Plant DREB transcription factor and its coding gene and use
A technology of transcription factors and coding genes, applied in the field of plant biology, can solve problems such as the comprehensive improvement of stress resistance of difficult plants, and achieve the effects of extensive practical significance and far-reaching theoretical significance
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Embodiment 1
[0031] Embodiment 1, the acquisition of DMY-DREB and its coding gene
[0032] Rice was extracted by CTAB method (Steiner JJ, Poklemba CJ, Fjellstrom RG, Elliott LF., A rapidone-tube genomic DNA extraction process for PCR and RAPD analyses. Nucleic Acids Res. 1995 Jul 11; 23(13): 2569-70.) Grass genome DNA, the specific method is as follows: take Spartina leaves, grind with liquid nitrogen, add 5 ml of CTAB solution preheated at 68°C (add 10mM mercaptoethanol before use), and heat at 60°C for 30 minutes per 1 gram of plant material . Then add an equal volume of chloroform:isoamyl alcohol, centrifuge at 15000g for 15 minutes, and take the supernatant. Add 2 / 3 times the volume of isopropanol to the supernatant, mix well, use the needle tip of the injection needle to stir out the DNA filament and place it in a new tube, then add 75% ethanol to wash the precipitate and the wall of the centrifuge tube, and then add ethanol Blot dry, air dry remaining ethanol, add TE to dissolve DN...
Embodiment 2
[0036] Embodiment 2, the construction of DMY-DREB plant expression vector and the stress tolerance experiment of transgenic tobacco
[0037] The Arabidopsis rd29A gene promoter sequence was amplified by Rd29A-S and Rd29A-A PCR methods using CTAB method to extract Arabidopsis genomic DNA as a template. Among them, Rd29A-S: 5'-TCTAGACGCATGATTTGATGGAGGAG-3', Rd29A-A: 5'-TCCAAAGATTTTTTTCTTTCCAA-3'. 25ul PCR reaction system contains 100ng genomic DNA, 1.5mM MgCl 2 , 20 mM Tris-HCl (pH8.4), 50 mM KCl, 0.8 mM dNTP mixture, 1 μM Rd29A-S and 1 μM Rd29A-A, 1 U of Pfu enzyme (Shanghai Shenyou Biotechnology Company). Carry out PCR cycling in a PCR-thermal cycler (Eppendorf, Germany) according to the following scheme: first 94°C for 5 minutes; then 94°C for 1 minute, 54°C for 1 minute, 72°C for 1 minute, a total of 30 cycles; finally 72°C 10 minutes. Use the gel recovery kit (Beijing Tianwei Times Technology Co., Ltd.) to recycle the amplified DNA fragment of about 944bp according to th...
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