烟草脱外壁花粉的电激基因转移

EXINE-DETACHED POLLEN OF NICOTIANA TABACUM AS AN ELECTROPORATION TARGET FOR GENE TRANSFER

以 β-葡糖苷酸酶 GUS 基因作为报告基因 ,通过瞬间表达的检测 ,比较了烟草 Nicotianatabacum L . 脱外壁花粉、未萌发与萌发花粉的电激导入效果 ,探讨了不同电激条件及启动子对外源基因瞬间表达的影响 .结果表明 :当脉冲时间常数为 13 ms时 ,导致脱外壁花粉和萌发花粉生活力下降约50 %的电场强度分别为 750 V/ cm和 12 50 V/ cm,在此条件下电激 ,二者的导入效果最好 .脱外壁花粉的GUS基因表达水平约为萌发花粉的 5倍、花粉粒的 30倍 .玉米花粉特异启动子 Zm13- 2 60 能启动 GUS基因在脱外壁花粉和萌发花粉中高效表达 ,而 Ca MV

In developing alternative systems for plant transformation the authors investigat ed the use of male gametophyte as the foreign gene receptor.However,delivery of foreign DNA into pollen is difficult because of the existence of a thick exine,therefore a new experi mental system was developed using exine detached pollen (EDP) of Nicotiana tabacum as an electroporation target which was also compared with germinating pollen (GP) and pollen grains (P). A transient GUS expression assay was conducted to analyze the efcts of differ ent electroporation conditions and promoter activity.The pollen specific promoter Zm13 from Zea mays mediated high level of GUS gene expression but CaMV 35S only had very low activity in both EDP and GP. The optimal field strength for gene transfer was obtained at 750 V/cm for EDP and 1250 V/cm for GP when the time constant of pulse was 13 ms.The GUS activity in EDP had a 5 fold increase as compared with GP and P respectively.The level of GUS gene expression was slightly increased when adding 10% PEG into the electro poration buffer.This result indicates that pollen deprived of exine responds much better to foreign gene transfer than the previously used intact pollen grains and may be a better vector to introduce,via pollen tube, genes into the egg cell and offsprings.