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。
[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 eDNA sequences of H-FABP from mammals. Technique of rapid amplification of eDNA ends （RACE） was employed to clone the full length cDNA ofH-FABP gene in Lanzhou fat-tailed sheep [ Results ] About 425 bp 5＇-RACE eDNA and 231 bp 3＇-RACE eDNA was obtained by 5＇-RACE and 3＇-RACE, respectively, using skeletal muscle RNA transcribed eDNA as template. Nest PCR was performed to clone 177 bp intermediate fragment. The fall length eDNA 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 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.
出处
《中国农业科学》
CAS
CSCD
北大核心
2013年第3期639-646,共8页
Scientia Agricultura Sinica
基金
国家自然科学基金(31160440
31260533)
甘肃省科技支撑计划项目(1011NKCA051)
甘肃省农业生物技术研究与应用开发项目(GNSW-2009-13)
国家民委科研项目(2009-158-09XB02)
兰州市科技计划项目(2011-1-113)
