Calcium dependent protein kinase CPK32, coding gene and application thereof
A protein kinase, encoding gene technology, applied in the field of genetic engineering, to achieve the effect of great application value
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Embodiment 1
[0058] Example 1 Cloning of CPK32 protein and its coding gene and genome nucleic acid sequence
[0059] (1) Cloning of CPK32 protein coding gene
[0060] Trizol (Invitrogen) method was used to extract the total RNA of Arabidopsis thaliana seedlings (100-200 mg) cultured in a long-day photoperiod growth chamber (16 hours of light / 8 hours of darkness) for 7 days, and the integrity of the RNA was checked by formaldehyde-denatured RNA agarose gel electrophoresis sex. According to SUPERSCRIPT II instructions for synthesizing single-stranded cDNA. The synthesized single-stranded cDNA was diluted 10 times and used as template DNA, and the primer pair consisting of Primer 1 and Primer 2 was used for PCR reaction.
[0061] Primer 1: 5'-gctctagaATGGGTAATTGTTGCG-3'; (SEQ ID NO.5)
[0062] Primer 2: 5'-cgagctcTCATCTTGTATCACCA-3' (SEQ ID NO. 6).
[0063] PCR system (50 μL): 5 μL 10×Ex-taq PCR Buffer, 4 μL 2.5mMdNTP mix, 2.5 μL Primer 1 (10 μM), 2.5 μL Primer 2 (10 μM), 1 μL template D...
Embodiment 2
[0075] Example 2 Functional verification of CPK32 protein
[0076] 1. Construction of recombinant plasmids
[0077]1. Using the pMD18-T-CPK32-GENOME plasmid DNA molecule as a template, the primer pair (shown in SEQ ID NO.7-8) composed of Primer 3 and Primer 4 was used to perform PCR amplification to obtain the PCR amplification product, which was recovered about 4300bp DNA fragment.
[0078] 2. Digest the DNA fragment obtained in step 1 with restriction endonucleases BamHI and SalI, and recover the digested product.
[0079] 3. Digest pCAMBIA1300 vector (this plasmid is also called plant expression vector pCAMBIA1300-AT, GENBANK ACCESSION NO.FJ362601, VERSION FJ362601.1, GI: 213876770) with restriction endonucleases BamHI and SalⅠ, and recover the vector of about 10000 bp skeleton.
[0080] 4. Ligate the digested product of step 2 with the vector backbone of step 3 to obtain recombinant plasmid A. According to the sequencing results, the structure of the recombinant plasmi...
Embodiment 3
[0103] Example 3 Detection of FLC expression
[0104] The plants to be tested are: T of strain 1 3 Generation plant, T of line 2 3 Generation plant, T of line 3 3 Generation plants, Arabidopsis thaliana cpk32 mutant and Columbia ecotype Arabidopsis Col-0.
[0105] After the experimental materials were grown for 7 days in a long-day photoperiod (16 hours of light / 8 hours of darkness), total RNA was extracted by the Trizol method.
[0106] (1) Formaldehyde denaturing gel 1.2% agarose gel in 1×MOPS (MOPS 0.2M, NaAc
[0107] (2) Sample preparation: Take 15-20μg RNA into a 1.5mL RNase-free centrifuge tube, add DEPC water to 20μL, add 20μL sample buffer, heat at 65°C for 5 minutes, cool on ice, add 3μL Loading buffer, Mix well.
[0108] (3) Put the gel into the electrophoresis tank, cover the gel with 1×MOPS solution, and pre-electrophoresis for 15 minutes.
[0109] (4) Spotting, 100-120V (5V / cm), electrophoresis for 2-2.5 hours.
[0110] (5) During electrophoresis, prepare t...
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