Computational protocol: Overexpressing the Multiple-Stress Responsive Gene At1g74450 Reduces Plant Height and Male Fertility in Arabidopsis thaliana

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Protocol publication

[…] In order to identify potential knockout lines for the five intronless genes that were selected for this study, the following SALK T-DNA insertion lines were ordered from the Nottingham Arabidopsis Stock Centre (NASC): SALK_020993 (At1g18740), SALK_145820 (At1g74450), SALK_071404 (At4g27652), SALK_088755 (At4g29780) and SALK_024636 (At5g12010) []. For backcrossing and knockout line comparisons to wildtype, the Arabidopsis background wildtype line that was used to generate the SALK T-DNA stocks [] was also ordered from NASC. This line, which carried NASC stock number N60000, is currently referred to as Col-0 but was formerly listed as Col-8.The locations of the exonic T-DNA insertions were confirmed by PCR using MangoTaq DNA Polymerase and dNTPs (Bioline). Genomic DNA was extracted from N60000 (Col-0) and mutant leaves according to the Shorty method described previously []. lists the left and right genomic primers that were chosen for each line based on outputs from the T-DNA primer design programme (http://signal.salk.edu/tdnaprimers.2.html). In addition, the left T-DNA border primer LBb1.3 was used in combination with each right genomic primer to detect the T-DNA insertions.Absence of relevant mRNA in each line was confirmed by RT-PCR (OneStep RT-PCR Kit, Qiagen). RNA was extracted from Col-0 and homozygous mutant leaves with the RNeasy Plant Mini Kit (Qiagen) and treated with RNase-Free DNase (Qiagen).Gene specific primers were chosen based on genomic DNA sequence information (NCBI) and outputs from the Primer3 programme. The selected left and right primers for each T-DNA insertion line are listed in . Alpha-(α)-Tubulin was used as a constitutive control with primers as published previously [].The confirmed knockout lines were backcrossed to Col-0 four times prior to self-fertilization and selection of homozygous knockout lines. Subsequently, a double knockout line was created by crossing single knockout lines for the related At1g18740 and At1g74450 genes. […]

Pipeline specifications

Software tools SHORTY, Primer3
Application qPCR
Organisms Arabidopsis thaliana, Homo sapiens
Chemicals Ruthenium