Selection of reference genes from two leafhopper species challenged by phytoplasma infection, for gene expression studies by RT-qPCR

BMC Research Notes

Cristina Marzachì¹  Domenico Bosco²  Luciana Galetto¹ 

[1] Istituto di Virologia Vegetale, CNR, Strada delle Cacce 73, 10135 Torino, Italy;Dipartimento di Scienze Agrarie, Forestali e Alimentari, Università degli Studi di Torino, Via Leonardo Da Vinci 44, 10095 Grugliasco, TO, Italy

关键词: Normfinder;    geNorm;    BestKeeper;    Housekeeping genes;    Insect vectors;    Macrosteles quadripunctulatus;    Euscelidius variegatus;    “Candidatus Phytoplasma asteris”;   

Others  :  1141373
DOI  :  10.1186/1756-0500-6-409

 received in 2013-05-10, accepted in 2013-09-19,  发布年份 2013

【 摘 要 】

Background

Phytoplasmas are phloem-limited phytopathogenic wall-less bacteria and represent a major threat to agriculture worldwide. They are transmitted in a persistent, propagative manner by phloem-sucking Hemipteran insects. For gene expression studies based on mRNA quantification by RT-qPCR, stability of housekeeping genes is crucial. The aim of this study was the identification of reference genes to study the effect of phytoplasma infection on gene expression of two leafhopper vector species. The identified reference genes will be useful tools to investigate differential gene expression of leafhopper vectors upon phytoplasma infection.

Results

The expression profiles of ribosomal 18S, actin, ATP synthase β, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and tropomyosin were determined in two leafhopper vector species (Hemiptera: Cicadellidae), both healthy and infected by “Candidatus Phytoplasma asteris” (chrysanthemum yellows phytoplasma strain, CYP). Insects were analyzed at three different times post acquisition, and expression stabilities of the selected genes were evaluated with BestKeeper, geNorm and Normfinder algorithms. In Euscelidius variegatus, all genes under all treatments were stable and could serve as reference genes. In Macrosteles quadripunctulatus, BestKeeper and Normfinder analysis indicated ATP synthase β, tropomyosin and GAPDH as the most stable, whereas geNorm identified reliable genes only for early stages of infection.

Conclusions

In this study a validation of five candidate reference genes was performed with three algorithms, and housekeeping genes were identified for over time transcript profiling of two leafhopper vector species infected by CYP. This work set up an experimental system to study the molecular basis of phytoplasma multiplication in the insect body, in order to elucidate mechanisms of vector specificity. Most of the sequences provided in this study are new for leafhoppers, which are vectors of economically important plant pathogens. Phylogenetic indications were also drawn from sequence analysis of these genes.

【 授权许可】

   
2013 Galetto et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

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