利用PMI选择标记进行杨树转基因体系的研究  

Poplar Transgenic System by Using PMI as Biosafety Selective Marker

作　　者：查琳[1,2] 王伟东[1] 续晨[3] 诸葛强[1] 

机构地区：[1]南京林业大学南方现代林业协同创新中心,江苏南京210037 [2]无锡市林木种苗管理站,江苏无锡214000 [3]南京晓庄学院生命科学系,江苏南京210000

出　　处：《林业科学研究》2016年第2期221-226,共6页Forest Research

基　　金：国家科技部"863计划"课题(2013AA102703);国家国际科技合作专项(2014DFG32440);江苏高校优势学科建设工程项目(PAPD)

摘　　要：[目的]利用不同于抗生素的PMI为选择标记基因的遗传转化体系,对杨树进行双抗虫基因(Bt和Cp TI)的转基因研究。建立杨树以PMI为安全标记基因的转基因体系,为安全、高效的林木转基因育种研究提供实验依据。[方法]选择已有的转Cp TI抗虫基因(利用Kmr选择标记获得)的美洲黑杨杂种优良无性系南林895杨(Populus×euramericana‘Nanlin895’)为受体材料,对杨树叶片、叶片分化芽、茎段生根的甘露糖敏感性等筛选优化,采用农杆菌介导的方法进行双抗虫基因的研究。[结果]较为适合的杨树叶片筛选培养基为:甘露糖8 g·L^(-1)和蔗糖22 g·L^(-1);叶片分化芽筛选培养基为:甘露糖10 g·L^(-1)和蔗糖20 g·L^(-1);茎段生根筛选培养基:甘露糖8 g·L^(-1)和蔗糖22 g·L^(-1)。在此基础上,获得了转Bt和Cp TI双抗虫基因的植株8株。[结论]初步建立了以PMI为安全标记基因的杨树转基因体系:确定了PMI筛选的最适选择压,成功构建了含PMI选择标记基因的植物抗虫表达载体,通过农杆菌介导的遗传转化最终获得了转双抗虫基因植株。[Objective]Using the PMI different from antibiotic marker gene as selective marker gene to study poplar transgenic system with two insect-resistant genes（ Bt and Cp TI） and provide experimental data for high-efficient and safe transgenic tree breeding. [Method] Populus × euramericana ‘Nanlin895 ＇genetically modified by Cp TI obtained by using Kmrselective marker was chosen as a receipt,and the mannose sensitivity of poplar leaves,bud growth and stem rooting in a medium were tested for studying poplar transgenic system with two insect-resistant genes by way of Agrobacterum-mediated transformation. [Result] The suitable composition for genetic transformation of poplar leaves was： Mannose 8 g·L^（-1）＋ Sucrose 22 g·L^（-1）; for bud growth： Mannose 10 g·L^（-1）＋ Sucrose 20 g·L^（-1）; and for stem rooting： Mannose 8 g·L^（-1）＋ Sucrose 22 g·L^（-1）. Based on the results,eight transgenic plants with Bt and Cp TI genes we obtained. [Conclusion]The transgenic system of poplar was initially established by use of biosafety selective marker PMI. The best selection pressure was determined. The plant anti-insect expression vector containing PMI selective marker gene was established,and the transgenic poplar with two insect-resistant geneswas obtained by Agrobacterum-mediated transformation.

关 键 词：PMI 选择标记 南林895杨 转基因体系 抗虫 

分 类 号：S792.119[农业科学—林木遗传育种]