DOI: 10.5061/dryad.3xsj3txb0
title: Expression Analysis and Functional Characterization of CER1 Family Genes Involved in Very-LongChain Alkanes Biosynthesis in Brachypodium distachyon

description: Cuticular wax accumulation and composition affects drought resistance in
 plants. Brachypodium distachyon plants subjected to water deficit and
 polyethylene glycol treatments resulted in a significant increase in total
 wax load, in which very-long-chain (VLC) alkanes were more sensitive to
 these treatments than other wax compounds, implying that VLC alkanes
 biosynthesis plays a more important role in drought resistance in B.
 distachyon. ECERIFERUM1 (CER1) has been reported to encode a core enzyme
 involved in VLC alkanes biosynthesis in Arabidopsis (Arabidopsis
 thaliana), but few corresponding genes are investigated in B. distachyon.
 Here, we identified eight CER1 homologous genes in B. distachyon, namely
 BdCER1-1 to BdCER1-8, and then analyzed their sequences feature,
 expression patterns, stress induction, and biochemical activities. These
 genes had similar protein structure to other reported CER1 and CER1-like
 genes, but displayed closer phylogenetic relationship to the rice OsGL1
 genes. They were further found to exhibit various tissue expression
 patterns after being induced by abiotic stresses. Among them, BdCER1-8
 gene showed extremely high expression in leaves. Heterologous introduction
 of BdCER1-8 into the Arabidopsis cer1 mutant rescued VLC alkanes
 biosynthesis. These results indicate that BdCER1 genes are likely to be
 involved in VLC alkanes  biosynthesis of B. distachyon. Taken
 together, BdCER1-8 seems to play an explicit and predominant 
 role in VLC alkanes biosynthesis in leaf. Our work
 provides important clues for further characterizing
 function of CER1 homologous genes in B. distachyon and
 also an option to improve drought resistance of cereal crops.
description: The total RNA was extracted using the HiPure Plant RNA Mini Kit
 (Magen, Guangzhou, China) following standard protocol. Before reverse
 transcription, about 1–2 μg total RNA was treated with gDNA Eraser to
 remove genomic DNA, and then the first-strand cDNA was synthesized by
 PrimeScript RT Enzyme Mix I (Takara, Dalian, China) according to the
 manufacturer’s instruction. Finally, the products were diluted 10-fold
 with sterile distilled water. Quantitative real-time PCR (qRT-PCR) was
 carried out by using an ABI StepOnePlus instrument (Applied Biosystems).
 Each reaction volume of 25 µl contained 12.5 μl SYBR® Green I Mix (ToYoBo,
 Osaka, Japan), 2.5 μl cDNA, 1 μl 10 μM forward primer, 1 μl 10 μM reverse
 primer, and 8 µl ddH2O. The PCR cycle used was as follows: 95°C for 1 min,
 40 cycles of 95°C for 15 s, 58°C for 30 s, and 72°C for 45 s. Data were
 collected during the extension step. Dissociation curve analysis was
 performed as follows: 95°C for 15 s, 60°C for 1 min, and 95°C for 15 s.
 Specific primers of BdCER1 genes were listed in Supplementary Table 7.
 BdACTIN gene (Bradi_1g10630) was selected as an endogenous control. To
 check expression levels of BdCER1-8 in leaves and stems of different
 transgenic Arabidopsis lines, both semi-quantitative RT-PCR and qRT-PCR
 assays were performed using AtACT8 gene (At1g49240) as an endogenous
 control. The relative expression levels were analyzed using the 2−ΔΔCT
 method (Livak and Schmittgen, 2001). Expression levels of the BdCER1
 homologous genes under abiotic stresses were analyzed using the software
 package of Heatmap on the BMKCloud platform (www.biocloud.net).