国槐离体再生及抗虫基因sck的转导

In Vitro Regeneration and Agrobacterium Transformation of Sophora japonica with sck Gene

以国槐叶片作为外植体,筛选出诱导不定芽分化的最佳培养基;利用根癌农杆菌介导法将经过修饰的广谱抗虫基因豇豆胰蛋白酶抑制剂基因sck(Signal-CpTI-KDEL)导入国槐。国槐叶片与农杆菌共培养结束后,转移至筛选培养基MS+BA3 mg·L-1+IAA0·05 mg·L-1+G418 8 mg·L-1+Cef 500 mg·L-1上。不定芽在培养基1/2MS+IBA1·0 mg·L-1+G418 10 mg·L-1+Cef 100 mg·L-1上进一步培养获得完整再生植株。通过PCR和Southern blotting检测证明,修饰的豇豆胰蛋白酶抑制剂基因sck已成功导入国槐细胞。

Sophora japonica is a valuable gardening tree that has been planted in the North of China. However, a problem hampering the application of its full potential is susceptibility to insect. In this study, taking leaves of Sophora japonica as the explants, the optimum medium for the induction of adventitious bud differentiation was selected. Modified cowpea trypsin inhinitor（CpTI） gene sck （Signal-CpTI-KDEL）, an insecticidal gene, was introduced into S. japonica by Agrobacteriummediated genetic transformation. After cocultivation of leaves of S. japonica with Agrobacterium tumefaciens harbored sck gene, kanamycin resistance shoots were transferred to selective MS medium containing 3 mg·L^- 1 BA, 0.05 mg· L^- 1 IAA, 8 mg·L^- 1 G418 and 500 mg·L^- 1 cefotaxime. Transgenic plants were obtained after rooting on half-strength MS medium supplemented with 1.0 mg·L^-1 indole-3-butyrique acide （.IBA）, 10 mg·L^-1 G418 and 100 mg·L^-1 cefotaxime. PCR and Southern blotting confirmed that Modified CpTI gene（ sck）was successfully transferred into S. japonica mediated by A. tumefaciens.