农杆菌介导5种中草药叶片瞬时表达的研究  

Study on Transient Expression in Leaves of Five Chinese Medicinal Herbs Mediated by Agrobacterium tumefaciens

作　　者：孙敏 达晓伟 张悦婧 李颖 庞海龙[1,2] 冯汉青[1,2] SUN Min;DA Xiaowei;ZHANG Yuejing;LI Ying;PANG Hailong;FENG Hanqing(College of Life Science, Northwest Normal University, Lanzhou 730070, China;2New Rural Development Research Institute, Northwest Normal University, Lanzhou 730070, China)

机构地区：[1]西北师范大学生命科学学院,兰州730070 [2]西北师范大学新农村发展研究院,兰州730070

出　　处：《西北植物学报》2021年第7期1240-1247,共8页Acta Botanica Boreali-Occidentalia Sinica

基　　金：甘肃省引导科技创新发展专项资金(2019ZX-05);西北师范大学新农村发展研究院开放基金;国家自然科学基金(31870246);甘肃省重点研发计划(18YF1NA051)。

摘　　要：农杆菌介导的瞬时基因表达因其操作简单、可重复性高和实验成本低而成为研究基因功能、生产活性蛋白的有效方法。该研究以非病毒型(载体Ⅰ)和病毒型二元载体(载体Ⅱ)及两种农杆菌(EHA105、LBA4404)为介导,通过叶片渗透法在5种中草药(甘草、黄芩、黄芪、大黄、板蓝根)叶片中进行报告基因GFP的瞬时表达,并对影响表达的因素进行分析,以明确中药材中适宜瞬时表达技术应用的物种材料。结果显示:(1)农杆菌在5种中草药叶片中的瞬时转化效率受到载体、菌株和植物物种类型的影响。(2)在所选的5种中草药植物中,与无转化载体的菌株相比,当农杆菌重悬液OD600为0.8、叶片侵染5~7 d时,含非病毒型二元载体的EHA105菌株在甘草叶片中荧光强度最高,大黄次之;含病毒型二元载体的LBA4404菌株在黄芩叶片中GFP荧光表达量最高;含不同载体的不同菌液侵染的甘草、黄芪叶片仅有注射孔处检测出荧光或几乎无荧光蛋白表达,即未向四周扩散表达;在板蓝根叶片中检测出弱荧光或无荧光强度。研究表明,甘草和黄芩植物叶片可作为农杆菌介导基因瞬时表达的材料。Agrobacterium-mediated transient gene expression has become an effective method for studying gene function and producing active proteins due to its simple operation,high reproducibility and low experimental cost.In this study,the transient expression of the reporter gene GFP in the leaves of five Chinese herbal herbs[Glycyrrhiza uralensis Fisch.,Scutellaria baicalensis Georgi,Astragalus membranaceus var.mongholicus(Bge.)Hsiao,Rheum officinale Baill.,Isatis tinctoria L.]was conducted by means of leaf osmotic method,which was mediated by non-viral(vector I),viral binary vector(vectorⅡ)and two kinds of Agrobacterium tumefacients(EHA105,LBA4404).The factors affecting the expression were analyzed in order to identify the suitable materials for the application of transient expression technology in Chinese medicinal materials.The results showed that:(1)the transient transformation efficiency of Agrobacterium tumefaciens in the leaves of five Chinese herbal medicines was affected by vectors,strains and plant species types.(2)Among the five selected Chinese herbal plants,compared with the strains without transformation vector,when the OD600 of Agrobacterium resuspend was 0.8 and the leaves were infected for 5-7 days,the fluorescence intensity of EHA105 strain containing non-viral binary vector was the highest in Glycyrrhiza uralensis Fisch.leaves,followed by Rheum officinale Baill..The strain LBA4404 containing the virus-type binary vector had the highest fluorescence expression of GFP in the leaves of Scutellaria baicalensis Georgi.Fluorescence or almost no fluorescent protein expression was detected in the leaves of Glycyrrhiza uralensis Fisch.and Astragalus membranaceus var.mongholicus(Bge.)Hsiao membranaceus infected by different bacterial fluids containing different vectors,that is,they were not diffused to the surrounding areas.Weak fluorescence or no fluorescence intensity was detected in the leaves of Isatis tinctoria L..The results showed that the leaves of Glycyrrhiza uralensis Fisch.and Scutellaria baicalensi

关 键 词：农杆菌 瞬时表达 二元载体(非病毒型和病毒型) 中草药 GFP 

分 类 号：Q785[生物学—分子生物学] Q786