Plant stress tolerance related protein gmnf-yc17 and its coding gene and application
A plant stress tolerance, coding gene technology, applied to plant stress tolerance-related protein GmNF-YC17 and its coding gene and application fields, can solve problems such as comprehensive improvement of stress resistance of difficult plants, and achieve the goal of improving drought tolerance. Effect
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Embodiment 1
[0060] Embodiment 1, the acquisition of gene GmNF-YC17
[0061] 1. Acquisition of the gene GmNF-YA15
[0062] Soybean Tiefeng No. 8 grown in hydroponics for about 10 days (National Germplasm Bank, No. ZM242; recorded in the following documents: Wang Caijie, Sun Shi, Wu Baomei, Changruzhen, Han Tianfu. Since the 1940s in China Pedigree Analysis of Soybean Varieties Planted by Area. Chinese Journal of Oil Crops. 2013, 35(3):246-252, available to the public from the Institute of Crop Science, Chinese Academy of Agricultural Sciences) Seedlings at the four-leaf stage were drought-treated for 2 hours, quick-frozen with liquid nitrogen, Store at -80°C for later use. mRNA was isolated using QuikprepMicro mRNA Purification Kit (Pharmacia). First-strand cDNA synthesis was performed with Reverse Transcriptase XL (AMV). The ds cDNA was synthesized by SMART method, and the PCR products were detected by 1.0% agarose gel electrophoresis.
[0063] Sequence 2 in the full-length sequence l...
Embodiment 2
[0086] Example 2, Application of GmNF-YC17 as a transcription factor
[0087] The main principle of using the yeast one-hybrid system to demonstrate the activation properties of transcription factors is as follows: image 3 As shown, the CCAAT cis-acting element and the mutant CCAAT cis-acting element were constructed to the upstream of the basic promoter Pmin (minimal promoter) of the pHISi-1 vector and the pLacZi vector, respectively, and the reporter genes (His3, LacZ and URA3). When the expression vector YEP-GAP (without activation function) connected with the target gene encoding the transcription factor is transformed into the yeast cells connected with the CCAAT cis-acting element and the mutant CCAAT cis-acting element, if the mutant CCAAT The reporter gene in yeast cells with cis-acting elements cannot be expressed, but the reporter gene in yeast cells with specific CCAAT cis-acting elements can be expressed, indicating that the transcription factor can bind to CCAAT...
Embodiment 3
[0138] Embodiment 3, the application of GmNF-YC17 in improving the stress tolerance of plants
[0139] 1. Obtaining transgenic GmNF-YC17 Arabidopsis
[0140] 1. Construction of recombinant vector
[0141] 1) Cloning of GmNF-YC17 gene
[0142] A pair of primers (GmNF-YC17-121F and GmNF-YC17-121R) were designed according to the sequence of the GmNF-YC17 gene.
[0143] GmNF-YC17-121F: 5'-TCCCCCGGGATGAGACAAGCTGGTGCATA-3';
[0144] GmNF-YC17-121R: 5'-GACTAGTAGAGCAATCTATAGTTTCCACC-3'.
[0145] Using Glycine max L. Tiefeng 8 intact plant cDNA as a template, PCR amplification was performed with GmNF-YC17-121F and GmNF-YC17-121R, and a PCR product (GmNF-YC17 gene) with a size of about 372bp was obtained .
[0146] The above PCR product was recovered.
[0147] 2) Construction of recombinant vector
[0148] ① Digest the PCR product recovered in step 1 with restriction enzymes SmaI and SpeI, and recover the 372bp digested product;
[0149] ② Digest pBI121 (purchased by Clontech) w...
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