超声波对烟草花粉的生物学效应及其诱导外源基因向烟草花粉的转移  

The Biological Effect and Direct Gene Transfer to Tobacco Pollen by Sonication

作　　者：董云洲[1] 

机构地区：[1]中国农业科学院生物技术研究中心,北京100081

出　　处：《应用基础与工程科学学报》1999年第4期393-401,共9页Journal of Basic Science and Engineering

摘　　要：用超声波法处理烟草花粉的生物学效应及它诱导ＧＵＳ基因转化烟草花粉，获得了短暂表达的研究结果．单核晚期和双核早中期的烟草游离花粉，置于含０．４ｍ ｏｌ／Ｌ甘露醇的缓冲液中，随着超声强度提高或／和处理时间的延长，对烟草花粉的活力和萌发率具有显著影响．声强０．５Ｗ／ｃｍ ２、时间１０～２０ｍ ｉｎ 是比较理想的参数．将游离花粉与质粒ｐＢⅠ１２１ 混合，以声强为０．５Ｗ／ｃｍ ２ 的脉冲超声波处理１０ｍ ｉｎ， 检测ＧＵＳ基因的平均短暂表达频率（ＴＴＦ％ ），单核晚期花粉为１８．６％ ，双核早中期花粉为３５．７％ ．对超声转化的不同发育时期的烟草花粉，分别以两条不同途径操作和筛选转基因植株：（１） 单核晚期的花粉：直接进行体外培养、植株再生和筛选，以期获得单倍体转基因植株．在卡那霉素选择培养基上，花粉细胞进行两次或多次分裂后即行停止；（２） 双核早中期的花粉，在体外培养成熟后授粉，收获种子，检测种子中外源基因是否存在．在所收３４１ 粒种子中，没能筛选到卡那霉素抗性植株．就如何筛选超声波转化花粉、获得转基因植株的技术途径进行了讨论．The biological effect and direct gene transfer to tobacco pollen by sonication were described in this paper. The isolated tobacco pollen was sonicated with an ultrasonic pulse generator in a sonication buffer containing 5% DMSO, 15mmol/L NaCl, 1.5mmol/L Sodium citrate, 0.4mol/L Mannitol. The acoustic power and duration of sonication had a remarkable effect on the viability and percentage of germination of pollen grains. The optimizeded parameters were 0.5W/cm 2 & 10~20min treatment. In the transformation of tobacco pollen, 40μg/ml Salmon DNA and 20μg/ml plasmid pBⅠ121, which containing GUS reporter gene and nptⅡ selective marker gene, were added into the sonication buffer. At 0.5W/cm 2 acoustic power for 10min, the transient expression frequency of GUS gene was 18.6% and 35.7% in Late uninucleate and Early/Mid binucleate pollen respectively. The transformed Late uninucleate pollen was used for inducing cell division and plant regeneration. In SCD tissue culture medium with 100mg/l kanamycin (Kan), the pollen underwent several times cell division and formed about 100 cell Kan resistance (Kan R) aggregates. Unfortunately, the cell division stopped. On the other hand, the transformed Early/Mid binucleate pollen was cultured for in vitro maturation and pollination, and totally 341 seeds were harvested. All the seeds were screened on MS medium with 100mg/l kanamycin, no Kan R plantlet was generated in the selective medium. As for producing trangenic plant by the combination between sonication and pollen was discussed.

关 键 词：超声波 烟草 花粉 转基因 

分 类 号：Q947[生物学—植物学]