Full-Length CDNA Cloning Of H-FABP Gene And Its Bioinformatics Analysis In Lanzhou Fat-Tailed Sheep

Fat acid binding proteins (FABPs) are involved in fatty acid transport from the plasma membrane to the sites of β-oxidation and triacylglycerol or phospholipid synthesis, and they play a very important role in animal fat metabolism. Using SMARTerTM RACE technique in this research, the full length cDNA of H-FABP in Lanzhou fat-tailed sheep was cloned and its bio informatics were analysed. The results are as follows:1. Total RNA was extracted from tail fat in Lanzhou fat-tailed sheep and cDNA was reverse transcripted using RNAiso Plus kit. A231bp fragment of5’-cDNA and a425bp fragment of3’-cDNA of FABP was cloned by SMARTerTM RACE cDNA technique with the pairs of primer GPS2and NGSP2, GPS1and NGSP1, respectively. The middle cDNA of177bp was amplified by nest PCR using primers of GPS1and GPS2. The full length cDNA of748bp of FABP gene was finally spliced (GenBank Accession No. JQ780322).2. The ORF was402bp (110nt-511nt), coding133amino acids; initiator codon for ATG was located110nt-112nt, and5’-UTR was109bp (1nt-109nt); terminator codon for TGA was located509nt-511nt, and3’-UTR was237bp (512nt-748nt).3.FABP protein molecular weight was14761.8Da, and its formulas was C655H1055N173O205S4, and its theory isoelectric point was6.11, and the protein belonged to the hydrophilic stability protein, and in Lanzhou fat-tailed sheep, H-FABP had as similar3D structure as other species, which was formed of two alpha helix and ten reverse parallel β folding.4.Through the amino acid sequence alignment and homology analysis of H-FABP in Lanzhou fat-tailed sheep with that of other species, it was showed that the nucleotide sequence homology of H-FABP gene in Lanzhou fat-tailed sheep with that of Xuhuai breed capra hircus, Taiwan capra hircus, Tianfu breed capra hircus, Canadian ordinary cattle (gi:74354539), Chinese Black cattle (gi:224760716), Canis lupus familiaris, Oryctolagus cuniculus, Rattus norvegicus and Mus musculus is99.3%,99.0%,99.0%,97.4%,96.8%,87.4%,84.5%,82.5%and80.8%, respectively; and its corresponding amino acid sequence homology of H-FABP in the above species is100%,100%,100%,97.0%,96.2%,94.0%,88.0%,87.2%and86.5%, respectively. In the amino acid sequences of six ruminant animal, G(glycine) located in the34th position, and M(methionine) in the109th position, and T(threonine) in the122nd position and Q(glutamine) in the132nd position of H-FABP amino acid sequence in Bos taurus is respectively replaced by A(alanine), I(isoleucine), S(serine), E(glutamic acid) in the same position in Ovis aries; and there is V(valine) in the50th position in Ovis aries and Canadian ordinary cattle (gi:74354539), but there is I (isoleucine)in the same position of Chinese Black cattle (gi:224760716).5. H-FABP gene in Lanzhou fat-tailed sheep contains24restriction enzyme cutting sites, and H-FABP is not a transmembrane protein, and the possibility of such protein existing in the cytoplasm is21%; it does not contain disulfide bond in its amino acid sequence; and eight amino acids including three serine,five threonine and one tyrosine may become the phosphorylation sites of protein kinase.