Application of Arabidopsis at5g10290 Gene and Its Mutants in Improving Plant Drought Tolerance
An AT5G10290, Arabidopsis technology, applied in the application of improving plant drought tolerance, in the field of Arabidopsis AT5G10290 gene and its mutants, can solve problems such as unidentified tissue-specific genes
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[0033] In the present invention, the preparation method of the transgenic plant containing the Arabidopsis AT5G10290 gene or the overexpression vector preferably comprises the following steps:
[0034] Inserting the Arabidopsis AT5G10290 gene into the base vector to obtain the Arabidopsis AT5G10290 gene overexpression vector;
[0035] The Arabidopsis AT5G10290 gene overexpression vector was transformed into plants by Agrobacterium-mediated transformation, and transgenic plants were obtained after verification. Compared with the wild type, the transgenic plants carrying the Arabidopsis AT5G10290 gene are hypersensitive to ABA; at the same time, the obtained transgenic plants are significantly higher than the wild type in terms of leaf wilting, leaf water content and MDA content after drought stress, This shows that the transgenic plants have achieved stronger drought resistance.
[0036] The present invention also provides the application of the Arabidopsis AT5G10290 gene and ...
Embodiment 1
[0041] Construction method of AT5G10290 gene overexpression vector (35S::AT5G10290)
[0042] The AT5G10290 gene and the pEGAD vector were digested with EcoRI and SmaI, respectively, and cut into fragments with cohesive ends. The two fragments were ligated overnight at 16°C, and the resulting ligation products were transformed into E. coli DH5α. The transformed Escherichia coli was screened in glufosinate-resistant LA solid medium, single colonies were picked, plasmids were extracted, and the insertion of foreign fragments was identified by single enzyme digestion, double enzyme digestion, and PCR. The pEGAD vector contains an EGFP reporter gene and a herbicide resistance gene, which are independently regulated by the CaMV 35S promoter at the front of each. The specific operation steps are as follows:
[0043] 1. The AT5G10290 gene fragment and the pEGAD vector were double-digested with EcoRI and SmaI respectively, and cut into sticky ends. The double-digestion system is as fo...
Embodiment 2
[0091] Agrobacterium Transformation and Identification of AT5G10290 Gene Overexpression Vector
[0092] 1. Preparation method of Agrobacterium EHA105 competent cells
[0093] (1) Agrobacterium EHA105 was inoculated in 20 ml of LB liquid medium, and cultured overnight at 28° C. with shaking at 220 rpm.
[0094] (2) The next day, take 1ml of the activated bacterial solution and inoculate it into 100ml LB liquid medium, and cultivate it at 28°C and 220rpm to OD 600 The value is 0.4 to 0.5.
[0095] (3) Place the bacterial solution in an ice bath for 30 minutes.
[0096] (4) Centrifuge at 12000 rpm for 10 min at 4°C to collect the bacteria.
[0097] (5) Suspend the precipitate with 20ml of 0.5M pre-cooled NaCl solution, and ice-bath for 20min.
[0098] (6) Centrifuge at 12,000 rpm for 10 min at 4°C to collect bacteria.
[0099] (7) Discard the supernatant and use 2ml of 20mM ice-cold CaCl 2 The solution was suspended and preserved by adding glycerol to a final concentration ...
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