光核桃AmUspA蛋白的表达特性及功能研究  

A Preliminary Study on Expression Characteristics and Functions of Recombinant AmUspA protein from Amygdalusmira

作　　者：任梓齐 毕雪奇 张力文[1] 陈宇[1] 罗秋香[1] REN Zi-qi;BI Xue-qi;ZHANG Li-wen;CHEN Yu;LUO Qiu-Xiang(Key Laboratory of Saline-alkali Vegetation Ecology Restoration,Ministry of Education/College of Life Sciences,Northeast Forestry University,Harbin 150040,China)

机构地区：[1]东北林业大学东北盐碱植被恢复与重建教育部重点实验室/生命科学学院,哈尔滨150040

出　　处：《沈阳农业大学学报》2022年第1期24-33,共10页Journal of Shenyang Agricultural University

基　　金：中央高校基本科研业务基金项目(2572017CA10);国家自然科学基金项目(31500317)。

摘　　要：为探究UspA蛋白在光核桃(Amygdalusmira 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)的基因序列设计一对特异性上下游引物,通过PCR扩增技术从光核桃叶片中克隆出普遍胁迫蛋白基因AmUs⁃pA。生物信息学分析结果表明:该基因ORF全长528bp,编码175个氨基酸,无跨膜区域,为亲水性稳定蛋白。蛋白序列分析和系统进化分析表明,AmUspA具有Usp家族典型的UspA结构域,并且与其他植物的Usp蛋白具有较高的同源性。随后构建了pET 21a-AmUspA融合表达载体,优化了蛋白表达条件,即在37℃、IPTG浓度为0.1mmol·L^(-1),诱导8h时可获得较高浓度的重组蛋白,经纯化脱盐后共得到浓度为0.91mg·mL^(-1)的融合蛋白25mg并制备抗体。最后,通过模拟各种非生物胁迫条件考察AmUspA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AmUspA的菌株抗逆性均优于空载体,并且在400mmol·L^(-1)NaCl、40mmol·L^(-1)NaHCO_(3)、10μmol·L^(-1)H_(2)O_(2)、100mmol·L^(-1)甘露醇胁迫下优势显著,进一步表明UspA可能在光核桃的防御反应中发挥作用。该研究为探索AmUspA蛋白的生物学作用奠定了基础,同时也为优化桃属种质资源提供了新的思路。To investigate the conditions of UspA protein expression in Amygdalusmira and its regulatory role in the defence response to adversity stress,a pair of specific upstream and downstream primers was designed using the gene sequence of Prunus persica in the database and the universal stress protein gene AmUspA was cloned from Amygdalusmira leaves by PCR amplification.Bioinformatics analysis showed its ORF is 528bp,which encoding a hydrophilic and stable protein with 175 amino acids,without trasmembrane.Protein sequence analysis and phylogenetic analysis indicated that AmUspA has the typical UspA domain of the Usp family and shares high homology with Usp proteins from other plants species.Subsequently,the pET-21a-AmUspA fusion expression vector was constructed,and the conditions for protein expression were optimized.The higher concentration of recombinant protein could be obtained at 37℃with an IPTG concentration of 0.1mmol·L^(-1)for 8 hours of induction.A total of 25mg of the fusion protein with a concentration of 0.91mg·mL^(-1)was yielded and antibodies were prepared after protein purification and desalting.Finally,the expression pattern of the AmUspA-transformed strains in E.coli was investigated by simulating various abiotic stress conditions,suggesting that the stress resistance of AmUspA-transformed strains was better than empty vector under various stress conditions.It was significantly advantageous that under 400mmol·L^(-1)NaCl,40mmol·L^(-1)NaHCO_(3),10μmol·L^(-1)H_(2)O_(2),and 100mmol·L^(-1)mannitol stress,further indicating that UspA may play a role in the defense response of Amygdalusmira.This study laid a foundation for exploring the biological role of AmUspA protein,and also provided a new idea for optimizing germplasm resources of peach.

关 键 词：光核桃 普遍胁迫蛋白 非生物胁迫 

分 类 号：Q945.79[生物学—植物学]