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

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1 0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 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 nueleotide 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 a-helix,10 antiparallel [3-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 ofH-FABP gene in sheep.