亳菊EDT1抗旱基因遗传转化体系建立与耐旱性初步评价

Establishment Transformation System of Drought-resistant Transgenic Chrysanthemum morifolium with EDT1 Gene and Preliminary Evaluation of Drought Tolerance

目的:建立亳菊EDT1抗旱基因遗传转化体系,并初步评价其耐旱性。方法:以亳菊茎段外植体为受体材料,抗草甘膦除草剂为筛选基因,通过发根农杆菌介导法转入EDT1抗旱基因。通过农杆菌介导,对影响亳菊遗传转化效率的四个因素进行讨论:侵染时间、农杆菌菌液浓度、乙酰丁香酮浓度和共培养时间,以及草甘膦最佳筛选浓度的研究。结果:建立了农杆菌介导的亳菊高效遗传转化体系,菌液浓度(A600)为0.6~0.8,乙酰丁香酮浓度150μmol/L,侵染时间25 min,共培养3 d,除菌培养后,可得到15 d最大转化效率95.6%。草甘膦对亳菊组培苗致死率的研究表明其最佳筛选浓度为100 mg/L。通过聚合酶链式反应(PCR)检测,初步得到5株转基因植株。结论:在干旱胁迫下,叶片中抗氧化酶活性和MDA含量测定表明,转基因植株具有较高的耐旱性。

Objective：To establish transformation system of drought-resistant transgenic Chrysanthemum morifolium with EDT1 gene, and to evaluate the drought tolerance. Methods：Chrysanthemum morifolium with stem explant was used as receptor material, and resistanted to glyphosate herbicide as the filter gene and transfered the drought-resisitantgene（ EDT1 ） into Chrysanthemum morifolium by using Agrobacterium rhizogenes. Four factors influence efficiency of gene transformation for Chrysanthemum morifolium, which included the time of infection,the concentration of Agrobacterium rhizogenes bacterial and acetosyringone,the time of co-cultured were studied, and screened the best concentration of glyphosate. Results ：Transformation system of drought-resistant transgenic Chrysanthemum morifo- lium with EDTI gene was established. The concentration of Agrobacterium rhizogenes bacterial （A600 ） was 0. 6 - 0. 8, the concentration of acetosyringone was 150 μmol/L,the infection time was 25 rain, and co-cultured time was 3 d. After 15 days＇s sterilization culture, the maximum conversion efficiency was 95.6% , the best selected concentration of glyphosateis was 100 mg/L. The regenerated plants by PCR testing, five transgenic plants were obtained. Conclusion：Antioxidant enzyme activity and MDA content measurement under drought stress indicate that the transgenic plants have higher drought tolerance.