Yunnan red pear PybHLH 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 less research, and achieve the effect of strong advantages, accurate experimental results, and convenience for subsequent experiments.

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

There are many previous studies on MYB, but less on bHLH and WD40 proteins in plants

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Yunnan red pear of the present invention PyBH Gene cloning, prokaryotic expression, purification and antibody preparation, the specific steps are as follows:

[0036] (1) Primer design

[0037] According to the coding frame of apple MdbHLH33 gene (GenBank accession number is DQ266451.1), design a pair of specific primers PybHLH-F: 5' -GGATCC atggctcagaatcatgagagggtg-3' and PybHLH-R:5'- CTCGAG gcacttaccagcaattttccaaagc-3', BamHI and XhoI restriction sites were added to the 5' ends of the upstream and downstream primers respectively (the underlined part is the restriction site).

[0038] (2) Extraction of total RNA

[0039] a. After fully grinding 0.1 g of the red pericarp of Yunhongli No. 1 into powder with liquid nitrogen, add 1 ml of RNA extraction buffer (4 M guanidine isothiocyanate, 25 mM sodium citrate, 0.5 % (w / v) Sodium lauryl sarcosinate, 2% (w / v) PVP, β-mercaptoethanol), transferred to a 2 ml centrifuge tube, grinded evenly, and then added 1 / ...

Embodiment 2

[0070] Example 2: Western blotting of PybHLH protein-specific antibody and detection of transgenic tobacco

[0071] In order to detect the effectiveness of the PybHLH protein-specific antibody, the PybHLH protein antibody was used to detect the induced PybHLH prokaryotic expression protein and transgenic tobacco, and the specific process was as follows:

[0072] The instruments used in this example are vertical protein electrophoresis instrument, PVDF membrane (millipore), Semi-Dry Transfer Cell (BIO-RAD) transfer membrane system;

[0073] Reagents are acrylamide, dimethylacrylamide, 10% APS, TEMED, 1×glycine Buffer, 1×PBS, 1×PBT, skimmed milk powder, Whitman 3MM filter paper and other SDS-PAGE and Western test reagents. 】,

[0074] A. Western blotting detection of PybHLH prokaryotic expression protein

[0075] Protein sample preparation: protein sample and 1 / 5 volume of 5× protein loading buffer (Tris-Cl (pH 6.8) 250 mM, SDS 10%, bromophenol blue 0.5%, glycerol 50%, β-me...

Embodiment 3

[0112] Example 3: Co-immunoprecipitation and Far Western blotting analysis of the interaction between PyMYB and PybHLH

[0113] 1. Use the soluble protein (500ug) extract of Yunhong pear No. 1 peel for co-immunoprecipitation analysis, add 5ug specific antibody anti-PyMYB to the total peel protein solution (PyMYB specific antibody is completed by our laboratory, specific The steps are as follows: Clone to obtain Yunhongli No. 1 PyMYB gene, construct a prokaryotic expression vector, and obtain PyMYB recombinant protein; purify the obtained PyMYB recombinant protein, collect the purified protein, and send the purified PyMYB protein to the antibody preparation company to prepare polyclonal antibody) and anti-GST (GST antibody was purchased from Kangwei Century), shake and incubate at room temperature for 2 hours; then add 20ul protein A / G agarose to the protein solution and incubate overnight at 4°C with shaking; after incubation, the protein mixture is centrifuged at 3500 g f...

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Abstract

The invention discloses a Yunnan red pear PybHLH gene through anthocyanin biosynthesis, trichome development and a salt stress regulatory protein and a prokaryotic expression vector thereof. The PybHLH gene is cloned from red fruit peel of Yunnan red pear NO.1 by using a RT-PCR (reverse transcription-polymerase chain reaction) technology, then the prokaryotic expression vector pGEX-4T-PybHLH is constructed, PybHLH protein is expressed in escherichia coli Rosetta (DE3) by pGEX-4T-PybHLH vector and then is purified, and Yunnan red pear PybHLH purified protein is obtained; the prokaryotic expression vector of the PybHLH gene is applied to preparing a pybHLH specific antibody, and the obtained pybHLH specific antibody is also used for researching the interaction of the protein and protein as well as the protein and DNA(deoxyribonucleic acid) and the detection to PybHLH transgenic plant. The obtained pybHLH specific antibody contains glutathione S transferase (GST) and is applied to researching the interaction of the protein through Far Western blotting and co-immunoprecipitation and accurately separating and obtaining the protein and DNA segment which are interacted with PybHLH.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and specifically relates to a Yunnan red pear anthocyanin biosynthesis, trichome development and salt stress regulation protein gene PyBH The prokaryotic expression thereof and the application of the prokaryotic expression vector in the preparation of PybHLH protein and specific antibody. Background technique [0002] Red peel 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 regul...

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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