Application of protein ghccoaomt7 in regulation of plant heat tolerance
A technology of protein and heat resistance, applied in the fields of application, plant peptides, plant products, etc., can solve the problems of cotton bud shedding, boll development deformity, cotton production reduction, etc.
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Embodiment 1
[0060] Cloning of the coding gene (GhCCoAOMT7 gene) of embodiment 1, protein GhCCoAOMT7
[0061] 1. Using the Trizo1 method to extract the total RNA from the leaves of upland cotton TM-1 seedlings grown to 14 days, and then using reverse transcriptase AMV to reverse transcribe the first-strand cDNA to obtain the cDNA of upland cotton TM-1.
[0062] 2. Using the cDNA of Upland Cotton TM-1 obtained in step 1 as a template, PCR amplification was performed using a primer pair consisting of Forward Primer: 5'-ATGATTTGTTTCTCAAGCATATATTTG-3' and Reverse Primer: 5'-TCAACGAATTCGCCTGCA-3' to obtain approximately 750bp double-stranded DNA molecule.
[0063] The reaction system is 50 μL, consisting of 2 μL template (containing 1 μg of upland cotton TM-1 cDNA), 1 μL Phusion enzyme, 2.5 μL Forward Primer aqueous solution (concentration is 10 μM), 2.5 μL Reverse Primer aqueous solution (concentration is 10 μM), 10 μL 5×PhusionBuffer, 10 μL dNTP mixture (the concentrations of dATP, dTTP, dGT...
Embodiment 2
[0066] Example 2, the acquisition and identification of Arabidopsis transgenic GhCCoAOMT7
[0067] 1. Acquisition of recombinant plasmids 35S::GhCCoAOMT7-EYFP and GV3101 / 35S::GhCCoAOMT7-EYFP
[0068] 1. Insert the double-stranded DNA molecule shown in position 1 to 744 of Sequence 1 in the sequence table between the recognition sequences of restriction enzymes Sal I and BamH I of the vector pEZR-LNY to obtain a recombinant plasmid 35S::GhCCoAOMT7-EYFP.
[0069] The recombinant plasmid 35S::GhCCoAOMT7-EYFP expresses the protein GhCCoAOMT7 shown in Sequence 2 in the Sequence Listing.
[0070] 2. The recombinant plasmid 35S::GhCCoAOMT7-EYFP was introduced into Agrobacterium tumefaciens GV3101 to obtain the recombinant Agrobacterium, named GV3101 / 35S::GhCCoAOMT7-EYFP.
[0071] The vector pEZR-LNY was introduced into Agrobacterium tumefaciens GV3101 to obtain the recombinant Agrobacterium, named as GV3101 / pEZR-LNY.
[0072] 2. Acquisition of transgenic Arabidopsis thaliana
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Embodiment 3
[0107] Example 3, Acquisition and Identification of Upland Cotton TM-1 Silent Strain
[0108] Infection solution: containing 10mM MES, 10mM MgCl 2 and 200 mM acetosyringone in water.
[0109] 1. Construction of recombinant plasmid PTRV2-GhCCoAOMT7 and acquisition of recombinant Agrobacterium
[0110] 1. Using the Trizo1 method to extract the total RNA from the leaves of upland cotton TM-1 seedlings grown to 14 days, and then using reverse transcriptase AMV to reverse transcribe the first-strand cDNA to obtain the cDNA of upland cotton TM-1.
[0111] 2. Using the cDNA of upland cotton TM-1 obtained in step 1 as a template, a primer pair consisting of primer F: 5'-CGACGACAAGACCCTAAAGATAATGAAGGGAGTTTTGATTTT-3' and primer R: 5'-GAGGAGAAGAGCCCTTCTATGATCGACCTCCTCGATT-3' was used for PCR amplification to obtain PCR amplified product.
[0112] The reaction system was 50 μL, consisting of 2 μL template (containing 1 μg of upland cotton TM-1 cDNA), 1 μL Phusion enzyme, 2.5 μL primer ...
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