农杆菌GV3101中反式玉米素合成基因促进烟草再生

Trans-zeatin Synthesizing Gene in Agrobacterium tumefaciens GV3101 Strain Stimulates the Explant Regeneration of Nicotiana

胭脂碱型农杆菌GV3101已经被广泛用于植物遗传转化研究。已有的研究结果证明,农杆菌GV3101株系含有的反式玉米素合成(trans-zeatin synthesizing,tzs)基因编码产物会影响烟草细胞器的形态及细胞的生理状态。然而,有关tzs基因对遗传转化过程外植体再生的影响研究却少有报道。本文在前期研究工作的基础上,以2种烟草、4个农杆菌株系为组培实验材料,验证了胭脂碱型农杆菌tzs基因产物的生理活性。结果表明:以外源添加生长调节物质的外植体为阳性对照,在不添加任何生长调节剂的培养基上,与GV3101菌株共培养的烟草外植体能分化再生,并发育成完整植株;外植体再生与GV3101携带的质粒种类无关;外植体与农杆菌GV3101培养液共培养24 h,烟草再生效果较好;与GV3101株系共培养24 h,将外植体烟草叶片匀浆,经亲和柱分离纯化后,检测出烟草外植体叶片中高达0.78 ng/g FW-1的反式玉米素含量。菌落PCR扩增结果证实,农杆菌GV3101株系有tzs基因序列。以上结果表明,农杆菌GV3101株系内的tzs基因的表达产物有生理学活性,能够促进烟草外植体再生,调节细胞生长。

Ti plasmid of nopaline type Agrobacterium contains a trans-zeatin synthesizing gene.Agrobacterium strain GV3101 is widely used in plant genetic transformation experiment.The previous studies show that Agrobacterium GV3101 strain contains tzs genes which affect the organelle morphology and physiological state of the cells of tobacco.However,there are few studies on the influence of tzs genes on the regeneration of explants.Based on the previous findings,two tobacco varieties and four strains of Agrobacterium were used as tissue cultural materials in this research to testify the biological activity of tzs gene products.The leaf explants grew on MS medium with plant growth regulator were used as positive control.The tissue culture results indicate that explants co-cultured with GV3101 strains could regenerate and reproduce integrated plant in the medium without plant growth regulators; explant regeneration is independent on the types of plasmids in GV3101; explant regeneration ratio is better when the explants were co-cultured with Agrobacterium GV3101 supernatant for 24 hours; the content of anti-tzsin the explants was 0.78 ng/g FW-1 and high level of tzs was detected in the leaves of tobacco explants after co-culture with GV3101 for 24 hours and purification of the homogenization of tobacco explants leaves by affinity column.Colony PCR amplification showed that Agrobacterium tumefaciens GV3101 strain has tzs gene sequence.These results showed that expression products of tzs gene in Agrobacterium tumefaciens GV3101 strain has physiological activity which improves cell growth and explants regeneration.