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Reference Gene Selection for RT-qPCR Analysis in Maize Kernels Inoculated with Aspergillus flavus.

ABSTRACT: Resistance against infection by the fungus Aspergillus flavus Link in commercial maize (Zea mays L.) is the topic of many studies, but few studies have investigated the effects of A. flavus infection on gene expression levels in ear kernels. A crucial component of gene expression profiling by RT-qPCR is having a reliable set of reference genes that show relatively constant expression across the treatments and phenotypes under study. Currently, however, there is no published information on reference genes suitable for measuring changes in kernel gene expression levels after infection with A. flavus. Thus, in this study, six candidate reference genes (ACT1, β-Tub2, eIF4A2, TATA, EFIα, and GAPDH) were evaluated and ranked according to their expression stability. The genes were amplified from first-strand cDNA samples synthesized from kernels of two susceptible and two resistant maize lines that were either inoculated with A. flavus or water or not inoculated. Three software packages were used to calculate and rank the stability of expression for these genesgeNorm, NormFinder, and BestKeeper. The analysis revealed that the most stable genes to normalize expression levels from maize kernels responding to A. flavus inoculation and wounding were ACT1, EFIα, and eIF4A2.

SUBMITTER: Alves Oliveira D

PROVIDER: S-EPMC8229600 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Transcriptional changes in Aspergillus flavus-infected maize kernels

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Aspergillus flavus infection induces transcriptional and physical changes in developing maize kernels.

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Project description:Aflatoxin contamination caused by the opportunistic pathogen A. flavus is a major concern in maize production prior to harvest and during storage. Previous studies indicate that both constitutive and induced resistance are involved in maize kernel defense against A. flavus infection, little is known about molecular mechanisms of mature kernels in response to fungal infection. The purpose of this study is to determine gene expression differences in maize kernels between resistant and susceptible lines in response to A. flavus challenge. To avoid the environmental effects in the field inoculation, a laboratory based inoculation technique Kernel Screening Assay (KSA) was used to challenge kernels with A. flavus. After 72 hours incubation of inoculated and noninculated mature kernels, gene expression profiles of two Near Isogenic Lines (NIL) of Eyl25 (A. flavus resistant) and Eyl31 (A. flavus susceptible) were compared using oligonucleotide array.

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Project description:BACKGROUND: Planthoppers not only severely affect crops by causing mechanical damage when feeding but are also vectors of several plant virus species. The analysis of gene expression in persistently infected planthoppers might unveil the molecular basis of viral transmission. Quantitative real-time RT-PCR (RT-qPCR) is currently the most accurate and sensitive method used for quantitative gene expression analysis. In order to normalize the resulting quantitative data, reference genes with constant expression during the experimental procedures are needed. RESULTS: Partial sequences of the commonly used reference genes actin (ACT), ?1-tubulin (TUB), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), elongation factor 1 alpha (EF1A), ribosomal protein S18 (RPS18) and polyubiquitin C (UBI) from Delphacodes kuscheli, a planthopper capable of persistently transmitting the plant fijivirus Mal de Río Cuarto virus (MRCV), were isolated for the first time. Specific RT-qPCR primers were designed and the expression stability of these genes was assayed in MRCV-infective and naïve planthoppers using geNorm, Normfinder and BestKeeper tools. The overall analysis showed that UBI, followed by 18S and ACT, are the most suitable genes as internal controls for quantitative gene expression studies in MRCV-infective planthoppers, while TUB and EF1A are the most variable ones. Moreover, EF1A was upregulated by MRCV infection. CONCLUSIONS: A RT-qPCR platform for gene expression analysis in the MRCV-infected planthopper vector Delphacodes kuscheli was developed. Our work is the first report on reference gene selection in virus-infected insects, and might serve as a precedent for future gene expression studies on MRCV and other virus-planthopper pathosystems.

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2010-10-01 | E-GEOD-19883 | biostudies-arrayexpress

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