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Plant induction type promoter and application of plant induction type promoter

A promoter and inducible technology, applied in the field of plant molecular biology, can solve the problems of not being able to meet the requirements of transgenic breeding and less research, and achieve the effect of broad application space and market prospects

Active Publication Date: 2016-04-27
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although some stress-inducible promoters have been cloned, they still cannot meet the requirements of different transgenic breeding of different plants, and new stress-inducible promoters still need to be discovered and studied
In particular, there are relatively few studies on cloning stress-inducible promoters from plants in special habitats

Method used

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  • Plant induction type promoter and application of plant induction type promoter
  • Plant induction type promoter and application of plant induction type promoter
  • Plant induction type promoter and application of plant induction type promoter

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Embodiment 1, the acquisition of HbHAK2 promoter

[0052] 1. Cultivation of wild barley

[0053] Salt pasture - wild barley (Hordeumbrevisubulatum (Trin.) Link), collected from the saline grassland of Inner Mongolia. At room temperature, wild barley seeds were soaked in deionized water for 12 hours, and then placed in a refrigerator at 4°C overnight. Rinse three times with sterilized deionized water, put them in a culture dish with wet gauze, and germinate at 25°C. After 4-5 days after most of it germinates, transfer to a sterilized glass bottle filled with 1 / 2 Hoagland culture solution (wrapped with opaque paper), at a temperature of 22-23°C and a light intensity of 1000- 3000μmolm -2 the s -1 , grow under the conditions of light 12h / day and darkness 12h / day, and take materials for later use when they grow to two leaves and one heart.

[0054] 2. Cloning of HbHAK2 promoter

[0055] Genomic DNA of wild barley was extracted by the CTAB method, and PCR amplification...

Embodiment 2

[0060] Embodiment 2, the acquisition of transgenic strains of Arabidopsis thaliana

[0061] 1. Construction of proHbHAK2:GUS fusion expression vector

[0062] The PCR amplified product obtained in Example 1 and the vector pYBA1121 were double-digested with SacI and HindIII, respectively, and the digested fragment containing the HbHAK2 promoter and the backbone fragment of the vector pYBA1121 were respectively recovered, and the two were ligated to obtain a recombinant expression vector. It was confirmed by enzyme digestion and sequencing that the recombinant expression vector was pYBA1121( figure 1 The DNA fragment between the SacI and HindIII sites of ) was replaced by the DNA fragment shown in SEQIDNo.1 in the sequence table (ie, the promoter HbHAK2), and the recombinant expression vector was named proHbHAK2:GUS. The T-DNA region of the recombinant expression vector proHbHAK2:GUS contains the expression cassette proHbHAK2+GUS+polyA.

[0063] 2. Obtaining of transgenic Arab...

Embodiment 3

[0072] Example 3, Functional verification of the HbHAK2 promoter in transgenic Arabidopsis

[0073] 1. Treatment and cultivation of transgenic Arabidopsis plants

[0074] The seeds of transgenic proHbHAK2:GUS Arabidopsis strains were treated with 10% sodium hypochlorite, rinsed with sterile water for 3-5 times, deposited on 4°C for 2 days, and then sowed on MS plates, cultured at 25°C for 10 days, and germinated Consistent seedlings were subjected to no treatment, ABA treatment and adversity stress treatment. The ABA treatment was treated with ABA solution containing 100 μM and 200 μM for 2 and 4 days respectively; the adversity stress treatment was specifically treated with 300 mM and 400 mM NaCl solution and 10% and 20% PEG for 2 and 4 days respectively, and the seedlings were placed Treated at low temperature (4°C) and high temperature (42°C) for 2 and 4 days, respectively.

[0075] 2. Analysis of HbHAK2 promoter activity

[0076] The above-mentioned untreated, ABA-treat...

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Abstract

The invention discloses a plant induction type promoter and application of the plant induction type promoter. The nucleotide sequence of the promoter is the nucleotide sequence shown by SEQ ID No.1 in a sequence table. Experiments prove that under the conditions of different concentrations and different processing time of NaCl, PEG and ABA, the promoter HbHAK2 can drive the GUS gene expression; and under the non-processed (comparison), low temperature (4 DEG C) and high temperature (42 DEG C) stress, the HbHAK2 cannot drive the GUS gene expression. The result shows that the HbHAK2-744bp promoter is a novel induction type promoter; and high promoter activity can be realized under the salt or drought stress or the ABA induction; and the target gene expression can be inducted for adapting to the environment. The promoter can be applied to stress-tolerance gene engineering vector construction, plant stress-tolerance molecular mechanism study and crop salt-resistant and drought-resistant transgenic breeding. An economic fast and effective path is provided for plant genetic modification.

Description

technical field [0001] The invention relates to a plant-inducible promoter in the field of plant molecular biology and its application. Background technique [0002] Soil salinization, drought and other adversities are the main environmental limiting factors for plant growth and development. Plants cannot escape adversity, they can only cope. Therefore, plants have evolved a set of fine-tuning mechanisms for sensing adversity stress, transmitting adversity signals, and responding at the molecular, cellular, and physiological levels. Many studies have shown that various stresses act on plant cells, not only at the post-transcriptional level, but also at the transcriptional level, inducing the expression of stress-responsive genes to adapt to the environment. [0003] Plant-inducible promoters can fine-tune the expression of genes in plants under external stimuli. The study of inducible promoters has always been a hot issue in the regulation of plant transcription and expre...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/82C12N5/10A01H5/00
CPCC07K14/415C12N15/8273C12N15/8293
Inventor 李瑞芬常聪聪魏建华
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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