Application of Asian cotton GaNCED3 gene in improving drought resistance of plants
A technology of drought resistance and plants, applied in the fields of application, plant products, genetic engineering, etc., can solve the problems of limited quantity and complex mechanism of drought resistance response, and achieve the effect of enriching understanding
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Embodiment 1
[0048] Cloning and expression analysis of GaNCED3 gene
[0049] 2.1 Total RNA extraction and cDNA first-strand synthesis
[0050] The Shixiya No. 1 seeds after velvet were sown in wet sand and cultured in an incubator at 27°C with a photoperiod of 16h light / 8h dark. After 5 days, the cotton seedlings were transferred to a hydroponic box containing 1 / 2 Hoagland nutrient solution, and the culture conditions remained unchanged. When the cotton seedlings have 3 true leaves, carry out the drought stress treatment simulated by PEG. The treatment method is to add 17% PEG6000 (w / v) to the hydroponic nutrient solution. Cut the young leaves and roots of cotton seedlings, and use the RNAprep Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit to extract total RNA. cDNA synthesis using reverse transcription kit PrimeScript TM RT reagent Kit, then refer to the fluorescent quantitative PCR kit TB Green TM Premix Ex Taq TM II instructions for real-time fluorescent quant...
Embodiment 2
[0062] GaNCED3 gene silencing reduces drought resistance in cotton plants
[0063] 1. Construction of GaNCED3 gene VIGS silencing vector
[0064] The cDNA of leaves treated with PEG for 2 hours was used as a template, and GaNCED3-V-F and GaNCED3-V-R were used as primers for PCR amplification, and a PCR product with a length of 300bp was obtained.
[0065] GaNCED3-V-F: 5'-GAGTAAGGTTACCGAATTCTCTTCATTTGCCTAAGCAACAATCAC-3', SEQ ID NO: 3;
[0066] GaNCED3-V-R:5'-TGAGCTCGGTACC GGATCC TAGACTCCTTGTATGCAATCCGG-3', SEQ ID NO: 4; where underlined GAATTC and GGATCC The recognition sequences of restriction endonucleases EcoRI and BamHI are indicated respectively, and GAGTAAGGTTACC and TGAGCTCGGTACC are protection bases.
[0067] The PCR product was double-digested with restriction endonucleases EcoRI and BamHI, and the digested product was recovered. The pTRV-RNA2 vector was double-digested with restriction endonucleases EcoRI and BamHI, and the vector skeleton was recovered. Liga...
Embodiment 3
[0082] Overexpression of GaNCED3 gene can improve the drought resistance of transgenic Arabidopsis
[0083] 1. Construction of recombinant plasmids
[0084] The cDNA of leaves treated with PEG for 2 hours was used as a template, and GaNCED3-F and GaNCED3-R were used as primers for PCR amplification.
[0085] GaNCED3-F:5’-GAACACGGGGGAC TCTAGA ATGGCTTCATCAACAGCAGC-3', SEQ ID NO: 6; GaNCED3-R: 5'-TGAACGATCGGGGAAATTC GAGCTC TTAGGCCTGTTTTTCCAAGTCC-3', SEQ ID NO: 7; where underlined TCTAGA and GAGCTC The recognition sequences of restriction endonucleases XbaI and ScaI are indicated respectively, and GAACACGGGGGAC and TGAACGATCGGGGAAATTC are protected bases.
[0086] The above PCR product and pBI121 vector were digested with restriction endonucleases XbaI and ScaI. Ligate the recovered enzyme-digested product and the vector backbone to construct a recombinant plasmid, and perform sequencing verification on the recombinant plasmid.
[0087] The results showed that the recomb...
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