NF-Y nuclear transcription factor gene ZmNF-YA13, protein encoded by gene and application of gene
A nuclear transcription factor, NF-Y technology, applied in the fields of application, genetic engineering, plant gene improvement, etc., can solve the problem of crop yield reduction and other issues
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Embodiment 1
[0026] Example 1 Cloning and analysis of ZmNF-YA13 gene cDNA
[0027] (1) Maize total RNA extraction
[0028] Weigh 50 mg Zheng 58 fresh corn leaves and grind them into powder in liquid nitrogen. After the liquid nitrogen has completely evaporated, quickly transfer it to a centrifuge tube containing 1 mL of pre-cooled Trizol reagent, mix well, and let stand at room temperature for 10 minutes; add 0.2 mL of chloroform, mix well, and let stand at room temperature for 5 minutes; centrifuge at 4°C Centrifuge at 12000r / min for 15min in the machine, transfer the supernatant to a new centrifuge tube; add an equal volume of isopropanol, mix well, and let stand for 10min at room temperature; centrifuge at 12000r / min at 4℃ in a low-temperature centrifuge 15min; Discard the supernatant, add 1.0mL of pre-cooled 75% ethanol (prepared with DEPC water), fully wash the pellet, centrifuge at 4°C, 7500rpm for 5min; discard the supernatant, blow-dry it in an ultra-clean workbench until it becomes tr...
Embodiment 2
[0051] Example 2 Analysis of ZmNF-YA13 gene expression pattern in corn
[0052] The corn material variety used in this experiment was Zheng 58.
[0053] Corn treatment methods used to study stress treatment: place the corn seeds in water at 65°C, stir well, and when the water reaches room temperature, place the seeds in a humid and dark place to germinate. After the seeds germinate for 5 days, plant the corn seedlings in The culturing conditions in a flowerpot with an equal amount of quartz sand in a constant temperature light incubator are temperature 24°C / 20°C (day / night), photoperiod 16h / 8h (day / night), and air humidity 60%. After the corn seedlings grow to the three-leaf one-heart stage, select the same plants for drought, NaCl, Na 2 CO 3 , Cold, ABA and potassium deficiency stress treatments. This experiment uses 25% PEG6000 for 24h to simulate natural drought ( Figure 4 A, B); watering with hoagland nutrient solution containing 200mM NaCl for salt stress induction ( Figure...
Embodiment 3
[0059] Example 3 Obtainment of ZmNF-YA13 gene-transformed tobacco
[0060] (1) Construction of ZmNF-YA13 gene plant expression vector
[0061] 1) Obtaining the plant expression vector pTF101: The plasmid of the pTF101 plant expression vector is extracted by using the plasmid small quantity extraction kit of Shenggong Bioengineering Co., Ltd. See the instructions for the specific method. Using two restriction enzymes, SmaI and SpeI, the pTF101-35s plant expression vector was subjected to double enzyme digestion reaction to linearize it, and the digested product was detected by agarose gel electrophoresis, and the coagulation at the target area was cut off. Gel and use gel recovery kit for recovery and purification.
[0062] 2) Obtaining the coding region of ZmNF-YA13: Amplify the coding region of ZmNF-YA13 gene with ZmNF-YA13-S and ZmNF-YA13-35S-A as the forward and reverse primers using the single-stranded cDNA obtained by reverse transcription as the template. . The PCR results w...
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