A protein related to plant stress tolerance and its coding gene scmyb3r1 and application
A technology for encoding genes and plants, applied in the direction of plant gene improvement, application, plant peptides, etc.
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Embodiment 1
[0045] Embodiment 1, the acquisition of ScMTB3R1
[0046] 1. Acquisition of ScMTB3R1
[0047] 1. Treat the seedlings of rye AR132 grown for 2 weeks in a refrigerator at 4°C for 5 hours to obtain the treated seedlings; extract the total RNA of the treated seedlings, and construct a cDNA library according to the instructions of Bao Bio cDNA Library Construction Kit; use 12% agar Sugar gel electrophoresis detection, recovery and purification.
[0048] 2. Connect the purified cDNA to the NTT vector, transform E.coli strain DH5α, collect all the grown E. coli colonies, extract the mixed plasmids and transform yeast EGY48, and spread the auxotrophic plate (SD / His2 / Leu2), 30°C Culture for 2-4 days, and use NTT to screen for nuclear localized proteins.
[0049] 3. Extract yeast plasmids from the screened yeast clones and transform them into Escherichia coli. A batch of clones are obtained after sequencing. One of the proteins contains the MYB domain. The protein was compared at NCBI...
Embodiment 2
[0063] Example 2, Acquisition and Functional Analysis of Transgenic ScMTB3R1 Arabidopsis
[0064] 1. Construction of recombinant expression vector
[0065] 1. Extraction of RNA
[0066] The seedling stage rye AR132 was used as the experimental material, and the total RNA was extracted by the TRNzol method, and the qualified RNA was reverse-transcribed into cDNA with the reverse transcription kit of TaKaRa Company, and stored at -20°C for future use.
[0067] 2. PCR amplification
[0068] The cDNA obtained above was used as a template, and primers ScMYB3R1-F2 and ScMYB3R1-R2 were used for PCR amplification to obtain the target gene ScMYB3R1. The primer sequence is as follows: ScMYB3R1-F2: 5'-TCC CCCGGG ATGGGGGCCATGGCGGAGG-3' (the underlined part is the recognition site of Sma I);
[0069] ScMYB3R1-R2: 5'-GG ACTAGT TTAGGTTACATCCATGTTTGTCG-3' (the underlined part is the recognition site of Spe I).
[0070] PCR reaction system: 2×GC Buffer 25μl, dNTP Mix 4μl, upstream and ...
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