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Promoter with both plant overground tissue organ specificity and photoinduced specificity and application thereof

A tissue organ and promoter technology, applied in the field of plant genetic engineering, can solve problems such as gene silencing, death, and plant metabolic imbalance, and achieve good regulation and wide application value

Inactive Publication Date: 2015-05-20
JIANGXI AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The purpose of the present invention is to solve the problem that in the existing plant genetic engineering technology, due to the use of a constitutive promoter, the foreign gene overexpresses the heterologous protein or metabolite in the transgenic plant, which makes the original metabolism of the plant unbalanced and hinders the growth of the plant. Normal growth and development, even death, and repeated use of the same constitutive promoter to drive the expression of multiple foreign genes may cause gene silencing

Method used

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  • Promoter with both plant overground tissue organ specificity and photoinduced specificity and application thereof
  • Promoter with both plant overground tissue organ specificity and photoinduced specificity and application thereof
  • Promoter with both plant overground tissue organ specificity and photoinduced specificity and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: Cloning with Plant Aboveground Organs and Light-Inducible Specific Promoters

[0035] First, using the Arabidopsis genomic DNA as a template, the PCR method was used to amplify a 1851bp promoter with both plant aboveground tissues and organs and light-induced specificity, and the amplified product was recovered and TA cloned.

[0036] (1) PCR amplification of the target fragment:

[0037] ①According to known Arabidopsis AtcpSecY2 Design specific primers for the sequence of the gene promoter region, and introduce in the upstream primer Xba I restriction site, introduced in the downstream primer Nco I restriction site.

[0038] Upstream primer: 5'- TCTAGA CTTGTCGACAACTACTGATCCG-3' (introduced Xba I restriction site)

[0039] Downstream primer: 5'- CCATGG CGAGAGGATGAGGAGAGAAA-3' (introduced Nco I restriction site)

[0040] ② Extract Arabidopsis genomic DNA according to conventional methods, use the genomic DNA as a template, and use the above ...

Embodiment 2

[0059] Embodiment 2: utilize pCAMBIA1301 carrier (containing GUS Reporter gene) construction "both plant above-ground tissue organs and light-inducible specific promoter- GUS "Fusion gene

[0060] (1) Extract vector pCAMBIA 1301 plasmid (purchased from CAMBIA Company) from Escherichia coli, use Xba I / Nco Reclaim the large carrier fragment (which contains GUS reporter gene sequence).

[0061] (2) Extract the plasmid from the TA clone prepared in Example 1, use Xba I / Nco I double enzyme digestion, recovery by agarose gel electrophoresis (same as Example 1) Arabidopsis thaliana has both aerial tissues and organs and light-induced specific promoter fragments.

[0062] (3) The above two fragments were ligated overnight at 16°C under the catalysis of ligase to complete the "both plant aerial tissue and organ and light-inducible specific promoter- GUS "Fusion gene construction.

[0063] Connection system:

[0064] Reagent Amount added (μl) B...

Embodiment 3

[0073] Preparation of transgenic Arabidopsis plants

[0074] (1) The "both plant aboveground tissue and organ and light-inducible specific promoter" constructed in Example 2- GUS "The fusion gene was transformed into Arabidopsis thaliana. The specific transformation method used the method of Floral dip mediated by Agrobacterium (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.

[0075] (2) PCR detection of transgenic plants: cut out the leaves of transgenic plants and wild-type plants respectively, and extract genomic DNA from leaves by referring to the "Molecular Cloning Experiment Guide (Third Edition)" (Huang Peitang et al., 2002), and perform PCR with the following primers Reaction, reaction system is as embodiment 1:

[0076] Upstream primer: 5'- TCTAGACTTGTCGACAACTACTGATCCG-3'

[0077] Downstream primer: 5'-TACAGTCTTGCGCGACATGCG-3'

[0078] The PCR...

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Abstract

The invention relates to a newly discovered DNA sequence, which can be used as a promoter to regulate the specific expression of the target gene in the tissues and organs of the aboveground part of the plant in the form of light induction. The present invention clones the promoter sequence of the AtcpSecY2 gene from the model plant Arabidopsis thaliana, and then confirms in the transgenic Arabidopsis that the promoter can drive the GUS reporter gene to be specific in the tissues and organs of the aboveground parts of the plant in the form of light induction Express. The promoter of the present invention is used to construct a "promoter-target gene to be expressed" fusion gene, which can be transformed into a plant to obtain a transgenic plant with both plant aboveground tissues and organs and light-induced specific expression of the target gene. This not only helps to study the transcriptional regulation and expression mode of plant genes under light conditions, but also can be applied in plant genetic engineering to make exogenous genes moderately expressed under light conditions accompanied by plant physiological states, and at the same time accurately and effectively change the physiological state of plants. Photosynthetic characteristics and agronomic traits, to achieve strain improvement of crops, has a wide range of application value.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering, and specifically uses molecular biology technology to obtain a promoter having both plant aboveground tissue and organ and light-induced specificity and its application in transgenic plants. Background technique [0002] A promoter is a DNA sequence located upstream of a structural gene that recognizes, binds, and initiates transcription of RNA polymerase. The promoter is like a "switch" that can control the initiation time and intensity of transcription of its downstream structural gene. The regulation of gene expression in higher plants is one of the central contents of plant genetic engineering research. As an important molecular regulatory element at the transcription level, the study of promoters has become a hot spot in molecular biology (Wang Ying et al., 2003; Li Jie et al. , 2006). [0003] Promoters in plants can be divided into three categories according to their mode and func...

Claims

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Application Information

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