Brachypodium distachyon Drought Tolerance Tolerance Gene And Its Encoded Protein And Its Application
A gene and drought technology, which is applied to Brachypodium erinaceus drought resistance and salt resistance gene and expression vector and its encoded protein and application fields, can solve problems such as deterioration, quality decline, crop yield reduction, etc., and achieves reduction of oxidative damage and improvement of tolerance. the effect of enhancing stability
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Embodiment 1
[0036] Example 1: Isolation of Brachypodium distachyon BdGF14a gene
[0037] 1. Cloning of BdGF14a gene
[0038] The 14-3-3 gene sequence (BRADI1G11290.1, GenBank: KU933262.1) obtained by Phytozome v12.1 (https: / / phytozome.jgi.doe.gov / pz / portal.html) Blast was submitted to NCBI (https: / / www.ncbi.nlm.nih.gov) for BLAST comparison analysis. Use the Primer6 primer design software to specifically amplify the primers for Brachypodium distachyon BdGF14a. The primer sequences are shown in SEQ ID NO.3 and SEQ ID NO.4. The PCR reaction system is shown in Table 1. The PCR program is as follows: pre-denaturation at 98°C for 3 minutes; Denaturation at 98°C for 15s; annealing at 65°C for 5s; extension at 72°C for 30s; final extension at 72°C for 10min; storage at 4°C. Among them, the three consecutive steps of denaturation, annealing and extension set up 35 cyclic reactions.
[0039] Table 1 PCR reaction system
[0040]
[0041] 2. Gel recovery of target fragments
[0042] Spot th...
Embodiment 2
[0065] Example 2: Construction of pBI121-BdGF14a-GFP eukaryotic expression vector
[0066] 1. PCR amplification of BdGF14a gene
[0067] Using the cloning vector pMD18-T-BdGF14a containing the correctly sequenced Brachypodium distachyon BdGF14a gene as a template, use the Oligo7 primer design software to design the 5' end with pBI121 vector multi-cloning site region XbaI restriction site and BamHI enzyme The gene-specific primers at the cleavage site were used for PCR amplification. The primer sequences are shown in Tables SEQ ID NO.5 and SEQ ID NO.6. The PCR reaction system is shown in Table 3 / same as Table 1. The PCR program is as follows: pre-denaturation at 98°C for 2 minutes; 98°C Denaturation at ℃ for 10s; annealing at 65℃ for 5s; extension at 72℃ for 30s; final extension at 72℃ for 10min; storage at 4℃. Among them, the three consecutive steps of denaturation, annealing and extension set up 35 cyclic reactions.
[0068] Table 3 PCR reaction system
[0069]
[0070]...
Embodiment 3
[0116] Example 3: Subcellular localization of Brachypodium distachyon BdGF14a protein
[0117] 1. Preparation and transformation of Agrobacterium competent
[0118] All the operation steps of the preparation and transformation experiment of Agrobacterium competent cells were carried out aseptically in the ultra-clean workbench. The specific process is as follows:
[0119] 1) Take the preserved Agrobacterium strain EHA105 from a -80°C ultra-low temperature refrigerator and inoculate it on the YEB solid medium containing 50 mg / L Str (streak), and culture it in the dark at 28°C for 2-3 days.
[0120] 2) Pick large and round monoclonal colonies, inoculate them into 1 mL of YEB liquid medium containing 50 mg / L Str, and incubate in the dark at 200 rpm at 28°C for 12 to 16 hours.
[0121] 3) Take 1 mL of the above-mentioned bacterial solution and transfer it to 100 mL of YEB (containing 50 mg / L streptomycin), expand the culture on a shaker at 28 ° C at 200 rpm, and measure its OD va...
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