Protein CkWRKY33 and coding gene and application thereof
A protein and coding technology, applied in the fields of application, genetic engineering, plant gene improvement, etc., can solve the problem of little research on stress-related genes
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Embodiment 1
[0063] Example 1. Cloning of the coding gene of protein CkWRKY33 (i.e. CkWRKY33 gene)
[0064] 1. Sow the seeds of Caragana korshinskii on sandy soil and manage the normal growth to obtain a Caragana korshinskii plant that has grown for one month; then stop watering for 20 days to obtain a drought-treated Caragana korshinskii plant.
[0065] The drought-treated Caragana korshinskii plant has a well-developed root system, white leaves and thick epidermal hairs, which have strong drought resistance.
[0066] 2. Take the leaves of the drought-treated Caragana korshinskii plant obtained in step 1, extract total RNA first, and then reverse transcription to obtain the cDNA of Caragana korshinskii.
[0067] 3. After completing step 2, use the cDNA of Caragana korshinskii as a template, and use primer F: 5'-ATGACTATGGATGATCATAACTG-3' and primer R: 5'-TTAGAAGTCCTTTGACATAAAT-3' to perform PCR amplification, and then recover About 1614bp PCR amplification product.
[0068] 4. Sequencing the PCR a...
Embodiment 2
[0071] Example 2. Obtainment of transgenic CkWRKY33 gene Arabidopsis and identification of drought resistance
[0072] 1. Obtaining the recombinant plasmid pBinGlyRed3-CkWRKY33
[0073] 1. Cut pBinGlyRed3 vector with restriction enzymes EcoRI and XmaI (Zhang C, Iskandarov U, Klotz ET, et al. Athraustochytrid diacylglycerol acyltransferase 2 with broadsubstrate specificity strongly increases oleic acid content in engineered Arabidopsis thaliana seeds. Journal of Experimental Botany, 2013 , 64(11):3189-3200.), and recover a vector backbone of about 9kb.
[0074] 2. Using the cDNA of Caragana korshinskii obtained in step 2 in step 1 as a template, using primer FF: 5’-ACGGGGGACT GA ATTC ATGACTATGGATGATCATAACTG-3' (underlined is the recognition site of restriction enzyme EcoRI) and primer FR: 5'-CCGCCTCGAG CCCGGG A primer pair consisting of TTAGAAGTCCTTTGACATAAAT-3' (underlined is the recognition site of restriction enzyme XmaI) was subjected to PCR amplification, and a PCR amplificat...
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