Computational protocol: miR397/Laccase Gene Mediated Network Improves Tolerance to Fenoxaprop-P-ethyl in Beckmannia syzigachne and Oryza sativa

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Protocol publication

[…] MiR397 has been implicated in the network for lignin biosynthesis via putative interactions with several transcription factors (TFs) and laccases in Populus trichocarpa (). The possible mechanism of miR397/laccase in regulating plant resistance to fenoxaprop-P-ethyl was assessed by constructing B. syzigachne RNA-seq libraries using RNA from the S population and three R populations (R1, R2, and R3) (). The four libraries were assayed for quality and quantity and sequenced by Beijing BioMarker Technologies (Beijing, China). The mRNA-Seq libraries were constructed and sequence data were analyzed, assembled, and annotated as described by . Gene names of specific TFs were assigned based on the best Basic Local Alignment Search Tool hit (highest score), excluding any uninformative descriptions.The results of the comparison between S and R libraries suggested that RSEM could be used to quantify the reads identified as TFs (). Transcript levels were quantified by counting reads per kilobase of exon model per million mapped reads (RPKM) (). The RPKM method of read density reflects the molar concentration of a transcript by normalizing for RNA length and the total number of reads. The EBSeq software was used to determine P-values for the samples with no biological replicates. EBSeq employs an empirical Bayes hierarchical model (). A two-fold change, a P-value of <0.01, and a minimum RPKM of 2 in at least one of the treatments were used as the criteria to classify a response as up- or down-regulation. TFs were found to be consistently up- or down-regulated in R1, R2, and R3.The downstream effect of laccase down-regulation on the up-regulated pathways that contribute to NTSR against fenoxaprop were further confirmed by conducting additional experiments that detected the expressions of these TFs and oxidase/peroxidase genes in WT and OXmiR397 rice. Primers used for detecting their expression are listed in Supplementary Table . […]

Pipeline specifications

Software tools BLASTN, RSEM, EBSeq
Application RNA-seq analysis
Organisms Oryza sativa, Nicotiana tabacum