抗菌肽融合表达提高油菜抗病性的分子机制研究

Sclerotinia stem rot is one of the most destructive diseases on the oil crops like oilseed rape, soybean and sunflower. However, the progress on the resistance breeding in the last decades got little progress in case of no resistance gene resource found. During the time, Antimicrobial peptides had been aroused to be one of the hottest spots for crop disease resistance improvement due to its high activity, pollution-free and pathogen selectiveless. However, large amount of researches showed that plant expressed antimicrobial peptides were unstable in plant cell so gave low level of accumulation and produced small effects in transgenic plants. In order to resolve this problem, a novel strategy to produce cleavable tandem repeat antimicrobial peptide multimer in plant cell was designed in this proposal. According to this strategy, antimicrobial peptide would be firstly expressed in transgenic plant cell as tandem repeat fused multimers, then be introduced to the intercellular space by signal peptide and lastly the multimers would be digested into single functional antimicrobial peptide units on the special designed cleavage sites. This strategy was concentrated to improve antimicrobial peptide expression efficiency and stability so as to improve its effects on the pathogen. In theory, the disease resistance is correlated with the number of fused antimicrobial peptide units in transgenic plants. In order to verify this strategy, the special designed transgenic vectors will be firstly introduced to Arabidopsis for theoretical research and then introduced to Brassica napus to improve the sclerotinia sclerotiorum resistance.The relationship between Thanatin fused number and expression amount, cleavage efficiency of fused Thanatin, and fungicide activity on S. sclerotiorum will be investigated to disclose the mechanism.

抗菌肽具有高效、环保、不使病源菌产生耐药性等优点，成为植物抗病基因工程研究和应用的热点。但大量的研究结果表明抗菌肽转入植物后遇到的主要问题是抗菌肽易被植物体内的蛋白酶降解，表达丰度偏低，限制了抗菌肽功效的发挥。为解决这一问题，在上一个国家自然科学基金支持下设计了一种在植物体内可剪切的多拷贝抗菌肽串联融合表达技术，来提高抗菌肽的表达效率和稳定性。前期工作中将1-5拷贝的的Thanatin抗菌肽基因转入拟南芥或油菜，显著提高了转基因株系对菌核病的抗性，本项目通过研究抗菌肽拷贝数与表达量的关系、融合抗菌肽剪切效率、剪切抗菌肽的活性等内容来揭示抗菌肽融合表达的作用机理，为进一步完善抗菌肽融合表达设计，提高抗菌肽表达丰度提供理论依据。同时通过菌核病诱导型启动子对抗菌肽的表达进一步改良，使抗菌肽在病原菌侵染时有选择性表达，优化基因能量利用效率，提高抗病效果的同时不影响其它农艺性状。

油菜是我国的主要油料作物之一，其生产上的病害主要是菌核病等真菌病害，但由于缺乏抗原，长期以来在油菜菌核病育种方面进展缓慢。抗菌肽具有高效、环保、不使病原菌产生耐药性等优点，成为植物抗病基因工程研究和应用的热点。但大量的研究结果表明抗菌肽转入植物后遇到的主要问题是抗菌肽易被植物体内的蛋白酶降解，表达丰度很低，限制了抗菌肽功效的发挥。为解决这一问题，本研究设计了一种在植物体内可自动剪切的多拷贝抗菌肽融合表达技术，来提高抗菌肽的表达效率和稳定性。结果表明，串联融合表达的抗菌肽拷贝数越多，抗菌肽表达丰度越高，且融合表达的抗菌肽在体内迅速剪切为单个有活性的抗菌肽单元，剪切效率非常高，未剪切的前体蛋白含量很低。五拷贝抗菌肽融合表达的转基因油菜显示其抗菌核病性显著提高。从而证明这一技术在应用上切实可行。为进一步优化该技术，筛选了EXL3和LTP2两个菌核病诱导型启动子，驱动6种不同抗菌肽串联融合表达，转基因油菜显示菌核病诱导后EXL3和LTP2启动子驱动的外源基因转录水平比35S高3倍以上，6种不同的抗菌肽融合表达抗病效果比5拷贝单一抗菌肽融合表达更好。