CN111139257A - Pear PMEI protein in-vitro expression method and application thereof - Google Patents
Pear PMEI protein in-vitro expression method and application thereof Download PDFInfo
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Abstract
The invention discloses an in vitro expression and purification method of pear PMEI protein and application thereof. Cloning pear PMEI gene by designing primer; constructing an escherichia coli recombinant expression plasmid PMEI-pCold-TF; transforming the constructed recombinant expression plasmid PMEI-pCold-TF into escherichia coli Rosetta (DE3) to construct a pear PMEI protein recombinant expression strain; carrying out protein induction expression on the recombinant strain, and purifying the recombinant pear PMEI protein by using a nickel column affinity chromatography; thereafter, the pear pollen tube and the Arabidopsis root were treated with the obtained recombinant pear PMEI protein. By the method, the first in-vitro expression and purification of the pear PMEI protein are realized, the expression efficiency is high, the purified protein stably exists, and the purity can completely meet the requirements of related experiments; the growth of the pear pollen tube and the root of the arabidopsis is promoted by treating the pear PMEI protein on the pear pollen tube and the root of the arabidopsis.
Description
Technical Field
The invention belongs to the technical field of bioengineering, relates to a pear PMEI protein in-vitro expression method and application thereof, and particularly relates to a pear PMEI protein cloned and purified from pear pollen, wherein the pear PMEI protein is used for treating pear pollen tubes and arabidopsis roots respectively, so that the growth of the pear pollen tubes and the arabidopsis roots is promoted.
Background
Pectin is a major constituent of the primary cell wall of dicotyledonous and monocotyledonous plants other than the family Gramineae, as well as of the pollen wall and pollen tube wall. During the growth and development of plants, the degradation and modification of pectin require the participation of a series of enzymes, wherein Pectin Methyl Esterase (PMEs) is an important hydrolytic enzyme in the degradation and metabolism process of pectin, so that methyl esterified pectin is subjected to methyl esterification, and the hardness and elasticity of a cell wall are adjusted. Pectin methylesterase inhibitors (PMEIs) are proteins which regulate the activity of PME, can form a reversible 1:1 non-covalent complex with PME, and inhibit the activity of PME by covering active sites. The inhibition of PMEI on PME regulates the methyl esterification degree of pectin, thereby having influence on cell wall mechanics. Research shows that PMEI plays an indispensable role in seed germination, hypocotyl elongation, pollen germination and pollen tube growth, fruit ripening, and coping with various biotic and abiotic stresses. The separation and purification of PMEI protein will facilitate the further study of its biological function and mechanism of action. PMEI is widely present in various tissues and growth stages of plants, but separation and purification of natural PMEI proteins is difficult, and chemical synthesis of PMEI is costly. Therefore, the preparation of recombinant PMEI protein by genetic engineering techniques is an effective way to solve the above problems.
The method for preparing recombinant protein by gene engineering technology is widely applied. To date, various protein expression systems have been constructed, such as prokaryotic expression systems, eukaryotic expression systems, cell-free expression systems, and the like. The colibacillus expression system is the most common prokaryotic expression system and has the advantages of simple genetic background, clear biochemical characteristics, high growth speed, low economic cost, high expression level, easy separation and purification of products, convenient industrial production and the like. Since the construction, numerous recombinant proteins have been produced by E.coli expression systems. The PMEI protein purified by the genetic engineering technology is used for treating the root parts of the pear pollen tube and the arabidopsis thaliana, and the promotion effect of the PMEI protein on the root growth of the pear pollen tube and the root growth of the arabidopsis thaliana can be observed.
Disclosure of Invention
The invention aims to provide a preparation method for pear PMEI protein in vitro expression, which has low cost, high yield and easy purification.
The invention also aims to provide application of the pear PMEI protein in promoting growth of pear pollen tubes and roots of arabidopsis thaliana.
The purpose of the invention can be realized by adopting the following technical scheme:
a method for in vitro expression of pear PMEI protein comprises the following steps:
(1) extracting total RNA of pear pollen, carrying out reverse transcription to obtain cDNA, and designing a primer to carry out PCR amplification on pear PMEI gene by taking the cDNA generated by the reverse transcription as a template;
(2) constructing escherichia coli prokaryotic expression recombinant plasmid of pear PMEI protein;
(3) transforming the recombinant plasmid constructed in the step (2) into an escherichia coli strain Rosetta (DE3) by a heat shock method, and constructing a prokaryotic expression recombinant strain of pear PMEI protein;
(4) culturing the recombinant Escherichia coli strain constructed in the step (3) to OD6000.4-0.6, adding IPTG to induce the expression of pear PMEI protein after low-temperature treatment;
(5) purifying the pear PMEI protein subjected to induced expression;
(6) and (3) identifying the recombinant pear PMEI protein.
As a preferred technical scheme, the forward primer used for PCR amplification of the pear PMEI gene in the step (1) is as follows: 5'-ATGGAGCTCGGTACCCTCGAGAGAGCGTCTCAATTAGTTAGGAGTGTTTG-3', the reverse primer is: 5'-AGCAGAGATTACCTATCTAGACTAGATATTTAATAACTGGGAAGTAAC-3' are provided.
Further preferably, in the step (1), the reaction system for PCR amplification of the pear PMEI gene is as follows: ddH2O19. mu.l, 2.5. mu.l each of the upstream and downstream primers, 1. mu.l of cDNA, 2 XSuperPfx MasterMix 25. mu.l; the PCR reaction conditions are as follows: pre-denaturation at 98 ℃ for 3min, then at 98 ℃ for 10s, 60 ℃ for 30s, and 72 ℃ for 1min for 30 cycles, and finally extension at 72 ℃ for 10 min.
Preferably, in the step (2), the escherichia coli prokaryotic expression vector used in constructing the prokaryotic expression recombinant plasmid of the pear PMEI protein is pCold-TF, and the enzyme cutting sites of the pear PMEI gene insertion are Xho I and Xba I sites. As a detailed technical scheme, in the step (2), the product of PCR amplification of the pear PMEI gene is detected by 1% agarose gel electrophoresis and is cut and recovered, and the recovered product is connected to an Escherichia coli prokaryotic expression vector pCold-TF which is subjected to double enzyme digestion by Xho I and Xba I restriction enzymes by using homologous recombinase.
Further preferably, in the step (4), the low-temperature treatment is performed by standing on ice for 5min and then standing at 15 ℃ for 40 min, the final concentration of the inducer IPTG is 0.5mmol/L, and the condition of induced expression is 15 ℃ and the time is 24 hours. The detailed process for inducing the expression of pear PMEI protein comprises the following steps: escherichia coli prokaryotic expression recombinant vector of pear PMEI proteinInoculating to 100ml LB liquid culture medium at a ratio of 1:50, shake culturing at 37 deg.C and 220rpm overnight, transferring to 300ml LB liquid culture medium at a ratio of 1:50, shake culturing at 37 deg.C and 220rpm until bacterial liquid OD6000.4-0.6, rapidly standing on ice for 5min, and standing at 15 deg.C for 40 min; adding inducer IPTG with final concentration of 0.5mmol/L, shake culturing at 15 deg.C and 220rpm, and inducing expression for 24 hr; wherein the LB liquid medium contains 100. mu.g/ml ampicillin.
Further preferably, in the step (5), the detailed process for purifying the pear PMEI protein for inducing expression is as follows: after the induction expression is finished, centrifuging at 4 ℃ and 10000rpm for 10 minutes, then discarding the supernatant, collecting thalli sediment, and adding 20ml of lysate into the sediment for resuspension; carrying out ultrasonic crushing on the resuspension until the resuspension is clarified, centrifuging the resuspension for 10 minutes at 4 ℃ and 10000rpm, collecting supernatant, filtering the supernatant by a 0.45-micron filter membrane, and purifying the pear PMEI protein by using a nickel column affinity chromatography; before purifying the protein, balancing the nickel column affinity chromatography medium by using a 10-time column volume of balancing buffer solution; collecting purified protein, concentrating and desalting with 30kDa ultrafiltration tube at 4 deg.C and 6000rpm, and storing at-80 deg.C;
wherein the ultrasonic power of the ultrasonic crushing method is 240w, and the conditions are that the ultrasonic crushing method is started for 3s and stopped for 7s for 27 minutes;
the formula of the lysis solution is as follows: 140mM sodium chloride, 2.7mM potassium chloride, 10mM disodium hydrogen phosphate, 1.8mM potassium dihydrogen phosphate, 50 × EDTA-free protease inhibitor cocktail III, pH 7.3;
the formula of the equilibrium buffer solution is as follows: 500mM sodium chloride, 20mM tris (hydroxymethyl) aminomethane, 5mM imidazole, pH 7.3.
The N end of the pear PMEI protein is provided with 6 histidine tags (His-Tag).
Further preferably, in the step (6), the recombinant pear PMEI protein is identified by SDS-PAGE electrophoretic analysis. The specific process for identifying the recombinant pear PMEI protein by the SDS-PAGE electrophoretic analysis method comprises the following steps: taking 50 mu l of purified sample, adding 25 mu l of 2 xSDS-PAGE sample buffer, taking 10 mu l of mixed solution to perform SDS-PAGE electrophoresis, and identifying pear PMEI protein after Coomassie brilliant blue staining.
Application of pear PMEI protein in promoting growth of pear pollen tube and root of Arabidopsis thaliana is provided.
In the application, the recombinant pear PMEI protein expressed by the method is used for treating pear pollen tubes or arabidopsis roots. And measuring and counting the change of the length of the pear pollen tube and the length of the root of the arabidopsis thaliana, and verifying the promotion effect of the recombinant pear PMEI protein on the growth of the pear pollen tube and the root of the arabidopsis thaliana. In order to evaluate the effect of pear PMEI protein on the growth of pear pollen tubes and root parts of arabidopsis thaliana, pear pollen tubes cultured in a pear culture medium without pear PMEI protein and arabidopsis thaliana grown on an MS culture medium without the pear PMEI protein are taken as negative controls at the same time, and the changes of the lengths of the pear pollen tubes and the root parts are measured and counted.
As a preferred technical scheme, the process for treating the pear pollen tube by using the recombinant pear PMEI protein comprises the following steps: taking a proper amount of pear pollen in a pollen culture medium, and carrying out dark culture for 1 hour in an incubator at 25 ℃ and 120 rpm; adding recombinant pear PMEI protein with final concentration of 10mmol, and culturing in an incubator at 25 deg.C and 120rpm for 3 hr; the length of the pear pollen tube was measured and counted at 2, 3, 4 hours.
The formula of the pear pollen culture medium is as follows: 10% sucrose, 0.01% boric acid, 0.03% calcium diphosphate tetrahydrate, 0.58% MES, 10% PEG, adjusted to pH 6.3 with Tris.
As a preferred technical scheme, the process for treating the root of arabidopsis by using the recombinant pear PMEI protein comprises the following steps: coating 30 mu l of 0.1mg/ml recombinant pear PMEI protein on a nonreactive MS culture medium; transferring the arabidopsis thaliana seedlings with the seedling age of 10 days to a nonreactive MS culture medium coated with pear PMEI protein, and vertically placing the plates; the Arabidopsis thaliana seedlings were further cultured in a greenhouse at an average temperature of 25 ℃ and an average humidity of 65% for 16/8 hours in light/dark treatment, and the length of Arabidopsis thaliana roots was measured and counted on days 2, 4, 6, and 8 after the transfer.
Wherein the formula of the MS culture medium is as follows: 2% sucrose, 0.43% MS, 0.75% agar, adjusted to pH 5.8 with KOH.
The method for expressing pear PMEI protein in vitro and the application experiment process comprise the following steps:
(1) sequence analysis and cloning of pear PMEI gene
(1.1) a pear PMEI (Pbr003471.1) gene, the nucleotide sequence of which is SEQ ID NO:1, and the protein sequence of which is SEQ ID NO: 2. The signal peptide sequence SEQ ID NO 3 was analyzed and removed using the sequence analysis website SignalP 4.1 Server (http:// www.cbs.dtu.dk/services/SignalP /).
(1.2) extracting the total RNA of the pollen of the Dangshan pear according to the instruction of the total plant RNA extraction kit, and carrying out reverse transcription on the total RNA of the pollen into cDNA by using a reverse transcription kit.
(1.3) primer design and PCR reaction: the forward primer (PMEI-pCold-Xho I-F) was designed to be 5' -ATGGAGCTCGGTACCCTCGAGAGAGCGTCTCAATTAGTTAGGAGTGTTTG-3 '(in italics the front homology arm of pCold-TF vector, underlined Xho I cleavage site), reverse primer (PMEI-pCold-XbaI-R) 5' -AGCAGAGATTACCTATCTAGACTAGATATTTAATAACTGGGAAGTAAC-3’
(the rear homology arm of the Pcoled-TF vector is in italics, and the Xba I restriction site is underlined); the PCR reaction system is as follows: ddH2O19. mu.l, 2.5. mu.l each of the upstream and downstream primers, 1. mu.l of cDNA, 2 XSuperPfx MasterMix 25. mu.l; the PCR reaction conditions are as follows: pre-denaturation at 98 ℃ for 3min, then at 98 ℃ for 10s, 60 ℃ for 30s, and 72 ℃ for 1min for 30 cycles, and finally extension at 72 ℃ for 10 min.
(1.4) PCR amplification of the product of pear PMEI gene was detected by 1% agarose gel electrophoresis and recovered by cutting the gel. The recovered product was ligated to the Escherichia coli prokaryotic expression vector pCold-TF which had been digested with Xho I and Xba I restriction enzymes by the homologous recombinase (see the vector map of TAKARA).
(1.5) the accuracy of the recombinant expression vector PMEI-pCold-TF was verified by DNA sequencing of Jinzhi Biotechnology Ltd, Suzhou.
(2) Construction of recombinant expression E.coli Strain
The recombinant expression vector PMEI-pCold-TF with correct sequencing was transformed into E.coli strain Rosetta (DE3) by heat shock method, spread on LB solid plate (containing 100. mu.g/ml ampicillin), cultured overnight in 37 ℃ incubator, picked up single colony into 500. mu. lLB liquid medium (containing 100. mu.g/ml ampicillin), and cultured with shaking at 37 ℃ and 220rpm for 4 hours. After the bacterial liquid is verified to be correct by PCR, the ratio of 7: 3 (bacteria liquid: glycerol) and 50% (v/v) of sterilized glycerol are added, mixed evenly, frozen by liquid nitrogen and stored in a refrigerator at minus 80 ℃.
(3) Induced expression and purification of pear PMEI protein
(3.1) inducible expression of Pear PMEI protein
Inoculating the recombinant expression strain stored in the step (2) into 100ml of LB liquid culture medium according to the volume ratio of 1:50, performing shake culture at 37 ℃ and 220rpm overnight, inoculating into 300ml of LB liquid culture medium according to the ratio of 1:50, and performing culture at 37 ℃ and 220rpm until the OD of bacterial liquid is obtained600Rapidly placing on ice for 5 minutes when the concentration reaches 0.4-0.6, standing at 15 deg.C for 40 minutes, adding IPTG with final concentration of 0.5mmol/L, culturing at 15 deg.C and 220rpm with shaking, and inducing expression for 24 hours. Wherein the LB liquid medium contains 100. mu.g/ml ampicillin.
(3.2) purification of Pear PMEI protein
After the induction expression of the pear PMEI protein is finished, centrifuging at 4 ℃ and 10000rpm for 10 minutes, discarding the supernatant, collecting the bacterial pellet, adding 20ml of lysis solution (140mM sodium chloride, 2.7mM potassium chloride, 10mM disodium hydrogen phosphate, 1.8mM potassium dihydrogen phosphate, 50X EDTA-free protease inhibitor cocktail III (Germany Merck Michelson), pH 7.3) into the pellet, and re-suspending the pellet; the resuspension solution is subjected to ultrasonication (the ultrasonic power is 240w, the conditions are that the opening is carried out for 3s, the stopping is carried out for 7s, and the total time is 27 minutes) until the resuspension solution is clarified, the centrifugation is carried out for 10 minutes at 4 ℃ and 10000rpm, the supernatant is collected, and the PMpear EI protein is purified by using a nickel column affinity chromatography after being filtered by a 0.45 mu m filter membrane. Prior to protein purification, the nickel column affinity chromatography medium was equilibrated with 10 column volumes of equilibration buffer (500mM sodium chloride, 20mM tris (hydroxymethyl) aminomethane, 5mM imidazole, pH 7.3); the purified protein was collected, concentrated and desalted at 6000rpm at 4 ℃ using an ultrafiltration tube (cut-off: 30 kDa). Taking 50 mu l of purified sample, adding 25 mu l of 2 xSDS-PAGE sample buffer, taking 10 mu l of mixed solution to perform SDS-PAGE electrophoresis, identifying pear PMEI protein after Coomassie brilliant blue staining, and storing at-80 ℃ for later use.
(4) Application of recombinant pear PMEI protein
And (3) determining the protein concentration of the PMEI protein of the purified recombinant pear according to a BCA protein concentration determination kit of Shanghai Biotech company. And (3) treating the roots of the Dangshan pear pollen tube and the Columbia-0 wild type Arabidopsis by using the purified recombinant pear PMEI protein, and measuring and counting the length changes of the pollen tube and the roots. Taking a proper amount of pear pollen in 200 mul pollen culture medium, carrying out dark culture in an incubator at 25 ℃ and 120rpm for 1 hour, adding recombinant pear PMEI protein with the final concentration of 10mmol, and continuing to carry out dark culture in the incubator at 25 ℃ and 120rpm for 3 hours. At 2, 3 and 4 hours, the growth of the pollen tube was observed by using a microscope Nikon TE100, and the length of the pear pollen tube was measured and counted by using software Image-Pro Plus 6.0. Three biological replicates were set up for each treatment, 100 pollen tube lengths were measured each time, and the average was calculated. The formula of the pear pollen culture medium is as follows: 10% sucrose, 0.01% boric acid, 0.03% calcium diphosphate tetrahydrate, 0.58% MES, 10% PEG, adjusted to pH 6.3 with Tris.
Coating 30 mu l of 0.1mg/ml recombinant pear PMEI protein on a nonreactive MS culture medium; arabidopsis seedlings grown on the non-resistant MS medium and having a seedling age of 10 days were transferred to the non-resistant MS medium coated with pear PMEI protein, the plates were placed vertically, then the Arabidopsis seedlings were continuously cultured in a greenhouse at an average temperature of 25 ℃ and an average humidity of 65% for 16/8 hours in light/dark treatment, and the length of Arabidopsis roots was measured and counted on days 2, 4, 6, and 8 after the transfer. Three biological replicates were set for each treatment, and the length of 50 Arabidopsis roots was measured and averaged. Wherein the formula of the MS culture medium is as follows: 2% sucrose, 0.43% MS, 0.75% agar, adjusted to pH 5.8 with KOH.
In order to evaluate the effect of pear PMEI protein on the growth of pear pollen tubes and root parts of arabidopsis thaliana, pear pollen tubes cultured in a pear culture medium without pear PMEI protein and arabidopsis thaliana grown on an MS culture medium without the pear PMEI protein are taken as negative controls at the same time, and the changes of the lengths of the pear pollen tubes and the root parts are measured and counted.
The invention has the beneficial effects that:
(1) the invention adopts the genetic engineering technology, expresses and purifies the pear PMEI protein in vitro for the first time, and has high expression efficiency and good purification effect.
(2) The methods of the invention for in vitro expression and purification of proteins are applicable to all PMEI proteins in pear.
(3) The pear PMEI protein expressed and purified by the invention stably exists under the in vitro condition and has protein activity, thereby providing a research basis for further researching the biological functions exerted in the pear growth and development process.
(4) The pear PMEI protein expressed by the invention can directly promote the growth of the Dangshan pear pollen tube and the root of Columbia-0 wild type Arabidopsis under in vitro conditions, and provides reference for the functional research of the pear PMEI protein.
Drawings
FIG. 1 is a diagram of the prediction of the signal peptide of the pear PMEI protein sequence.
FIG. 2 is a diagram of agarose gel electrophoresis of PCR amplified pear PMEI gene;
wherein M is the mass of the DNA standard molecule; 1 is PCR amplification product.
FIG. 3 is a SDS-PAGE representation of the induced expression and purification of recombinant pear PMEI proteins;
wherein M is a protein standard molecular weight; 1 is total protein of thallus induced by 0.5mmol/L IPTG; 2, the purified recombinant pear PMEI protein; 3 is total protein of induced thallus without IPTG.
FIG. 4 is the effect of recombinant pear PMEI protein on the growth of Dangshan pear pollen tube;
wherein, FIG. 4a is the pear pollen tube after 3 hours of untreated pear pollen tube and recombinant pear PMEI protein treatment; FIG. 4b is the change in length of untreated pear pollen tube and of pear pollen tube after treatment with recombinant pear PMEI protein within 1-3 hours.
FIG. 5 is a graph of the effect of recombinant pear PMEI protein on Columbia-0 wild type Arabidopsis root growth;
wherein FIG. 5a is an untreated Arabidopsis root and a recombinant Pear PMEI protein treated Arabidopsis root on day eight; FIG. 5b is the change in length of the untreated Arabidopsis roots and of the Arabidopsis roots after recombinant pear PMEI protein treatment within 0-8 days.
Detailed Description
The following describes embodiments of the present invention in detail. The embodiments described below with reference to the accompanying drawings are illustrative only for the purpose of explaining the present invention, and are not to be construed as limiting the present invention.
Example 1: construction and identification of PMEI-pCold-TF recombinant plasmid
Taking fresh Dangshan pear pollen, and extracting total RNA of the pollen by using a plant total RNA extraction kit; using total RNA of pollen as a template, and carrying out reverse transcription by using a reverse transcription kit to obtain pollen cDNA; as shown in figure 1, analyzing and removing the signal peptide sequence of pear PMEI gene, designing primers, adding homologous arms of vector pCold-TF and enzyme cutting sites of Xba I and Xho I at two ends of the primers, and carrying out PCR amplification on the PMEI gene by taking pollen cDNA as a template, wherein the PCR reaction system is as follows: ddH2O19. mu.l, 2.5. mu.l each of the upstream and downstream primers, 1. mu.l of cDNA, 2 Xsuper Pfx MasterMix 25. mu.l; the PCR reaction conditions are as follows: pre-denaturation at 98 ℃ for 3min, then at 98 ℃ for 10s, 60 ℃ for 30s, and 72 ℃ for 1min for 30 cycles, and finally extension at 72 ℃ for 10 min. The PCR product was identified by electrophoresis on a 1% agarose gel. As shown in FIG. 2, there is a clear bright band at 500-750bp, which coincides with the length of PMEI gene.
The PCR product is recovered by using a Gel extraction kit of Cornish century company, obtaining a recovered PMEI gene, the recovered PMEI gene is connected to a pCold-TF expression vector which is also subjected to double enzyme digestion by Xba I and Xho I through homologous recombinase, the recovered PMEI gene is reacted for 30 minutes at 37 ℃ to construct a recombinant plasmid PMEI-pCold-TF, the recombinant plasmid is transformed into an Escherichia coli strain DH5 α through a heat shock method, the recombinant plasmid is coated on an LB solid plate (containing 100 mu g/ml ampicillin) and cultured for 16 hours at 37 ℃, a single bacterium is selected to fall into a 500 mu lLB liquid culture medium (containing 100 mu g/ml ampicillin), the mixed solution is subjected to shake culture at 37 ℃ and 220rpm for 4 hours, PCR identification is carried out, the correct single clone strain is identified to be sent to Suzhou jin Zhi Biotechnology Limited company DNA sequencing to verify the sequence correctness of the sequence of the coding region, the sequence of the obtained pear EI gene is named as PMEI plasmid sequence conforming to a reference series, the sequence of the PMEI gene is identified as a sequence of the pCold-TF, and the sequence of the amino acid sequence of the SEQ ID TF., the sequence of the recovered PMEI gene is obtained SEQ ID 2, and the sequence of.
Example 2: expression of pear PMEI gene in Escherichia coli
1. Obtaining recombinant expression strain for expressing pear PMEI
The correctly sequenced monoclonal strains were inoculated into 4ml of LB liquid medium (containing 100. mu.g/ml ampicillin), and cultured overnight with shaking at 220rpm at 37 ℃. The next day, the recombinant Plasmid PMEI-pCold-TF was extracted according to the instructions of the Quickpure Plasmid Mini kit Rapid Small extraction kit, Kangji Shiji corporation. 1ng of the recombinant plasmid PMEI-pCold-TF was transformed into an Escherichia coli strain Rosetta (DE3) by a heat shock method, spread on an LB plate (containing 100. mu.g/ml ampicillin) to select recombinants, and cultured overnight at 37 ℃ to obtain a genetically engineered bacterium expressing the PMEI of pear. Taking single colony of the obtained genetically engineered bacteria, streaking on LB plate (containing 100. mu.g/ml ampicillin), inoculating a small amount of streaked bacteria into 1ml LB (containing 100. mu.g/ml ampicillin) liquid medium, shaking-culturing at 37 ℃ and 220rpm for 4 hours, and performing 7: 3 (bacterial liquid: glycerol) is added with 50% (v/v) of sterilized glycerol, and after being uniformly mixed, the mixture is frozen and stored in a refrigerator at minus 80 ℃ by liquid nitrogen, thus obtaining the recombinant expression strain for expressing the pear PMEI protein.
2. Expression of pear PMEI recombinant protein
The stored recombinant expression strain of pear PMEI protein was inoculated into 100ml LB (containing 100. mu.g/ml ampicillin) liquid medium at a volume ratio of 1:50, and cultured with shaking overnight at 220rpm at 37 ℃ to activate the recombinant expression strain. Then inoculating the activated recombinant expression strain into 300ml LB liquid culture medium (containing 100. mu.g/ml ampicillin) according to a ratio of 1:50, culturing, shaking at 37 ℃ and 220rpm until OD600Is 0.4-0.6. Taking 5ml of bacterial liquid as a negative control, rapidly placing the rest bacterial liquid on ice for 5 minutes, standing for 40 minutes at 15 ℃, finally adding IPTG with the final concentration of 0.5mmol/L, performing shake culture at 15 ℃ and 220rpm, and performing induced expression for 24 hours. 5ml of the expressed bacterial solution was centrifuged at 4 ℃ and 10000rpm for 4 minutes, and the bacterial pellet was collected. To the pellet and the negative control, 200. mu.l of 10% SDS (10g/100ml) was added, respectively, mixed well and boiled in a water bath for 10 minutes, ice-cooled for two minutes, and then centrifuged at 10000rpm for 10 minutes at 4 ℃ to obtain 50. mu.l of each supernatant, and 25. mu.l of 2 Xprotein loading buffer was added. Taking 15 mul of each, performing 12% conventional SDS-PAGE electrophoresis, staining with Coomassie brilliant blue, decolorizing, and detecting recombinant protein expressionThe results are shown in lanes 1 and 2 of FIG. 3.
Example 3: separation, purification and identification of recombinant pear PMEI protein
Subjecting the residual recombinant expression bacteria liquid to induced expression for 24 hours and detecting the correctness by SDS-PAGE electrophoresis to centrifuge at 4 ℃ and 10000rpm for 10 minutes, discarding the supernatant and collecting the bacterial pellet, adding 20ml of lysis solution (140mM sodium chloride, 2.7mM potassium chloride, 10mM disodium hydrogen phosphate, 1.8mM potassium dihydrogen phosphate, 50 × EDTA-free protease inhibitor cocktail III (Merck Mich, Germany) into the pellet to resuspend the pellet at pH 7.3); the resuspension was sonicated (sonication power 240w, conditions 3s on, 7s off, 27 min total) to clear, centrifuged at 10000rpm for 10min at 4 ℃, the supernatant was collected and filtered through a 0.45 μm filter to remove impurities, and the pear PMEI protein was purified using Ni-NTA Sepharose affinity chromatography packing from Mercury, Germany.
Before purifying the protein, the nickel column affinity chromatography medium was equilibrated with 10 column volumes of equilibration buffer (500mM sodium chloride, 20mM tris (hydroxymethyl) aminomethane, 5mM imidazole, pH 7.3) at a controlled flow rate of 1 ml/min; purifying 20ml of protein supernatant sample filtered by the filter membrane by a column, and controlling the flow rate to be 1 ml/min; washing the impure proteins in the column with 20 column volumes of a wash solution containing 40mM imidazole (500mM sodium chloride, 20mM tris (hydroxymethyl) aminomethane, 30mM imidazole, pH 7.3) at a flow rate of 1 ml/min; eluting the purified protein in a 10-fold column volume elution buffer (500mM sodium chloride, 20mM tris (hydroxymethyl) aminomethane, 80mM imidazole, pH 7.3) containing 60mM imidazole at a flow rate of 1ml/min, collecting the eluate to obtain the purified protein, concentrating at 6000rpm at 4 ℃ using an ultrafiltration tube (cut-off of 30kDa), desalting, and storing at-80 ℃. A small amount of the protein was analyzed for SDS-PAGE purity, and the results are shown in lane 3 of FIG. 3.
Example 4: application of recombinant pear PMEI protein
And (3) determining the protein concentration of the PMEI protein of the purified recombinant pear according to a BCA protein concentration determination kit of Shanghai Biotech company. And (3) treating the roots of the Dangshan pear pollen tube and the Columbia-0 wild type Arabidopsis by using the purified recombinant pear PMEI protein, and measuring and counting the length changes of the pollen tube and the roots. Taking a proper amount of pear pollen in 200 mul pollen culture medium, carrying out dark culture in an incubator at 25 ℃ and 120rpm for 1 hour, adding recombinant pear PMEI protein with the final concentration of 10mmol, and continuing to carry out dark culture in the incubator at 25 ℃ and 120rpm for 3 hours. At 2, 3 and 4 hours, the growth of the pollen tube was observed by using a microscope Nikon TE100, and the length of the pollen tube was measured and counted by using software Image-Pro Plus 6.0. Three biological replicates were set up for each treatment, 100 pollen tube lengths were measured each time, and the average was calculated. The formula of the pear pollen culture medium is as follows: 10% sucrose (g/100ml), 0.01% boric acid (g/100ml), 0.03% calcium diphosphate tetrahydrate (g/100ml), 0.58% MES (g/100ml), 10% PEG (g/100ml) adjusted to pH 6.3 with Tris.
Coating 30 mu l of 0.1mg/ml recombinant pear PMEI protein on a nonreactive MS culture medium; arabidopsis seedlings grown on the non-resistant MS medium and having a seedling age of 10 days were transferred to the non-resistant MS medium coated with pear PMEI protein, the plates were placed vertically, then the Arabidopsis seedlings were continuously cultured in a greenhouse at an average temperature of 25 ℃ and an average humidity of 65% for 16/8 hours in light/dark treatment, and the length of Arabidopsis roots was measured and counted on days 2, 4, 6, and 8 after the transfer. Three biological replicates were set for each treatment, and the length of 50 Arabidopsis roots was measured and averaged. Wherein the formula of the MS culture medium is as follows: 2% sucrose (g/100ml), 0.43% MS (g/100ml), 0.75% agar (g/100ml) adjusted to pH 5.8 with KOH.
In order to evaluate the effect of pear PMEI protein on the growth of pear pollen tubes and root parts of arabidopsis thaliana, pear pollen tubes cultured in a pear culture medium without pear PMEI protein and arabidopsis thaliana grown on an MS culture medium without the pear PMEI protein are taken as negative controls at the same time, and the changes of the lengths of the pear pollen tubes and the root parts are measured and counted.
Sequence listing
<110> Nanjing university of agriculture
<120> pear PMEI protein in-vitro expression method and application thereof
<160>5
<170>SIPOSequenceListing 1.0
<210>1
<211>567
<212>DNA
<213> Pear (Pyrus spp)
<400>1
atggcctcct caatcagtta tgcaatgttc attgcttcct tgctcttatg tctctcgtcg 60
tctccagcac catttgcaaa tgcaagagcg tctcaattag ttaggagtgt ttgcaagcaa 120
acccacgaag aattcggcta cagctatagg cagtgcgtaa aatctctttg gaaagatatt 180
ccaattagat cggcaactaa tctcaaagat cttgatatag ccattcttaa attagcagca 240
gcaaatgcag cacaaaccaa agctacgttt gaaaaagctt tcaacgccac caacaagaac 300
actaatggca cggcagctat aaagcagtgt gtagattcgt atgattttgc gttaggggct 360
ttcgtttttg cagtgcgagg ggtcaatgac ggtgacaaat cggtcaccaa aatcctcaca 420
cagacccaag atgaccttgt tcgttgccaa agagcattgg cctctgttga agttcagctt 480
cctatgccag tatcgacgac aaacttttgg gtcatgttat atagggatgt tgcattcctt 540
gttacttccc agttattaaa tatctag 567
<210>2
<211>188
<212>PRT
<213> Pear (Pyrus spp)
<400>2
Met Ala Ser Ser Ile Ser Tyr Ala Met Phe Ile Ala Ser Leu Leu Leu
1 5 10 15
Cys Leu Ser Ser Ser Pro Ala Pro Phe Ala Asn Ala Arg Ala Ser Gln
20 25 30
Leu Val Arg Ser Val Cys Lys Gln Thr His Glu Glu Phe Gly Tyr Ser
35 40 45
Tyr Arg Gln Cys Val Lys Ser Leu Trp Lys Asp Ile Pro Ile Arg Ser
50 55 60
Ala Thr Asn Leu Lys Asp Leu Asp Ile Ala Ile Leu Lys Leu Ala Ala
65 70 75 80
Ala Asn Ala Ala Gln Thr Lys Ala Thr Phe Glu Lys Ala Phe Asn Ala
85 90 95
Thr Asn Lys Asn Thr Asn Gly Thr Ala Ala Ile Lys Gln Cys Val Asp
100 105 110
Ser Tyr Asp Phe Ala Leu Gly Ala Phe Val Phe Ala Val Arg Gly Val
115 120 125
Asn Asp Gly Asp Lys Ser Val Thr Lys Ile Leu Thr Gln Thr Gln Asp
130 135 140
Asp Leu Val Arg Cys Gln Arg Ala Leu Ala Ser Val Glu Val Gln Leu
145 150 155 160
Pro Met Pro Val Ser Thr Thr Asn Phe Trp Val Met Leu Tyr Arg Asp
165 170 175
Val Ala Phe Leu Val Thr Ser Gln Leu Leu Asn Ile
180185
<210>3
<211>84
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>3
atggcctcct caatcagtta tgcaatgttc attgcttcct tgctcttatg tctctcgtcg 60
tctccagcac catttgcaaa tgca 84
<210>6
<211>50
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>6
atggagctcg gtaccctcga gagagcgtct caattagtta ggagtgtttg 50
<210>7
<211>48
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>7
agcagagatt acctatctag actagatatt taataactgg gaagtaac 48
Claims (10)
1. A method for in vitro expression of pear PMEI protein is characterized by comprising the following steps:
(1) extracting total RNA of pear pollen, performing reverse transcription to form cDNA, designing a primer, and performing PCR amplification on pear PMEI gene by taking the cDNA generated by the reverse transcription as a template;
(2) constructing escherichia coli prokaryotic expression recombinant plasmid of pear PMEI protein;
(3) transforming the recombinant plasmid constructed in the step (2) into an escherichia coli strain Rosetta (DE3) by a heat shock method, and constructing a prokaryotic expression recombinant strain of pear PMEI protein;
(4) culturing the recombinant Escherichia coli strain constructed in the step (3) to OD6000.4-0.6, adding IPTG to induce the expression of pear PMEI protein after low-temperature treatment;
(5) purifying the pear PMEI protein subjected to induced expression;
(6) and (3) identifying the recombinant pear PMEI protein.
2. The method for in vitro expression of pear PMEI protein according to claim 1, wherein in the step (1), the forward primers for PCR amplification of pear PMEI gene are: 5' -
ATGGAGCTCGGTACCCTCGAGAGAGCGTCTCAATTAGTTAGGAGTGTTTG-3', the reverse primer is: 5'-AGCAGAGATTACCTATCTAGACTAGATATTTAATAACTGGGAAGTAAC-3' are provided.
3. The method for in vitro expression of pear PMEI protein according to claim 1 or 2, wherein in the step (1), the reaction system for PCR amplification of pear PMEI gene is: ddH2O19. mu.l, 2.5. mu.l each of the upstream and downstream primers, 1. mu.l of cDNA, 2 Xsuper PfxMasterMix 25. mu.l; the PCR reaction conditions are as follows: pre-denaturation at 98 ℃ for 3min, then at 98 ℃ for 10s, 60 ℃ for 30s, and 72 ℃ for 1min for 30 cycles, and finally extension at 72 ℃ for 10 min.
4. The method for in vitro expression of pear PMEI protein according to claim 1, wherein in the step (2), the Escherichia coli prokaryotic expression vector used in constructing the prokaryotic expression recombinant plasmid of pear PMEI protein is pCold-TF, and the restriction enzyme cutting sites for inserting pear PMEI gene are Xho I and Xba I sites.
5. The method for in vitro expression of pear PMEI protein according to claim 1, wherein in step (4), said low temperature treatment is a step of standing on ice for 5min and then at 15 ℃ for 40 min, said final concentration of inducer IPTG is 0.5mmol/L, and said condition of induced expression is 15 ℃ for 24 hours.
6. The method for in vitro expression of pear PMEI protein according to claim 1 or 5, wherein in the step (4), the detailed process for inducing expression of pear PMEI protein is as follows: inoculating Escherichia coli prokaryotic expression recombinant vector of pear PMEI protein into 100ml LB liquid culture medium according to the proportion of 1:50, performing shake culture at 37 ℃ and 220rpm overnight, then transferring into 300ml LB liquid culture medium according to the proportion of 1:50, performing shake culture at 37 ℃ and 220rpm until bacterial liquid OD6000.4-0.6, rapidly standing on ice for 5min, and standing at 15 deg.C for 40 min; adding inducer IPTG with final concentration of 0.5mmol/L, shake culturing at 15 deg.C and 220rpm, and inducing expression for 24 hr;
wherein the LB liquid medium contains 100. mu.g/ml ampicillin.
7. The method for in vitro expression of pear PMEI protein according to claim 1, wherein in the step (5), the detailed process for purifying the pear PMEI protein for inducing expression is as follows: after the induction expression is finished, centrifuging at 4 ℃ and 10000rpm for 10 minutes, then discarding the supernatant, collecting thalli sediment, and adding 20ml of lysate into the sediment for resuspension; carrying out ultrasonic crushing on the resuspension until the resuspension is clarified, centrifuging the resuspension for 10 minutes at 4 ℃ and 10000rpm, collecting supernatant, filtering the supernatant by a 0.45-micron filter membrane, and purifying the pear PMEI protein by using a nickel column affinity chromatography; before purifying the protein, balancing the nickel column affinity chromatography medium by using a 10-time column volume of balancing buffer solution; collecting purified protein, concentrating and desalting with 30kDa ultrafiltration tube at 4 deg.C and 6000rpm, and storing at-80 deg.C;
wherein the ultrasonic power of the ultrasonic crushing method is 240w, and the conditions are that the ultrasonic crushing method is started for 3s and stopped for 7s for 27 minutes;
the formula of the lysis solution is as follows: 140mM sodium chloride, 2.7mM potassium chloride, 10mM disodium hydrogen phosphate, 1.8mM potassium dihydrogen phosphate, 50 × EDTA-free protease inhibitor cocktail III, pH 7.3;
the formula of the equilibrium buffer solution is as follows: 500mM sodium chloride, 20mM tris (hydroxymethyl) aminomethane, 5mM imidazole, pH 7.3.
8. The method for in vitro expression of pear PMEI protein according to claim 1, wherein in the step (6), the identification method of the recombinant pear PMEI protein is SDS-PAGE electrophoretic analysis, and the specific process of the SDS-PAGE electrophoretic analysis for identifying the recombinant pear PMEI protein is as follows: taking 50 mu l of purified sample, adding 25 mu l of 2 xSDS-PAGE sample buffer, taking 10 mu l of mixed solution to perform SDS-PAGE electrophoresis, and identifying pear PMEI protein after Coomassie brilliant blue staining.
9. Application of pear PMEI protein in promoting growth of pear pollen tube and root of Arabidopsis thaliana is provided.
10. Use according to claim 9, wherein the recombinant pear PMEI protein produced according to the method of claims 1-8 is used to treat pear pollen tube or arabidopsis root.
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| CN113481211A (en) * | 2021-08-02 | 2021-10-08 | 中国农业科学院棉花研究所 | Pectin methylesterase inhibitory factor GhPMEI39 and application of encoded protein thereof |
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| CN113481211A (en) * | 2021-08-02 | 2021-10-08 | 中国农业科学院棉花研究所 | Pectin methylesterase inhibitory factor GhPMEI39 and application of encoded protein thereof |
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