Promoter of wheat dehydrin gene and application thereof
A promoter and dehydrin technology, applied in the field of plant genetic engineering, can solve the problems of wheat dehydrin gene promoter cloning and application that have not been reported and the like
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Embodiment 1
[0026] Embodiment 1, wheat dehydrin gene TT expression analysis of
[0027] 1.1 Material Handling
[0028]The seeds of wheat variety Shanrong 3 germinated normally, and the endosperm was removed after one week. They were cultured in Hangload medium for two weeks, and transferred to Hangload medium containing 200mM NaCl for stress treatment. At 0, 0.5, 3, 12, 24, and 48 hours after the treatment, young leaves and roots were taken and stored in liquid nitrogen.
[0029] 1.2 Extraction of wheat Total RNA by Trizol method
[0030] 1. Put the tissue material into a liquid nitrogen pre-cooled mortar, and fully grind it into powder in liquid nitrogen;
[0031] 2. After the liquid nitrogen evaporates to dryness, transfer it to a 2ml centrifuge tube immediately, add about 1ml of Invitrogen’s TRIzol extract for every 100mg of material, shake and mix the sample vigorously to fully lyse the sample, and place it at room temperature for 5 minutes;
[0032] 3. Add 0.2ml of chloroform,...
Embodiment 2
[0082] Embodiment 2, wheat dehydrin gene promoter DHNP clone
[0083] 2.1 Extraction of wheat genomic DNA
[0084] 1. Using CTAB method to extract wheat genomic DNA. The steps are:
[0085] (1) Take about 0.5g of wheat root system and grind it in liquid nitrogen, put it in a 1.5ml centrifuge tube, add 700μl extraction buffer (100mmol / L Tris-HCl, 50mmol / L EDTA-Na 2 , 100mmol / L NaCl, 1.5% CTAB, 2% mercaptoethanol), 65 o Incubate in C water bath for 30-60min, during which time mix by inverting 3-4 times;
[0086] (2) Take out the centrifuge tube, add 700 μl phenol: chloroform: isoamyl alcohol (25:24:1) solution to each tube, and mix for 10 minutes;
[0087] (3) Centrifuge at 10000rpm for 10min;
[0088] (4) Slowly draw the supernatant into another 1.5ml centrifuge tube with the tip of the pipette (cut off the front 1 / 3);
[0089] (5) Add an equal volume of chloroform:isoamyl alcohol (24:1) and repeat the above extraction process. Slowly remove the supernatant;
[0090...
Embodiment 3
[0146] Embodiment 3, wheat dehydrin gene promoter expression vector ( pCAM1391Z / TaDHNP) build
[0147] According to the promoter DHNP The sequence design contains Hind III and EcoR The promoter upstream and downstream primers of I, the primer name and sequence are Dhy2p1: 5'-CCCAAGCTTCCCGGGCTGGTAAAAACGAAG-3'( Hind III) Dhy2p2: 5′-CGGAATTCCTTGCAGACTTGCACAGGTGTTG-3′ ( EcoR I). The plasmid containing the promoter sequence was used as a template for PCR amplification. After electrophoresis and recovery, the PCR amplification product was ligated with the pGEM-T easy vector, transformed into Escherichia coli, and the plasmid was extracted. use Hind III and EcoR I respectively for the pCAM1391Z vector and containing DHNP The pGEM-T easy vector of the promoter was double digested, and the large fragment of the pCAM1391Z vector and the DHNP Small promoter fragments were ligated with T4 DNA ligase and then transformed into Escherichia coli DH5α competent cells. The rec...
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