水牛甲状旁腺素1受体基因CDS区克隆与生物信息学分析

试验旨在对水牛甲状旁腺素1受体（parathyroid hormone 1 receptor，PTH1R）基因CDS区进行克隆，并对所获序列进行生物信息学分析，以期丰富水牛PTH1R基因研究的基础数据。提取水牛基因组DNA，以牛PTH1R基因为参考序列（GenBank登录号：NM_001075332.1），应用Primer Premier 5.0软件设计引物序列，运用PCR扩增及测序获得水牛完整CDS区序列，使用DNAMAN、ProtParam、SOPMA、PSORT Ⅱ Prediction等在线分析软件分析PTH1R的一级结构、二级结构、三级结构与理化性质，并进行同源性分析及构建系统进化树。结果显示，试验成功克隆了水牛PTH1R基因完整编码序列，该序列长为2 283 bp，CDS区长1 770 bp，序列已提交GenBank，登录号：MF380401，可编码589个氨基酸。PTH1R基因CDS区核苷酸序列与黄牛、猪、马、山羊、绵羊和骆驼同源性比对结果显示，其同源性分别为99.4%、93.2%、93.5%、95.3%、98.1%和93.9%，物种之间同源性较高，进化树分析结果与其亲缘关系远近一致，表明水牛PTH1R基因编码区在进化过程中比较保守。蛋白理化性质分析显示，水牛PTH1R蛋白分子式为C₂₉₉₆H₄₆₁₆N₇₉₂O₈₂₃S₃₀，分子质量为65 860.22 u，半衰期为30 h，理论等电点（pI）为8.37，水溶液在280 nm处的消光系数为117 770，肽链N端为M（Met），不稳定系数为43.36，属于碱性不稳定蛋白。脂肪系数为88.61，总平均亲水性为0.007，该蛋白属于不可溶性蛋白。二级结构分析显示，水牛PTH1R基因蛋白中包含有218个α-螺旋、114个延伸链、44个β-转角、213个无规则卷曲，与三级结构预测结果相一致。综上所述，PTH1R基因编码区在长期生物进化过程中具有较强的保守性，PTH1R基因的成功克隆及分析为进一步揭示其遗传特性提供了理论依据。

Cloning and Bioinformatics Analysis on PTH1R gene CDS in Buffalo

In order to enrich the basic data of buffalo parathyroid hormone 1 receptor (PTH1R) gene, the CDS region of buffalo PTH1R was cloned, and the sequence was analyzed by bioinformatics method in this study. Primers sequence were designed according to cDNA sequence of cattle PTH1R gene in GenBank (accession No:NM_001075332.1) by Primer Premier 5.0 software. The CDS region in buffalo PTH1R gene was cloned by PCR amplification and nucleic acid sequencing technology. The primary structure, secondary structure, tertiary structure, physicochemical properties, homology were analyzed and phylogenetic tree of PTH1R was constructed by DNAMAN, ProtParam, SOPMA and PSORTⅡ prediction softwares. The results showed that the length of PTH1R gene in buffalo was 2 283 bp, the CDS region was 1 770 bp, GenBank accession No.was MF380401, encoding 589 amino acids. The homology of buffalo PTH1R gene CDS region compared to cattle, pigs, horses, goats, sheep and camels were 99.4%, 93.2%, 93.5%, 95.3%, 98.1% and 93.9%, respectively. There was a high homology among different species, and phyletic evolution was in accordance with their genetic relationship. The research indicated that PTH1R gene was conservative in the course of evolution. Analysis of physicochemical properties of protein showed that PTH1R protein molecular formula was C₂₉₉₆H₄₆₁₆N₇₉₂O₈₂₃S₃₀, the molecular weight was 65 860.22 u, the half-life was 30 h, theoretical electrical points (pI) was 8.37, the extinction coefficient of water solution at 280 nm was 117 770, the peptide chain N-terminal was M (Met), the unstable coefficient was 43.36, belong to the basic unstable protein. The fat coefficient of PTH1R protein was 88.61, and the total average hydrophilicity was 0.007, belongs to insoluble protein. The secondary structure analysis showed that the buffalo PTH1R gene protein contained 218 α-helixes, 114 extended strands, 44 β-turns and 213 random colis, and consistent with the tertiary structure prediction. PTH1R gene coding region was conservative in the course of long-term biological evolution. This results provided a theoretical basis for revealing the genetic characteristics of buffalo PTH1R gene.