以基因枪介导获转ps1-barnase基因的工程雄性不育水稻植株

Engineered Male Sterile Transgenic Plants of Rice (Oryza sativa L.) with ps1-barnase Gene Transformation by Particle Bombardment

以ps1-barnase(brn)为目的基因,pHcintG(PG)为选择/标记基因进行共转化,以PDS-1000-氦气基因枪介导,将brn及PG基因转化到水稻台北309及秋光的核DNA中,得到了转ps1-barnase基因的工程雄性不育植株。以悬浮细胞作为基因枪轰击的靶材料,转化植株再生频率较初级愈伤组织的为高。转brn基因植株的其他主要性状与供体亲本无显著差异,但却表现不育。其不育的程度在不同的植株之间表现不同。在转brn基因植株中观察到全不育(占全部brn阳性植株的40.6％)、高不育(占15.6％)及半不育的个体(占43.7％)。全不育的转基因植株自交完全不能结实(结实率为零),除个别植株外,花粉完全不被I-KI染色;而人工授以正常的花粉则可以获得杂交种子。而brn基因的阴性植株及未进行转化的对照植株则完全可育,表明转基因植株之雄性不育乃brn基因所致。结果表明,brn基因在水稻中是完全可以正常表达的,其表达的时期推测在花粉母细胞减数分裂前至花粉形成之间的整个时期。

Barnase-ps1 and pHcintG genes were co-transferred into rice genomic DNA, and engineering male sterile rice plants were induced in Tai Pei 309 and Qui Guang (japonica rice). Primary callus and suspension cells as the target tissues were bombarded by the particle bombardment of Biolistic PDS-1000 / He Particle Delivery System (BIO-RAD) with the plasmid DNA mixed to Gold Particle. The efficiency of regenerated plant from resistant clones in suspension cells was higher than that in primary callus.In transgenic plants positive for barnase-ps1 gene, three different level of male sterile were observed: complete male sterile making up 40.6% of the total transgenic plant positive barnase gene; high male sterile making up 15.6% and half male sterile making up 43.7%. The pollen grains in complete male sterile of transgenic plants were not stained by I-KI solution, shrunk and quite irregular in shape. No seed was set in the condition of self control pollination. When the normal pollens from wild type rice plants were pollinated on these transgenic male sterile plants, the hybrid seeds were set showing that the female organ of the ms plant was normal. Therefore these transgenic plants were demonstrated to be male sterile. The fertility of negative T_0Plants was normal, even though they were suffered all of the treatments like the positive plant. So the male sterile in positive transgenic plants was induced by ps1-barnase gene. This experiment demonstrated that the expression of barnase-ps1 expressed gene in rice plant. It is hypothesized that the expressive stage of this gene might be from before meiosis of pollen mother cell to pollen formation after meiosis.