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Salt stress-induced specific promoter of plant leaf and application thereof

A technology of plant leaves and promoters, applied in the field of plant genetic engineering, can solve problems such as death, plant metabolic imbalance, hindering normal growth and development of plants, etc., achieve wide application value and improve salt tolerance

Inactive Publication Date: 2014-10-29
JIANGXI AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is to solve the problem of using constitutive promoters to overexpress heterologous proteins or metabolites in transgenic plants due to the use of constitutive promoters in the existing plant genetic engineering technology, which makes the original metabolism of plants unbalanced and hinders the normal growth of plants development, and even death

Method used

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  • Salt stress-induced specific promoter of plant leaf and application thereof
  • Salt stress-induced specific promoter of plant leaf and application thereof
  • Salt stress-induced specific promoter of plant leaf and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1: Cloning of a leaf-specific promoter induced by salt stress in Arabidopsis

[0033] First, using Arabidopsis genomic DNA as a template, the 1444 bp salt stress-induced Arabidopsis leaf-specific promoter was amplified by PCR method, and the amplified product was recovered and TA cloned.

[0034] (1) PCR amplification of the target fragment:

[0035] ①According to known Arabidopsis AtPGK2 Design specific primers for the sequence of the gene promoter region, and introduce in the upstream primer Bam H I restriction site, introduced in the downstream primer Nco I restriction site:

[0036] Upstream primer: 5'-CGC GGATCC AATATCACACCACGGATTGGG-3'

[0037] Downstream primer: 5'-CATG CCATGG GTGAAGTCAACAGATAGTGA-3'

[0038] ② Extract Arabidopsis thaliana genomic DNA according to a conventional method, use the genomic DNA as a template, and use the above primers to perform PCR amplification to prepare the Arabidopsis leaf-specific promoter fragment ind...

Embodiment 2

[0057] Example 2: Using the pCAMBIA1301 vector (containing GUS reporter gene) to construct the "salt stress-induced plant leaf-specific promoter-GUS" fusion gene

[0058] (1) Extract vector pCAMBIA 1301 plasmid (purchased from CAMBIA Company) from Escherichia coli, use Bam H I / Nco Recover the large carrier fragment (which contains GUS reporter gene sequence).

[0059] (2) Extract the plasmid from the TA clone prepared in Example 1, and use Bam H I / Nco I double-enzyme digestion, and recovered (same as Example 1) plant leaf-specific promoter fragments induced by salt stress by agarose gel electrophoresis.

[0060] (3) Ligate the above two fragments under the catalysis of ligase overnight at 16°C to complete the construction of the "salt stress-induced plant leaf-specific promoter-GUS" fusion gene on the pCAMBIA 1301 vector.

[0061] Connection system:

[0062] Reagent Amount added (μl) Salt stress-induced plant leaf-specific promoter fragme...

Embodiment 3

[0069] Example 3: Preparation of transgenic Arabidopsis plants

[0070] (1) Transform Arabidopsis thaliana with the "salt stress-induced plant leaf-specific promoter-GUS" fusion gene constructed in Example 2. The specific transformation method uses the Agrobacterium-mediated Floral dip method (Clough and Bent, 1998) , the obtained seeds were subjected to 50 mg l -1 For hygromycin resistance screening, normal growing plants were transferred to soil for culture.

[0071] (2) PCR detection of transgenic plants: Cut the leaves of transgenic plants and wild-type plants respectively, refer to the method of "Molecular Cloning Experiment Guide (Third Edition)" (Huang Peitang et al., 2002) to extract genomic DNA from leaves, and use the following primers for PCR Reaction, reaction system is as embodiment 1:

[0072] Upstream primer: 5'-CGCGGATCCAATATCACACCACGGATTGGG-3'

[0073] Downstream primer: 5'-TACAGTCTTGCGCGACATGCG-3'

[0074] The PCR products were subjected to agarose gel ...

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Abstract

The invention relates to a salt stress-induced specific promoter of a plant leaf and an application of the salt stress-induced specific promoter. A promoter sequence of an AtPGK2 gene is cloned from a model plant Arabidopsis, and then, in transgenic Arabidopsis, the promoter can drive a GUS (Glucuronidase) reporter gene to be specifically expressed in the plant leaf in a salt stress-induced form. By using the promoter provided by the invention, a promoter-target gene fused gene is established and obtained, and a transgenic plant having a target gene which can be specifically expressed in the plant leaf in a salt stress-induced form can be obtained by using the promoter-target gene fused gene into plant transformation. Therefore, the promoter not only is beneficial to research on the molecular mechanism of the plant responding to salt stress, but also is applied to plant genetic engineering so that the exogenous gene regulates specific expression of the target gene in the plant leaf in a way of salt stress induction, and meanwhile, the salt resistance of the plant is improved with a clear target. The promoter has a wide application value.

Description

Technical field [0001] The present invention is a plant genetic engineering field. Specifically, it involves a kind of plant leaves that are induced by salt coercion. Background technique [0002] Salt coercion is one of the non -biological stress suffered by plants, which seriously affects the growth and development of plants and the output and quality of crops (Parvaiz and Satyawati (2008).my country's salt soil is widely distributed, large area, and many types, accounting for about 10%of the land area, and the area of ​​salt soil is continuously expanded each year, threatening the sustainable development of agricultural production (Sun Jianchang et al., 2008).Therefore, it is important to cultivate new varieties of crops with strong salt resistance. [0003] The promoter is a regulatory sequence located in the upstream of the structural gene. It is like a "switch" and can regulate the time and space specificity of its downstream gene expression.The promoter of higher plants is...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/10C12N15/82A01H5/00
Inventor 刘栋李卫春马利霞程建峰
Owner JIANGXI AGRICULTURAL UNIVERSITY
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