Screening and evaluation of reference genes for Candidatus Liberibacter asiaticus

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.

Key words:Citrus Huanglongbing, “Candidatus Liberibacter asiaticus”, Real-time quantitative PCR, Reference gene