Application of birch bpspl2 gene in regulating adventitious root development of birch
A technology of root development and white birch, applied in application, genetic engineering, plant genetic improvement, etc., can solve the problems of gene function redundancy, loss of single gene function, failure of homologous gene function compensation, high homology of coding sequence, etc.
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Embodiment 1
[0037] Construction of Inhibition Fusion Expression Vector pROKⅡ-BpSPL2-SRDX
[0038] 1. Obtaining of BpSPL2-SRDX fusion fragment
[0039]The 1563bp sequence after the stop codon TGA was removed from the white birch BpSPL2 gene, then with the pMD18-T plasmid containing the BpSPL2 gene ORF sequence as a template, BpSPL2F (SEQ ID NO: 3) and BpSPL2-SRDXR (SEQ ID NO: 4) as primers, amplified by PCR The target fusion fragment of BpSPL2-SRDX containing BamHI and KpnI restriction sites was added. 1% agarose gel electrophoresis to detect the PCR band to obtain the target fragment.
[0040] PCR amplification reaction system: sterile water 30.75 μL, TaqBuffer 5 μL, MgCl 23 μL, dNTPs 4 μL, primers F and R 2 μL, template 2 μL, Taq enzyme 1.25 μL.
[0041] Reaction program: pre-denaturation at 98°C for 3min, denaturation at 98°C for 30s, annealing at 60°C for 30s, extension at 72°C for 2min, extension at 72°C for 7min, a total of 35 cycles.
[0042] 2. Ligation of BpSPL2-SRDX sequence a...
Embodiment 2
[0052] Obtaining pROKⅡ-BpSPL2-SRDX and pGWB5-BpSPL2-GFP Transgenic Birch
[0053] 1. Genetic transformation by leaf disc method
[0054] (1) Preparation of Agrobacterium engineering bacteria liquid: pick pROKⅡ-BpSPL2-SRDX or pGWB5-BpSPL2-GFP (preserved in our laboratory) Agrobacterium single colony one day before the experiment, cultivate overnight at 28°C with LB liquid medium, and then Determination of bacterial liquid OD 600 When it is 0.8-1.0, dilute the bacterial solution 20 times, and continue to cultivate at 28°C until the OD of the bacterial solution 600 between 0.6-0.8, resuspend the bacteria with liquid WPM (6-BA 2.0mg / L+NAA 0.2mg / L) as the infection engineering bacteria solution.
[0055] (2) Selection of explants: choose the leaves of rooted seedlings with bright green leaves and larger leaf area, generally the third and fourth leaves at the top.
[0056] (3) Cutting of explants: the part with the strongest meristematic ability is near the base of the petiole 0....
Embodiment 3
[0067] Phenotype Observation of Adventitious Root Occurrence Regulated by Transgenic Birch
[0068] The wild-type, overexpression (OE3, OE5) and suppressed expression (DR1, DR4) transgenic white birch microcuttings with consistent growth status were placed in WPM medium containing 0.4mg / L IBA, and the number of adventitious roots and rooting time were observed after 4 weeks , rooting rate and adventitious root phenotype. Three independent experiments were performed per line, and each experiment was biologically replicated 40 times. Counting the number of indefinite roots, the results are as follows Figure 6 As shown, the average number of adventitious roots of BpSPL2-SRDX suppressed plants was 8, and the average number of adventitious roots of WT was 5, indicating that the number of adventitious roots of BpSPL2-SRDX plants was more than that of wild type.
[0069] In terms of adventitious root formation time, BpSPL2-SRDX transgenic plants began to develop adventitious roots...
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