农杆菌介导的MsDUF基因转化紫花苜蓿的遗传转化体系建立及优化  

Establishment and Optimization of Agrobacterium-Mediated Genetic Transformation System of MsDUF Gene into Alfalfa

作　　者：安明珠 马向丽[1] 周凯 段新慧[1] 姜华[1] 韩博[1] An Mingzhu;Ma Xiangli;Zhou Kai;Duan Xinhui;Jiang Hua;Han Bo(College of Animal Science and Technology,Yunnan Agricultural University,Kunming650201,China)

机构地区：[1]云南农业大学动物科技学院,云南昆明650201

出　　处：《山东农业科学》2022年第3期22-27,共6页Shandong Agricultural Sciences

基　　金：云南省基础研究面上项目(202101AT070206);云南省科技计划项目(2019FD098)。

摘　　要：为建立适宜MsDUF基因转化紫花苜蓿的遗传转化体系,本试验在已知的紫花苜蓿再生体系的基础上,采用农杆菌介导法,以保定紫花苜蓿(MedicagosativaL.cv.Baoding)下胚轴和子叶为转化受体,将MsDUF基因导入到紫花苜蓿中,通过对愈伤诱导、分化及生根培养阶段抗生素浓度的筛选建立了优化的遗传转化体系,同时对筛选获得的转基因紫花苜蓿进行PCR和RT-PCR检测。结果表明,农杆菌介导的MsDUF基因遗传转化的最佳条件为:将预培养2d的紫花苜蓿下胚轴用含pCAMBIA1301-MsDUF质粒的农杆菌菌液(OD_(600)为0.3~0.5)侵染10min,共培养暗处理2d后转入含30mg/LHyg和500mg/LCef的愈伤诱导培养基和分化培养基上诱导分化,待抗性芽长到2~3cm高时转入含30mg/LHyg和300mg/LCef的生根培养基上,得到6株经PCR检测为阳性的抗性植株;选取5株进行RT-PCR检测,均检测出大小与目的条带相吻合的清晰条带,证明MsDUF基因已成功转入保定紫花苜蓿中。本试验成功建立了农杆菌介导的高效MsDUF基因转化紫花苜蓿的遗传转化体系,为进一步研究MsDUF基因功能奠定了基础,同时为紫花苜蓿育种和种质创新提供了基础材料。In order to establish a genetic transformation system suitable for transformation of MsDUF gene into alfalfa,based on the known regeneration system,MsDUF gene was introduced into the hypocotyl and cotyledon of Medicago sativa L.cv.Baoding by Agrobacterium-mediated method.The optimized genetic transformation system was established by screening antibiotic concentration during callus induction,differentiation and rooting culture,and PCR and RT-PCR were used to detect the transgenic alfalfa.The results showed that the optimal conditions for Agrobacterium-mediated genetic transformation of MsDUF gene were as follows:the hypocotyls of alfalfa pre-cultured for 2 days were inoculated with Agrobacterium solution containing pCAMBIA1301-MsDUF plasmid(OD_(600) as 0.3~0.5)for 10 min;after cocultured for 2 days in dark,they were transferred onto the medium containing 30 mg/L Hyg and 500 mg/L Cef for induction and differentiation;when resistant buds grew to 2~3-cm height,they were transferred onto the rooting medium containing 30 mg/L Hyg and 300 mg/L Cef.Six resistant plants were obtained after detected by PCR.Five strains were selected for RT-PCR detection,and all detected out clear bands with the size consistent with target bands,which proved that MsDUF gene was successfully transferred into alfalfa.In this study,a high-efficiency genetic transformation system for MsDUF gene into alfalfa mediated by Agrobacterium was successfully established,which laid foundations for further study the function of MsDUF gene and provided basic materials for breeding and germplasm innovation of alfalfa.

关 键 词：紫花苜蓿 MsDUF基因 农杆菌介导 遗传转化体系 PCR检测 RT-PCR检测 

分 类 号：S551.7[农业科学—作物学] Q785[生物学—分子生物学]