Protein of adjusting plant tolerant to low-phosphorus stress and its coding gene and application thereof
A gene-encoded, low-phosphorus-resistant technology, applied in the field of bioengineering, can solve problems such as unknown effects
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Embodiment 1
[0028] Embodiment 1, the acquisition of MAX4 and its coding gene
[0029] 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 SynthesisKit (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'-TGCCCTCTTTCCAATACACTG-3', primer 2: 5'-AACTGTATCCACCCAATCCG-3'. Pre-denatured at 94°C for 3 minutes, then 30 sec at 94°C, 30 sec at 54°C, and 60 sec at 72°C for a total of 35 cycles, and finally extended at 72°C...
Embodiment 2
[0030] Example 2, Cultivation of Arabidopsis thaliana with Enhanced Low Phosphorus Tolerance
[0031] 1. The acquisition of homozygous mutants max4-7 and max4-8 with the MAX4 gene knocked out
[0032] Two T-DNA insertion mutant seeds were obtained from the American Arabidopsis Germplasm Center (SALK_072708, max4-7; SALK_147538, max4-8; http: / / www.arabidopsis.org / servlets / TairObject?type=germplasm&id =4626493). In order to identify homozygous mutants whose MAX4 gene has been knocked out, the seeds of SALK_072708 and SALK_147538 were planted on soil culture, the RNA of a single plant was extracted, and after reverse transcription into cDNA, the cDNA was used as a template to perform PCR with the following primers Amplification: max4-7, primer 1 LBb1: 5'-GCGTGGACCGCTTGCTGCAACT-3, primer 2: MAX4-7LP 5'-AGTACGACGACGTATTGTCCG-3', primer 3: MAX4-7RP 5'-GGATTGATGCTGCACATATCC-3'; max4-8, primer 1LBb1: 5'-GCGTGGACCGCTTGCTGCAACT-3, primer 4: MAX4-8LP 5'-LPAGACAAACCCCATTCATCATG-3', prim...
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