Chinese medicine composition anti-wrinkle multi-function liquid, its preparation and application
A gene and protein technology, which is applied to plant resistance-related protein ATSAR42 and its encoding gene and application fields, can solve the problems of increasing salicylic acid, increasing the synthesis level of salicylic acid, which have not yet been found, and achieves the effect of enhancing disease resistance.
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Embodiment 1
[0061] Embodiment 1, the acquisition of ATSAR42 protein and its coding gene
[0062] 1. According to the existing NCBI database and literature, set a pair of primers as follows:
[0063] F1 (forward primer): 5'-ccg ctcgag tttgctttgtatgctcaacaatt-3' (XhoI restriction recognition sequence is underlined);
[0064] R1 (reverse primer): 5'-cg ggatcc atctaggagccaactctgtg-3' (the BamHI restriction recognition sequence is underlined).
[0065] 2. Genomic DNA was extracted from leaves of Arabidopsis thaliana ecotype Columbia.
[0066] 3. Using genomic DNA as a template, use the primer pair designed in step 1, and perform PCR amplification with PrimeSTAR HS high-fidelity enzyme from TaKaRa Company.
[0067] 4. The PCR amplification product is sequenced, as shown in sequence 2 of the sequence listing.
[0068] The protein shown in Sequence 1 of the sequence listing is named ATSAR42 protein. The gene encoding the ATSAR42 protein is named as the ATSAR42 gene, and its genomic DNA is...
Embodiment 2
[0069] Embodiment 2, the cloning of each gene and the construction of its recombinant expression vector
[0070] 1. Acquisition of ATSAR42 gene and construction of recombinant plasmid 326-ATSAR42-FLAG
[0071] 1. Genomic DNA was extracted from leaves of Arabidopsis thaliana ecotype Columbia.
[0072] 2. Using the genomic DNA in step 1 as a template, use the primer pair composed of F1 and R1 to carry out PCR amplification under the action of TaKaRa's PrimeSTAR HS high-fidelity enzyme to obtain PCR amplification products. The agarose gel electrophoresis of the PCR amplification product is shown in figure 1 (M represents a nucleotide marker, which is DL2000 from TaKaRa Company).
[0073] 3. The PCR amplified product of step 2 was double-digested with restriction endonucleases XhoI and BamH I, and the digested product was recovered.
[0074] 4. Digest the 326-FLAG expression vector with restriction endonucleases XhoI and BamH I, and recover the vector backbone of about 3827bp.
...
Embodiment 3
[0096] Embodiment 3, the application of ATSAR42 gene in inducing ICS1 gene promoter (ProAtICS1) to start gene expression
[0097] The recombinant plasmid 326-ATSAR42-FLAG and recombinant plasmid 326-Pro constructed in Example 2 AtICS1 ::GFP co-transformed Arabidopsis protoplasts (recombinant plasmid 326-T 7 -FLC is used as a negative control of the recombinant plasmid 326-ATSAR42-FLAG; the 326-FLAG expression vector is used as another negative control of the recombinant plasmid 326-ATSAR42-FLAG), the specific steps are as follows:
[0098] 1. Germinate Colombian ecotype Arabidopsis seeds on MS medium, transplant them into soil when the roots grow to 1-3 cm, and cultivate them in a greenhouse at 23° C. (12 hours of light per day, light intensity of 150 μE).
[0099] 2. Add 20ml of double distilled water to a 90mm petri dish, then add 1.82g of D-mannitol and dissolve it.
[0100] 3. Take the unbolted leaves (about 90 pieces) that have been cultured for 4 weeks in step 1, cut t...
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