柑橘黄龙病病原菌nrdB基因的原位杂交分析

In Situ Hybridization Analysis of Distinctive Ribonucleotide Reductase β Subunit Gene from Candidatus Liberibacter Asiaticus of Citrus

柑橘植株是否感染黄龙病,可通过qPCR检测其病原韧皮部杆菌(CandidatusLiberibacter Asiaticus,CLas)来判断,但是不同组织样本影响检测结果,因此有必要研究韧皮部杆菌在不同组织中的定位。以柑橘黄龙病韧皮部杆菌的核糖核苷酸还原酶(RibonucleotideReductase,RNR)β亚基基因序列为依据,设计和制备123 bp的RNA探针,经原位杂交试验,在显微镜下能清晰地观察到阳性植株筛管和伴胞中的可视化蓝色杂交信号,导管呈环纹结构。CLas在不同器官中的分布存在显著差异,与新生组织根尖和茎尖相比,RNR基因在枝皮、叶柄、叶脉的韧皮部中的定位更准确。原位杂交的可视化结果与qPCR检测结果一致。

Candidatus Liberibacter asiaticus(CLas),can be determined by qPCR detection.However,the selection of different tissue samples will affect the results of qPCR detection.Therefore,it is necessary to study the location of CLas in citrus tissues.Based on the specific gene sequence of ribonucleotide reductase(RNR)βsubunit of CLas of citrus Huanglongbing disease(HLB),123 bp antisense RNA probe and sense RNA probe was designed and prepared.In situ hybridization analysis was performed on the pathogen gene of CLas in shoot tip,leaf vein,petiole,clade and root tip,meanwhile,the pathogens were detected by qPCR.The results showed that the visual blue hybridization signals in sieve tube and companion cells of phloem in CLas-infected plants could be clearly observed under the microscope and the distribution of pathogens in the different organs was significantly different,compared with root tip and shoot-tip,RNR gene distribution in phloem of bark,petiole and vein was more accurate locating.The HLB-infected condition detected by qPCR and the visualization results of in situ hybridization shared the consistency in citrus nursery plants.