柑橘黄龙病菌内参基因的筛选与评估

Screening and evaluation of reference genes for Candidatus Liberibacter asiaticus

摘要:

【背景】柑橘黄龙病是世界柑橘生产上最具毁灭性的病害之一，主要由候选韧皮部杆菌属亚洲种(“Candidatus Liberibacter asiaticus”，CLas)引起。CLas全基因组测序已经完成，因而该病原菌的基因表达研究和功能验证得以进行。【目的】筛选CLas内参基因并评估其不同侵染时期和在不同品种植物寄主中的表达稳定性。【方法】基于基因功能类别，利用实时荧光定量PCR技术分析23个CLas的候选内参基因相对表达情况(16S rRNA基因作为参照基因)。结合Ct值标准差和geNorm、NormFinder、RefFinder软件，评价内参基因的表达稳定性。【结果】在感染黄龙病不同时期和不同品种的植物寄主样本中，14对引物表现出较强的特异性和稳定性。内参基因稳定性排名：ftsZ>gyrA>rpoB1>ftsA>secA>gap>zapE>gmk2>rpoD>secY>rpoO>ftsW>gmk1>recA，根据geNorm配对变异值Vn/n+1选择稳定性最好的ftsZ和gyrA作为内参基因作进一步评估。以ftsZ+gyrA以及16S rRNA基因分别作为内参基因检测柑橘黄龙病菌致病基因LasΔ5313的表达水平，所得的表达模式相同。【结论】柑橘黄龙病菌中涉及DNA复制和细胞分裂功能的管家基因表达较稳定，在CLas的基因表达研究中可选择ftsZ+gyrA的基因组合作为内参。本研究为后续利用实时荧光定量PCR分析CLas基因表达及研究CLas致病机理奠定基础。

Abstract:

[Background] Citrus Huanglongbing (HLB), one of the most devastating diseases in citrus production, is mainly caused by “Candidatus Liberibacter asiaticus” (CLas). With the available of CLas whole genome sequence, the gene expression study and functional verification of CLas genes become possible. [Objective] To screen the reference genes of CLas and assess the expression of reference genes in different infection stage and different plant hosts. [Methods] Twenty-three reference genes of CLas were screened according to the categories of gene function and analyzed by real-time quantitative PCR (16S rRNA gene as the control). Combined analysis of the standard deviation of Ct values, geNorm, NormFinder and RefFinder were used to evaluate the expression stability of the reference genes. [Results] Fourteen pairs of primers showed superior specificity and stability in plant sample collected from different infection stage of CLas and different citrus cultivars. The stability of reference gene were ranked as: ftsZ>gyrA>rpoB1>ftsA>secA>gap>zapE>gmk2>rpoD>secY>rpoO>ftsW>gmk1>recA. Based on the geNorm pairing variation value Vn/n+1, ftsZ and gyrA were selected as reference genes for further evaluation. By using ftsZ+gyrA and 16S rRNA gene as reference genes, a CLas pathogenic gene (LasΔ5313) showed similar expression patterns among above reference genes. [Conclusion] The expression of housekeeping genes associated with DNA replication and cell division function of CLas were relatively stable. ftsZ and gyrA can be used as reference genes for the expression analysis of CLas genes. This study provides foundation for the analysis of CLas genes by using real-time PCR and the pathogenic mechanism of CLas.