Plant stomata specific promoter and application thereof
A promoter, plant technology, applied in applications, plant products, climate change adaptation, etc., can solve problems such as lack of stomatal expression specificity
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Embodiment 1
[0091] Embodiment 1, the cloning and isolation of sea island cotton stomatal-specific promoter SLSP (GbSLSP)
[0092] 1. Extraction of sea island cotton total DNA
[0093] The material was taken from Gossypium barbadense L. variety "SHZ2-214" (purchased from Shihezi University in Xinjiang), 1-2g of young leaves, and the total DNA was extracted by CTAB method (Molecular Cloning, third edition). Take 1-5 μl DNA sample, measure its concentration and purity with a UV spectrophotometer, and use 0.8% agarose gel electrophoresis to detect the DNA purity and integrity. Store the extracted DNA at -20°C.
[0094] 2. PCR amplification and recovery of amplified fragments
[0095] The following two primers (primer 1 and primer 2) were designed and synthesized to amplify the stomatal-specific promoter of Gossypium japonicus. In order to facilitate subsequent cloning and construction, restriction endonuclease Hind III was added to the 5' ends of the two primers and BamH I recognition sequ...
Embodiment 2
[0104] Embodiment 2, sea island cotton stomatal-specific promoter (GbSLSP) drives the construction of GUS gene or GFP gene plant expression vector
[0105] Two-step ligation method: pBS-GbSLSP is double-digested with restriction enzymes Hind III and BamH I, electrophoresed on 1.0% agarose gel, and two target bands of about 650bp and about 350bp are cut out, and DNA fragment recovery reagents are used The box (product of Beijing Dingguo Company) was recovered and purified. The target band (target band 1) of about 650 bp after purification is connected with the large fragment (about 13.9 kb) of the plant expression vector pBI121 plasmid (formerly Clontech company product) that has been digested with Hind III and BamH I to obtain a recombinant plasmid It was named pBI-Gb650; the purified target band (target band 2) of about 350 bp was connected to the recombinant plasmid pBI-Gb650 that had been digested with Hind III to obtain the recombinant plasmid pBI-Gb650+350 for transformat...
Embodiment 3
[0108] Embodiment 3, the acquisition and identification of transgenic GbSLSP tobacco and Arabidopsis
[0109] 1. Transformation of Agrobacterium and identification of transformants
[0110] Competent cells of Agrobacterium tumefaciens strains LBA4404 and GV3101 (products of Life Technology, USA) were prepared by the CaCl2 method (Molecular Cloning, third edition). The two recombinant expression vectors (pBI-GbSLSP-GUS and pBI-GbSLSP-GFP) obtained in Example 2 were transferred into the prepared LBA4404 competent cells by the freeze-thaw method ("Molecular Cloning" third edition), and at the same time, the pBI -GbSLSP-GUS vector was transformed into GV3101 competent cells. The transformed LBA4404 bacterial strain is inoculated into the YEB solid medium containing streptomycin (Str) 100mg / L and kanamycin (Kan) 50mg / L, the transformed GV3101 bacterial strain is inoculated into the YEB solid medium containing rifampicin (Rif) 25mg / L. L and Kanamycin (Kan) 50mg / L YEB solid medium,...
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