A kind of NF-Y type nuclear transcription factor gene zmnf-ya13, its coded protein and its application
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) Extraction of corn total RNA
[0028] Weigh 50 mg of Zheng 58 fresh corn leaves and grind them into powder in liquid nitrogen. After the liquid nitrogen has evaporated completely, quickly transfer it to a centrifuge tube containing 1 mL of pre-cooled Trizol reagent, mix well, and let it stand at room temperature for 10 minutes; add 0.2 mL of chloroform, mix well, and let it stand at room temperature for 5 minutes; In a centrifuge, centrifuge at 12000 r / min for 15 minutes, transfer the supernatant to a new centrifuge tube; add an equal volume of isopropanol, mix thoroughly, and let stand at room temperature for 10 minutes; Centrifuge for 15 min; discard the supernatant, add 1.0 mL of pre-cooled 75% ethanol (prepared with DEPC water), fully wash the precipitate, and centrifuge at 4°C, 7500 rpm for 5 min; discard the supernatant, dry it in an ultra-clean workbench until it becomes transparent, add Appropr...
Embodiment 2
[0051] Example 2 Analysis of ZmNF-YA13 gene expression pattern in maize
[0052] The corn variety used in this experiment was Zheng 58.
[0053] The maize treatment method used to study the stress treatment: put the maize seeds in water at 65°C, stir well, and when the water reaches room temperature, put the seeds in a humid and dark place to germinate. The cultivation conditions in a constant temperature light incubator in a flowerpot equipped with an equal amount of quartz sand are a temperature of 24°C / 20°C (day / night), a photoperiod of 16h / 8h (day / night), and an air humidity of 60%. After the corn seedlings grew to the stage of three leaves and one heart, the plants with the same growth were selected and subjected to drought, NaCl and NaCl respectively. 2 CO 3 , cold, ABA and potassium deficiency stress treatments. In this experiment, 25% PEG6000 was used to process 24h to simulate natural drought ( Figure 4 A, Figure 4
[0054] B); Irrigate with the hoagland nutri...
Embodiment 3
[0060] Example 3 Obtaining of Transgenic ZmNF-YA13 Gene Tobacco
[0061] (1) ZmNF-YA13 gene plant expression vector construction
[0062] 1) Acquisition of the plant expression vector pTF101: The plasmid of the pTF101 plant expression vector was extracted using the plasmid mini-extraction 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.
[0063] 2) Obtaining the coding region of ZmNF-YA13: the single-stranded cDNA obtained by reverse transcription was used as a template, and ZmNF-YA13-S and ZmNF-YA13-35S-A were used as forward and reverse primers respectively to amplify the coding region of ...
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