第三方(3)


2017519(金)

The transgenic T1 populations were screened


The transgenic T1 populations were screened on MS agar media containing 25 mg l-1 Hygromycin and 25 mg l-1 Basta. To generate GFP-PPKs lines, the coding sequences of PPKs were PCR-amplified and cloned into a Ti plasmid (pFGFP) modified from pCambia3301 by the In-Fusion cloning method, resulting in expression of GFP-PPKs driven by the ACTIN2 promoter (pACT2::GFP-PPKs). Ti plasmids expressing recombinant proteins were introduced into rdr6-11 allele, which suppresses gene silencing, by the floral-dip method51. The transgenic T1 populations were screened on compound soil sub-irrigated with the Basta solution. Plants were grown in walk-in growth chambers at 22 °C, 65% relative humidity under cool white fluorescent tubes. Long-day (LD) photoperiod is defined as 16 h light/8 h dark. Light-emitting diode was used to obtain monochromatic blue light (peak 450 nm; half-bandwidth of 20 nm), red light (peak 660 nm; half-bandwidth of 20 nm) or far-red light (peak 730 nm; half-bandwidth of 20 nm).



2017519(金)

Yeast two-hybrid assays


The prey vector pGADT7 expressing PPK or PPKC fused to the GAL4 activation domain and the Bait vector pBridge expressing CRY2 fused to the GAL4 DNA binding domain were used. The pairs of plasmids were co-transformed into yeast strain AH109. Colonies were selected on plates (SD-LW). After culture in SD-LW medium for 16 h (Dark), transformants were subcultured into fresh SD-LW medium and kept in dark or irradiated with blue light (30 mol m-2 s-1). β-galactosidase activity was measured using chlorophenol red-β-D-galactopyranoside as substrate and Miller Units were calculated according to the Clontech Yeast Protocols Handbook.



2017519(金)

The transgenic T1 populations


The transgenic T1 populations were screened on MS agar media containing 25 mg l-1 Hygromycin and 25 mg l-1 Basta. To generate GFP-PPKs lines, the coding sequences of PPKs were PCR-amplified and cloned into a Ti plasmid (pFGFP) modified from pCambia3301 by the In-Fusion cloning method, resulting in expression of GFP-PPKs driven by the ACTIN2 promoter (pACT2::GFP-PPKs). Ti plasmids expressing recombinant proteins were introduced into rdr6-11 allele, which suppresses gene silencing, by the floral-dip method51. The transgenic T1 populations were screened on compound soil sub-irrigated with the Basta solution. Plants were grown in walk-in growth chambers at 22 °C, 65% relative humidity under cool white fluorescent tubes. Long-day (LD) photoperiod is defined as 16 h light/8 h dark. Light-emitting diode was used to obtain monochromatic blue light (peak 450 nm; half-bandwidth of 20 nm), red light (peak 660 nm; half-bandwidth of 20 nm) or far-red light (peak 730 nm; half-bandwidth of 20 nm).



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