02682naa a2200265 a 450000100080000000500110000800800410001902200140006002400510007410000150012524502510014026000090039152017410040065000220214165300250216365300180218865300240220665300200223070000210225070000230227170000240229470000190231870000170233777300620235420837082018-01-03       2017    bl uuuu     u00u1 u    #d  a1572-96997 ahttps://doi.org/10.1007/s10482-017-0906-72DOI1 aPOLESE, V.  aSelection and validation of reference genes for RT-qPCR indicates that juice of sugarcane varieties modulate the expression of C metabolism genes in the endophytic diazotrophic Herbaspirillum rubrisubalbicans strain HCC103.h[electronic resource]  c2017  aQuantitative reverse transcription PCR (RT-qPCR) is an important tool for evaluating gene expression. However, this technique requires that specific internal normalizing genes be identified for different experimental conditions. To date, no internal normalizing genes are available for validation of data analyses for Herbaspirillum rubrisubalbicans strain HCC103, an endophyte that is part of the sugarcane consortium inoculant. This work seeks to identify and evaluate suitable reference genes for gene expression studies in HCC103 grown until middle log phase in sugarcane juice obtained from four sugarcane varieties or media with three different carbon sources. The mRNA levels of five candidate genes (rpoA, gyrA, dnaG, recA and gmK) and seven target genes involved in carbon metabolism (acnA, fbp, galE, suhB, wcaA, ORF_0127.0101 and _0127.0123) were quantified by RT-qPCR. Analysis of expression stability of these genes was carried out using geNorm and Normfinder software. The results indicated that the HCC103 dnaG and gyrA genes are the most stable and showed adequate relative expression level changes among the different sugarcane juices. The highest expression level was seen for ORF_0127.0101, which encodes a sugar transporter, in juice from sugarcane variety RB867515 and glucose as the carbon source. The suhB gene, encoding SuhB inositol monophosphatase, had a higher relative expression level on 0.5% glucose, 100% sugarcane juice from variety RB867515 and 0.5% aconitate. Together the results suggest that dnaG and gyrA genes are suitable as reference genes for RT-qPCR analysis of strain HCC103 and that juice from different sugarcane varieties modulates the expression of key genes involved in carbon metabolism.  anitrogen fixation  aInternal normalizing  aOrganic acids  aRelative expression  aSugar transport1 aSOARES, C. de P.1 aSILVA, P. R. A. da1 aARAUJO, J. L. S. de1 aBALDANI, J. I.1 aVIDAL, M. S.  tAntonie van Leeuwenhoekv, 110gn. 12, p. 1555-1568, 2017.