光核桃AmRanBP1重组蛋白表达特性及其抗逆性初步研究

A Preliminary Study on the Expression Characteristics and Stress Resistance of AmRanBP1 Recombinant Protein in Amygdalus mira

光核桃为蔷薇科桃属落叶乔木,具有耐寒、耐旱等优良特性。为探究RanBP1蛋白在光核桃[Amygdalus mira(Koehne)]中的表达条件及其在逆境胁迫防御反应中的调控作用,利用数据库中碧桃(Prunus persica)的基因序列采用同源克隆得到AmRanBP1。生物信息学分析结果表明,该基因开放阅读框ORF全长696 bp,编码231个氨基酸,无跨膜区域,为亲水性稳定蛋白,无信号肽结构。蛋白序列分析和系统进化分析表明,AmRanBP1与其他植物的RanBP1蛋白具有较高的同源性,例如苹果(Malus domestica)、大豆(Glycine max)。构建了pET-21a-AmRanBP1融合表达载体,优化了蛋白表达条件,即在40℃、IPTG浓度为2.00 mmol·L^(-1),诱导4.0 h时可获得较高浓度的重组蛋白,对表达产物进行纯化用以制备单克隆抗体。最后,通过模拟各种非生物胁迫条件考察AmRanBP1转化菌株在大肠杆菌中的表达模式,发现各种胁迫条件下转AmRanBP1的菌株抗逆性均优于空载体,并且在高温、NaCl与CuSO_(4)胁迫下优势显著,进一步表明RanBP1可能在光核桃的防御反应中发挥作用。

Amygdalus mira(Koehne)is a deciduous tree of Rosaceae,which has excellent characteristics such as cold resistance and drought resistance.In order to explore the expression conditions of RanBP1 protein in A.mira Koehne and analyze its regulatory role in stress defense response,AmRanBP1 was obtained by homologous cloning using the gene sequence of Prunus persica in the database.The results showed the ORF of this gene was 696 bp in length,encoding 231 amino acids.Bioinformatics analysis results indicated it has no transmembrane region,stable hydrophilic,and no signal peptide structure.Protein sequence and phylogenetic analysis showed that AmRanBP1 had high homology with RanBP1 proteins of other plants,such as Malus domestica,Glycine max.The pET-21a-AmRanBP1 fusion expression vector was constructed,and the protein expression conditions were optimized.The recombinant protein with higher concentration was obtained at 40℃,IPTG concentration of 2.00 mmol·L^(-1) and induction time of 4.0 h.Monoclonal antibody was prepared after purification of AmRanBP1 fusion protein.Finally,the expression pattern of AmRanBP1-transformed strains in E.coli was investigated by simulating various abiotic stress conditions.The results showed that the stress resistance of AmRanBP1-transformed strains was better than that of empty vector under various stress conditions,and the advantages were significant under high temperature,NaCl and CuSO_(4) stress,further indicating that RanBP1 may play a role in the defense response of A.mira Koehne.This study laid a foundation for revealing the biological role of AmRanBP1 protein.