天麻抗真菌蛋白基因(gafp)转化彩色棉的研究

Study of Transferring the Gene of Gastrodia Antifungal Protein (gafp) into Color Cotton to Enhance Wilt Resistance

天麻抗真菌蛋白 (gastrodiaantifungalprotein简称GAFP)是从我国传统中药天麻 (GastrodiaelataBl.)中分离到的一种具有广谱抗真菌活性的蛋白质 ,它对许多植物真菌病包括棉花枯萎病、黄萎病等的致病菌离体具有很强的抑制作用 ,因此 ,在植物抗真菌病基因工程上有很重要的应用价值。本研究通过花粉管通道法 ,将GAFP的基因gafp转入 3个新疆彩色棉品种中 ,通过田间抗病筛选和分子检测 ,得到了高抗黄萎病的转基因植株 ,两株Southern杂交阳性植株LB_5_8和ZB_1_49对黄萎病表现整株免疫。RT_PCR的结果显示 ,LB_5_8和ZB_1_49中均有gafp的正确转录 ;离体的抑菌实验也表明 ,它们的蛋白粗提物对棉花黄萎病致病菌离体有明显的抑制 ,表明了gafp在转基因植株中的正确表达 ,翻译的产物具有活性。经过进一步选育和扩繁 ,发现转基因彩色棉后代具有稳定的、较强的抗黄萎病能力 ,本研究为通过植物抗病基因工程的方法防治棉花黄萎病提供了一条新的途径。

Gastrodia antifungal protein (GAFP) isolated from traditional Chinese medicinal herb Gastrodia elata Bl. is a small protein with a broad spectrum of antifungal activity. As it shows strong resistance to many pathogenic fungi including Vecticillium dahliae kleb and Fusarium oxysporum f. Sp. vasinfectum which cause cotton wilt, it possesses important application potential in plant antifungal genetic engineering. In this study the gene of GAFP ( gafp ) was transferred into three cultivars of color cotton using the method of pollen_tube_channel. After antifungal selection in the field and molecular assay, we obtained transgenic cotton plants with high level of resistance to wilt. Two Southern_blot_positive plants LB_5_8 and ZB_1_49 showed immunity to wilts. Result of RT_PCR denoted the correct transcription of gafp in LB_5_8 and ZB_1_49. Furthermore, in vitro antifungal activity assay demonstrated that crude protein extraction from them showed obvious resistance to Vecticillium dahliae kleb, which proved the correct expression of gafp in transgenic plants, and suggested the product of translation has antifungal activity. After two years' breeding, the offspring of these two transgenic plants still showed stable and strong resistance to cotton wilts. Present study provided an effective approach for the prevention and control of cotton wilt by genetic engineering which possess significant applied potential in cotton production.