Yunnan red pear PybMYB gene as well as prokaryotic expression vector and application thereof

A prokaryotic expression and gene technology, applied in the field of genetic engineering, can solve problems such as lack of scientific theory, and achieve the effect of strong specificity and efficient separation

Inactive Publication Date: 2013-06-12
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But, the scientific theory deficiency of the red pear coloring of instruction breeding, achievement of the present invention will lay the foundation for the molecular breeding of fruit trees and flowers

Method used

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  • Yunnan red pear PybMYB gene as well as prokaryotic expression vector and application thereof
  • Yunnan red pear PybMYB gene as well as prokaryotic expression vector and application thereof
  • Yunnan red pear PybMYB gene as well as prokaryotic expression vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 : Prokaryotic expression vector pGEX-4T- PyMYB build

[0040] (1) Primer design

[0041] According to the pear in the NCBI database myb Gene (Accession Number: EU153575.1) sequence and prokaryotic expression vector pGEX-4T-1 multiple cloning restriction site, design a pair of specific primers PyMYB-F: 5'- GGATCC GTCTACATGGAGGGATATAACGTTAACTTG-3' and PyMYB-R:5'- GTC GAC GATATCTTCTTCTTTTGAATGATTCCAAAGG-3', added at the 5' ends of the upstream and downstream primers Bam HI and Sal I enzyme cleavage site (the underline is the enzyme cleavage site).

[0042] (2) Extraction of total RNA

[0043] The total RNA of Yunhong pear No. 1 peel was extracted by the guanidine isothiocyanate method, and the electrophoresis detection results were as follows figure 1 .

[0044] (3) RT-PCR

[0045] Use Yunhongli No. 1 total RNA as a template, use M-MLV reverse transcriptase to synthesize cDNA and use it PyMYB -F and PyMYB -R for amplification to obtain PyMYB f...

Embodiment 2

[0051] Example 2: Prokaryotic expression vector pGEX-4T- PyMYB Prokaryotic expression of

[0052] The recombinant plasmid pGEX-4T- PyMYB Transformed into E. coli Rosetta (DE3) Competent cells, spread on LB + Amp solid plate, pick pGEX-4T- PyMYB The recombinant colonies were cultured overnight in LB + Amp liquid medium at 37 °C and 200 rpm on a shaker, and then inoculated on the same LB medium at a ratio of 1:100 and cultivated to OD 600 0.6-0.8, add IPTG to a final concentration of 1 mM, culture at 16 °C and 37 °C for 0, 2, 4, 6, and 8 h, and collect the bacterial liquid for total protein analysis; the collected bacterial liquid is 4 °C , centrifuged at 12 000 rpm for 1 min, discarded the supernatant, and precipitated with 100 μl SDS gel loading buffer (Tris-HCl 50 mM, pH 6.8; SDS 2 %; DTT 100 mM; bromophenol blue 0.1 %; glycerol 10 % ) was resuspended, boiled for 5 min, centrifuged at 12 000 rpm for 1 min, and 20 μl of the supernatant was taken for SDS-PAGE detection; ...

Embodiment 3

[0053] Example 3: Purification of PyMYB protein, the specific steps are as follows:

[0054] a. Bacterial cell disruption: 1 L of bacterial cells induced to express in large quantities at 16 °C and 1 mM IPTG were ultrasonically disrupted (working for 5 s, resting for 8 s) for 10 min;

[0055] b. Collect the supernatant and precipitate: centrifuge the broken cell solution at 4 °C and 12 000 rpm for 20 min, and keep the supernatant and precipitate respectively;

[0056] c. Filter the protein crushing supernatant with a 0.45 μm filter to remove impurities;

[0057] d. Pretreatment of GST gel chromatography column: Gently mix the GST gel until it is completely resuspended; pipette an appropriate amount of GST gel into the chromatography column, and use 10 times the column bed volume of PBS Buffer (10mM Na 2 HPO 4 , 1.8mM KH 2 PO 4 , 140mM NaCl, 2.7mM KCl, adjust the pH to 8.0) to wash the column;

[0058] e. Protein sample loading: add the PBS solution containing GST fusio...

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Abstract

The invention discloses a Yunnan red pear PyMYB gene through pigment synthesis regulation and trichome development regulatory protein as well as a prokaryotic expression vector thereof and an application of the prokaryotic expression vector. The PyMYB gene is cloned from red fruit peel of Yunnan red pear NO.1 by using a RT-PCR (reverse transcription-polymerase chain reaction) technology, and then the gene is expressed in escherichia coli Rosetta (DE3) by using the prokaryotic expression vector and is purified by a GST (Glutathione S-transferase) gel column purification method to obtain a Yunnan red pear PyMYB purified protein, wherein the Yunnan red pear PyMYB purified protein is used for the preparation of a PyMYB specific antibody and the application thereof in a PyMYB protein expression detection and immunoprecipitation, a chromatin co-immunoprecipitation and a fusion protein sedimentation experiment. The antibody prepared by the invention is strong in specificity, can accurately detect subcellular localization of the PyMYB protein, and is used for verifying protein interaction in the co-immunoprecipitation experiment, efficiently separating the protein and DNA (deoxyribonucleic acid) segment combined by the PyMYB protein and detecting the expression of the antibody in a transgenic plant.

Description

technical field [0001] The invention belongs to the field of genetic engineering, in particular to a Yunnan red pear pigment and trichome development regulation protein gene PyMYB The prokaryotic expression vector thereof, and the application of the prokaryotic expression vector and the PyMYB antibody. Background technique [0002] Red skin is an important trait index in pear molecular breeding. Due to its unique geographical and climatic conditions, Yunnan Province has more germplasm resources of red skin pears. Since 1986, four cultivars, Zaobaimi, Meirensu, Mantianhong and Yunhongli No. 1, have been bred successively. However, except for Yunhongli No. 1, the other three varieties will have poor coloration or even no coloration due to climate change. Therefore, it is necessary to study the coloration mechanism of red pear peel. Previous studies have shown that the synthesis of plant anthocyanins is regulated by the transcription complex MYB / bHLH / WD40. Previous studies o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/70C07K14/415C07K16/16
Inventor 李昆志崔道雷张晓东樊磊陈丽梅舒群苏俊
Owner KUNMING UNIV OF SCI & TECH
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