农杆菌介导的CFL(Cucumber-FLO-LFY)基因遗传转化大岩桐

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

桐

张明哲;王利琳;叶丹;吴志毅;陈曦;张晓峰;边红武;韩凝;朱睦元

【期刊名称】《农业生物技术学报》【年(卷),期】2011(19)3

【摘要】建立和优化了农杆菌(A grobacterium tumefaciens)介导的大岩桐(Sinningia speciosa)遗传转化方法,司时将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/L hyg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10 s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26～32 d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 g1oxinia. 【总页数】7页(P455-461)

【作者】张明哲;王利琳;叶丹;吴志毅;陈曦;张晓峰;边红武;韩凝;朱睦元【作者单位】浙江出入境检验检疫局,杭州,310016;浙江大学生命科学学院,杭州,310058;杭州师范大学,杭州,310036;浙江大学生命科学学院,杭州,310058;浙江出入境检验检疫局,杭州,310016;浙江出入境检验检疫局,杭州,310016;浙江出入境检验检疫局,杭州,310016;浙江大学生命科学学院,杭州,310058;浙江大学生命科学学院,杭州,310058;浙江大学生命科学学院,杭州,310058 【正文语种】中文

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