鸡FKBP5基因的克隆、生物信息学及组织表达谱分析

本研究旨在对鸡FK506结合蛋白5(FK506 binding protein 5,FKBP5)基因进行克隆和生物信息学分析,并检测其在鸡不同组织中的表达量。以鸡脾脏cDNA为模板,通过PCR方法扩增和克隆鸡FKBP5基因完整CDS区序列,并进行同源性比对及系统进化树构建;使用在线软件分析FKBP5蛋白的理化性质、疏水性、跨膜结构、信号肽、二级结构和三级结构;利用实时荧光定量PCR检测其在肢体内外翻畸形(valgus-varus deformity,VVD)组肉鸡和正常组肉鸡组织中的表达量,并绘制组织表达谱。结果显示,鸡FKBP5基因CDS区序列全长1350 bp,编码449个氨基酸;同源性比对和进化树分析表明,鸡FKBP5基因氨基酸序列与鹌鹑、鸭、壁虎、中华鳖、小鼠、人、猪、斑马鱼的同源性分别为98.4%、96.2%、85.8%、87.4%、81.1%、85.2%、86.1%和61.2%,与鹌鹑、鸭的亲缘关系最近,爬行类和哺乳类动物次之,与斑马鱼(鱼类)亲缘关系最远。鸡FKBP5蛋白分子质量为50.43 ku,理论等电点(pI)为5.94,半衰期为30 h,肽链N端为蛋氨酸(Met),不稳定系数为23.80,属于稳定蛋白;跨膜区和信号肽预测结果显示,FKBP5蛋白不属于跨膜蛋白和分泌型蛋白。结构域分析结果表明,该蛋白包括2个FKBP型肽基脯氨酰异构酶(FKBP-type peptidylprolyl isomerases)、3个四肽重复(TPR)序列,主要包含α-螺旋(46.77%)、延伸链(12.47%)和无规则卷曲(40.76%),且该蛋白与HSPA2、PPID、STIP1、HSP90AA1和HSP90AB1等互作蛋白具有很强的相关性。实时荧光定量PCR结果显示,FKBP5基因在鸡各组织中广泛表达,其中在软骨和法氏囊中表达量较高,在发病组肉鸡组织中的表达量均高于正常组肉鸡,且在心脏、腿肌、法氏囊、胸腺、软骨中表达量差异显著(P<0.05),在脾脏中表达量差异极显著(P<0.01)。本试验结果可为肉鸡骨骼疾病及腿部健康选育提供参考。

Cloning,Bioinformatics and Tissue Expression Profile Analysis of FKBP5 Gene in Chickens

The purpose of this study was to perform cloning and bioinformatics analysis of FK506 binding protein 5(FKBP5)gene in chickens,and detect its expression of different tissues in chickens.The spleen tissue cDNA in broiler was used as a template,the complete CDS region of FKBP5 gene in broilers was amplified and cloned,the homology was compared and phylogenetic tree was constructed.The physical and chemical properties,hydrophobicity,transmembrane structure,signal peptide,secondary structure and tertiary structure of FKBP5 protein were analyzed by online software.Real-time quantitative PCR was used to detect the expression of FKBP5 gene in different tissues of valgus-varus deformity(VVD)broilers and normal broilers.The results showed that the total CDS region sequence of FKBP5 gene in chickens was 1350 bp,which encoded 449 amino acids.The homology of amino acid sequence of FKBP5 in chickens with Coturnix japonica,Anas platyrhynchos,Gekko japonicus,Pelodiscus sinensis,Mus musculus,Homo sapiens,Sus scrofa and Danio rerio were 98.4%,96.2%,85.8%,87.4%,81.1%,85.2%,86.1%and 61.2%,respectively.The genetic relationship with Coturnix japonica and Anas platyrhynchos was the closest,followed by reptiles and mammals,and it had the farthest relationship with Danio rerio.The molecular weight of chicken FKBP5 protein was 50.43 ku,its theoretical isoelectric point was 5.94,the half-life was 30 h,the N-terminus of the peptide chain was methionine,the instability coefficient was 23.80,belonging to the stable protein.The prediction results of transmembrane region and signal peptide showed that FKBP5 protein was no transmem brane structure and signal peptide.Domain analysis showed that the protein contained two FKBP-type peptidylprolyl isomerases and three tetrapeptide repeats(TPR).FKBP5 protein structure was mainly consisted of alpha helix(46.77%),extended chain(12.47%)and random coil(40.76%),it had strong correlation with HSPA2,PPID,STIP1,HSP90AA1 and HSP90AB1.Real-time quantitative PCR results revealed that FKBP5 gene in chickes was expressed in all tissues.The expression of tissues in VVD broilers were higher than that of normal broilers,and the expression in heart,leg muscle,bursa,thymus and cartilage was significantly different(P<0.05),the expression in spleen was extremely significantly different(P<0.01).The results of this experiment provided reference for broiler bone disease and leg healthy breeding.