兰州大尾羊心脏型脂肪酸结合蛋白（H-FABP） 基因克隆及其同源性比较

doi:10.3864/j.issn.0578-1752.2013.03.021

摘要/Abstract

摘要： 【目的】克隆兰州大尾羊心脏型肪酸结合蛋白（H-FABP）基因全长cDNA序列，为研究绵羊H-FABP生物学作用和生产应用提供理论依据。【方法】根据已知哺乳动物H-FABP基因 cDNA 序列，设计5'和3'特异引物，运用cDNA 末端快速扩增（RACE）技术获得兰州大尾羊H-FABP基因全长 cDNA 序列。【结果】扩增获得兰州大尾羊5'端425 bp、3'端231 bp片段和 177 bp中间片段，拼接获得748 bp兰州大尾羊H-FABP基因全长cDNA 序列（GenBank登录号：JQ780322）。兰州大尾羊H-FABP基因ORF长 402 bp，编码 133 个氨基酸。核苷酸序列分析显示兰州大尾羊H-FABP基因序列与大多数哺乳动物相似，但其第66位发生的碱基转换（T←→G）引起所编码的第22位天门冬氨酸（N）不同于其它所有物种的赖氨酸（K）。构建的基因进化树分析结果显示兰州大尾羊与山羊亲缘关系最近。预测兰州大尾羊H-FABP蛋白质的空间结构与山羊和牛H-FABP类似，由2个α螺旋和10个反向平行的β折叠组成，10 个折叠片围成一个桶状结构，疏水性残基位于桶内，用于结合脂肪酸。【结论】克隆了兰州大尾羊H-FABP基因，为进一步研究该基因的功能奠定了基础。

关键词: 兰州大尾羊 , H-FABP基因 , cDNA 末端快速扩增 , 序列分析

Abstract: 【Objective】To clone the full length cDNA of heart fatty acid-binding protein (H-FABP) gene in Lanzhou fat-tailed sheep for providing a theoretical basis to study its biological function and application in sheep. 【Method】 The 5′- and 3′- gene specific primers were designed according to the alignment of known cDNA sequences of H-FABP from mammals. Technique of rapid amplification of cDNA ends (RACE) was employed to clone the full length cDNA of H-FABP gene in Lanzhou fat-tailed sheep. 【Results】 About 425 bp 5′-RACE cDNA and 231 bp 3′-RACE cDNA was obtained by 5′-RACE and 3′-RACE, respectively, using skeletal muscle RNA transcribed cDNA as template. Nest PCR was performed to clone 177 bp intermediate fragment. The full length cDNA of 748 bp H-FABP gene was spliced (GenBank Accession Number JQ780322). The open reading frame of sheep H-FABP gene is 402 bp in length, encoding a mature protein H-FABP of 133 amino acids and a resulting Mr=14 761. Phylogenetic analysis showed that H-FABP gene in Lanzhou fat-tailed sheep is more close to goat, Capra hircus. Alignment comparison indicated that nucleotide homology of H-FABP gene in sheep is more similar with mammals. However, the base transition from T to G in sixty-six of nucleotide sequence leading to the change from asparagines (N) to lysine (K) in twenty-second of amino acid sequence, which is different from other species. It is predicted that tertiary structure of H-FABP protein is very similar to H-FABP of C. hircus, having 2 α-helix, 10 antiparallel β-pleated sheets that form barrel. 【Conclusion】The full length cDNA of 748 bp H-FABP gene was first to be cloned by RACE. This finding may provide basic data for further studying the role of H-FABP gene in sheep.

Key words: Lanzhou fat-tailed sheep , H-FABP gene , rapid amplification of cDNA ends (RACE) , sequence analysis

徐红伟, 柏家林, 冯玉兰, 曹忻, 蔡勇, 金方圆, 达小强, 杨具田, 臧荣鑫. 兰州大尾羊心脏型脂肪酸结合蛋白（H-FABP）基因克隆及其同源性比较[J]. 中国农业科学, 2013, 46(3): 639-646.

XU Hong-Wei, BAI Jia-Lin, FENG Yu-Lan, CAO Xin, CAI Yong, JIN Fang-Yuan, DA Xiao-Qiang, YANG Ju-Tian, ZANG Rong-Xin. Cloning and Sequence Analysis of the Full-Length cDNA of H-FABP Gene in Lanzhou Fat-Tailed Sheep[J]. Scientia Agricultura Sinica, 2013, 46(3): 639-646.

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