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Stress tolerance related transcription factor protein NAC and application thereof

A transcription factor and stress tolerance technology, applied in the field of plant genetic engineering, can solve the problems of low iron solubility and biologically available iron, and achieve the effects of improving drought resistance, iron absorption ability and quality

Inactive Publication Date: 2017-05-31
INST OF SOIL SCI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in an aerobic soil environment, the solubility of iron is very low, and there is little bioavailable iron available to plants. The severe iron deficiency in plants, especially the low iron content in edible parts of plants, is the main source of iron deficiency in animals and humans.

Method used

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  • Stress tolerance related transcription factor protein NAC and application thereof
  • Stress tolerance related transcription factor protein NAC and application thereof
  • Stress tolerance related transcription factor protein NAC and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Embodiment 1: Cloning of NAC gene of Ilex japonica transcription factor and construction of plant expression vector

[0020] According to the results of the laboratory's previous sequencing of the transcriptome of A. japonicus, the AmNAC gene was selected from the A. japonicus transcriptome database, and the PCR product of Gateway homologous recombination-specific primers was designed to be 1005 bp. Upstream primer F1: 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGGAGTTGCAGAGAAAGA-3′, downstream primer R1: 5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCTCATTGCCTAAACCCAAACC-3′. The total RNA of Ilex japonicus grown in hydroponic culture for 30 days was extracted and reverse-transcribed using Oligo(dT)18 as a primer to obtain the reverse transcription product cDNA. Using this cDNA as a template, PCR amplification was carried out with specific primers F1 and R1. Gateway cloning was then performed with the pDnor222.1 vector.

[0021] The AmNAC gene fragment recovered from the PCR product was l...

Embodiment 2

[0022] Embodiment 2: Preparation of transgenic Arabidopsis

[0023] The steps to transform Arabidopsis thaliana with GV3101+psoup::AmNAC-pGreen are as follows:

[0024] (1) Cultivation of Agrobacterium: Pick the positive colony of Agrobacterium GV3101+pSoup with the AmNAC target gene from the plate and inoculate it in a culture medium containing 25mg / L rifampicin, 50mg / L tetracycline and 50mg / L kanamycin. In LB liquid medium, 28°C, 200rpm shaking culture OD 600nm = 1.0, dilute the bacterial solution 10 times with LB liquid medium, and continue shaking culture for 4 h; pour the bacterial solution into a sterile screw cap centrifuge tube, cover the tube cap, and centrifuge at 4000 rpm for 10 min; discard the supernatant, Invert the centrifuge tube for 1 min, drain the residual liquid; collect the bacteria.

[0025] (2) Agrobacterium-mediated transformation of Arabidopsis thaliana: the flower buds were cut off at the first flowering of Arabidopsis to promote the proliferation ...

Embodiment 3

[0027] Example 3: Detection of Drought Tolerance and Iron Content in the AmNAC Gene Transgenic Arabidopsis

[0028] (1) Determination of drought tolerance of transgenic Arabidopsis:

[0029] Determination of stomatal opening: select the rosette leaves of Arabidopsis thaliana that have been cultured for 4 weeks, with the back of the leaves facing up, and place them in openingbuffer (5mM KCl, 50μM CaCl 2 , 10mM MES-KOH, pH6.5) light (150μmol m -2 the s -1) for 3 hours to fully open the stomata. Add 10 μM ABA to the opening buffer for 2 hours, and add an equal volume of absolute ethanol to the control group as a control. The crushed leaves were observed under a Nikon microscope, photographed, and the stomatal opening was measured with ImageJ software. At least 30 stomata were measured and counted for each treatment in each experiment. The experimental results showed that when no ABA was added, the stomatal openings of wild-type and overexpressed Arabidopsis had no significan...

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Abstract

The invention discloses stress tolerance related transcription factor protein NAC and application thereof, wherein the amino acid sequence is shown by SEQ ID No.1. According to the invention, a transcription regulation factor NAC protein is cloned from extreme drought tolerant plant ammopiptanthus mongolicus, and a plant expression vector is established and introduced into arabidopsis through agrobacterium-mediated transformation; and through the means such as preliminary screening of hygromycin, molecular detection, drought stress and in-vivo iron content measurement, a transgenic arabidopsis strain with remarkably improved drought resistance and iron absorbability is obtained. A new variety of transgenic plant capable of efficiently absorbing iron and resisting drought is obtained, which is of great significance in improving the crop quality and enhancing the plant tolerance to adversity stress.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering, in particular, it relates to stress tolerance-related transcription factor protein NAC and its application in cultivating soil drought-resistant and high-efficiency iron-absorbing plants. Background technique [0002] Through long-term evolution, plants have formed a series of perfect adversity stress response mechanisms, including tolerance at the cellular level and regulation at the overall plant level. In the regulatory process, transcriptional regulation is particularly important. The tolerance of plants to adversity stress and the strength of nutrient element absorption often do not depend on a single factor, and their traits are affected by many genes. NAC transcription factor is a kind of plant-specific transcription factor, its biological function is diversified, and it participates in the regulation of multiple life phenomena, such as seed development, lateral root development...

Claims

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Application Information

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IPC IPC(8): C07K14/415C12N15/29C12N15/82A01H5/00
CPCC07K14/415C12N15/8243C12N15/8273
Inventor 苏彦华金曼杨顺瑛
Owner INST OF SOIL SCI CHINESE ACAD OF SCI
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