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Shepherd's purse peroxidase gene promoter and application thereof in improving plant cold tolerance

A shepherd's purse peroxide and enzyme gene technology, applied in improving plant cold resistance, can regulate the promoter of cold-induced genes, in the field of plant low-temperature-induced promoters, can solve the problem of affecting crop yields, and has not yet found that shepherd's purse is too high Oxidase gene promoter and other problems, to achieve the effect of improving cold resistance

Active Publication Date: 2016-12-07
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, when these genes are transferred to plants for expression, due to the use of the constitutive CaMV35S promoter, the genes are extensively expressed in plants. Phenotypes such as dwarfing, growth delay, and late flowering appear in the normal environment without low temperature, which will also affect the yield of crops (Zhou MQ, Xu M, Wu LH, Shen C, Ma H, Lin J. CbCBF from Capsella bursa-pastoris enhances coldtolerance and restrains growth via antagonist with gibberellin and affecting cell cycle signaling in tobacco, PlantMol Biol, 2014, 85:259-275)
At present, there is no report on the use of shepherd's purse peroxidase gene promoter in cultivating cold-resistant plants

Method used

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  • Shepherd's purse peroxidase gene promoter and application thereof in improving plant cold tolerance

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Isolation and identification of shepherd's purse peroxidase gene promoter

[0033] In this example, the promoter sequence of the shepherd's purse peroxidase gene was obtained by genome walking technology. The experimental operation process was carried out in accordance with the instructions of the Universal Genome Walker TM Kit (CLONTECH).

[0034] 1. Cultivation of Shepherd's Purse Seedlings

[0035] The shepherd's purse seeds used for sowing were purchased from Shanghai Seed Company, and treated at 4°C for 3-7 days before sowing, that is, vernalization treatment was performed to keep the germination of the seeds consistent. The vernalized seeds are sterilized with 75% ethanol for 5-10 minutes, and then go to the sterile operating table for the next step. Change the seeds in 75% ethanol to a small amount of absolute ethanol solution, then pour the seeds along with the absolute ethanol onto sterilized filter paper. After the absolute ethanol evaporates and ...

Embodiment 2

[0051] Example 2 Expression characteristics of shepherd's purse peroxidase gene under cold induction and cold acclimatization

[0052] In this example, it is confirmed that the shepherd's purse peroxidase gene has low-temperature induction properties.

[0053] 1. Processing of shepherd's purse seedlings

[0054]The cultivation of shepherd's purse seedling is identical with the procedure of embodiment 1. After the shepherd's purse seedlings grew to 4w, cold acclimatization and cold induction were carried out. The cold acclimation was treated continuously according to the temperature gradient of 12°C (4d), 4°C (4d), and 0°C (2h). 4h, 8h, 24h time gradient treatment, and then collect leaves, stems and roots and other materials, and store them at -70°C for later use.

[0055] 2. RNA extraction of shepherd's purse seedlings

[0056] The plant RNA extraction kit (CW0588) (Beijing Kangwei Century Biotechnology Co., Ltd.) was used to extract total RNA from three tissues of shepherd...

Embodiment 3

[0061] Example 3 GUS Analysis of Low Temperature Inducible Activity of Shepherd's Purse Peroxidase Gene Promoter

[0062] In this embodiment, the promoter sequence of the cloned shepherd's purse peroxidase gene is added with restriction site Bgl II and Pst Ⅰ. By replacing the position of the CaMV35S promoter and linking it to the pCAMBA1301 vector (provided by CAMBIA [the Center of the Application of Molecular Biology to International Agriculture, Australia]), a plant transformation vector that drives the expression of the GUS gene was constructed. The experiment of transforming Arabidopsis showed that the promoter of shepherd's purse peroxidase gene can enhance the expression of GUS gene under low temperature induction. Therefore, the promoter of shepherd's purse peroxidase gene has a potential application prospect in cultivating cold-resistant crops by means of genetic engineering.

[0063] 1. Construction of expression vector

[0064] In this embodiment, the constructe...

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Abstract

The invention belongs to the technical field of plant molecular biology, and in particular discloses a shepherd's purse peroxidase gene promoter and an application thereof in improving plant cold tolerance. The nucleotide sequence of the shepherd's purse peroxidase gene promoter is that of the 1st-1021st bases in SEQ ID No.1. A gene driven by the promoter can code a peroxidase gene which is inducible by low temperature and is capable of regulating reactive oxygen in plant cells. Under an induction condition of 4 DEG C, the expression amount of the promoter is up-regulated, and meanwhile, the expression amount of the promoter is also up-regulated in leaves and stems. An inducible plant expression vector, which is constructed by virtue of promoter fragments, can start the expression of a target gene in the case that plants are under low-temperature stress. The invention also provides an application of the promoter in breeding cold-tolerance plants, so that the cold tolerance of crop varieties can be improved.

Description

technical field [0001] The invention belongs to the technical field of plant molecular biology, and in particular relates to a plant low-temperature-induced promoter and its application, in particular to a shepherd's purse low-temperature-induced promoter capable of regulating cold-induced genes, and the application of the promoter in improving plant cold resistance . Background technique [0002] Substantial changes in external environmental factors such as temperature, moisture, light, and soil ion concentration will have adverse effects on plants, not only affecting the growth and yield of crops, but also limiting their temporal and spatial distribution (KasugaM, Liu Q, Miura S, Yamaguchi-Shinozaki K, Shinozaki K. Improving plant drought, salt, and freezing tolerance by gene transfer of a single stress-inducible transcription factor. Nat. Biotechnol., 1999, 17:287-291). However, many plants in low-temperature areas have undergone a long-term adaptation process to improve...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/10C12N15/82A01H5/00
CPCC12N9/0065C12N15/8273C12Y111/01
Inventor 林娟李伟伟陈虎魏东晖
Owner FUDAN UNIV
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