鹅MLPH基因分子克隆、组织表达及SNP检测

Molecular Cloning, Tissue Expression and SNP Identification of MLPH Gene in Goose

研究采用RT-PCR和克隆的方法获得鹅黑素亲和素(MLPH)基因的cDNA和基因组DNA序列,采用半定量RT-PCR的方法检测MLPH基因在鹅组织中的表达,并利用DNA池的方法扫描MLPH基因的多态位点。结果显示:鹅MLPH基因cDNA长2 147 bp(GenBank登录号:KU904428),包括17 bp的5′非翻译区、1 992 bp的开放阅读框和138 bp的3′非翻译区。生物信息学分析表明,鹅MLPH基因开放阅读框编码663个氨基酸残基,其分子量为74.62 ku,理论等电点为5.94。推导的MLPH氨基酸序列具有4个卷曲螺旋结构、1个锌指结构域和1个RAB结合域。鹅MLPH基因组DNA长20 540 bp(GenBank登录号:KU904431),按照"GT-AG"剪接法则,划分为16个外显子和15个内含子。进化树结果分析表明,鹅MLPH与家禽有密切的亲缘关系,特别是与绿头鸭的亲缘关系密切。半定量RT-PCR结果表明,鹅MLPH mRNA在不同的色素沉着组织中表达,如眼睛、背部皮肤、腹部皮肤和脚蹼,而在其他组织中未检测到表达。同时,鹅MLPH基因在外显子11上存在2个碱基突变(c.1506C>T、c.1542A>G),且分别导致编码的氨基酸改变(p.497Pro>Ser、p.509Lys>Glu)。该研究结果为鹅MLPH基因的研究奠定基础,为鹅羽毛颜色形成的研究提供参考。

In this study, the cDNA and genomic DNA sequences of goose melanophilin （MLPH）gene were cloned by RT-PCR, and the expression of MLPH gene in goose tissues was detected by semi-quantitative RT-PCR, as well as the SNP was screened by DNA pools. The results showed that the length of goose MLPH cDNA was 2 147 bp （GenBank accession number：KU904428）. The goose MLPH cDNA consisted of a 17 bp Y-terminal untranslated region （UTR）, a 1 992 bp open reading frame （ORF）, and a 138 bp 3＇-UTR. Bioinformatics analysis indicated that the ORF encoded a protein of 663 amino acid residues with a molecular weight of 74.62 ku and an isoelectric point （pI） of 5.94. The deduced MLPH amino acid sequence contained four coiled-coil domains,a zinc finger domain and a RAB binding domain. The length of goose MLPH genomic DNA was 20 540 bp （GenBank accession number： KU904431 ）, including sixteen exons and fifteen introns by ＂GT-AG＂ consensus splice site rule. Phylogenetic analysis displayed that the goose MLPH protein was closely related to that of poultry, especially in Arias platyrhynchos. Semi-quantitative RT-PCR displayed that the goose MLPH mRNA was differentially expressed in the pigment deposition tissues, including eye, skin of back, skin of abdomen and flipper,and no expression was detected in other tissues. Furthermore,two single nucleotide polymorphisms （SNPs） （c.1506C〉T,c.1542A〉G）were identified in the exon 11 of goose MLPH gene, leading to the changes of amino acid sequence （p.g97Pro〉Ser, p.509Lys〉Glu）. Those findings were the basis of the further study of goose MLPH gene,and served as important references for the research of goose feather color.