【作者】 马春花；

【导师】 王华；

【作者基本信息】 西北农林科技大学 ， 果树学， 2004， 硕士

【摘要】 植物基因工程技术为培育抗病植物品种开辟了一条全新而有效的途径。近年来，在植物抗病虫害方面取得了很大的进展，已经克隆和鉴定了不少与抗真菌病害有关的基因，并获得了许多能稳定遗传的抗病转化植株。由于酿酒葡萄品种主要是欧洲葡萄（Vitis vinifera L.），其丰产、优质，但抗病性差。尤其是我国大多数酿酒葡萄种植区域在葡萄生长期降雨偏多，导致葡萄霜霉病、白粉病等真菌病害严重，对酿酒葡萄的产量和质量影响很大。故培育优质抗病的酿酒葡萄品种对我国酿酒葡萄的生产和葡萄酒质量的提高具有重要意义。本研究利用基因工程技术构建了含有几丁质酶（Chi.）、β-1,3-葡聚糖酶（Glu.）、萝卜抗真菌蛋白（Rs-AFP2）的三价植物表达载体，并通过农杆菌直接转化法将三价植物表达载体转入农杆菌EHA105。以酿酒葡萄“梅尔诺”叶柄为材料，通过控制激素水平，光照和温度等，对建立再生体系的器官发生途径和体胚发生途径进行了研究，获得以下实验结果：通过控制激素水平，光照和温度等，对建立再生体系的器官发生途径和体胚发生途径进行了研究。试验结果表明，获得梅尔诺单芽增殖生根的培养基配方为1/2MS＋IBA0.1mg/L，获得了梅尔诺离体叶柄的再生不定芽，最佳培养基配方为MS+TDZ2.0mg/L，再生率为62.42%。同时获得了体胚萌发芽的再生不定芽最佳培养基配方为MS+TDZ4.0mg/L，再生率为52.25%，在此培养基上可直接诱导出绿色不定芽。二者的再生不定芽增殖的最佳培养基配方均为MS+BA0.5mg/L。试验结果表明，细胞分裂素TDZ对梅尔诺离体叶柄再生不定芽效果显著，较嫩的叶柄容易再生出不定芽，并且不同的暗培养时间对不定芽再生有很大的影响，暗培养时间30d时效果较好。本研究利用基因工程技术成功地构建了两种不同组合的含潮霉素（Hyg.）的几丁质酶、β-1，3-葡聚糖酶与萝卜抗真菌蛋白基因三价植物表达载体。用直接导入法将两种三价表达载体转化农杆菌EHA105，获得了农杆菌工程菌株，为下一步转化酿酒葡萄，获得对真菌有更广泛抗病性的新品系奠定基础。本研究为下一步转化酿酒葡萄，提高抗生素对酿酒葡萄转化细胞的有效筛选，获得优质、对真菌有更强抗病性和更广泛抑菌范围的酿酒葡萄新品系奠定了基础。更多还原

【Abstract】 Plant gene engineering techniques offer new and efficient methods for breeding cultivars resistant to disease. In recent years, great achievements have been obtained in genetically engineering plants resistant to disease and pests. Several genes for resistance to fungal disease have been cloned and demonstrated, leading to many disease resistant plants with stable heredity.Most cultivars used in viticulture are Vitis vinifera L., productive and high-quality but with poor disease resistance. In China especially, most areas suitable for cultivation have high rainfall, which results in fungal diseases such as powdery mildew and downy mildew affecting both the productivity and quality of wine grapes. So, it is very important to grow wine grape cultivars of high-quality and resistance to disease.This paper describes construction of trivalent-expression vectors containing Chitinase (Chi.), β-1,3-Glucanase (Glu.) and Raphnus sativus - antifungal protein (Rs-AFPs) by gene engineering techniques. By manipulating hormone levels, light intensities and temperature, we have developed two successful regeneration systems for the winegrape varieties Merlot Noir. The results were as follows:By manipulating hormone levels, light intensities and temperatures, we have developed two successful regeneration systems for the winegrape varieties Merlot Noir, For Merlot Poir, The best rooting culture medium was: ?MS + IBA (0.1mg/L). The optimum medium for adventitious bud regeneration from petioles was MS+TDZ2.0mg/L, and the germination rate was 62.42%. The optimum medium for adventitious buds regeneration from germination of somatic embryos of Merlot Noir was MS+TDZ(4.0mg/L), and the germination rate was 52.25%. The best adventitious bud propagation culture medium was MS + BA (0.5mg/L).In this study, TDZ was best for inducing adventitious bud from petioles in vitro, and it was easy to regenerate adventitious bud from the younger petiole. Maintaining cultures in dark had an important effect on regenerated adventitious bud, 30 days is best.The trivalent-expression vectors containing Chitinase(Chi.)/β-1, 3-Glucanase (Glu.)/Raphnus sativus-antifingal protein (Rs-AFPs), were constructed and transferred into Agrobacterium tumefaciens EHA105 through direct transformation by gene engineering techniques.This work will help us to: research genetic transformation of grapevines; select transgenic cells using antibiotics; develop new wine grape cultivars of high quality and with resistance to disease.更多还原