Arabidopsis thaliana floral organ specificity promoter and application thereof

A technology of Arabidopsis thaliana flower and promoter, which is applied in the field of plant genetic engineering and achieves the effect of wide application value

Inactive Publication Date: 2014-02-19
JIANGXI AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] At present, there is no promoter specifically expressed in the floral organs of the molecular biology model plant Arabidopsis thaliana, so there is an urgent need in the field to screen and develop promoters that are conducive to the specific expression of Arabidopsis or other plant floral organs

Method used

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  • Arabidopsis thaliana floral organ specificity promoter and application thereof
  • Arabidopsis thaliana floral organ specificity promoter and application thereof
  • Arabidopsis thaliana floral organ specificity promoter and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1: Arabidopsis floral organ-specific promoter cloning

[0033] First, using Arabidopsis genomic DNA as a template, the 2085bp floral organ-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 AtcpSecY1 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.

[0036] Upstream primer: 5'- TCTAGA GAACCATTGGCCATGTCGCTCA-3' (introduced Xba I restriction site)

[0037] Downstream primer: 5'- CCATGG CCTTATCCTTCTTCTCCTTC-3' (introduced Nco I restriction site)

[0038] ② The Arabidopsis genomic DNA was extracted according to conventional methods, and the genomic DNA was used as a template to perform PCR amplification using the above primers to prepare floral organ-specific prom...

Embodiment 2

[0058] Embodiment 2: utilize pCAMBIA1301 carrier (containing GUS reporter gene) to construct "Arabidopsis flower organ-specific promoter- GUS "Fusion gene

[0059] (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).

[0060] (2) Extract the plasmid from the TA clone prepared in Example 1, use Xba I / Nco I double enzyme digestion, recovery (same as Example 1) Arabidopsis flower organ-specific promoter fragment by agarose gel electrophoresis.

[0061] (3) The above two fragments were ligated overnight at 16°C under the catalysis of ligase to complete the "Arabidopsis floral organ-specific promoter- GUS "Fusion gene construction.

[0062] Connection system:

[0063] Reagent Amount added (μl) Floral organ-specific promoter fragment (50 ng μl -1 ) 2.0 Large fragment of pCAMBIA1301 vector (50 ng μl -1 ...

Embodiment 3

[0071] Embodiment 3: Preparation of transgenic Arabidopsis plants

[0072] (1) "Arabidopsis flower organ-specific promoter- 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.

[0073] (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:

[0074] Upstream primer: 5'-TCTAGAGAACCATTGGCCATGTCGCTCA-3'

[0075] Downstream primer: 5'-TACAGTCTTGCGCGACATGCG-3'

[0076] The PCR products were subjected to agarose gel electrophore...

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Abstract

The invention relates to a newly discovered DNA sequence which can be used as a promoter for regulating and controlling a target gene to be specifically expressed in a floral organ of arabidopsis thaliana. An AtcpSecY1 gene promoter sequence is cloned from a model plant arabidopsis thaliana; subsequently in a transgenic arabidopsis thaliana, the promoter is confirmed to be capable of driving a GUS reporter gene to be specifically expressed in the floral organ of arabidopsis thaliana. With the application of the promoter, a 'floral organ specificity promoter-target gene to be expressed' fusion gene is obtained by construction, arabidopsis thaliana is transformed, and the transgenic plant having the target gene specifically expressed in the floral organ can be obtained. The promoter not only provides important molecular elements for in-depth research of plant floral organ differentiation, formation, growth and development molecular mechanisms and other theory researches, but also provides important molecular regulating and controlling elements for plant genetic engineering seed breeding, especially the gene engineering seed breeding for regulating and controlling flower shapes, flower colors, flower aromas, ornamental life and the like of famous and valuable flowers, and has wide application value.

Description

[0001] technical field [0002] The invention belongs to the field of plant genetic engineering, in particular to obtaining an Arabidopsis floral organ-specific promoter by using molecular biology technology and its application in transgenic plants. Background technique [0003] A promoter is a specific nucleic acid sequence located upstream of a structural gene, which can control the initiation time and intensity of transcription of its downstream structural gene. Promoters are like "switches" that determine the activity of structural genes. Much of the regulation of gene expression in higher plants occurs at the transcriptional level, and is coordinated by various cis-acting elements and trans-acting factors. Plant gene promoters are important cis-acting elements (Li Jie et al., 2006). [0004] Promoters in plants can be divided into three categories according to their modes and functions of transcription initiation: constitutive promoters, tissue or organ-specific promot...

Claims

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Application Information

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