桃果实PpARF1蛋白的原核表达及多克隆抗体制备  

Prokaryotic expression and polyclonal antibody preparation of PpARF1 protein in peach fruit

作　　者：管丹 史梦雅[1] 胡晓[1] 关伟[2] 刘悦萍 GUAN Dan;SHI Mengya;HU Xiao;GUAN Wei;LIU Yueping(PlantSrience andTechnology College,Beijing University of Agriculture Beijing102206,China;College of Biological Science and Engineering/Key Laboratory of Urban Agriculture (North) Ministry of Agriculture,Beijing University of Agriculture Beijing102206China;Beijing Pinggu District People's Government Fruit Office101200,China)

机构地区：[1]北京农学院植物科学技术学院,北京101200 [2]北京市平谷区人民政府果品办公室,北京101200 [3]北京农学院生物科学与工程学院/农业部华北都市农业重点实验室,北京102206

出　　处：《北京农学院学报》2018年第1期1-5,共5页Journal of Beijing University of Agriculture

基　　金：2015年度新型生产经营主体科技能力提升工程项目(20150203-01)

摘　　要：【目的】为了探讨转录因子ARF1(Auxin Response Factor)对桃果实发育调控的机制。【方法】以‘24号’桃果实为试验材料,构建桃ARF1基因的原核表达载体pET32a-PpARF1,转化到BL21(DE3)中诱导表达菌体蛋白,筛选出重组蛋白的最适表达条件,按照最适条件大量摇菌诱导表达蛋白,并进行重组蛋白的纯化,最后用纯化诱导后的重组蛋白制备多克隆抗体及WB检测。【结果】SDS-PAGE电泳检测得到pET32a-PpARF1重组蛋白的位置大约在95.0kD,在37℃,转速250r/min,IPTG 0.10mmol/L,诱导4h,诱导出的蛋白表达量最大。免疫印迹试验显示所得抗血清能很好检测到目的蛋白。【结论】ARF转录因子参与生长素调控桃果实发育成熟的过程。【Objective】In order to expore the mechanism of transcription factor ARF on peach fruit development,we constructed the expression vector pET32a-PpARF1.【Methods】The peach fruits of‘24’were used as materials.The prokaryotic expression vector pET32a-PpARF1was constructed and transformed into BL21(DE3)expression vector.We screened out the optimal transduction conditions of recombination protein,and then recombination protein was induced expression according to the optimal transduction conditions.After the purification of recombinant protein,the polyclonal antibody was prepared and Western Blot was performed.【Results】The location of pET32a-PpARF1recombination protein was about95.0kD by SDS-PAGE.Conditions of maximal protein induction expression were37℃,250rmp/min and0.10mmol/L IPTG for4h.The Western Blot showed that the target protein could be detected by antiserum.【Conclusion】ARF transcription factors are involved in regulating the ripening and development of peach fruit,and this study provide a basis for PpARF1study.

关 键 词：桃果实 ARF 原核表达 目的蛋白 westernblot(WB)检测 

分 类 号：S662.1[农业科学—果树学]