Coca María, Bortolotti Cristina, Rufat Mar, Peñas Gisela, Eritja Ramón, Tharreau Didier, del Pozo Alvaro Martinez, Messeguer Joaquima, San Segundo Blanca
Departamento de Genética Molecular, Instituto de Biología Molecular de Barcelona, CID-CSIC, Jordi Girona 18, 08034 Barcelona, Spain.
Plant Mol Biol. 2004 Jan;54(2):245-59. doi: 10.1023/B:PLAN.0000028791.34706.80.
The Aspergillus giganteus antifungal protein (AFP), encoded by the afp gene, has been reported to possess in vitro antifungal activity against various economically important fungal pathogens, including the rice blast fungus Magnaporthe grisea. In this study, transgenic rice ( Oryza sativa ) constitutively expressing the afp gene was generated by Agrobacterium -mediated transformation. Two different DNA constructs containing either the afp cDNA sequence from Aspergillus or a chemically synthesized codon-optimized afp gene were introduced into rice plants. In both cases, the DNA region encoding the signal sequence from the tobacco AP24 gene was N-terminally fused to the coding sequence of the mature AFP protein. Transgenic rice plants showed stable integration and inheritance of the transgene. No effect on plant morphology was observed in the afp -expressing rice lines. The inhibitory activity of protein extracts prepared from leaves of afp plants on the in vitro growth of M. grisea indicated that the AFP protein produced by the trangenic rice plants was biologically active. Several of the T(2) homozygous afp lines were challenged with M. grisea in a detached leaf infection assay. Transformants exhibited resistance to rice blast at various levels. Altogether, the results presented here indicate that AFP can be functionally expressed in rice plants for protection against the rice blast fungus M. grisea.
由afp基因编码的巨大曲霉抗真菌蛋白(AFP)已被报道在体外对包括稻瘟病菌Magnaporthe grisea在内的多种具有重要经济意义的真菌病原体具有抗真菌活性。在本研究中,通过农杆菌介导的转化产生了组成型表达afp基因的转基因水稻(Oryza sativa)。将含有来自曲霉的afp cDNA序列或化学合成的密码子优化的afp基因的两种不同DNA构建体导入水稻植株。在这两种情况下,编码来自烟草AP24基因信号序列的DNA区域在N端与成熟AFP蛋白的编码序列融合。转基因水稻植株显示出转基因的稳定整合和遗传。在表达afp的水稻株系中未观察到对植物形态的影响。从afp植株叶片制备的蛋白提取物对稻瘟病菌体外生长的抑制活性表明,转基因水稻植株产生的AFP蛋白具有生物活性。在离体叶片感染试验中,用稻瘟病菌对几个T(2)纯合afp株系进行了挑战。转化体对稻瘟病表现出不同程度的抗性。总之,这里给出的结果表明,AFP可以在水稻植株中功能性表达,以抵御稻瘟病菌Magnaporthe grisea。