Computational protocol: DCA1 Acts as a Transcriptional Co-activator of DST and Contributes to Drought and Salt Tolerance in Rice

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Protocol publication

[…] All rice genetic stocks used in this study were in the Zhonghua 11 (ZH11) japonica variety background except for dca1, which is in the Nipponbare (japonica variety) background. 35S::DCA1 plants were generated via transformation with a construct produced by inserting an amplified ORF fragment containing DCA1 from ZH11 cDNA between the CaMV35S promoter and the 3’OCS terminator. 35S::DST plants were generated using a similar method. The dca1 mutant (NF7038) was obtained from the Rice Genome Resource Center.We generated knockdown transgenic plants using an artificial microRNA (designed by http://wmd3.weigelworld.org/cgi-bin/webapp.cgi). Target site on DCA1 was 5’- acgtgatgagagcgcatcatc-3’. The construction method was modified from Norman Warthmann et al. []. For the miRNA construct, three modification PCRs were performed with primers G-11491 + DmiR II, DmiR I + DmiR IV and DmiR III + G-11494 on ZH11 cDNA as template, yielding three fragments, respectively (). The three resulting fragments were gel purified (Qiagen) and then fused by one PCR with the two flanking primers G-11491 + G-11494 on a mixture of 1 ul from each of the previous three PCRs as template. The fusion product was again gel purified (Qiagen), cloned into the overexpression vector to generate DCA1 knockdown transgenic plants.Seeds were submerged in water at room temperature for 48 h, followed by germination for 18 h at 37°C. Seeds were then sown in a bottomless 96-well plate that was placed in a container of Yoshida’s culture solution and incubated in a growth chamber under a 13 h light (25°C)/11 h dark (23°C) photoperiod. For salt treatment, 20-day-old seedlings were transferred to a culture solution containing 100 mM NaCl. For PEG treatment, 20-day-old seedlings were transferred to a culture solution containing 18% (w/v) PEG4000. For soil drought experiments, 2-week-old seedlings that were cultured in the growth chamber were transplanted to containers with soil and grown in a greenhouse at 24–30°C and 50–60% relative humidity. About 50 days later, water was removed from the containers for the dehydration treatment. [...] Peptide sequences were obtained from Phytozome (Phytozome v9.1, http://www.phytozome.net/) and sequence alignment was performed using the Clustal Omega program (http://www.ebi.ac.uk/Tools/msa/clustalo/). […]

Pipeline specifications

Software tools WMD, Clustal Omega
Databases Phytozome RGRC
Applications Phylogenetics, Non-coding RNA analysis
Organisms Oryza sativa
Chemicals Hydrogen Peroxide