From c1a2b4f66d3a0fc0b7b166bd31cf4edf00f7c521 Mon Sep 17 00:00:00 2001 From: vipet103 <vipet103@hhu.de> Date: Mon, 18 Mar 2024 12:53:56 +0100 Subject: [PATCH] add protocols to psp study --- studies/psp_DayNight_Cd/protocols/Generation_of_psp.md | 3 +++ .../psp_DayNight_Cd/protocols/Plant_material_and_growth.md | 5 +++++ 2 files changed, 8 insertions(+) create mode 100644 studies/psp_DayNight_Cd/protocols/Generation_of_psp.md create mode 100644 studies/psp_DayNight_Cd/protocols/Plant_material_and_growth.md diff --git a/studies/psp_DayNight_Cd/protocols/Generation_of_psp.md b/studies/psp_DayNight_Cd/protocols/Generation_of_psp.md new file mode 100644 index 0000000..7bc92c4 --- /dev/null +++ b/studies/psp_DayNight_Cd/protocols/Generation_of_psp.md @@ -0,0 +1,3 @@ +## Generation and Selection of the Arabidopsis *PSP* Knock-Down Mutant + +The RNAi construct for the generation of PSP knock-down plants was designed according to Guo et al. (2003) using the intron-containing vector pSK-int as intermediate vector for cloning. The 660 bp long DNA fragments encoding for the sense and antisense RNA of the *Arabidopsis thaliana PSP* gene (At1g18640, nt 3-663 on cDNA) were generated using primer pairs P1/P2 and P3/P4, respectively, and were integrated into the pSK-Int vector via internal restriction sites (Supplementary Table S1). The complete construct was cloned into the final plant vector pUT-Kan encoding an Ubiquitin 10 promoter for expression of the construct and a kanamycin resistance gene for plant selection (Supplementary Figure S1A). Transformation in *Arabidopsis thaliana* Col-0 wildtype plants was carried out using the floral dip method (Clough and Bent, 1998). Transformed plants were selected on half-strength Murashige-and-Skoog medium plates containing 50 μg/ml kanamycin. Correctness of insert was verified via PCR on genomic DNA using primer P5 and P6. Selection was done out of 8 independent lines and was based on reduction of *PSP* transcript abundance. Knockdown of *PSP* mRNA amount was measured by RT-PCR on cDNA of T1 plants using primer P7 and P8. RT-PCR of *psp* mutant lines revealed a strong reduction of the *PSP* transcript abundance in mutant line #17, while mutant line #18 showed a weaker reduction in *PSP* transcript abundance. Detailed results of *PSP* knockdown amount for mutant line #17 and #18 in comparison to the WT at day and night generated via transcriptome analysis are shown in Supplementary Figure S1B. Homozygous T3 plants of *psp* mutant lines #17 and #18 were used for further characterization of the mutants. \ No newline at end of file diff --git a/studies/psp_DayNight_Cd/protocols/Plant_material_and_growth.md b/studies/psp_DayNight_Cd/protocols/Plant_material_and_growth.md new file mode 100644 index 0000000..095cd96 --- /dev/null +++ b/studies/psp_DayNight_Cd/protocols/Plant_material_and_growth.md @@ -0,0 +1,5 @@ +## Plant Material and Growth Conditions + +For the characterization of the mutant, chlorine gas surface-sterilized seeds of *Arabidopsis thaliana* Col-0 wildtype and *psp* mutant lines #17 and #18 were stratified at 4°C for 2 days. Germination was carried out on half-strength Murashige-and-Skoog (MS) medium (Duchefa Biochemie) containing 0.8% agar (w/v) in growth chambers (12 h light/12 h dark, 22°C day temperature/18°C night temperature, 100 μmol photons m-2 s-1 light intensity, ambient air). After pre-incubation of *psp* mutant plants and Col-0 wildtype control plants on ½ MS plates for 14 days, plants were transferred to soil. Further plant growth occurred in growth chambers under long day conditions (16 h light/8 h dark, 22°C day temperature/18°C night temperature, 100 μmol photons m-2 s-1 light intensity) in high CO2 (3000 ppm CO2). The growing condition allowed us to compare results of *psp* mutants with the *bou-2* mutant. 24 h before harvest, plants were shifted to ambient air conditions. Other growth parameters remained unchanged. + +To examine the significance of the phosphorylated pathway of Ser biosynthesis for Cys synthesis under normal conditions and upon Cd treatment, *Arabidopsis thaliana* Col–0 and *psp* mutant line #17 were used. Prior to cultivation, seeds were surface sterilized with 70% (v/v) ethanol containing 1% (v/v) Triton X-100, twice with 100% ethanol, sown on soil, and incubated at 4°C for at least 2 days to break dormancy. After incubation, seeds were transferred to growth chambers and kept under controlled conditions (12 h light/12 h dark, 22°C day temperature/19°C night temperature, 100–150 μmol photons m-2 s-1, light intensity, 60% relative humidity), at ambient CO2. After emergence of two fully developed leaves, each plant was transferred into a separate pot with new soil substrate and cultivated 4 weeks under the conditions indicated above. One week before harvest, one set of plants was watered with 50 mL 2mM CdCl2 solution in one dose. Experiments were performed with 5-week old plants. \ No newline at end of file -- GitLab