Computational protocol: CmMYB19 Over-Expression Improves Aphid Tolerance in Chrysanthemum by Promoting Lignin Synthesis

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Protocol publication

[…] Total RNA was isolated from NX leaves using the RNAiso reagent (TaKaRa, Tokyo Japan) following the manufacturer’s protocol. A 1 µg aliquot of the resulting RNA was included as the template for first cDNA strand synthesis, using SuperScript III reverse transcriptase (Invitrogen, Carlsbad, CA, USA). The primer pair CmMYB19-M-F/R () was designed to amplify a fragment of the CmMYB19 sequence, based on a previously acquired sequence [], and RACE (random amplification of cDNA ends) PCR was then used to obtain the full length cDNA. For the 3′-RACE, oligo (dT) was used to synthesize the first cDNA strand, followed by a nested PCR using the adaptor primer (J-R) and CmMYB19-3-1/2 (). For the 5’-RACE, the AAP and AUAP primers provided with the 5’-RACE System kit v2.0 (Invitrogen) were used in a nested PCR, along with CmMYB19-5-1/2 (). The PCR products, purified using a Biospin Gel Extraction kit (Bio Flux, Hangzhou, China), were introduced into pMD19-T (TaKaRa) for sequencing. Finally, the CmMYB19 open reading frame (ORF) was amplified using CmMYB19-F/R (). The CmMYB19 protein sequence was aligned with its homologs using ClustalW software (available on: http://www.ebi.ac.uk/Tools/msa/ clustalw2/) [] and a phylogenetic tree was generated using MEGA 5 software (available on: http://www.megasoftware.net) [] based on the neighbor-joining method and 1,000 bootstrap replicates following previous descriptions [,]. A. thaliana MYB polypeptides sequences were obtained from the Plant TFDB website (available on: planttfdb.cbi.edu.cn/). […]

Pipeline specifications

Software tools Clustal W, MEGA
Databases PlantTFDB TFdb Full Length cDNA
Application Phylogenetics
Organisms Saccharomyces cerevisiae, Allium cepa
Diseases Tick Infestations
Chemicals Amino Acids