榨菜BjuFIP蛋白纯化及非生物胁迫下基因表达模式分析  

Purification of protein BjuFIP in tuber mustard and its gene expression pattern under abiotic stress

作　　者：颜论 陈国建 陈明飞 游经番 彭姣姣 程春红[1] YAN Lun;CHEN Guojian;CHEN Mingfei;YOU Jingfan;PENG Jiaojiao;CHENG Chunhong(College of Life Science and Technology,Yangtze Normal University,Chongqing 408100,China)

机构地区：[1]长江师范学院生命科学与技术学院,重庆408100

出　　处：《福建农林大学学报（自然科学版）》2022年第5期606-612,共7页Journal of Fujian Agriculture and Forestry University:Natural Science Edition

基　　金：重庆市科技局项目(cstc2018jcyjAX0628);重庆市教育委员会项目(KJQN202101432);重庆市高校创新研究群体项目(CXQT21029).

摘　　要：采用荧光定量PCR技术分析BjuFIP在不同非生物逆境胁迫下的表达模式.通过无缝克隆技术将BjuFIP的CDS序列构建到原核表达载体pGEX4T-1上,测序正确后转化大肠杆菌表达菌株BL21,对BjuFIP-GST蛋白进行体外诱导表达纯化.结果表明,BjuFIP-1显著受低温(4℃)、高温(37℃)和NaCl的诱导表达,BjuFIP-2显著受低温(4℃)、高温(37℃)、ABA、NaCl以及Mannitol的诱导表达,说明BjuFIP在调控榨菜响应非生物逆境胁迫过程中具有重要作用.SDS-PAGE电泳检测结果显示,在16、25和37℃以及1 mmol·L^(-1)IPTG诱导条件下,重组菌株均能够表达出分子质量约为55 ku的BjuFIP-GST融合蛋白,并且16℃诱导条件下,BjuFIP-GST蛋白较多以可溶性蛋白的形式存在,通过Glutathione Beads亲和层析柱纯化后,获得了大量纯度较好的BjuFIP-GST融合蛋白.BjuFIP显著受非生物逆境胁迫的诱导表达,在调控榨菜响应非生物逆境胁迫过程中具有重要作用,并且获得了大量纯度较高的BjuFIP-1-GST和BjuFIP-2-GST融合蛋白,为后期进一步探究BjuFIP在调控榨菜抗逆过程中的功能提供依据.To explore the regulatory mechanism of BjuFIP in response to abiotic stresses in tuber mustard, the expression pattern of BjuFIP in tuber mustard were analyzed under various abiotic stress conditions by qPCR. Then the coding sequence of BjuFIP was seamlessly cloned into the expression vector pGEX4 T-1 and transformed into BL21 for subsequent prokaryotic expression and purification of protein BjuFIP. The results of qPCR showed the induced expressions of BjuFIP-1 under 4 and 37 ℃ and NaCl, and the induced expression of BjuFIP-2 by abscisic acid and mannitol, indicating the important role of BjuFIP in response to abiotic stresses. SDS-PAGE showed that the BjuFIP-GST fusion protein(55 ku) was expressed in BL21 under 16, 25 and 37 ℃ and isopropyl-β-d-thiogalactoside(IPTG)(1 mmol·L^(-1)), and mainly existed as soluble protein under 16 ℃. A large quantity of BjuFIP-GST fusion proteins with high purity can be obtained by affinity chromatography column with glutathione beads. To summarize, BjuFIPs were induced by various abiotic stresses in tuber mustard, which indicated the important roles in tackling abiotic stresses. The successful expression and purification of BjuFIP-GST fusion protein were contributed to the illumination of the function of BjuFIP in regulating the stress response of tuber mustard.

关 键 词：榨菜 F-BOX蛋白 BjuFIP 基因表达 蛋白纯化 

分 类 号：S637.3[农业科学—蔬菜学]