柑橘响应黄龙病菌侵染的NAC基因的克隆及表达分析

Gene Cloning and Expression Analysis of NAC Gene in Citrus in Response to Huanglongbing

挖掘柑橘抗黄龙病(Huanglongbing,HLB)基因是抗病育种的基础和关键。以感染黄龙病菌亚洲种Candidatus Liberibacter asiaticus(CLas)早期(2个月)锦橙根和叶片中脉比较转录组数据为基础,筛选到9个响应柑橘黄龙病侵染诱导的NAC基因,从中选3个差异表达水平较高的基因克隆,分别命名为Cs NAC21/22、Cs NAC68和Cs NAC78。生物信息分析表明3个基因均符合NAC基因家族的特征;烟草亚细胞定位结果表明,Cs NAC68定位在细胞核,Cs NAC21/22和Cs NAC78定位在细胞核和细胞质。实时荧光定量PCR(q RT-PCR)分析表明,3个候选基因在易感HLB的锦橙、耐病的马蜂柑和高耐病的九里香的组织和病原菌诱导表达特征呈现明显差异。以健康植株为对照,Cs NAC68和Cs NAC78主要在锦橙的根中响应CLas感染,显著上调表达,Cs NAC68在九里香叶肉和马蜂柑根中响应CLas感染,显著上调表达;Cs NAC21/22主要在锦橙根和马蜂柑叶肉中显著下调表达。以‘锦橙’叶片为试材通过q RT-PCR分析候选基因响应SA、JA、ABA、ETH诱导的表达特征,结果表明,3个基因可能参与ABA的信号转导途径,Cs NAC68可能参与SA和JA的信号转导途径,而Cs NAC78可能参与ETH的信号转导途径。

Citrus Huanglongbing(HLB)has become a major disease and limiting factor of production in citrus areas that have become infected.It can cause production extinction when the damage to citrus industry is serious,and there is no effective prevention and treatment method for this disease.The key to solve this problem is to excavate citrus resistance genes for disease resistance breeding.Therefor,in this study,based on the transcriptome data of Candidatus Liberibacter asiaticus(CLas)in the root and midrib in the early stage of infection(2 months),nine NAC(NAM,ATAF1/2,CUC2)genes were screened in response to HLB infection,and three genes with high differential expression were cloned,named as Cs NAC21/22,Cs NAC68 and Cs NAC78,respectively.Bioinformatics analysis showed that Cs NAC21/22,Cs NAC68 and Cs NAC78 encoded 306,503 and 152 amino acids respectively,and the isoelectric point(p I)was 5.43,6.59 and 8.39,respectively.The conserved domain analysis showed that Cs NAC21/22,Cs NAC68 and Cs NAC78 contained five subdomains of the conserved domain A–E,and the A,C and D parts were relatively conserved,which was consistent with the characteristics of the NAC gene family.Phylogenetic tree analysis showed that Cs NAC21/22 was closely related to Glycine max and Populus trichocarpa,and Cs NAC68 and Cs NAC78 were closely related to Arabidopsis thaliana and Populus trichocarpa.Subcellular localization analysis showed that Cs NAC68 located in the nucleus,and Cs NAC21/22 and Cs NAC78 located in the nucleus and cytoplasm.Real-time fluorescent quantitative PCR(q RT-PCR)analysis showed that the three candidate genes showed significantly different tissue and pathogen induced expression characteristics in HLB susceptible Jincheng(Citrus sinensis)and HLB resistant Mafenggan(C.hystrix)and Jiulixiang(Murraya exotica).With healthy plants as the control,the expression levels of Cs NAC68 and Cs NAC78 were significantly up-regulated in the Jincheng root in response to CLas infection,while Cs NAC68 was in response to CLas infection in the mesophyll of Jiulixiang and the root of Mafenggan significantly up-regulated expression.The expression of Cs NAC21/22 was significantly down-regulated in the root of Jincheng and the mesophyll of Mafenggan.Using Jincheng leaves as the test material,the expression characteristics of candidate genes in response to plant hormone induction were analyzed by q RT-PCR.The results showed that the three genes may be involved in the signal transduction pathway of abscisic acid(ABA),and Cs NAC68 may be involved in the signal transduction pathway of salicylic acid(SA)and jasmonic acid(JA),and Cs NAC78 may be involved in the signal transduction pathway of ethylene(ETH).