Promoter with both plant overground tissue organ specificity and photoinduced specificity and application thereof
A tissue organ and promoter technology, applied in the field of plant genetic engineering, can solve problems such as gene silencing, death, and plant metabolic imbalance, and achieve good regulation and wide application value
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Embodiment 1
[0034] Example 1: Cloning with Plant Aboveground Organs and Light-Inducible Specific Promoters
[0035] First, using the Arabidopsis genomic DNA as a template, the PCR method was used to amplify a 1851bp promoter with both plant aboveground tissues and organs and light-induced specificity, and the amplified product was recovered and TA cloned.
[0036] (1) PCR amplification of the target fragment:
[0037] ①According to known Arabidopsis AtcpSecY2 Design specific primers for the sequence of the gene promoter region, and introduce in the upstream primer Xba I restriction site, introduced in the downstream primer Nco I restriction site.
[0038] Upstream primer: 5'- TCTAGA CTTGTCGACAACTACTGATCCG-3' (introduced Xba I restriction site)
[0039] Downstream primer: 5'- CCATGG CGAGAGGATGAGGAGAGAAA-3' (introduced Nco I restriction site)
[0040] ② Extract Arabidopsis genomic DNA according to conventional methods, use the genomic DNA as a template, and use the above ...
Embodiment 2
[0059] Embodiment 2: utilize pCAMBIA1301 carrier (containing GUS Reporter gene) construction "both plant above-ground tissue organs and light-inducible specific promoter- GUS "Fusion gene
[0060] (1) Extract vector pCAMBIA 1301 plasmid (purchased from CAMBIA Company) from Escherichia coli, use Xba I / Nco Reclaim the large carrier fragment (which contains GUS reporter gene sequence).
[0061] (2) Extract the plasmid from the TA clone prepared in Example 1, use Xba I / Nco I double enzyme digestion, recovery by agarose gel electrophoresis (same as Example 1) Arabidopsis thaliana has both aerial tissues and organs and light-induced specific promoter fragments.
[0062] (3) The above two fragments were ligated overnight at 16°C under the catalysis of ligase to complete the "both plant aerial tissue and organ and light-inducible specific promoter- GUS "Fusion gene construction.
[0063] Connection system:
[0064] Reagent Amount added (μl) B...
Embodiment 3
[0073] Preparation of transgenic Arabidopsis plants
[0074] (1) The "both plant aboveground tissue and organ and light-inducible specific promoter" constructed in Example 2- GUS "The fusion gene was transformed into Arabidopsis thaliana. The specific transformation method used the method of Floral dip mediated by Agrobacterium (Clough and Bent, 1998). The obtained seeds were subjected to 50 mg l -1 For hygromycin resistance screening, normal growing plants were transferred to soil for culture.
[0075] (2) PCR detection of transgenic plants: cut out the leaves of transgenic plants and wild-type plants respectively, and extract genomic DNA from leaves by referring to the "Molecular Cloning Experiment Guide (Third Edition)" (Huang Peitang et al., 2002), and perform PCR with the following primers Reaction, reaction system is as embodiment 1:
[0076] Upstream primer: 5'- TCTAGACTTGTCGACAACTACTGATCCG-3'
[0077] Downstream primer: 5'-TACAGTCTTGCGCGACATGCG-3'
[0078] The PCR...
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