Transgenic expression of trypsin inhibitor CMe from barley in indica and japonica rice, confers resistance to the rice weevil Sitophilas oryzae
Transgenic expression of trypsin inhibitor CMe from barley in indica and japonica rice, confers resistance to the rice weevil Sitophilas oryzae
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DOI：
10.1023/a:1022176207180
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发表时间：
2003-02-01
影响因子：
3
通讯作者：
Díaz, I
中科院分区：
文献类型：
--
作者：
Alfonso-Rubí, J;Ortego, F;Díaz, I
籼稻和粳稻（Oryza sativa L.）通过用编码大麦胰蛋白酶抑制剂BTI-CMe的Itr 1基因和玉米泛素启动子进行粒子轰击转化植物，所述Itr 1基因在赋予胚乳特异性的自身启动子的控制下。从38个独立的转基因株系的籼稻（育种系IR 58）和15个粳稻（CV Senia）的选择，22和11，分别表示大麦抑制剂在可检测的水平。如蛋白质印迹分析所示，转基因被正确翻译，在R-3种子中的表达水平高达总提取蛋白质的0.31%（IR 58）和0.43%（Senia）。通过在液体培养基中的胰蛋白酶活性测定和通过用种子提取物进行的活性染色凝胶证实了BTI-CMe的功能完整性。与未转化的对照相比，在表达BTI-CMe的纯合转基因籼稻和粳稻种子上饲养的稻象甲（Sitophilus pasta，鞘翅目：象甲科）的存活率的显著降低，以及昆虫粗中肠提取物的胰蛋白酶样活性的降低，证实了该蛋白酶抑制剂基因用于控制重要储藏害虫的效用。
Indica and japonica rice (Oryza sativa L.) plants were transformed by particle bombardment with the Itr1 gene encoding the barley trypsin inhibitor BTI-CMe, under the control of its own promoter that confers endosperm specificity, and the maize ubiquitin promoter. From 38 independent transgenic lines of indica (breeding line IR58) and 15 of the japonica (cv Senia) selected, 22 and 11, respectively, expressed the barley inhibitor at detectable levels. The transgene was correctly translated as indicated by western blot analysis with a level of expression in R-3 seeds up to 0.31% (IR58) and 0.43% (Senia) of the total extracted protein. The functional integrity of BTI-CMe was confirmed by trypsin activity assays in liquid media and by activity staining gels, performed with seed extracts. The significant reduction of the survival rate of the rice weevil (Sitophilus oryzae, Coleoptera: Curculionidae) reared on homozygous transgenic indica and japonica rice seeds expressing the BTI-CMe, compared to non-transformed controls, and the decrease in the trypsin-like activity of insect crude midgut extracts, confirmed the utility of this proteinase inhibitor gene for the control of important storage pests.