Application of Arabidopsis thaliana At-ACA8 gene in plant stress tolerance and regulation of plant growth and development
A technology for plant growth and transgenic plants, which is applied in the application field of Arabidopsis thaliana At-ACA8 gene in plant stress resistance and regulation of plant growth and development, so as to achieve the effect of improving the ability of plants to resist stress
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Embodiment 1
[0041] Screening of At-ACA8 gene homozygous mutant aca8 and detection of gene expression:
[0042] (1) Screening of At-ACA8 gene homozygous mutant aca8:
[0043] The Arabidopsis thaliana T-DNA insertion mutant (seed number is CS859889) of the At-ACA8 gene (sequence list is shown in Table 1, Table 2, and Table 3), and the homozygous mutant strain was identified by PCR, such as Figure 2A As shown, the primers used are as follows: 5' end primer: 5'-CTTCAACGCCTTTGTTCTCTG-3'; 3' end primer: 5'-CCAGTAATCCAAAGTTGCTCG-3'; LBpROK2: 5'-ATTTTGCCGATTTCGGAA C-3', further sequencing verification found that the The mutant originates from the insertion of T-DNA into exon 34, resulting in gene mutation. The result is as Figure 2B As shown, the identified CS889 were all homozygous.
[0044] (2) Semi-quantitative RT-PCR identification of At-ACA8 gene transcription levels in leaves of Col-0 and mutant aca8:
[0045] Total RNA was extracted from leaves of Arabidopsis thaliana seedlings grown...
Embodiment 2
[0175] Stress resistance of mutant aca8 to low temperature stress:
[0176] (1) Low temperature stress phenotype test of wild-type plants and mutant aca8:
[0177] The Col-0 and aca8 seeds were sterilized and sowed in MS medium containing 1.5% sucrose, vernalized at 4°C for 3 days, and then germinated normally (temperature 22°C, humidity 50%-60%, light Cycle 10h light / 14h dark), the aseptic seedlings after 14 days of culture were subjected to low temperature freezing and thawing experiments. The treatment conditions are: cold acclimation at 4°C for 3 days, rapid cooling to -6°C for 3.5h-4h, heating to 4°C for recovery for one day, and then normal culture at 22°C. Col-0 can be observed after about 3-5 days Phenotypic differences from aca8. like Figure 3A As shown, the survival rate of the sterile seedlings of aca8 was higher after low temperature treatment, and the results showed that the sterile seedlings of mutant aca8 were more resistant to low temperature stress.
[01...
Embodiment 3
[0182] Stress resistance of mutant aca8 to high temperature stress:
[0183] (1) High temperature stress phenotype test of wild-type plants and mutant aca8:
[0184] The Col-0 and aca8 seeds were sterilized and sown in MS medium containing 1.5% sucrose, vernalized at 4°C for 3 days, and then germinated and grew normally (temperature 22°C, humidity 50%-60%, photoperiod 10h of light / 14h of darkness), the aseptic seedlings after 14 days of cultivation were subjected to high-temperature heat tampering and heat shock treatment. Placed at 22°C to resume culture. After about 3-5 days, the phenotypic difference between wild-type Col-0 and mutant aca8 can be observed, such as Figure 4A As shown, the survival rate of aca8 sterile seedlings after high temperature treatment is higher, indicating that the mutant aca8 sterile seedlings are more resistant to high temperature, and the recovery ability after heat shock is stronger than that of the wild type.
[0185] At the same time, high...
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