水牛骨形态发生蛋白1基因的克隆及其在不同组织中的表达研究

Cloning of Buffalo BMP1 Gene and its Expression Pattern Investigation in Different Tissues

本研究克隆了水牛骨形态发生蛋白1(bone morphogenetic protein-1,BMP1)基因序列,并运用生物信息学方法对其核苷酸序列的保守性和氨基酸的理化性质、蛋白质结构进行了系统分析,此外还运用实时荧光定量PCR技术对BMP1基因在水牛不同组织中的表达差异进行了检测。结果表明,应用RT-PCR技术克隆得到水牛BMP1基因的cDNA序列长度为3 195bp,其中包含完整的2 967bp的开放读码框(ORF),编码988个氨基酸。经序列相似性分析显示,水牛BMP1基因与牛、猪、马、人和小鼠相应氨基酸序列相似性分别为99%、96%、96%、96%和95%,具有很强的保守性。结合系统进化树分析结果推测,BMP1基因在不同物种及进化的过程中具有高度的保守性。对水牛BMP1蛋白的结构域预测结果发现,其存在1个信号肽区、1个前肽区、1个金属蛋白酶区、5个CUB区和2个EGF-like功能区。定量表达分析结果显示,BMP1基因在水牛心脏组织中相对表达量最高,睾丸、卵巢和生殖嵴等性腺器官表达量次之,骨头等其他组织表达量较低,肝脏表达量最低。

Buffalo BMP1 gene was cloned in the present study,the BMP1 sequence was systemically analysed by bioinformatics techniques,and the expression level of BMP1 gene in different tissues were also assayed with Real-time fluorescence quantitative PCR（QRT-PCR）.The results showed that with RT-PCR a 3 195 bp buffalo BMP1 gene was cloned and sequenced,including the whole ORF of 2 967bp（coding 988 amino acid）.The sequence multialigned results showed that buffalo BMP1 gene shared 99%,96%,96%,96% and 95% of similar amino acid sequence with Bos taurus,Sus scrofa,Equus,Homa sapiens and Mus musclus,respectively.It was predicted that buffalo BMP1 protein contained a signal peptide domain,apreregion,a metalloproteinases domain,five complement-Uegf-BMP-1domain（CUB domain）and two epitheloid growth factor-like domain（EGF-like domain）.In addition,we also analyzed the expression level in different tissues through QRT-PCR,the results showed that BMP1 gene mRNA existed in all nine tissues with the most abundant expression in heart,followed by testis,ovary,genital ridge,while with lower amount of bone and other tissues,the minimal expression in liver was observed.