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Specific expression carrier of bec gene in floral organ

A technology for expressing vectors and flower organs, which is applied in genetic engineering, plant genetic improvement, and the introduction of foreign genetic material using vectors, etc. It can solve the problems of low yield and complicated extraction process, and achieve the effect of changing color and improving ornamental value

Inactive Publication Date: 2006-01-04
NANJING FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] Indigo is one of the earliest natural dyes discovered, with bright and durable colors. It is usually extracted from indigo-containing plants, but the extraction process is complicated and the yield is low. Therefore, most of the indigo pigments currently on the market are chemically synthesized

Method used

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  • Specific expression carrier of bec gene in floral organ
  • Specific expression carrier of bec gene in floral organ

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1: Extraction of Arabidopsis total DNA.

[0019] 1. Take Arabidopsis thaliana Columbia ecotype seeds, inoculate them on the culture medium after aseptic treatment, and wait for the leaves to grow.

[0020] 2. Weigh 4 grams of fresh leaves as plant material, grind them into powder in liquid nitrogen, put them into a 50ml centrifuge tube, add 20ml of ice-bathed extraction buffer, and mix well.

[0021] 3. Centrifuge at 2,700g for 20 minutes, take the supernatant and add 8ml of lysis buffer, vortex to mix well, and incubate at 65oC for 20-30 minutes.

[0022] 4. Add 10 ml of chloroform / isoamyl alcohol (24:1), invert and mix well, centrifuge at 12000 rpm for 5 minutes, take the upper phase and transfer it to a new centrifuge tube. Repeat this several times until the interface is clear.

[0023] 5. Pipette the upper phase into another new Eppendof tube, add 2 / 3 volume of ice-cold isopropanol (about 5.6ml), and let stand at room temperature for 20-30 minutes.

[00...

Embodiment 2

[0030] Example 2: Cloning of a floral organ-specific promoter.

[0031] 1. Floral organ-specific promoter Pchs primers.

[0032] Upstream primer 5'-TAG GAG TTA AGT ATG CAC GTG TAA GAA CT-3'

[0033] Downstream primer 5'-CGC TAT AGT TAT CAC CAA CTT GG-3'.

[0034] 2. PCR amplification: The high-fidelity PCR reaction system is as follows: 10×ExBuf (Mg2+free) 2μl; 2mM dNTPMixer 2μl; 25mM Mg 2+ 1 μl; 0.2 μl of ExTaq DNA Polymerase, ie 1u; 1 μl of upstream primer, 30 pmol; 1 μl of downstream primer, 30 pmol; 1 μl of template (Arabidopsis total DNA about 10-250ng), add sterile ddH2O to make up 20 μl. Reaction program: pre-denaturation at 94°C for 3 minutes - (94°C for 40 seconds - 55°C for 30 seconds - 72°C for 40 seconds) × 35 cycles - 72°C for 10 minutes.

[0035] 3. After the reaction is completed, take 10 μl of the sample and go through 1.0% agarose gel electrophoresis, carefully cut out the specific fragment at about 530 bp with a disposable blade under ultraviolet light, re...

Embodiment 3

[0037] Example 3: Construction of floral organ-specific promoter gus expression vector (pESII).

[0038] 1. Take 7 μl of the PCR recovered product and connect it with 1 μl pGEM-T vector, transform Escherichia coli DH5α, and sequence and identify pGEM-T-Pchs.

[0039] 2. Digest pGEM-T-Pchs with PstI and NcoI, and connect the resulting fragment to the pUC-T-GAFP vector fragment that has been digested with PstI and NcoI, transform and identify, and obtain the intermediate vector pSI.

[0040] 3. The size of the positive clone was identified by double enzyme digestion with PstI and NcoI, and a fragment with a size of about 0.54 KB was excised.

[0041] 4. The Pchs promoter fragment obtained by digesting the intermediate vector pSI with HindIII and EcoRV was connected to the pBI121 vector fragment digested with HindIII and SmaI to obtain the floral organ-specific promoter gus expression vector pES II. Positive clones were identified by PCR, and a band of about 0.53 kb was seen, wh...

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Abstract

The present invention discloses one specific expression vector of bec gene in floral organ, and the vector contains promoter chalcone synthetase gene chs promoter Pchs, indole dioxygenase gene bec, terminator nos, screening marker gene NPTII and LB sequence and RB sequence expressed specifically in floral organ. The vector features that the bec gene is set under the regulation of floral organ specifically expressing promoter Pchs. Loading the vector into flower can create indigoidine specifically in floral organ to alter the color of flower and raise the enjoying value of flower.

Description

1. Technical field [0001] The invention relates to gene recombination and expression regulation. Specifically, the indole dioxygenase gene bec is recombined with a promoter specifically expressed in flower organs to construct a carrier plasmid capable of expressing indole dioxygenase specifically in flower organs. 2. Background technology [0002] Indigo is one of the earliest natural dyes discovered, with bright and durable colors. It is usually extracted from indigo-containing plants, but the extraction process is complicated and the yield is low. Therefore, most of the indigo pigments currently on the market are chemically synthesized. Since the discovery in 1928 that microorganisms can synthesize indigo pigment, it has become possible to produce natural indigo pigment on a large scale through microbial fermentation. The benzene ring in the indigo molecule can introduce various substituents (the most important being halogen) to form various indigo dyes. In addition, the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/29C12N15/52
Inventor 陈英黄敏仁王明庥诸葛强王光萍潘惠新李火根徐立安
Owner NANJING FORESTRY UNIV
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