【摘要】 为建立根癌农杆菌介导的草莓褪黑素基因高效遗传转化体系,以草莓品种"早红"叶片和叶柄为外植体,优化了影响根癌农杆菌遗传转化的因素,包括农杆菌菌液浓度、侵染时间、共培养时间和预培养时间等,建立了适宜草莓"早红"的农杆菌遗传转化体系。结果表明,"早红"草莓适宜的转化条件是:外植体的最适预培养时间为2～3d,农杆菌侵染的最佳菌液浓度为OD6000.3～0.5,最适侵染时间为15min,最适共培养时间为3d。在此基础上,通过根癌农杆菌介导法将褪黑素合成关键酶N-乙酰转移酶(AANAT)和羟吲哚-O-甲基转移酶(HIOMT)基因AANAT和HIOMT转入草莓基因组,并对获得的抗性植株进行PCR和PCR-Southern blot检测分析,结果表明外源基因已经整合进草莓基因组中。RT-PCR分析证明外源基因已经在转录水平表达。更多还原

【Abstract】 In order to establish Agrobacterium tumefaciens-mediated melatonin synthetase genes transformation system for strawberry, leaves or petioles were used as explants, and some factors influencing the transformation efficiency, such as the concentration and infection duration of Agrobacterium, the time of both co-culture and pre-culture, were optimized to establish a stable Agrobacterium mediated gene transfomation protocol of strawberry. The results indicated that the optimal pre-culture time of explants was 2～3 d; concentration of Agrobacterium OD_ 600 0.3～0.5; infection time 15 min, and the co-culture time 3d. Then, melatonin synthetase genes of both AANAT(encoding Arylalkylamine N-acetyltransferase, AANAT) and HIOMT(encoding Hydroxyindole O-methyltransferase, HIOMT) were transferred into strawberry (Fragaria ananassa Duch cv. zaohong) mediated by Agrobacterium tumefaciens. Transgenic plants were regenerated and characterized by using PCR analysis, PCR-Southern blot and RT-PCR detection, which confirmed that the foreign genes had been integrated into strawberry genome and transcripted properly.更多还原