Method for improving salt stress resistance capacity of plants
A salt stress, plant technology, applied in the field of improving the ability of plants to resist salt stress, can solve the problems of reducing the performance of salt stress resistance of plants, and achieve the effect of improving the salt resistance of plants and improving the ability of plants to resist salt stress
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[0036] Example 1. Extraction of Arabidopsis genomic DNA
[0037] Reagents:
[0038] Lysis Buffer: Tris-HCl, pH8.0 (0.2M); Urea (7M); Sodium lauryl sarcosinate (2%); EDTA (0.05M) Steps:
[0039] (1) Put an appropriate amount of the plant's green leaves or flowers and other tissues in a 1.5ml centrifuge tube, add 200μl of lysis solution, grind it with a small stick, and then rinse the small stick with 400μl of lysis solution.
[0040] (2) Add 600μl of phenol-chloroform and shake vigorously for 20 seconds to denature the protein.
[0041] (3) Centrifuge at 12000rpm for 5 minutes and take the supernatant.
[0042] (4) Add 50μl sodium acetate and 600μl isopropanol, mix well, and centrifuge at 12000rpm for 5min.
[0043] (5) Remove the supernatant and dissolve the precipitate in 400μl TE (Tris-EDTA).
[0044] (6) Add 40μl 3mol / L NaAC(PH5.2), 1ml absolute ethanol, mix well, centrifuge at 12000rpm for 5min.
[0045] (7) Remove the supernatant, wash the precipitate with 70% ethanol once, centrifuge;...
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[0047] Example 2 (a), PCR method to obtain the target gene fragment
[0048] (1) Using the Arabidopsis DNA extracted in Example 1 as a template
[0049] The polynucleotide sequence of the URO gene is shown in sequence 1 in the sequence listing.
[0050] The polynucleotide sequence of the promoter is shown in sequence 2 in the sequence listing.
[0051] (2) Primer design
[0052] According to the URO gene and its promoter sequence, and the convenience of purchasing and cloning, design a pair of primers, URO-F and URO-R. The former has a restriction endonuclease site for Xhol, and the primer sequence is :
[0053] Upstream primer URO-F: 5'ctc gag atg aac cac cgg gac aaa c, as shown in sequence 3.
[0054] The downstream primer URO-R: 5'tta atg atg acg atg acc g is shown in sequence 4.
[0055] (3) PCR amplification of the target fragment
[0056] PCR reagent: KOD-Plus enzyme was purchased from TOYOBO
[0057] PCR reaction system:
[0058]
[0059] PCR reaction program:
[0060] 94℃ 2min
[0061]...
Example Embodiment
[0075] Example 3. Cloning of PCT product
[0076] Reagents: The T-Vector used is pMD18-T Simple Vector, purchased from Takara
[0077] Connection system:
[0078]
[0079] Place the ligation system at 4°C or 16°C overnight, or 25°C for two hours to obtain recombinant plasmids.
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