Plants cold-tolerant and drought resisting protein, encoding genes and use thereof
A gene-encoded, cold-resistant technology, applied in the field of bioengineering, can solve problems such as not found
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Embodiment 1
[0031] Example 1, the acquisition of LWT2-1 and its coding gene
[0032] About 50-100 mg of wild-type Colombian ecotype Arabidopsis seedlings were used as materials, the total RNA was extracted with Trizol, and the RNA concentration was detected with an ultraviolet spectrophotometer. Press RevertAid TM First Strand cDNA Synthesis Kit (Fermentas Company) kit manual, using the extracted total RNA as a template to synthesize the first strand of cDNA. Using the extracted first-strand cDNA as a template, perform the following PCR reaction: 20 μl reaction system, containing 2 μl of 10×PCR buffer, 0.4 μl of dNTPs (10 mM) mixture, 2 μl each of primer 1 and primer 2, and Tag enzyme (5 U / μl) 0.2μl, and add double distilled water to the rest to 20μl. Among them, primer 1: 5'-AAAACATCAAAATGGCTGAAAAAG-3', primer 2: 5'-TCATTAAACTGACTGCTGAGAAATG-3'. Amplification on the Life Express gene thermal cycler: first 94°C pre-denaturation for 3min, then 94°C for 30sec, 54°C for 30sec, 72°C for ...
Embodiment 2
[0033] Embodiment 2, cultivating Arabidopsis thaliana with enhanced cold resistance and drought resistance
[0034] 1. Obtaining the homozygous mutant lwt2-1 whose LWT2-1 gene has been knocked out
[0035]T-DNA insertion mutant seeds were obtained from the American Arabidopsis Germplasm Center (SALK_023905; http: / / www.arabidopsis.org / servlets / TairObject?type=germplasm&id=4626493). To identify homozygous mutants with knockout of the LWT2-1 gene, SALK_023905 seeds were sown and DNA was extracted from a single plant, which was used as a template for PCR amplification with 3 pairs of primers. Among them, primer 1 LBb1: 5'-GCGTGGACCGCTTGCTGCAACT-3, primer 2: LWT2-LP 5'-AAGCACAAACGTGGTTACCTG -3', primer 3: LWT2-RP5'-GTCGCTTTGCTATGCTCAAAG-3'. According to the results of agarose gel electrophoresis of PCR products, a homozygous mutant lwt2-1 in which the LWT2-1 gene was knocked out was obtained. From the phenotype point of view, there was no significant difference between the mutant...
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