Gene related to plant drought tolerance, and encoded protein and applications thereof
A plant and gene technology, applied in the direction of plant gene improvement, application, plant peptides, etc., can solve the problems of drought tolerance research that has not been reported, and achieve the effect of improving survival rate and improving drought tolerance.
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Embodiment 1
[0048] This example is the cloning of the NtDSR1 gene, including the following steps:
[0049] (1) Extraction of NtDSR1 total RNA
[0050] Take the K326 tobacco leaves that have been grown for 4 weeks under normal growth conditions and quickly grind them into powder in liquid nitrogen. Take about 100 mg of the powdered material and place it in a 1.5 mL centrifuge tube containing 1.0 mL TRIzol (Invitrogen), and place it in- Store at 80°C for later use.
[0051] Use TRIzol reagent extraction method to extract total RNA: take out the sample stored at -80℃ for later use, after thawing at room temperature, add 200μL of chloroform, shake and mix well, after centrifugation, carefully remove the upper aqueous phase, transfer it to another centrifuge tube, and add 500μL of isopropanol, precipitation, centrifugation to isolate RNA, and then washed with 75% alcohol, and slightly dried at room temperature, add an appropriate volume of RNase-free water to fully dissolve. The extracted RNA is tr...
Embodiment 2
[0068] This example is the construction of a recombinant expression vector, including the following steps:
[0069] (1) Recombinant vector construction
[0070] Refer to the in-fusion seamless connection manual of Clontech, and establish the connection reaction system according to the requirements of the kit, as shown in Table 3 below, unit: μL.
[0071] Table 3 Connection reaction system
[0072]
[0073] After connecting at 50°C for 15 minutes, put it on ice for the next conversion.
[0074] (2) The ligation product of heat shock method transforms E. coli competent cells
[0075] Take 5μL of the ligation product under aseptic conditions and add it to the competent cells, mix gently and then ice bath for 30min, heat shock at 42℃ for 90s, quickly transfer the centrifuge tube to the ice bath and place it in the ice bath for 2min (2-3min is acceptable), add 800μL of antibiotic-free LB medium, shake gently on a 37℃ shaker for 1h, apply 200μL of culture medium on LB solid medium containing ...
Embodiment 3
[0081] This embodiment is the transformation of Agrobacterium with expression vector, including the following steps:
[0082] The expression vector pDT1-NtDSR1 was transformed into Agrobacterium GV3101 (Agrobacterium GV3101 purchased from Tiangen Biochemical Technology Co., Ltd.) by the heat shock method, and the plasmid was extracted and identified by restriction enzyme digestion. The method was the same as the above-mentioned expression vector identification procedure. The expression vector pDT1-NtDSR1 has been successfully transformed into Agrobacterium.
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