柑橘黄龙病菌亚洲种重复序列分析及其检测引物筛选  

Repetitive sequence analysis and detection primer screening of Candidatus Liberibacter asiaticus

作　　者：蒋青岚 孙太安 李大志[1] 李娜[1] 王冰 邓子牛[1] 戴素明[1] JIANG Qinglan;SUN Taian;LI Dazhi;LI Na;WANG Bing;DENG Ziniu;DAI Suming(College of Horticulture,Hunan Agricultrual University,National Center for Citrus Improvement-Changsha,Changsha 410128;Huaihua City Agricultural Comprehensive Service Center;College of Plant Protection,Hunan Agricultrual University)

机构地区：[1]湖南农业大学园艺学院,国家柑橘改良中心长沙分中心,长沙410128 [2]怀化市农业综合服务中心 [3]湖南农业大学植物保护学院

出　　处：《中国果树》2024年第8期98-103,共6页China Fruits

基　　金：国家重点研发项目(2019YFE0104100);财政部和农业农村部:国家现代农业(柑橘)产业技术体系专项(CARS-26);怀化市柔性人才项目。

摘　　要：由柑橘黄龙病菌亚洲种(Candidatus Liberibacter asiaticus,CLas)引起的黄龙病(Huanglongbing,HLB)对柑橘产业危害严重,通过加强病原检测有利于减少病害传播。利用高拷贝重复序列位点设计引物,虽能提高黄龙病检测的灵敏度,但重复序列的多态性很难保障引物检测特征稳定。通过比较11个CLas全基因组的重复序列,以期获得保守的重复序列,利于开发通用型CLas检测引物。结果显示,CLas全基因组有4段长度>50 bp的高拷贝重复序列,其重复数量和序列在11个菌株中均具有保守性。基于4段重复序列开发了CLas特异的6对引物,均能稳定检测田间柑橘黄龙病样品,熔解曲线吸收峰单一,熔解温度稳定。比较6对引物的灵敏度,发现基于5个拷贝重复序列开发的IR16-fr灵敏度最高。使用IR16-fr和常规引物CQULas-FR对50份田间柑橘叶片样品进行检测,结果显示,CQULas-FR对CLas的检出率为20%,而IR16-fr的检出率为26%。新引物IR16-fr可为田间CLas高效率检测提供技术支撑。Huanglongbing(HLB),caused by Candidatus Liberibacter asiaticus(CLas),poses significant threat to citrus industry.Strengthening pathogen detection is beneficial to avoid disease spreading.Though primers coming from high-copy repetitive sequence is helpful to enhance the sensitivity of HLB detection,the polymorphism of repetitive sequence remains challenge to keep the detection instability.In this study,we identified conserved repetitive sequences among 11 CLas strains to develop universal primers of CLas detection.The results revealed there are four kinds of conserved repetitive sequence in CLas genome with sequence length above 50 base pairs and high-copy.Based on these sequences,we developed six pairs of CLas-specific primers,which were tested to have ability in keeping instability of HLB detection.The melting curve of these primers showed single absorption peak and maintained consistent melting temperature among different HLB samples.Among the six primer pairs,IR16-fr,derived from a five-copy repetitive sequence,displayed the highest sensitivity.Fifty field citrus leaf samples were tested using IR16-fr and the conventional primer CQULas-FR,and the results showed that the detection rate of C Las by CQULas-FR was 20%,while that of IR16-fr was 26%.The new primer IR16-fr can provide technical support to enhance the efficiency of C Las detection.

关 键 词：柑橘黄龙病 CLas检测 重复序列 引物 

分 类 号：S666[农业科学—果树学]