摘要
以杜梨(Pyrus betulaefolia)为研究材料,利用发根农杆菌(Agrobacterium rhizogenes)开发了一种简单、快速、高效的杜梨毛状根遗传转化方法。将含有mCherry基因的表达载体pROK2导入发根农杆菌K599,侵染杜梨幼苗,通过注射烟草叶片、常规PCR及体视显微镜观察,检测转化效率。结果表明,利用侵染烟草叶片瞬时表达的方法,证明mCherry可以正常表达;发根农杆菌侵染40株杜梨后,再生毛状根植株为34株,再生率达85%,通过PCR鉴定,转基因毛状根长势良好且鉴定为阳性的植株有6株,阳性率达到17.6%;经体视显微镜观察,可以在转基因根中直接观察到mCherry的红色荧光。通过发根农杆菌直接侵染杜梨苗的方法,成功实现了外源基因在杜梨新生不定根中的表达,操作简便,转化率高,周期较短,为梨树基因功能研究和遗传改良提供了新型工具,为提高梨树遗传转化成功率奠定了基础。
Pyrus betulaefoliawas used as the research material to establish a simple,rapid and efficient method for genetic transformation of pear hairy roots by Agrobacterium rhizogenes.The expression vector pROK2 containing mCherry gene was introduced into Agrobacterium rhizogenes K599 to infect the seedlings of Pyrus betulaefolia.The function of the expression vector was detected by the three methods,namely,injection of tobacco leaves,routine PCR and stereoscopic fluorescence microscope observation.By transient expression in infected tobacco leaves,it was proved that mCherry could be expressed in plant cells.After infected by Agrobacterium rhizogenes infected 34 Pyrus betulaefolia seedlings regenerated hairy roots and the regeneration rate was 85%.Through the identification by PCR,there were 6 of the 34 transgenic plants with positive transgenic hairy roots,with a positive rate of 17.6%.The red fluorescence of mCherry can be observed directly in the transgenic root by stereoscopic fluorescence microscope.In this study,by using the method of Agrobacterium rhizogenes directly infecting Pyrus betulaefolia,the expression of foreign gene in the new adventitious root of pear was successfully realized.This method overcomes the difficulties such as complex genetic background,long childhood,simple operation,high transformation rate and short cycle.It can transform roots stably in a relatively short time,which provides a new tool for pear gene function research and genetic improvement.This work provides a foundation for improving the success rate of genetic transformation of pear trees.
作者
郝紫微
戴雨沁
张绍铃
王鹏
HAO Zi-wei;DAI Yu-qin;ZHANG Shao-ling;WANG Peng(Pear Engineering and Technology Research Center,Nanjing Agricultural University,Nanjing 210095,China)
出处
《湖北农业科学》
2021年第1期151-154,159,共5页
Hubei Agricultural Sciences
基金
中央高校基本科研业务费基础前沿项目(JCQY201903)
国家现代农业(梨)产业技术体系项目(CARS-28)。
