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studies/psp_DayNight_Cd/protocols/Generation_of_psp.md

+## 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.
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