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Corn bHLH transcription factor gene ZmMIT1 and use thereof for coding protein

A technology for transcription factors and coding proteins, which can be applied in application, genetic engineering, plant genetic improvement, etc.

Inactive Publication Date: 2011-11-02
HENAN AGRICULTURAL UNIVERSITY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are no reports on the cloning and functional identification of bHLH transcription factor genes induced by manganese stress in maize.

Method used

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  • Corn bHLH transcription factor gene ZmMIT1 and use thereof for coding protein
  • Corn bHLH transcription factor gene ZmMIT1 and use thereof for coding protein
  • Corn bHLH transcription factor gene ZmMIT1 and use thereof for coding protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1: Material Handling

[0020] Maize (Zea mays L.) inbred line: Zheng 58; purchased from the Institute of Crops, Henan Academy of Agricultural Sciences.

[0021] Corn Material Handling:

[0022] Disinfection: pick plump corn seeds, soak them in distilled water for 24 hours, disinfect them with 0.1% mercuric chloride for 5 minutes and rinse them with distilled water 5 times.

[0023] A, germination: germinate the fully water-absorbing seeds at 28°C in the dark; the specific operation is: sow the fully water-absorbing seeds on a porcelain plate covered with a layer of water-absorbing cotton, and cover it with two layers of wet Absorbent gauze, absorb water for 3 days in the dark.

[0024] B, Stress treatment of different heavy metal ions: the germinated seeds were transferred to a petri dish containing distilled water, and cultivated in a light culture room with a temperature of 24-26°C, a humidity of 60%, and a light-dark cycle of 12h / 12h. When the seedlings gr...

Embodiment 2

[0025] Example 2: Cloning of the ZmMIT1 gene

[0026] A. Isolation and purification of total RNA from maize leaves

[0027] 1. Extraction of total RNA

[0028] (1) Grind the leaf with liquid nitrogen until it becomes powdery, take an appropriate amount and add it to a 2ml centrifuge tube, and add 1ml TRIZOL lysate at the same time, shake and mix well, and let stand at room temperature for 5 minutes.

[0029] (2) Centrifuge at 12,000 rpm for 15 minutes at 4°C, and take the supernatant into a new centrifuge tube.

[0030] (3) Add 200 μl of chloroform, shake vigorously for 15 seconds, and let stand at room temperature for 3 minutes.

[0031] (4) Centrifuge at 12,000 rpm for 15 minutes at 4°C.

[0032] (5) Pipette 400-600 μl of the upper aqueous phase into a new centrifuge tube.

[0033] (6) Add the same volume of isopropanol as the absorption solution in the previous step, mix gently, and let stand at room temperature for 5-10 minutes.

[0034] (7) Centrifuge at 12,000 rpm f...

Embodiment 3

[0072] Example 3: Expression Analysis of ZmMIT1 Gene

[0073] A. In this example, the fluorescent quantitative RT-PCR method was used to analyze the expression of the ZmMIT1 gene under the stress of different heavy metals (manganese, copper, iron, zinc) ions. 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.

[0074] According to the full-length cDNA sequence of the ZmMIT1 gene, specific primers for fluorescent quantitative PCR were designed as follows:

[0075] Upstream primer: 5'-GTGGGTCTTCGACTGTCCCCCTTAT-3' (SEQ ID NO: 4);

[0076] Downstream primer: 5'-GGATGCTTTTGTGCTTGATTCTGTA-3' (SEQ ID NO: 5).

[0077] BioRad iQ5 real-time quantitative PCR instrument was used for PCR amplification.

[0078] The PCR reaction system (25μl) consists of:

[0079] cDNA template (about 2...

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Abstract

The invention provides a corn bHLH transcription factor (Manganese-Induced Transcription Factor1) coding gene ZmMIT1 which expresses under induction of heavy metal manganese stress and use thereof for coding a protein. The gene can improve the resistance to heavy metal manganese stress in plants, in particular the manganese poisoning resistance in tobacco. The ZmMIT1 has a bright application prospect in field of heavy metal pollution resistance in corn.

Description

technical field [0001] The invention relates to the field of plant genetic engineering, in particular to the application of a maize bHLH transcription factor coding gene ZmMIT1 and its coding protein. Background technique [0002] With the growth of population and the development of industrialization, the discharge of waste residue and waste water and the increase of mine waste land, coupled with the widespread use of various chemical substances, the harm of heavy metal pollution to human health and crop production has become increasingly prominent. As a heavy metal widely distributed, manganese is an essential trace element for plant growth and development. It directly participates in the redox process of the electron transfer system in plant photosynthesis. However, excessive manganese will pollute the soil and cause stress and toxicity to plant growth and development. Currently, manganese toxicity has become the main limiting factor for crop yield and quality improvement ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C07K14/415C12N15/84A01H5/00
Inventor 夏宗良苏新宏吴建宇孙凯乐李志敏丁俊强
Owner HENAN AGRICULTURAL UNIVERSITY
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