Dip法获得转反义磷脂酶Dγ(PLDγ)基因拟南芥植株

TRANSGENIC ARABIDOPSIS TRANSFORMED WITH ANTISENSE PHOSPHOLIPASE Dγ(PLDγ) GENE OBTAINED BY DIP METHOD

在含反义PLDγ基因的质粒根癌农杆菌的介导下 ,以生长到初果期的健壮拟南芥植株为转基因材料 ,用Dip法进行转基因实验 ,对影响转化率的浸染条件进行了比较研究。适宜的转化条件是将拟南芥的整个花序浸泡于农杆菌悬浮液中 (含 5 %蔗糖 ,0 .0 4 %吐温 - 80 ) ,每次浸染 10min ,间隔 3天浸染一次 ,共浸染 3次。每次浸染后将植株横放于暗箱中 2 4h ,再置于 2 2～ 2 4℃的光照下正常生长。待种子收获后在含有卡那霉素 70mg L的选择培养基中选择转化植株。PCR及PCR -Southernblot分析检测 ,该方法的转化率每 10 0粒种子可高达 2 %。

A modified Dip transformation method for the effects of dipping time, frequency, interval of days between dips was studied by Agrobacterium tumefaciens carrying the construction plasmid containing anti-PLDγ in Arabidopsis when numerous immature floral buds and a few siliques were present .The suitable condition of this method was as follows. developing floral tissues were diped into a solution containing Agrobacterium tumefaciens ,5% sucrose, 0.04% Tween-80 for three times with a interval of three days between dips, lasting for 10 minutes at a time. And then dipped plants were removed to darkroom thwartwise for 24 hours before placing them to greenhouse at 22-24℃ for continuous growth. When siliques on plants were very dry, the seeds were harvested and selected in culture medium containing 70mg/L kanamycine. The frequency of transformants was up to 2%(two transformants for every 100seeds) by the analysis of PCR and PCR-Southern blot.