超声波法直接导入外源基因——高效烟草转化系统的建立  

ULTRASONIC DIRECT GENE TRANSFER——THE ESTABLISHMENT OF HIGH EFFICIENCY GENETIC TRANSFORMATION SYSTEM FOR TOBACCO

作　　者：章力建[1] 陈乐玫[1] 袁静[1] 李长公 贾士荣[1] 许宁[2] 赵南明[2] 

机构地区：[1]中国农业科学院生物技术研究中心,北京100081 [2]清华大学生物科学与技术系,北京100084

出　　处：《中国农业科学》1991年第2期83-89,共7页Scientia Agricultura Sinica

摘　　要：本文报告一种新的将外源基因导入带壁植物细胞的方法——超声波直接导入外源基因法。将烟草叶片小块置于含pBI121.1质粒的缓冲液中,用声强为0.5w/cm^2的脉冲超声波处理30分钟,培养一天后86%的叶块中检测到有GUS基因的短暂表达,叶片组织的平均转化面积高达60%-70%。处理叶块中的60%在含Kan 100mg/1的分化培养基上再生出Kan抗性芽,对照叶块则在此培养基上全部死亡。经检测Kan抗性小植株叶片中的GUS活性,按外植体总数计算,稳定转化率达22.3%,抽样检测NPTⅡ活性和DNA斑点杂交均显阳性,证明外源基因已整合到烟草DNA中。A novel ultrasonic direct gene transfer technique was developed and described by which the foreign gene could be introduced into plant cells. Leaf segments of Nicotiana tabacum var. White Burley were immerged in a buffer solution containing plasmid DNA pBI121.1 and sonieated salmon sperm DNA The samples were then sonicated with an ultrasonic pulse generator at 0.5w/cm^2 acoustic power for 30 rain. One day after sonica- tion, the transient expression of GUS gene was detected in 86% of the treated leafsegments and the average area of transformed tissue on leaf pieces reached60-70%. Kanamycin-resistant (Kan^R) shoots were regenerated from transformed leaf segments cultured on the differentiation medium containing Kan 100rag/l, while the leaf segments of the control were all dead on the same selection medium. The GUS activity was found in the leaves of regenerated Kan^R plantlets and a high frequency(22.3%) of stable transformation was obtained. The integration and expression of foreign gene were also confirmed by Dot-blot, and NPT II assay.

关 键 词：超声波 烟草 遗传转化 

分 类 号：S572.032[农业科学—烟草工业]