Application of CbDREB2AL gene in preparation of salt-tolerant transgenic plant
A transgenic and genetic technology, applied in the fields of applications, plant peptides, plant products, etc., can solve the problem of undiscovered CbDREB2AL gene transgenic plants, etc.
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Embodiment 1
[0022] Cloning of the sequence of CbDREB2AL coding gene of Ion mustard:
[0023] Utilize RNA extraction and separation reagent (Trizol, Invitrogen) to isolate the total RNA of the regenerated seedlings of Alpine mustard, and the specific method is: collect 100 mg of the regenerated seedlings of Alpine mustard, immediately place it in liquid nitrogen and grind it into powder, then add 1ml of Trizol reagent, and mix well , inhale into a 1.5ml centrifuge tube; place at room temperature for 5min; add 0.2ml of fresh chloroform, shake vigorously for 15s, and let stand at room temperature for 3min; centrifuge at 12000g for 15min at 4°C; transfer the supernatant to a new 1.5ml centrifuge tube , add 0.5ml of isopropanol and mix well, and centrifuge at 12000g for 10min at 4°C to precipitate RNA; wash the RNA pellet with 1ml of 75% ethanol, dissolve it in an appropriate amount of DEPC-treated water, and store it at -70°C for later use.
[0024] The full-length sequence of the gene was ob...
Embodiment 2
[0032] Obtaining of CbDREB2AL Transgenic Plants and Determination of Salt Tolerance
[0033] The construction of the plant overexpression vector of the CbDREB2AL gene of Ion mustard alpine: the fragment obtained in Example 1 obtained by sequencing verification by using the Gateway technology of Invitrogen Company through the BP reaction (BP II Enzyme mix, Invitrogen No.11789020) was recombined into the pDONR / Zeocin vector (Invitrogen No.12535-035), transformed into Escherichia coli DH5α competent cells, and the entry clone was obtained by screening with 20mg / L Zeocin, and then the plasmid was extracted and passed through Gateway The LR reaction (LR II Enzyme mix, Invitrogen No.11791100) the CbDREB2AL gene was recombined onto the PMDC32 carrier, transformed in Escherichia coli DH5α competent cells, and successfully recombined overexpression vector PMDC32-CbDREB2AL (such as image 3 ).
[0034] Agrobacterium-mediated transformation: transform the constructed overexpression pla...
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