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为了获得抗溃疡病的‘红阳’猕猴桃转基因植株,以红阳猕猴桃试管苗叶盘为转基因受体材料,通过根癌农杆菌介导将CaMV35S启动子调控下的LJAMP2基因导入红阳猕猴桃。450个叶盘与携带表达载体质粒pBI121的根癌农杆菌菌珠LBA4404共培养2 d后,转入含25 mg/L Kan的筛选培养基培养40 d,15 d转接1次,之后30 d继代1次。结果表明,在MS+3.0 mg/L BA+1.0 mg/L NAA筛选培养基中,Kanr芽率达85%以上,在1/2 MS+0.8 mg/L IBA培养基中,Kanr芽生根率达100%。共获得Kanr再生植株40株,经GUS组织染色和PCR分析证明,其中23株为转基因植株。阳性率为57.50%,转化率达5.11%。抗溃疡病基因LJAMP2已成功导入红阳猕猴桃,为红阳猕猴桃的抗病基因工程育种奠定了基础。
In order to obtain anti-ulcer disease ’Hongyang’ kiwifruit transgenic plants, the leaf discs of red kiwifruit in vitro culture medium were used as the transgenic receptor materials, and the LJAMP2 gene under CaMV35S promoter mediated by Agrobacterium tumefaciens was introduced into kiwifruit. 450 leaf discs were co-cultured with the Agrobacterium tumefaciens strain LBA4404 carrying the expression vector plasmid pBI121 for 2 days and then transferred to a screening medium containing 25 mg / L Kan for 40 days for 15 days and once for 30 days Subsequent 1 times. The results showed that the Kanr bud rate was over 85% in MS + 3.0 mg / L BA + 1.0 mg / L NAA medium, and the rooting rate of Kanr bud in 1/2 MS + 0.8 mg / L IBA medium reached 100%. A total of 40 Kanr regenerated plants were obtained. The results of GUS staining and PCR analysis showed that 23 of them were transgenic plants. The positive rate was 57.50% and the conversion rate was 5.11%. Anti-ulcer gene LJAMP2 has been successfully introduced into red kiwifruit, kiwifruit of Hongyang disease-resistant gene engineering breeding laid the foundation.