Application of Zea mays L. drought-induced protein (ZmDIP1) gene and its encoding protein
A corn and drought technology, applied in application, genetic engineering, plant genetic improvement, etc., can solve the problems of inhibiting root growth, research, and plant dwarfing, and achieve the effect of huge economic benefit potential and broad application prospects
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Embodiment 1
[0031] Example 1: Material Handling
[0032] Maize (Zea mays L.) inbred line: Zheng 58, purchased from the Institute of Crops, Henan Academy of Agricultural Sciences.
[0033] Corn Material Handling:
[0034] A. Seed disinfection: select plump corn seeds, sterilize them with 0.1% mercury chloride, and germinate them at 28°C in the dark.
[0035] B. Drought stress treatment: move the germinated seeds to a petri dish containing distilled water, and cultivate them in a light culture room with a temperature of 24-26°C, a humidity of 60%, and an alternating cycle of light and dark of 12h / 12h. When the seedlings grew to two leaves and one heart (about 10 days), the distilled water was poured out, and the stress treatment experiment of 20% PEG 6000 solution was carried out. The treatment time was 1, 3, 6, 12, 24, 48 hours respectively; the maize after stress treatment was used as the experimenter, and the maize seedlings without stress treatment were used as the control group.
Embodiment 2
[0036] Example 2: Cloning of ZmDIP1 (drought-induced protein 1) gene
[0037] A. Isolation and purification of total RNA from maize leaves
[0038] 1. Extraction of total RNA
[0039] Total RNA was extracted according to the instructions of TRIZOL (Invitrogen, USA) reagent.
[0040] 2. Purification of RNA (take 50 μl of total RNA as an example)
[0041] (1) Purification system
[0042]
[0043] (2) Incubate at 37°C for 30 minutes to remove DNA from RNA.
[0044] (3) Add DEPC-H 2 O to 300 μl.
[0045] (4) Add 300 μl of phenol / chloroform (1:1) for extraction.
[0046] (5) Add an equal volume of isopropanol, and precipitate at -20°C for 1-2 h.
[0047] (6) Add 30~40 μl DEPC-H 2 O dissolved.
[0048] B. RT-PCR Amplification of Maize ZmDIP1 Gene
[0049] 1. First-strand cDNA synthesis from maize leaves
[0050]Absorb 1-2 μg of the total RNA of corn leaves into a 1.5 ml centrifuge tube, follow the RevertAid of Fermentas Company TM First Strand cDNA Synthesis Ki...
Embodiment 3
[0062] Example 3: Expression Analysis of ZmDIP1 Gene
[0063] A. In this example, the fluorescent quantitative RT-PCR method was used to analyze the expression of the ZmDIP1 gene under 20% PEG 6000 stress. The experimental samples were corn leaves from the experimenter and the control group described in Example 1. In each sample, according to the method described in Example 2, 5 μg of total RNA was reverse-transcribed into first-strand cDNA as a template.
[0064] According to the full-length cDNA sequence of the ZmDIP1 gene, specific primers for fluorescent quantitative PCR were designed as follows:
[0065] Upstream primer (SEQ6): 5'-AGCAGATGGAACTGGCAAAACTA-3';
[0066] Downstream primer (SEQ7): 5'-GTATGAAGCATCCTCTTCACCGT-3'.
[0067] BioRad iQ5 real-time quantitative PCR instrument was used for PCR amplification.
[0068] The PCR reaction system (25 μl) consists of:
[0069] cDNA template (about 25 ng): 0.5 μl, upstream primer: 1 μl, downstream primer: 1 μl, 2×SYBR Gre...
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