发根农杆菌介导的虎杖转基因体系优化  

Optimization of Agrobacterium rhizogenes-mediated Polygonum cuspidatum Transgenic System

作　　者：段梦灵 李鲁汉 廖辉 陈新月 马钰玺 林艳丽 柳忠玉[1] 吴晓倩 潘佑找[2] 伍翔 DUAN Mengling;LI Luhan;LIAO Hui;CHEN Xinyue;MA Yuxi;LIN Yanli;LIU Zhongyu;WU Xiaoqian;PAN Youzhao;WU Xiang(College of Life Science,Yangtze University,Jingzhou Hubei 434025;College of Horticulture and Gardening,Yangtze University,Jingzhou Hubei 434025)

机构地区：[1]长江大学生命科学学院,湖北荆州434025 [2]长江大学园艺园林学院,湖北荆州434025

出　　处：《现代农业科技》2021年第4期46-50,共5页Modern Agricultural Science and Technology

基　　金：国家自然科学基金项目“虎杖中一个新的MYB转录因子在白藜芦醇合成中的功能研究”(81803670);湖北省大学生创新训练计划项目“虎杖PcMYB1反义植物表达载体构建及对虎杖毛状根的转化”(省级S202010489075;校级2019279);长江大学大学生创新训练计划项目“虎杖转录因子PcMYB2功能初步探究”(Yz2020265)。

摘　　要：为建立虎杖高效遗传转化体系,用携带有植物表达载体pCAMBIA1301的发根农杆菌ATCC15834侵染虎杖无菌苗获得毛状根。对影响虎杖毛状根诱导的因素(如外植体种类、农杆菌活力、培养基种类、超声波辅助时间)进行优化,以获得最佳培养条件;通过潮霉素筛选阳性毛状根,以确定最佳潮霉素浓度。结果表明:以叶片为外植体、1/2MS为培养基、菌液OD600=0.8、超声波辅助5 s时,虎杖遗传转化毛状根效果最佳;筛选出的毛状根经GUS染色和PCR分子鉴定,该方法的阳性转化率为92%。该研究优化了虎杖毛状根的培养条件,为后期试验探究建立了更加高效的虎杖转基因体系。In order to establish a highly efficient genetic transformation system of Polygonum cuspidatum,Agrobacterium rhizogenes ATCC15834 carrying gene expression vector pCAMBIA1301 was used to infect the aseptic seedling of Polygonum cuspidatum to obtain hairy roots.The factors affecting the induction of hairy roots were optimized,such as explants types,Agrobacterium rhizogenes activity,culture medium and ultrasonic assisted time.The positive hairy roots were screened by hygromycin and the optimal hygromycin concentration was determined.The results showed that,when leaves were used as explants,1/2MS was used as culture medium,OD600 was 0.8,ultrasonic-assisted time was 5 s,Polygonum cuspidatum genetic transformation effect was the best.The hairy roots were identified by GUS staining and PCR molecular identification,its positive transformation rate was 92%.This study optimized the culture conditions of Polygonum cuspidatum hairy roots,and established a more efficient transgenic system for later experimental exploration.

关 键 词：虎杖 发根农杆菌 毛状根 转基因 

分 类 号：S567.239[农业科学—中草药栽培]