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建立和优化了农杆菌(Agrobacterium tumefaciens)介导的大岩桐(Sinningia speciosa)遗传转化方法,同时将CFL(Cucumber-FLO-LFY)基因转入大岩桐中,期望获得提早开花的转基因植株。采用双酶切法将CFL基因连接到质粒载体pCAMBIA13011上,得到具有CaMV35S组成型启动子的植物表达载体pCA-CFL。以大岩桐无菌苗叶片为材料,进行潮霉素(hygromycin,Hyg)浓度梯度培养,确认20mg/Lhyg为筛选压。采用超声波处理、农杆菌液浸泡等多种方法进行大岩桐转基因,GUS检测结果表明,超声波处理10s的方法转化效率最高。转化后的叶片进行诱导培养并经过2轮Hyg筛选后,获得一批Hyg抗性再生植株。进一步用PCR、斑点杂交检测发现,CFL基因已整合到大岩桐基因组中。将转基因大岩桐移栽到盆中并在长日照的环境下生长直至开花。经分析表明,超过71%的转基因大岩桐比野生型提早26～32d开花,且大部分的转基因植株不形成侧枝而是直接在顶端成花。研究结果提示,CFL基因和LEAFY(LFY)基因在功能上是同源的。
Agrobacterium tumefaciens mediated genetic transformation of Sinningia speciosa was established and optimized. At the same time, CFL (Cucumber-FLO-LFY) gene was transferred into Sinningia davidiana, and transgenic plants with early flowering were expected to be obtained. The CFL gene was ligated into the plasmid vector pCAMBIA13011 by double digestion to obtain the plant expression vector pCA-CFL with CaMV35S constitutive promoter. The leaves of avermitilis seedlings were used for the hygromycin (Hyg) concentration gradient culture to confirm the screening pressure of 20mg / Lhyg. The results of GUS test showed that the method of ultrasonic treatment and Agrobacterium tumefaciens immersion were the most efficient. The transformed leaves were induced to culture and after 2 rounds of Hyg screening, a batch of Hyg-resistant regenerated plants were obtained. Further analysis by PCR and dot blot showed that the CFL gene was integrated into the genome of Ophiopogon japonicus. The genetically modified Ostrich was transplanted into pots and grown in long sunlight until flowering. The analysis showed that more than 71% of the genetically modified Big Tianyong flowering 26 ~ 32d earlier than the wild type, and most of the transgenic plants do not form collateral but directly into the top of the flowering. The results suggest that the CFL gene and the LEAFY (LFY) gene are functionally homologous.