinong Saanen goat FAS gene and the study of gene function.