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Yunnan red pear delta PyTTG1 gene, prokaryotic expression vector thereof and application of prokaryotic expression vector

A prokaryotic expression and gene technology, applied in the field of genetic engineering, can solve the problem of no subcellular localization

Inactive Publication Date: 2012-07-18
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the PyTTG1 gene in Yunnan red pear and apple has been cloned and transgenic, but it has not been specifically used to accurately detect the subcellular localization of PyTTG1 protein, efficiently purify PyTTG1 protein, and efficiently separate the protein and DNA fragments bound by PyTTG1 protein and antibodies to detect its expression in transgenic plants

Method used

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  • Yunnan red pear delta PyTTG1 gene, prokaryotic expression vector thereof and application of prokaryotic expression vector
  • Yunnan red pear delta PyTTG1 gene, prokaryotic expression vector thereof and application of prokaryotic expression vector
  • Yunnan red pear delta PyTTG1 gene, prokaryotic expression vector thereof and application of prokaryotic expression vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: Prokaryotic expression vector pET32a- ?PyTTG1 build

[0034] (1) Primer design

[0035] Yunnan red pear PyTTG1 Gene (GenBank accession number is HQ641374) coding frame and apple, Arabidopsis, corn, perilla, petunia WD40 Gene and prokaryotic expression vector pET-32a multiple cloning restriction site, design a pair of specific primers ?PyTTG1 -F: 5'- CCG CCATGG AGAACTCTACGCAAGAATCG-3′, ?PyTTG1 -R: 5'- CCG CTCGAG GTTCGGCTTTATTGAAAGGGTATCC-3', added at the 5' end of the upstream and downstream primers respectively Nco I and xho I Restriction site and protective base (the underlined part is the restriction site, and the italic part is the protective base).

[0036] (2) Extraction of total RNA

[0037] Use the guanidine isothiocyanate method to extract the total RNA of the red pear peel, and the electrophoresis detection results are as follows: figure 1 .

[0038] (3) RT-PCR

[0039] Using the total RNA of Yunnan red pear as a template, c...

Embodiment 2

[0044] Example 2: Prokaryotic expression vector pET32a- ?PyTTG1 Prokaryotic expression of

[0045] Using the heat stimulation method, the recombinant plasmid pET32a-Δ PyTTG1 Transformed into E.coli Rosetta (DE3) Competent cells, spread on LB + Amp solid plate, pick pET32a-Δ PyTTG1 The recombinant colonies were cultured in LB + Amp liquid medium at 37 °C, 200 rpm shaker overnight, 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 0.5 mM, culture at 16°C and 37°C for 0, 2, 4, and 6 hours, respectively, and collect the bacterial liquid for total protein analysis; the collected bacterial liquid is 4°C, 12 000 rpm for 1 min, discard the supernatant, and precipitate 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%) Suspended, boiled for 5 min, centrifuged at 12 000 rpm for 1 min, and 20 μl of the supernatant was taken for SDS-PAGE detec...

Embodiment 3

[0046] Example 3: Purification of ΔPyTTG1 protein, the specific steps are as follows:

[0047] a. Bacterial cell disruption: 1 L of bacterial cells induced to express in large quantities at 16 °C and 0.5 mM IPTG were ultrasonically disrupted (work for 3 s, rest for 6 s) for 5 min;

[0048] 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;

[0049] c. Filter the protein crushing supernatant with a 0.22 μm filter to remove impurities;

[0050] d. Pretreatment of the His-Trap HP column: wash the column with 5 times the column volume of pure water; equilibrate the column with 5 times the column volume of binding buffer (sodium phosphate buffer (pH7.4) 20 mM, NaCl 0.5M, imidazole 30 mM) , the flow rate is 1 ml / min;

[0051] e. Protein sample on the column: the flow rate is 1 ml / min, and the effluent is collected;

[0052] f. Column washing: use 5 times column v...

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Abstract

The invention discloses a Yunnan red pear pigment synthesis and trichome development regulatory protein PyTTG1 gene delta PyTTG1 and a prokaryotic expression vector thereof. The Yunnan red pear delta PyTTG1 purified protein is obtained by cloning the specific segment delta PyTTG1 of the PyTTG1 gene from the Yunnan red pear, constructing the prokaryotic expression vector of the gene and expressing the gene in the Escherichia coli. The Yunnan red pear delta PyTTG1 purified protein is applied to preparation of a PyTTG1 specific antibody, detection on PyTTG1 protein expression, immunoprecipitation, and chromatin immunoprecipitation. The antibody has high specificity and can accurately detect the subcellular localization of the PyTTG1 protein, efficiently purify the PtTTG1 protein, efficiently separate the protein bound with the PyTTG1 protein from the DNA segment, and detect the expression condition of the the PtTTG1 protein in transgenic plants.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and specifically relates to a Yunnan red pear pigment regulation and trichome development regulation protein gene ΔPyTTG1 The prokaryotic expression vector thereof, and the application of the prokaryotic expression vector in the preparation of ΔPyTTG1 protein and specific 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...

Claims

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Application Information

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IPC IPC(8): C12N15/29C07K14/415C12N15/70C07K16/16C12R1/19
Inventor 李昆志张晓东樊磊崔道磊陈丽梅舒群苏俊
Owner KUNMING UNIV OF SCI & TECH
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