Plant stress tolerance-associated protein TaNF-YA 1, coding genes thereof and applications
A technology for encoding genes and genes, applied in the field of plant stress tolerance-related protein TaNF-YA1 and its encoding genes and applications
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Embodiment 1
[0057] Embodiment 1, the cloning of TaNF-YA1 gene
[0058] 1. Isolation of RNA
[0059] Three-leaf seedlings of triticale wheat grown in hydroponic culture for about 10 days were subjected to drought treatment for 2 hours, quick-frozen with liquid nitrogen, and stored at -80°C for later use. RNA was isolated using RNA prep pure Plant Kit (DP432, Tiangen Biochemical Technology (Beijing) Co., Ltd.). Reverse Transcriptase XL (AMV) (TaKaRa) was used for first-strand cDNA synthesis. The dscDNA was synthesized by SMART method, and the PCR product was detected by 1.0% agarose gel electrophoresis.
[0060] 2. Acquisition of the full-length sequence of TaNF-YA1 gene
[0061] Obtain the nuclear transcription factor full-length cDNA sequence (as shown in sequence table sequence 2) of wheat CCAAT-box by 5'RACE and 3'RACE method, the coding gene of this transcription factor is named as TaNF-YA1 gene, and its open reading The framework is the 54th to 950th nucleotides at the 5' end of S...
Embodiment 2
[0062] Example 2, real-time fluorescent quantitative PCR analysis of the expression characteristics of TaNF-YA1 gene
[0063] 1. Coercion treatment
[0064] Be the small white wheat seedling of 10 days with potted seedling age, carry out following treatment:
[0065] (1) Drought treatment: take out the young white wheat seedlings to absorb the moisture on the roots, place them on dry filter paper, and cultivate them in a drought for 0.5 hours, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, and 24 hours, then take out the materials , quick-frozen with liquid nitrogen, and stored at -80°C for later use.
[0066] (2) High-salt treatment: place the young white wheat seedlings in 200mM NaCl aqueous solution, and cultivate them under light for 0.5 hour, 1 hour, 2 hours, 5 hours, 12 hours, and 24 hours, then take out the materials respectively, and quickly freeze them with liquid nitrogen. Store at 80°C for later use.
[0067] (3) High temperature treatment: place the white wheat se...
Embodiment 3
[0085] Example 3, TaNF-YA1 subcellular localization analysis
[0086] 1. Construction of recombinant expression vector
[0087] Primers TaNF-YA1 and TaNF-YA1-XI were designed according to the sequence of the TaNF-YA1 gene shown in the 54th to 947th positions of Sequence 2, and the ends of the primers were introduced into SalI and BamHI restriction sites respectively, and the cDNA of triticale was used as Template, PCR amplification to obtain TaNF-YA1 gene.
[0088] TaNF-YA1: 5'-CCT GTC GAC ATGGAGGATCATCCTGGCCATC-3';
[0089] TaNF-YA1-XI: 5'-CGT GGATCC CCTCATCATGGAAGCGCGCTGG-3'.
[0090] PCR amplification products were detected by 1.2% agarose gel electrophoresis. Recover the target product of about 900bp, digest it with SalI and BamHI, connect it to the subcellular localization vector hGFP between the SalI and BamHI sites, and transform E. coli DH5α by heat shock. Plasmid extraction was performed on positive single clones, PCR detection was performed with TaNF-YA1 and ...
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