农杆菌介导LJAMP2基因导入‘红阳’猕猴桃及分子鉴定

Agrobacterium-mediated transformation of LJAMP2 gene into ‘Red Sun’ kiwifruit and its molecular identification

摘要:

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。

Abstract:

Bacterial canker caused by Pseudomonas syringae pv. Actinidiae is one of the most important diseases of kiwifruit (Actinidia chinensis) and leads to considerable yield losses. In order to obtain transgenic plants with resistance for ‘Red Sun’ kiwifruit to canker disease, a non-specific lipid transfer protein-like antimicrobial protein gene (LJAMP2) from motherwort (Leonurus japonicus) was introduced into ‘Red Sun’ kiwifruit through Agrobacterium-mediated transformation. After two days of co-cultivation with A. tumefaciens strain LBA4404 harboring 35S:LJAMP2, the transformed explants were transferred to the selection medium containing 25 mg/L kanamycin+3.0 mg/L BA+1.0 mg/L NAA. The regeneration efficiency of kanamycin-resistant shoots reached to 85%. All (100%) of kanamycin-resistant shoots rooted on half-strength MS medium supplemented with 0.8 mg/L IBA and a total of 40 regenerated plantlets were obtained. PCR and histochemical GUS activity analysis show that 23 of 40 lines (57.50%) were positive, suggesting that the LJAMP2 gene was integrated into the genome of ‘Red Sun’ kiwifruit. Taken together, we established an efficient genetic transformation method for ‘Red Sun’ kiwifruit using A. tumefaciens and the transformation frequency reached 5.11%. This protocol will be useful for the genetic breeding of ‘Red Sun’ kiwifruit for improvement of disease resistance.