梅山猪PBD-1基因CDS区克隆、组织表达及生物信息学分析

Cloning,tissue expression detection and bioinformatics analysis of PBD-1 gene coding sequence(CDS)regions of Meishan pigs

为系统探究猪β防御素-1(PBD-1)基因的结构和功能,以梅山猪为试验材料,扩增PBD-1基因CDS区,应用生物信息学分析猪PBD-1基因编码蛋白的特性和功能,运用DnaSP和MEGA分析猪PBD-1基因与其他物种进化关系,同时利用实时荧光定量方法检测PBD-1基因在梅山猪不同组织中的表达水平。结果表明:猪PBD-1基因CDS区长195bp,编码64个氨基酸,为脂溶性疏水性、不稳定的分泌型蛋白,无跨膜结构,存在信号肽区域(第1～22位氨基酸);该蛋白存在3个特异性蛋白质激酶的结合位点(PKC、PKA、UNSP),二级结构以α螺旋和无规则卷曲为主;包含defensin-beta保守结构域(第28～62位氨基酸),且该蛋白可能与PBD-2、CCR6、NPG1、PAMP-23、ZCCHC3等蛋白存在相互作用关系。进化分析显示,猪PBD-1基因与驴、驯鹿具有较高的同源性,可聚为一类。组织表达谱显示,PBD-1基因在梅山猪不同组织中均有表达,尤其在胃、免疫组织(胸腺、淋巴)及肠道组织(空肠、回肠)中呈现高表达。这一研究为今后深入研究PBD-1基因功能提供了理论参考。

To systematically explore the structures and functions of porcineβdefensin-1（PBD-1）gene,the coding sequence（CDS）of PBD-1 from Meishan pigs was amplified and further analyzed by bioinformatic programs.DnaSP and MEGA were used to reveal the evolutionary relationship of PBD-1 gene across different species.The expression of PBD-1 gene in different tissues of Meishan pigs was detected by using real-time PCR.The results showed that PBD-1 CDS full length was 195 bp encoding 64 amino acids.The PBD-1 protein was fat-soluble hydrophobic,unstable and secretory,without transmembrane structure and had a signal peptide（1-22 amino acids）.Functional site analysis indicated that PBD-1 protein contained three specific protein kinase binding sites（PKC,PKA,UNSP）,and its secondary structure was mainly constituted by alpha helices and random coil.Moreover,analysis of the functional regions revealed that the PBD-1 protein contained the defensin-beta（28-62 amino acids）conserved domain and may interact with the PBD-2,CCR6,NPG1,PAMP-23,ZCCHC3 proteins.Lastly,evolutionary analysis demonstrated that the PBD-1 gene had higher homology with PBD-1 in Rangifer tarandus and Equus asinus,and they were clustered in the phylogenetic tree.Tissue expression profiles showed that PBD-1 gene was ubiquitously expressed in different tissues of Meishan pigs,and presented higher expression especially in the stomach,immune tissues（thymus lymph）and intestinal tissues（jejunum,ileum）.This study could provide theoretical references for the further study of PBD-1 gene function.