转抗病基因甘蔗遗传稳定性和抗病机制的研究

Sugarcane is one of the most important sugar and economic crops in China. The fungus diseases in sugarcane are increasing seriously. Cultivate new varieties with fungal disease resistance is one of the main goals of the sugarcane breeding. This project is based on genetic transformation of sugarcane and obtained the disease-resistant transgenic lines. Focus on solving this major problem of sugarcane production and sugar industry facing currently and in future, we will research the genetic stability and disease-resistant mechanism of transgenic sugarcane which were integrated with gene of Medicago sativa defensin 1, Medicago truncatula defensin 4 and Killer protein 4 through the techniques of molecular biology, plant physiology, pathology and genetics in combined. The stability and broad spectrum of transgenic sugarcane resistance to major fungal diseases will be evaluated by infecting spontaneously in the disease field, such as sugarcane smut. The physiological and biochemical mechanism for disease-resistant of transgenic sugarcane will be clarified by research on physiological and biochemical changes of sugarcane and the process of production and transduction of systemic acquired resistance signal cytokine after inoculated with pathogens. The molecular basis of interaction between sugarcane and pathogens will be explored by studying the proteins basis of the interactions and the relationship between target gene expression and disease resistance. It would lay theoretical foundation and provide high-quality materials for broad-spectrum disease-resistant sugarcane breeding with genetic engineering technology.

甘蔗是我国最重要的糖料作物和经济作物，真菌病害对其危害日益严重，培育抗真菌病害的新品种是甘蔗育种的一个主要目标。本项目是在对甘蔗遗传转化获得抗病转基因工程植株的基础上，从解决当前和今后甘蔗生产和制糖业所面临的重大问题出发，通过分子生物学、植物生理学、病理学、遗传学等多学科相结合，对转MsDef1、MtDef4和KP4基因甘蔗遗传稳定性和抗病机制进行深入研究。通过在病地自发侵染，评价转基因甘蔗对抵抗甘蔗黑穗病等主要真菌病害的稳定性和广谱性；通过检测接种病原菌后甘蔗的生理生化变化，系统获得性抗性信号因子的产生和转导过程的研究，阐明转基因甘蔗抗病生理生化机制；通过病原菌与甘蔗互作的蛋白质基础以及目的基因表达与抗病性关系的研究，探讨甘蔗与致病菌互作的分子基础，从而为进一步利用基因工程技术培育甘蔗广谱抗病品种奠定理论基础和提供优质材料。

甘蔗是我国最重要的糖料作物和经济作物，真菌病害对其危害日益严重，培育抗真菌病害的新品种是甘蔗育种的一个主要目标。本项目是在对甘蔗遗传转化获得抗病转基因工程植株的基础上，从解决当前和今后甘蔗生产和制糖业所面临的重大问题出发，通过分子生物学、植物生理学、病理学、遗传学等多学科相结合，对转MsDef1、MtDef4和KP4基因甘蔗遗传稳定性和抗病机制进行深入研究。.通过对转基因甘蔗T1至T3代植株进行PCR扩增、Southern杂交、RT-PCR、Q-RT-PCR、Bar免疫/检测试纸条等分子检测，证明外源基因MsDef1、MtDef4和KP4已经整合到甘蔗基因组中，拷贝数为1-3个，并能够正常表达翻译，外源基因在不同株系中的表达量不同，转基因的拷贝数与其相对表达量成正比。通过连续几年田间抗病性鉴定，证明外源基因MsDef1、MtDef4和KP4导入甘蔗提高了转基因植物对黑穗病的抗性且抗病性能稳定地进行遗传。经过筛选获得6个抗病性强并能稳定遗传的转基因甘蔗株系，并且其产量和蔗糖分与非转基因甘蔗无显著差异。通过分析转MsDef1、MtDef4和KP4基因甘蔗株系接种甘蔗黑穗病菌前后，植株中活性氧含量、防御酶系活性、次生代谢产物含量、内源激素含量等物质变化，发现接种黑穗病菌后，转基因甘蔗植株叶片中超氧阴离子自由基和过氧化氢的含量低于非转基因甘蔗，表明转基因甘蔗受活性氧的伤害小于非转基因甘蔗；转基因甘蔗植株叶片防御酶中POD、SOD、PAL的活性高于非转基因甘蔗，而CAT和PPO的活性与非转基因甘蔗没有显著差别；转基因甘蔗的次生代谢产物（总酚和类黄酮）和内源激素（水杨酸和乙烯）的含量变化与非转基因甘蔗无显著差别；转基因甘蔗的病程相关蛋白中几丁质酶活性与非转基因甘蔗无显著差异，但是β-1，3-葡聚糖酶活性却高于非转基因甘蔗。通过比较转MsDef1、MtDef4和KP4基因甘蔗与非转基因甘蔗接种甘蔗黑穗病菌后病原菌的生长增殖情况，发现转基因甘蔗植株中黑穗病菌的生长受到明显抑制。初步揭示了转基因甘蔗抗病的分子机制。本项目的完成为甘蔗抗病育种和抗病机制的研究奠定了坚实的基础。