发根农杆菌介导的柑橘黄龙病抗性快速评价体系优化及应用  

Optimization and Application of Rapid Evaluation System for Citrus Huanglongbing Resistance Mediated by Agrobacterium rhizogenes

作　　者：曹鹏 周金环 王新亮 李楚欣 李佳欣 姜培 刘金香[1] 宋震[1] CAO Peng;ZHOU JinHuan;WANG XinLiang;LI ChuXin;LI JiaXIN;JIANG Pei;LIU JinXiang;SONG Zhen(Citrus Research Institute,Southwest University,Chongqing 400712;National Agricultural Technology Extension and Service Center,Beijing 100125)

机构地区：[1]西南大学柑桔研究所,重庆400712 [2]全国农业技术推广服务中心,北京100125

出　　处：《中国农业科学》2024年第16期3182-3191,共10页Scientia Agricultura Sinica

基　　金：国家重点研发计划(2021YFD1400800);国家现代农业(柑橘)产业技术体系(CARS-26-05B)。

摘　　要：【目的】黄龙病(Huanglongbing,HLB)是一种毁灭性柑橘病害,主要由韧皮部杆菌属亚洲种(Candidatus Liberibacter asiaticus,CLas)侵染引起。本研究旨在探究侵染初期CLas在柑橘毛状根中的增长规律,优化发根农杆菌(Agrobacterium rhizogenes)介导的黄龙病抗性快速评价体系并应用于抗菌肽的抗性评价。【方法】基于植物表达载体pGNGM1300,通过发根农杆菌K599介导的毛状根转化体系转化不同柑橘品种,筛选毛状根诱导快、转化率高的品种;接种CLas后,利用实时荧光定量PCR(real-time quantitative PCR,qPCR)对毛状根中的CLas进行定期检测,揭示其增长规律;在优化黄龙病抗性评价体系的基础上,通过CLas和相关基因的(RT-)qPCR、胼胝质含量检测、显微结构观察和症状观察等开展抗菌肽的黄龙病抗性评价。【结果】供试的11个柑橘品种中,香橼(Citrus medica)的毛状根诱导最快(15 d)、诱导率最高(73.75%),并且毛状根中转基因阳性率高(53.54%)。qPCR定期监测结果显示,在接种后第20—30天(days post inoculation,dpi),CLas开始在根中定殖;在30—50 dpi,CLas含量呈显著上升的趋势;CLas含量在60—120 dpi时略有波动,但与50 dpi时差异不显著。抗菌肽MaSAMP(stable antimicrobial peptide)、HBD-4(homo sapiens defensin beta 4)和CB(cecropin B)的抗性评价结果分析显示,在50—120 dpi,MaSAMP和CB表达植株中CLas含量均低于对照,且差异显著,除90 dpi外HBD-4表达植株中CLas含量也显著低于对照。3种抗菌肽表达植株中的胼胝质含量均显著低于对照(60 dpi),韧皮部细胞壁增厚不明显,未出现明显的淀粉粒和胼胝体沉积,并且均未发现根部死亡等现象(90 dpi)。【结论】揭示了侵染早期CLas在柑橘毛状根中的增长规律;优化了发根农杆菌介导的黄龙病抗性快速评价体系;明确转基因过表达抗菌肽MaSAMP、HBD-4和CB可显著抑制CLas的增殖,减少胼胝质沉积,减轻黄龙病症状,具有用于黄龙病�【Objective】Citrus Huanglongbing(HLB) is a destructive citrus disease primarily caused by Candidatus Liberibacter asiaticus(CLas).The objective of this research is to investigate the growth pattern of CLas in citrus hairy roots at the initial stage of infection,optimize the rapid evaluation system of Agrobacterium rhizogenes-mediated HLB resistance and apply it to the resistance evaluation of antimicrobial peptides.【Method】A plant expression vector,pGNGM1300,was employed to transform different citrus varieties via the hairy root transformation system,which was mediated by A.rhizogenes K599.Subsequently,varieties with rapid induction and high transformation rate were then selected.Following inoculation with CLas,the presence of CLas in hairy roots was consistently demonstrated through the use of real-time quantitative PCR(q PCR),which allowed for the monitoring of its growth pattern.Further,the optimization of the HLB resistance evaluation system permitted the HLB resistance evaluation of antimicrobial peptides to be conducted via(RT-) qPCR analysis of CLas and related genes,quantification of callose deposition,microstructure observation,and symptom assessment.【Result】Among the 11 tested citrus varieties,citron(Citrus medica) exhibited the fastest induction of hairy roots(15 d),accompanied by the highest induction rate(73.75%) and a substantial transgene positivity rate(53.54%) in hairy roots.The results of regular q PCR monitoring indicated that CLas began to colonize roots on 20-30 days post-inoculation(dpi).At 30-50 dpi,there was a notable increase in the CLas content.The content of the CLas exhibited a slight fluctuation at a range of 60 to 120 dpi,though the discrepancy with 50 dpi was not statistically significant.The results of the antimicrobial peptide resistance evaluation,conducted on MaSAMP(stable antimicrobial peptide),HBD-4(homo sapiens defensin beta 4),and CB(cecropin B),revealed that at 50-120 dpi,the content of CLas in both MaSAMP-and CB-expressing plants was significantly lower than tha

关 键 词：柑橘黄龙病 抗菌肽 发根农杆菌 毛状根转化 抗性评价 

分 类 号：S436.66[农业科学—农业昆虫与害虫防治]