真空辅助农杆菌介导中华猕猴桃高效遗传转化体系的建立

Establishment of Efficient Genetic Transformation System of Actinidia chinensis by Vacuum Infiltration Assisted Method

以中华猕猴桃伏牛95-2(Actinidia chinensis‘Funiu 95-2')叶片为试验材料,对不同侵染方式、预培养时间、菌液浓度、共培养时间等影响β-葡萄糖苷酸酶基因(β-glucuronidase,GUS)瞬时表达率的因素进行了研究。结果表明,叶片预培养3天,菌液浓度A_(600)值为0.3,真空渗入方式侵染10 min,共培养4天条件下,GUS基因瞬时表达率最高,达到92.2%。对转基因抗性植株进行PCR检测和GUS组织化学染色,初步证明外源基因已整合到中华猕猴桃伏牛95-2的基因组中。

Factors affecting transient expression frequency of GUS gene, including different infection methods, days of pre-culture, concentration of Agrobacterium tumefaciens, days of co-culture were studied in genetic transformation, using leaves ofAcitnidia chinensis ＇Funiu 95-2＇ as explants. The results showed that the highest transient expression frequency （92.2%） was obtained when explants were firstly pre-cultured for 3 d, infected for 10 min with Agrobacterium tumefaciens （A600=0.3） by vacuum infiltration and finally co-cultured for 4 d. PCR detection and GUS histochemical staining proved initially that the foreign gene had been integrated into the Acitnidia chinensis genomes.