韧皮部特异表达抗菌肽基因提高柑桔黄龙病抗性的研究

Citrus huanglongbing is the most destructive disease in the world citrus industry.The disease is associated with a phloem-restricted α-proteobacteria (Candidatus Liberibacter sp.). it is shown that either the immune hemolymph or antimicrobial peptides purified from Chinese oak silkworm can efficiently kill Huanglongbing bacterium in vitro. However，it is not clear for the resistance mechanism and intensity of antimicrobial peptides against Huanglongbing bacteria in plants . Here, to further investigate the potential of antimicrobial peptide gene in the genetic breeding of citrus huanglongbing resistance, the effects of targeting Cecropin B to different subcellular locations (extracellular space,cytoplasm or endoplasmic reticulum) on expression and bactericidal activity of antimicrobial peptide are evaluated in transgenic plants， and then phloem-specific promoter is selected to regulate expression of Cecropin B gene for improving resistance to Huanglongbing in transgenic citrus. On these results, possible mechanisms to explain the resistance of transgenic plants against huanglongbing will be explored. This implementation of the project would offer novel strategies for breeding disease resistance of new citrus varieties.

柑桔黄龙病（Huanglongbing）是一种危害最严重的世界性柑桔病害，其病原物是一种韧皮部限制性细菌。研究表明，柞蚕免疫血淋巴及其纯化的抗菌肽对柑桔黄龙病病原菌分离物具有杀灭作用，但在植物体内对黄龙病细菌的抗性机制和强弱尚不清楚。本项目拟通过比较Cecropin B 在不同亚细胞部位（细胞质、细胞间隙或内质网）的表达情况和杀菌活性，明确显著提高抗菌肽杀菌效果的亚细胞定位方式；在此基础上，选取韧皮部特异启动子调控Cecropin B 基因的表达，提高抗菌肽的杀菌效率，进而改良柑桔对黄龙病的抗性，并阐明转基因植株抗性提高的机制，研究结果将为柑桔抗病基因工程育种开辟一条新的途径。

柑桔黄龙病(Citrus Huanglongbing, HLB)是世界柑桔生产上最具毁灭性的病害，几乎能危害所有的柑桔品种。昆虫来源抗菌肽已被广泛用于植物抗病基因工程研究中。本项目通过韧皮部特异启动子调控人工合成抗菌肽Cecropin B在柑橘中的表达，评价了抗菌肽亚细胞定位表达对转基因植株黄龙病抗性的影响。结果表明，转抗菌肽cecropin B 基因能显著增强柑橘对黄龙病的抗性。显微观察发现传毒后转基因植株韧皮部厚度及其细胞数目和细胞壁厚度均明显小于野生型，暗示了韧皮部超量表达cecropin B 基因能显著抑制HLB病原菌侵染引起寄主韧皮部细胞的病理性增生，从而增强植株的抗性。相对而言，分泌表达cecropin B更能有效增强转基因植株的抗病性。更为重要的是，我们发现将抗菌肽富集于黄龙病病原菌驻留的筛管细胞和伴胞中更有利于抗菌肽的杀菌效果。这些重要结果为进一步在柑橘中靶向特异表达抗菌肽增强柑橘黄龙病抗性，也为今后利用抗菌肽基因创制高抗黄龙病的柑橘新品种提供了有价值的理论参考和技术基础。