天山云杉ABI5基因的克隆与原核表达活性测定  

Cloning and Prokaryotic Expression Activity Determination of ABI5 Gene from Picea schrenkiana

作　　者：范晓聪 王佳琰 阮晓[2] 奚雨欣 杨丽[2] 吴庆飞 王强[1,2] Fan Xiaocong;Wang Jiayan;Ruan Xiao;Xi Yuxin;Yang Li;Wu Qingfei;Wang Qiang(College of Forestry and Technology,Zhejiang A&F University,Hangzhou,311300;School of Biological and Chemical Engineering,NingboTech University,Ningbo,315100)

机构地区：[1]浙江农林大学林业与技术学院,杭州311300 [2]浙大宁波理工学院生物与化学工程学院,宁波315100

出　　处：《分子植物育种》2024年第9期2898-2904,共7页Molecular Plant Breeding

基　　金：国家自然科学基金项目(31701781)资助。

摘　　要：ABI5又名植物脱落酸不敏感蛋白5(Abscisic acid-insensitive 5),作为ABA信号通路中重要的转录因子,协调参与了种子发育和萌发、植物生长、逆境胁迫等方面的调控。目前的研究多是以拟南芥为对象,而在天山云杉等木本植物上报道极少。本研究以天山云杉的cDNA为模板进行了PCR扩增,得到了天山云杉ABI5序列,经测序发现其全长为1071 bp,共编码了352个氨基酸。将ABI5基因连接pET-24a原核表达载体,得到了重组质粒pET-24a-ABI5。对IPTG浓度、诱导时间、培养基类型和孵育温度进行了优化,得到了最适培养条件为采用LB培养基,IPTG终浓度为0.2 mmol/L,20℃下诱导8 h。通过SDS-PAGE检测纯化产物发现大部分蛋白以包涵体形式存在,对部分包涵体蛋白进行了复性并利用孔雀石绿显色反应进行了活性的验证。ABI5,also known as abscisic acid-insensitive protein 5,is an important transcription factor in ABA signaling pathway,which plays a coordinating role in the regulation of seed development and germination,plant growth,and stress.At present,most of the studies are on Arabidopsis thaliana,but there are few reports on woody plants such as Picea schrenkiana.In this study,the cDNA of Picea schrenkiana was used as the template for PCR amplification,and the ABI5 sequence of Picea schrenkiana was obtained.The total length of ABI5 was 1071 bp,encoding 352 amino acids.The recombinant plasmid pET-24a-ABI5 was obtained by cloning ABI5 gene into pET-24a expression vector.IPTG concentration,induction time,medium type and incubation temperature were optimized,and the optimal culture condition was LB medium,the final concentration of IPTG was 0.2 mmol/L,and the incubation temperature was 20℃for 8 h.The purified products were detected by SDS-PAGE and most of the proteins were found to exist in the form of inclusion bodies.Some of the proteins were renaturated and their activity was verified by malachite green chromogenic reaction.

关 键 词：天山云杉 ABI5 分子克隆 原核表达 包涵体复性 

分 类 号：S791.18[农业科学—林木遗传育种]