Emani Chandrakanth, Garcia Juan Manuel, Lopata-Finch Emily, Pozo Maria Jose, Uribe Pedro, Kim Dong-Jin, Sunilkumar Ganesan, Cook Douglas R, Kenerley Charles M, Rathore Keerti S
Institute for Plant Genomics & Biotechnology, Norman Borlaug Center for Southern Crop Improvement, Texas A&M University, College Station, TX 77843-2123, USA.
Plant Biotechnol J. 2003 Sep;1(5):321-36. doi: 10.1046/j.1467-7652.2003.00029.x.
Mycoparasitic fungi are proving to be rich sources of antifungal genes that can be utilized to genetically engineer important crops for resistance against fungal pathogens. We have transformed cotton and tobacco plants with a cDNA clone encoding a 42 kDa endochitinase from the mycoparasitic fungus, Trichoderma virens. Plants from 82 independently transformed callus lines of cotton were regenerated and analysed for transgene expression. Several primary transformants were identified with endochitinase activities that were significantly higher than the control values. Transgene integration and expression was confirmed by Southern and Northern blot analyses, respectively. The transgenic endochitinase activities were examined in the leaves of transgenic tobacco as well as in the leaves, roots, hypocotyls and seeds of transgenic cotton. Transgenic plants with elevated endochitinase activities also showed the expected 42 kDa endochitinase band in fluorescence, gel-based assays performed with the leaf extracts in both species. Homozygous T2 plants of the high endochitinase-expressing cotton lines were tested for disease resistance against a soil-borne pathogen, Rhizoctonia solani and a foliar pathogen, Alternaria alternata. Transgenic cotton plants showed significant resistance to both pathogens.
事实证明，真菌寄生真菌是抗真菌基因的丰富来源，这些基因可用于对重要作物进行基因工程改造，使其对真菌病原体具有抗性。我们用一个编码来自真菌寄生真菌绿色木霉的42 kDa内切几丁质酶的cDNA克隆转化了棉花和烟草植株。从82个独立转化的棉花愈伤组织系中再生出植株，并分析转基因表达情况。鉴定出了几个内切几丁质酶活性明显高于对照值的初级转化体。分别通过Southern印迹分析和Northern印迹分析证实了转基因的整合和表达。在转基因烟草的叶片以及转基因棉花的叶片、根、下胚轴和种子中检测了转基因内切几丁质酶活性。在这两个物种中，用叶片提取物进行的荧光凝胶分析中，内切几丁质酶活性升高的转基因植物也显示出预期的42 kDa内切几丁质酶条带。对高表达内切几丁质酶的棉花品系的纯合T2植株进行了针对土传病原体立枯丝核菌和叶部病原体链格孢的抗病性测试。转基因棉花植株对这两种病原体均表现出显著抗性。
