柑橘黄龙病侵染相关抗病基因同源序列的克隆鉴定与表达分析

[目的]从黄龙病耐病寄主植物cDNA中筛选抗病基因相关序列并对其进行表达分析研究。[方法]根据已克隆的植物抗性基因表达产物NBS-LRR保守区域设计简并引物,以耐HLB的柑橘属柚cDNA为模板扩增RGAs,并进行实时荧光定量PCR。[结果]通过RFLP分析及克隆测序共得到5个NBS类抗病基因相似序列(RGAs)片段,在GenBank上登录号为HM777043～HM777047。通过Clustalx、DNAMAN等软件分析5个RGAs及其推导的氨基酸的相似性,结果显示它们均含有典型NBS-LRR类抗性基因所具有的保守区域:P-loop、Kinase-2a、GL-PLAL,其与已克隆的烟草N、亚麻L6、拟南芥RPS2、RPS5、RPP8、RPM1等抗病基因在保守区域氨基酸水平上的相似性为19.71%～42.86%。根据得到的序列设计特异性引物,对5个RGAs在HLB侵染过程中的表达进行定量PCR,结果显示嫁接病芽接穗后的8次连续采样中5个RGA的表达受到不同程度的调控。[结论]表明5个RGAs可能与黄龙病的侵染有关。

Isolation and expression analysis of resistance gene analogs related to infection of Citrus Huanglongbing

[Objective] Resistance gene analogs were isolated from cDNA of plant tolerant to HLB and analyzed by expression.[Method] The resistance gene analogs were amplified from cDNA of Citrus maxima using degenerated primers based on the conserved regions of NBS(Nucleotide Binding Site)-LRR(Leucine Rich Repeat) domain from plant resistance genes cloned,and the quantitative Real-time PCR was used to check the expression of 5 RGAs.[Result] A total of 5 RGA fragments were successfully obtained,with GenBank accession numbers of HM777043-HM777047.The deduced amino acid of these RGAs were analyzed by Clustalx and DNAMAN softwares.Sequence analysis showed that these RGAs contained the conserved domains P-loop,kinase-2a and GLPL,which were conserved in NBS-LRR type disease resistance genes.The 5 RGAs shared 19.71%-42.86% identity with the resistance genes of tobacco N,flax L6,and Arabidopsis thaliana RPS2,RPS5,RPP8 and RPM1.The quantitative Real-time PCR results showed that expression of the 5 RGAs was different.[Conclusion] Therefore,it suggested that the 5 RGAs might be related with pathogen infection.