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In-vitro expression method for pear PMEI (pectin mthylesterase inhibitor) protein, and application of pear PMEI

An in vitro expression and protein technology, applied in the field of bioengineering, can solve the problems of high cost of PMEI and difficulty of PMEI protein

Active Publication Date: 2020-05-12
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

PMEI widely exists in various tissues and growth stages of plants, but it is difficult to isolate and purify natural PMEI protein, and the cost of chemically synthesizing PMEI is high

Method used

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  • In-vitro expression method for pear PMEI (pectin mthylesterase inhibitor) protein, and application of pear PMEI
  • In-vitro expression method for pear PMEI (pectin mthylesterase inhibitor) protein, and application of pear PMEI
  • In-vitro expression method for pear PMEI (pectin mthylesterase inhibitor) protein, and application of pear PMEI

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1: Construction and Identification of PMEI-pCold-TF Recombinant Plasmid

[0065] Fresh Dangshansu pear pollen was taken, and total pollen RNA was extracted with a plant total RNA extraction kit; pollen total RNA was used as a template, and pollen cDNA was obtained by reverse transcription with a reverse transcription kit; figure 1 As shown, the signal peptide sequence of the pear PMEI gene was analyzed and removed, primers were designed, and the homology arms of the vector pCold-TF and the restriction sites of Xba I and Xho I were added to both ends of the primers, and the pollen cDNA was used as a template. PCR amplification of PMEI gene, PCR reaction system: ddH 2 O 19 μl, each 2.5 μl of upstream and downstream primers, 1 μl of cDNA, 2×Super Pfx MasterMix 25 μl; the PCR reaction conditions were: 98°C pre-denaturation for 3 minutes, followed by 98°C for 10 seconds, 60°C for 30 seconds, 72°C for 1 minute, a total of 30 cycles, and finally 72°C ℃ extension 10mi...

Embodiment 2

[0067] Embodiment 2: the expression of pear PMEI gene in escherichia coli

[0068] 1. Obtain a recombinant expression strain expressing pear PMEI

[0069] The monoclonal strain with correct sequencing was inoculated into 4 ml LB liquid medium (containing 100 μg / ml ampicillin), and cultured with shaking at 37° C. and 220 rpm overnight. The next day, the recombinant plasmid PMEI-pCold-TF was extracted according to the instructions of the QuickPure Plasmid Mini kit of Kangwei Century Company. Transform 1ng of the recombinant plasmid PMEI-pCold-TF into Escherichia coli strain Rosetta (DE3) by heat shock method, spread on LB plates (containing 100 μg / ml ampicillin) to select recombinants, and culture overnight at 37°C to obtain the expression pear Genetically engineered bacteria of PMEI. Get the single bacterium colony of the obtained genetically engineered bacteria and streak culture on the LB plate (containing 100 μg / ml ampicillin), inoculate a small amount of streaked culture ...

Embodiment 3

[0072] Embodiment 3: Separation, purification and identification of recombinant pear PMEI protein

[0073] The above-mentioned induced expression for 24 hours and the correct remaining recombinant expression bacterial liquid detected by SDS-PAGE electrophoresis were centrifuged at 4°C and 10000rpm for 10 minutes, the supernatant was discarded and the bacterial precipitate was collected, and 20ml of lysate (140mM sodium chloride) was added to the precipitate. , 2.7mM potassium chloride, 10mM disodium hydrogen phosphate, 1.8mM potassium dihydrogen phosphate, 50×EDTA-free protease inhibitorcocktail III (Merck Millipore, Germany), pH 7.3) to resuspend the precipitate; (Ultrasonic power is 240w, the conditions are 3s on, 7s off, 27 minutes in total) After clarification, centrifuge at 4°C and 10,000rpm for 10 minutes, collect the supernatant, and filter the supernatant through a 0.45μm filter membrane to remove impurities. Libo's Ni-NTA agarose affinity chromatography filler was use...

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Abstract

The invention discloses an in-vitro expression and purification method for a pear PMEI (pectin mthylesterase inhibitor) protein, and application of the pear PMEI. A primer cloning pear PMEI gene is designed; an escherichia coli recombinant expression plasmid PMEI-pCold-TF is constructed; the constructed recombinant expression plasmid PMEI-pCold-TF is converted into escherichia coli Rosetta (DE3) to construct a pear PMEI protein recombinant expression strain; the recombinant strain is subjected to protein induced expression, and a nickel column affinity chromatography method is used for purifying a recombinant pear PMEI protein; and then, the pollen tube of the pear and the root part of arabidopsis thaliana are processed by the obtained recombinant pear PMEI protein. Through the method disclosed by the invention, the first-time in-vitro expression and purification of the pear PMEI protein is realized, expression efficiency is high, a purified protein stably exists, and the purity of thepurified protein completely can meet the requirements of a related experiment. Through processing for the pollen tube of the pear and the root part of the arabidopsis thaliana by the recombinant pearPMEI protein, the pollen tube of the pear and the root part of the arabidopsis thaliana are accelerated to grow.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and relates to a method for expressing pear PMEI protein in vitro and its application, in particular to cloning and purifying a pear PMEI protein from pear pollen, and using the protein to treat pear pollen tubes and Arabidopsis thaliana respectively root, promoting the growth of pear pollen tubes and Arabidopsis roots. Background technique [0002] Pectin is the main component of the primary cell wall of dicotyledonous plants and monocotyledonous plants except Poaceae, and also the main component of pollen wall and pollen tube wall. In the process of plant growth and development, the degradation and modification of pectin requires the participation of a series of enzymes, among which pectin methylesterases (pectin mthylesterases, PMEs) are an important class of hydrolytic enzymes in the process of pectin degradation and metabolism, making Methylated pectin is demethylated, thereby regula...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/29C12P21/02C07K14/415C07K1/36C07K1/34C07K1/22C12N5/04A01N47/44A01P21/00C12R1/19
CPCC12N15/70C07K14/415C12N5/04C12N5/0025A01N47/44
Inventor 吴巨友朱晓璇汤超王鹏张绍铃
Owner NANJING AGRICULTURAL UNIVERSITY
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