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Promoter for plant gene specifically expressed in stoma and vascular bundle

A technology of vascular bundles and promoters, applied in the field of plant molecular biology and plant genetic engineering, can solve the problems of plant energy and nutrient waste, trait changes, affecting growth and development, etc., and achieve the effect of plant trait improvement

Inactive Publication Date: 2012-01-11
GRADUATE SCHOOL OF THE CHINESE ACAD OF SCI GSCAS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the continuous and efficient expression of exogenous genes in plants not only wastes plant energy and nutrients, but also often changes some traits and affects growth and development.

Method used

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  • Promoter for plant gene specifically expressed in stoma and vascular bundle
  • Promoter for plant gene specifically expressed in stoma and vascular bundle
  • Promoter for plant gene specifically expressed in stoma and vascular bundle

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1Bj

[0023] Example 1 Cloning of BjEXPA1 promoter sequence

[0024] Use the cloned expandin gene to design the primer sequence for genome walking (S2: 5'-TCACCACCGCCATAAAAGGTAGCAT-3'; S1: 5'-CTGTGACGACGATTGAGCCAGGGAG-3'), and use LA-PCR in vitro Cloning Kit (TAKARA) kit to start For the amplification of the sub-region, the primer sequence provided by the kit: C1: 5'-GTACATATTGTCGTTAGAACGCGTAATACGACTCA-3'; C1: 5'-CGTTAGAACGCGTAATACGACTCACTATAG GGAGA-3'. Specific steps are as follows:

[0025] 1. Extraction of Genomic DNA from Indian Mustard (CTAB Method)

[0026] 1.165℃ preheat 2×CTAB + Extraction buffer

[0027] 1.2 Weigh 1g of fresh plant tissue, put it in a mortar, grind it in liquid nitrogen, and distribute it into 10ml Eppendorf tubes;

[0028] 1.3 Add 600-700μl 65℃ to preheat 2×CTAB + Extract the buffer solution at 65°C for 30 min, invert several times during the period. Put it on ice for 5 minutes immediately after taking it out;

[0029] 1.4 Add an equal volume of chloroform-isoamy...

Embodiment 2

[0049] Example 2 Specific expression in guard cells and vascular bundles of BjEXPA1 promoter in transgenic tobacco

[0050] First, pCAMBIA1303 plant binary expression vector was digested with BamHI and NcoI to remove the 35S promoter on the vector. Using the LA-PCR method, we obtained a 1432bp promoter sequence upstream of the start codon and named it pBjEXPA1. According to the sequence, the promoter amplification and vector construction primers were designed: expproup: 5'-CGCGGATCCTCTAGAATCTAACTAACCCT-3'; expproup: 5 '-CATGCCATGGCTTGTTATCTTCTTAAAACCT-3'. Using this pair of primers to amplify the promoter sequence, after recovery, double digestion with BamHI and NcoI, and then connect with the digested pCAMBIA1303 vector to construct a hygromycin-resistant pBjEXPA1-gus recombinant vector. The recombined vector was transformed into Agrobacterium strain EHA105, and then used to infect tobacco leaf discs, and positive transgenic tobacco was screened with hygromycin. The specific i...

Embodiment 3

[0064] Example 3 Specific expression in guard cells and vascular bundles of BjEXPA1 promoter in transgenic Arabidopsis

[0065] The recombinant plasmid pBjEXPA1-gus was transformed into Arabidopsis thaliana using the pollen tube introduction method. The specific operation process is as follows:

[0066] 1. Preparation of bacterial liquid

[0067] Agrobacterium EHA105 carrying the recombinant plasmid pCAM-pBjEXPA1-gus was divided into a single colony on an LB plate (kanamycin 50μg / mL, rifampicin 40μg / mL), cultured at 28°C for about 48h. Pick a single colony in 2mL LB liquid medium containing antibiotics, 28°C, 200rpm, shaking culture for about 12h. Then transfer 2 mL of the bacterial solution to 200 mL of antibiotic-containing LB liquid medium, centrifuge at 28°C, 250 rpm, 12 h, 3000 g for 10 minutes to collect the bacteria, resuspend with Arabidopsis transformation permeate, and adjust OD 600 Value to about 0.8, used to infect Arabidopsis.

[0068] 2. Preparation of Arabidopsis

[006...

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Abstract

The invention discloses a promoter sequence for a gene specifically expressed in a stoma and a vascular bundle of a plant. The invention provides a promoter sequence of a Brassica juncea expansin gene (BjEXPA1) cloned by utilizing a genomic deoxyribonucleic acid (DNA) walking method, which is capable of driving a reporter gene to be specifically expressed in the stoma and the vascular bundle; the BjEXPA1 promoter sequence is fused with beta-glucuronidase (gus) gene to construct a plant expression vector; and the constructed recombinant vector is utilized to transform tobacco and arabidopsis to detect an obtained transgenic plant, and shown by a result, the BjEXPA1 promoter is capable of driving the GUS reporter gene to be expressed in the stoma and the vascular bundle of the plant.

Description

Technical field: [0001] The invention relates to the fields of plant molecular biology and plant genetic engineering, and relates to a promoter sequence that can drive the specific expression of a reporter gene in plant stomata and vascular bundles. Background technique: [0002] Gene expression is regulated and controlled by various physical and chemical factors in the organism, and is finely regulated at DNA level, transcription level, post-transcription level, translation level, post-translational processing, transportation and positioning. Promoter refers to a specific DNA sequence that RNA polymerase and some trans-acting factors recognize and bind to to initiate transcription correctly and effectively. Promoters generally consist of two parts: one part is required to form a universal transcription structure , Usually called the core promoter region, including the transcription start point and the adjacent TATA box; the other part is the region that determines the specificit...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/63A01H5/00
Inventor 柴团耀孙涛张玉秀刘戈宇
Owner GRADUATE SCHOOL OF THE CHINESE ACAD OF SCI GSCAS
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