Vacuolar hydrogen ion pyrophosphatase gene AlVP1 as well as encoded protein and application of gene
A technology of pyrophosphatase and tonoplast membrane, which can be applied in the field of genetic engineering and can solve the problems of relatively few studies on drought tolerance.
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Embodiment 1
[0025] Cloning and Analysis of Example 1 AlVP1 Gene cDNA
[0026] (1) Extraction of total RNA from Roe chinensis
[0027]Weigh 50 mg of 400 mM NaCl-treated roewort leaves and grind them into powder in liquid nitrogen. After the liquid nitrogen is completely evaporated, quickly transfer it to a centrifuge tube containing 1mL pre-cooled Trizol reagent, mix well, and let stand at room temperature for 10min; add 0.2mL chloroform, mix well, let stand at room temperature for 5min; centrifuge at 4°C In the machine, centrifuge at 12000r / min for 15min, transfer the supernatant to a new centrifuge tube; add an equal volume of isopropanol, mix thoroughly, and let stand at room temperature for 10min; centrifuge at 12000r / min in a low-temperature centrifuge at 4°C 15min; Discard the supernatant, add 1.0mL pre-cooled 75% ethanol (prepared with DEPC water), fully wash the precipitate, centrifuge at 4°C, 7500rpm for 5min; discard the supernatant, dry it in an ultra-clean bench until transpar...
Embodiment 2
[0048] Example 2 Analysis of the expression pattern of AlVP1 gene in Swertia chinensis
[0049] The materials used in this experiment were planted on the roof of Dalian University of Technology, and the layering method was used for asexual reproduction to ensure the consistency of its genetic background. After the seedlings are rooted in quartz sand (about 1-2 weeks), they are cut off from the total plant, and placed in a constant temperature light incubator to slow down the seedlings for 10 days. After slowing down the seedlings, the plants with the same growth were selected to be treated with drought, salt, cold, ABA and potassium deficiency stress respectively. In this experiment, the method of 30% PEG6000 treatment for 24 hours was used to simulate natural drought; the plants of Swertia chinensis were placed in a refrigerator at 4°C for low temperature induction treatment; 400mmol·L -1 NaCl solution irrigation for salt stress induction; 100 μmol L -1 ABA watering was use...
Embodiment 3
[0054] Example 3 Obtaining of Transgenic AlVP1 Gene Tobacco
[0055] (1) AlVP1 gene plant expression vector construction
[0056] ① Obtaining the plant expression vector PTF101: The plasmid of the plant expression vector PTF101 was extracted using the Plasmid Mini-prep Kit of Sangon Bioengineering Co., Ltd., and the specific method is shown in the instruction manual. The pTF101-35s plant expression vector was subjected to a double digestion reaction with two restriction enzymes, SmaI and SpeI, to make it linear, and the products after digestion were detected by agarose gel electrophoresis, and the condensate at the target region was excised. The gel was recovered and purified using a gel recovery kit.
[0057]②Acquisition of AlVP1 coding region: The single-stranded cDNA obtained by reverse transcription was used as a template, and AlVP1-S and AlVP1-A were used as forward and reverse primers respectively to amplify the coding region of AlVP1 gene. The PCR results were detecte...
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