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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) is a kind of broad-spectrum antifungal protein isolated from gastrodiaelata Bl., A traditional Chinese medicine. It is effective against many plant fungal diseases including Fusarium wilt, Verticillium wilt, Pathogenic bacteria in vitro has a strong inhibitory effect, therefore, in plant genetic engineering of antifungal disease has a very important value. In this study, pollen tube pathway was used to transfer GAFP gene gafp into three Xinjiang cotton varieties. Through field screening and molecular detection, transgenic plants with high resistance to Verticillium wilt were obtained. Two Southern hybridization plants, LB_5_8 And ZB_1_49 on the performance of Verticillium wilt immune. The result of RT-PCR showed that gafp was correctly transcribed in both LB_5_8 and ZB_1_49. In vitro bacteriostasis experiments also showed that their protein crude extracts inhibited the growth of Verticillium dahliae in vitro significantly, indicating that gafp Transgenic plants are correctly expressed and the translated product is active. After further breeding and propagation, it was found that the offspring of transgenic color cotton had a stable and strong ability of resistance to Verticillium wilt. This study provided a new way for controlling Verticillium dahliae by means of plant disease resistance genetic engineering.