桃毛根转化体系的建立与优化  

Establishment and optimization of peach hairy root transformation system

作　　者：张思佳 吴凡颖 李小姗 常明明[1,2] 刘悦萍 杨海清[3] ZHANG Sijia;WU Fanying;LI Xiaoshan;CHANG Mingming;LIU Yueping;YANG Haiqing(College of Biology,Resources and Environment,Beijing University of Agriculture,Beijing 100096,China;Key Laboratory of Urban Agriculture in North China,Ministry of Agriculture and Rural Affairs,Beijing 102206,China;Beijing Pinggu District Fruit Industry Service Center,Beijing 101200,China)

机构地区：[1]北京农学院生物与资源环境学院,北京100096 [2]农业农村部华北都市农业重点实验室,北京102206 [3]北京市平谷区果品产业服务中心,北京101200

出　　处：《北京农学院学报》2024年第3期63-68,共6页Journal of Beijing University of Agriculture

基　　金：中国农科院合作项目(中华人民共和国农业农村部项目)“土壤样品总盐和碱解氮、有效磷、速效钾等含量测试”。

摘　　要：【目的】为了解决桃遗传转化中易形成嵌合体、转化过程耗时和工作量大等问题,建立并优化了发根农杆菌介导的毛根转化体系。【方法】以三周龄无菌组培‘中桃24号’桃苗下胚轴、子叶和上部分植株为外植体,利用pCAMBIAsuper1300-GFP质粒转化的发根农杆菌MSU440介导转化。将侵染后的外植体转移到含有抗菌素(头孢菌素)的培养基上培养7 d左右。观察到愈伤组织和毛状根的形成。对有毛状根发出的外植体进行体式荧光显微镜检测,观察绿色荧光信号。提取有荧光信号的毛状根总RNA,PCR检测阳性毛状根,并筛选最适转化品种、侵染浓度和侵染时间。【结果】利用发根农杆菌MSU440侵染桃下胚轴,诱导形成毛状根,并通过转化载体表达的GFP快速筛选到转基因毛状根,PCR扩增到了RolB和eGFP基因片段;条件优化结果表明,在OD值为0.8的发根农杆菌侵染液中浸泡30 min为最佳转化条件,最高转化率为63.6%。【结论】在桃上建立了发根农杆菌介导的毛根转化体系,优化了转化条件,为桃基因功能验证和获得稳定转基因桃植株提供技术基础。[Objective]In order to solve the problems of chimera,time-consuming and heavy workload in peach genetic transformation,this experiment established and optimized the hairy root transformation system mediated by Agrobacterium rhizogenes.[Methods]The hypocotyl,cotyledons and upper parts of 3-week-old sterile tissue-cultured‘Zhongtao 24’peach seedlings were used as explants,and the transformation was mediated by Agrobacterium rhizogenes MSU440 transformed with pCAMBIAsuper1300-GFP plasmid.The infected explants were transferred to the medium containing antibiotics(cephalosporins)and cultured for about 7 d.The formation of callus and hairy roots was observed.The explants from hairy roots were detected by stereo fluorescence microscope to observe the green fluorescence signal.The total RNA of hairy roots with fluorescence signal was extracted,and the positive hairy roots were detected by PCR,and the optimum transformation varieties,infection concentration and infection time were screened.[Results]Agrobacterium rhizogenes MSU440 was used to infect the hypocotyl of peach,and hairy roots were induced.Transgenic hairy roots were quickly screened by GFP expressed in the transformation vector,and RolB and eGFP gene fragments were amplified by PCR.The results of optimization showed that soaking in the infection solution of Agrobacterium rhizogenes with OD value of 0.8 for 30 min was the best transformation condition,and the highest transformation rate was 63.6%.[Conclusion]The hairy root transformation system mediated by Agrobacterium rhizogenes was established in this experiment,and the transformation conditions were optimized,which provided a technical basis for verifying the function of peach genes and obtainingstable transgenic peach plants.

关 键 词：桃 遗传 转化 农杆菌 毛状根 

分 类 号：S662.03[农业科学—果树学]