农杆菌介导的CFL(Cucumber-FLO-LFY)基因遗传转化大岩桐  

Transformation of CFL(Cucumber-FLO-LFY) Gene in Gloxinia(Sinningia speciosa) Mediated by Agrobacterium tumefaciens

作　　者：张明哲[1,2] 王利琳[3] 叶丹[2] 吴志毅 陈曦 张晓峰 边红武[2] 韩凝[2] 朱睦元[2] 

机构地区：[1]浙江出入境检验检疫局,杭州3100163 [2]浙江大学生命科学学院,杭州310058 [3]杭州师范大学,杭州310036

出　　处：《农业生物技术学报》2011年第3期455-461,共7页Journal of Agricultural Biotechnology

基　　金：国家质检总局科技计划项目(No.2011IK249);浙江省科技厅面上项目(No.2009C32076);浙江出入境检验检疫局科研项目(No.ZK200958)共同资助

摘　　要：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。Different Agrobacterium-mediated protocols were used to evaluate the transformation efficiency of gloxinia（Sinningia speciosa）,and CFL（Cucumber-FLO-LFY） gene was introduced into gloxinia to investigate its potential to promote early flowering.The plant expression vector pCA-CFL was constructed by inserting CFL gene into the plant high-efficient expression vector pCAMBIA13011.To restrain the growth of untransformed cells,we confirm the hygromycin（Hyg） filtration pressure（20mg/L） through explants culture treated with different Hyg concentrations.CFL gene was transformed into leaves of gloxinia mediated by Agrobacterium tumefaciens and GUS（β-glucuronidase） test showed that the protocol of ultrasonic 10 s treatment was the most efficient.After two selection cultures,dozens of Hyg-resistant regenerated green shoots were obtained.PCR and Southern dot blot analysis revealed that CFL gene had been integrated into the gloxinia genome.Transgenic seedlings were grown under long-day condition until maturity.The analysis showed that most of transgenic gloxinia plants（71%） flowered 26~32 days earlier than wild-type plants,and had terminal flowers emerging directly from shoot apex with no inflorescence branches.Our results imply that CFL act as a functional homolog of LEAFY（LFY） in gloxinia.

关 键 词：CFL(Cucumber-FLO-LFY)基因 转基因大岩桐 农杆菌介导 

分 类 号：S513.03[农业科学—作物学]