CN110819649A

CN110819649A - In-vitro expression method of recombinant pectin methylesterase PbrPME, coding gene and application thereof

Info

Publication number: CN110819649A
Application number: CN201910951450.1A
Authority: CN
Inventors: 张绍铃; 汤超; 吴巨友; 王鹏; 朱晓璇
Original assignee: Nanjing Agricultural University
Current assignee: Nanjing Agricultural University
Priority date: 2019-10-08
Filing date: 2019-10-08
Publication date: 2020-02-21

Abstract

The invention discloses an in vitro expression method of recombinant pectin methylesterase PbrPME, and a coding gene and application thereof. Cloning pear PbrPME gene by designing primer; constructing an escherichia coli recombinant expression vector PbrPME-pCold-TF; transforming the constructed recombinant expression vector PbrPME-pCold-TF into escherichia coli Rosetta (DE3) to construct a recombinant strain for expressing pear PbrPME protein; expressing the recombinant strain, and purifying the recombinant pear PbrPME protein by using a nickel column affinity chromatography; and (3) treating the pear pollen tube by using the recombinant pear PbrPME protein, counting the length of the treated pollen tube, and verifying the activity of the recombinant pear PbrPME protein. The method realizes the in vitro expression and the application of the pear PbrPME protein and can completely meet the requirements of related experiments.

Description

In-vitro expression method of recombinant pectin methylesterase PbrPME, coding gene and application thereof

Technical Field

The invention relates to the technical field of bioengineering, in particular to an in vitro expression method of pear PbrPME protein and application thereof.

Background

Pectin exists in the cell walls of higher plants, is concentrated between the primary wall and the intercellular layer of the cell walls, and is interwoven with other cellulose, hemicellulose, lignin and cell wall extensin in the primary wall to form a hard structure.

Pectin is the main component in the pollen tube cell wall and is the basic polysaccharide in the pollen tube cell wall. Pectin is synthesized and secreted into the cell wall in a highly methyl esterified form, and then methyl esterified by Pectin Methyl Esterase (PME), two metabolites, namely hydrogen ions and methanol, are generated in the process, and carboxyl groups are exposed in the formed demethylated pectin. PME not only participates in pollen germination and pollen tube growth, but also participates in fruit ripening and cambium differentiation. The function of PME in the modification of cell wall structure has two aspects, namely that pectin generates a large amount of exposed carboxyl groups and extracellular Ca²⁺Binding, the cell wall becomes rigid, hindering cell spreading and growth; on the other hand, the occurrence of demethylation causes a decrease in extracellular pH, which leads to a relaxation of the cell wall structure, in many cases if the activities of the gum lyase and polygalacturonase are increased.

PME also plays an important role in the production and processing process of fruit and vegetable products such as pears and the like. For example, the hardness of fruits and vegetables is mainly influenced by pectin in peels, and PME can effectively improve the crispness of canned fruits and vegetables and pickled vegetables and improve the commodity value of the canned fruits and vegetables and the pickled vegetables. Therefore, the research on PME has double significance of theoretical value and practical value. To date, many studies have been made on pectin methylesterase at home and abroad, and one of the problems faced in this study is the expression and extraction of PbrPME. The preparation of recombinant protein by gene engineering technology is a technological means with wide prospect. To date, there are a variety of protein expression systems, 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 compared with the traditional method for extracting protein from living materials, the system has the advantages of clear genetic background and biochemical characteristics, fast growth, low cost, high expression quantity, relatively simple separation and purification of expression products, convenience for large-scale production and the like. There have been numerous cases of successful expression of recombinant proteins for a long time. In conclusion, the preparation of recombinant PME by genetic engineering technology is an effective way to solve the above problems.

Disclosure of Invention

The invention aims to provide a preparation method for in vitro expression of pear PbrPME protein with low cost, high yield and easy purification and application thereof.

The purpose of the invention can be realized by adopting the following technical scheme:

a method for in vitro expression of pear PbrPME protein comprises the following steps:

a. extracting total RNA of pear pollen, performing reverse transcription to obtain cDNA, and designing a primer PCR to amplify a pear PbrPME gene;

b. constructing a prokaryotic recombinant expression vector for expressing the pear PbrPME protein;

c. b, transforming the prokaryotic recombinant expression vector constructed in the step b into an escherichia coli strain Rosetta (DE3) to construct a PbrPME recombinant expression strain;

d. c, culturing the PbrPME recombinant expression strain constructed in the step c to OD₆₀₀Reaching 0.3-0.5, and after low-temperature treatment, adding an inducer IPTG to induce and express pear PbrPME protein;

e. purifying the pear PbrPME protein for induction expression.

f. The recombinant PbrPME protein is utilized to treat the pear pollen tube, and the influence of the recombinant PbrPME protein on the pollen tube is observed.

As a preferred technical scheme, in the step a, the forward primer for PCR amplification of the pear PbrPME gene is 5-

CCCTCGAGATGAAAAACAAAGGAGGAGGAG-3 ', and the reverse primer is 5'-GCTCTAGAAACGGAAACCATGCCAGC-3'.

Further preferably, in the step a, the PCR reaction system for PCR amplification of the pear PbrPME gene is: ddH₂O31. mu.l, 2.5. mu.l each of the upstream and downstream primers, 1. mu.l of cDNA, 10. mu.l of 5 XPisuion GC Buffer, 1. mu.l of 10mM dNTPs, 50mM MgCl₂solution 1. mu.l, Phusion DNA Polymerase 1. mu.l; the PCR reaction conditions are as follows: pre-denaturation at 98 ℃ for 30min, then at 98 ℃ for 10s, 60 ℃ for 30s, and 72 ℃ for 2min for 30 cycles, and finally extension at 72 ℃ for 10 min.

As a preferred technical scheme, in the step b, when a prokaryotic recombinant expression vector for expressing the pear PbrPME protein is constructed, the used prokaryotic expression vector is an escherichia coli expression vector pCold-TF, and the insertion site for cloning the pear PbrPME gene is between Xho I and Xba I enzyme digestion sites.

Further preferably, the PCR product is analyzed by electrophoresis in a 1% agarose gel and recovered by cutting the gel, and the recovered product is double-digested with XhoI and Xba I restriction enzymes and ligated to XhoI and Xba I sites of the same double-digested E.coli expression vector pCold-TF.

As a preferable technical scheme, in the step d, the low-temperature treatment is that the mixture is placed on ice for 5-10min and then is kept stand at 10-15 ℃ for 40-50min, the final concentration of the inducer IPTG is 0.3-0.5mmol/L, and the condition of induced expression is induced at 15 ℃ for 22-24 h.

Further preferably, the detailed process of inducing expression of the pear PbrPME protein in the step d is as follows: the constructed PbrPME recombinant expression strain is inoculated into 100ml LB liquid culture medium containing 100 mu g/ml ampicillin according to the proportion of 1:50, shake-cultured overnight at 37 ℃ and 200-250rpm, then transferred into 300ml LB liquid culture medium containing 100 mu g/ml ampicillin according to the proportion of 1:50, shake-cultured at 37 ℃ and 200-250rpm until OD of bacterial liquid₆₀₀Rapidly placing on ice for 5-10min when the temperature reaches 0.3-0.6, and standing at 15 deg.C for 40-50 min; then adding an inducer IPTG with the final concentration of 0.3-0.5mmol/L, carrying out shake culture at 15 ℃ and 200-250rpm, and carrying out induced expression for 22-24 h;

as a preferred technical scheme, the detailed process for purifying the pear PbrPME protein for induction expression in the step e comprises the following steps: after the induction expression is finished, centrifuging at 12000rpm for 10min at 4 ℃, then abandoning the supernatant, collecting the precipitate, and adding a lysate into the precipitate to resuspend the precipitate; and (3) carrying out ultrasonic crushing on the resuspended bacterial liquid with the power of 240W under the conditions: turn on for 3s, stop for 7s, and total 27 min. After the ultrasonication is finished, centrifuging at 12000rpm for 10min, collecting supernatant, filtering the supernatant by a filter membrane of 0.45 mu m, purifying the pear PbrPME protein by using a nickel column affinity chromatography method, and balancing a chromatography medium by using a balance buffer solution with 10 times of column volume before purifying the protein by using a nickel column affinity chromatography medium; collecting purified protein, concentrating and desalting at 8000rpm at 4 deg.C by use of 30kDa ultrafiltration tube, and storing at-80 deg.C;

further preferably, the detailed process of treating the pear pollen tube with the recombinant PbrPME protein in the step f is as follows: the protein concentration was determined using Nanodrop2000, and 5-8. mu.g of protein was taken to treat the pear pollen tube pre-cultured in 400. mu.l of liquid medium 200-. After 1-2h of treatment, 10-20 μ l of pear pollen tube samples were taken under an optical microscope to observe the pollen tube length and counted using Image-Pro Plus 6.0 software.

The formula of the lysis solution is as follows: 150mM sodium chloride, 50mM tris (hydroxymethyl) aminomethane-hydrochloric acid, 1mM ethylenediaminetetraacetic acid, 0.1% sodium dodecyl sulfate (w/v, g/100ml), 0.1% Triton X-100(v/v), 50 × EDTA-freeprotease inhibitor cocktail III (Merck Micke, Germany), 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 formula of the pollen liquid culture medium is as follows: 0.55mM calcium nitrate, 1.60mM boric acid, 1.60mM magnesium sulfate, 1.00mM potassium nitrate, 440.00mM sucrose and 5.00mM2- (N-morpholinyl) ethanesulfonic acid, 4-morpholinylethanesulfonic acid, pH 6.0-6.2.

The in vitro expression method of the pear PbrPME protein comprises the step of carrying 6 histidine tags (His-Tag) at the N end of the pear PbrPME protein.

The most preferable technical scheme of the pear PbrPME protein in-vitro expression method comprises the following steps:

(1) sequence analysis and cloning of pear PbrPME gene

(1.1) the pear PbrPME gene has a nucleotide sequence of SEQ ID.1 and an amino acid sequence of SEQ ID.2. The transmembrane domain sequence was analyzed and removed using the transmembrane domain analysis website TMHMM (http:// www.cbs.dtu.dk/services/TMHMM /).

(1.2) extracting the pollen RNA of the Dangshan pear according to the instruction of the total plant RNA extraction kit, and performing reverse transcription by using the reverse transcription kit to obtain cDNA.

(1.3) primer design and PCR reaction: design forward primer (PbrPME-pCold-Xho I-Lp) as 5' -CCCTCGAGATGAAAAACAAAGGAGGAGGAG-3 '(Xho I cleavage site underlined, start codon in bold) and 5' -GC for the reverse primer (PbrPME-pCold-Xba I-Rp)TCTAGAAACGGAAACCATGCCAGC-3' (Xba I cleavage site underlined); the PCR reaction system is as follows: ddH₂O31. mu.l, 2.5. mu.l each of the upstream and downstream primers, 1. mu.l of cDNA, 10. mu.l of 5 XPisuion GC Buffer, 1. mu.l of 10mM dNTPs, 50mM MgCl₂solution 1. mu.l, Phusion DNApolymerase 1. mu.l; the PCR reaction conditions are as follows: pre-denaturation at 98 ℃ for 30min, then at 98 ℃ for 10s, 60 ℃ for 30s, and 72 ℃ for 2min for 30 cycles, and finally extension at 72 ℃ for 10 min.

(1.4) the PCR product was analyzed by 1% agarose gel electrophoresis and recovered by cutting gel, as can be seen in FIG. 2, there was a clear bright band around 2000bp, which was consistent with the expected size. The recovered product was double-digested with Xho I and Xba I restriction enzymes and ligated to Xho I and Xba I sites of the same double-digested E.coli expression vector pCold-TF (see vector map of TAKARA).

(1.5) constructing a recombinant expression vector PbrPME-pCold-TF, and verifying the correctness of the enzyme digestion by DNA sequencing of Jinzhi Biotech, Suzhou, after the enzyme digestion verification is correct.

(2) Construction of expression Strain

Chemically converting a correctly verified recombinant expression vector PbrPME-pCold-TF into an escherichia coli strain Rosetta (DE3), culturing at 37 ℃ on an LB solid plate (containing 100 mu g/ml ampicillin), selecting a single colony to 1ml of LB liquid culture medium (containing 100 mu g/ml ampicillin), performing shake culture at 37 ℃ and 250rpm overnight, absorbing 1 mu l of bacterial liquid as a template, performing PCR verification to be correct, adding 300 mu l of sterilized 50% (v/v) glycerol into 700 mu l of bacterial liquid, uniformly mixing, quickly freezing by liquid nitrogen, and storing in a refrigerator at-80 ℃, wherein the stored strain is the recombinant expression strain.

(3) Induced expression and purification of pear PbrPME protein

(3.1) inducible expression of Pear PbrPME protein

Inoculating the PbrPME recombinant expression strain preserved in the step (2) into 100ml LB liquid culture medium containing 100 mu g/ml ampicillin according to the ratio of 1:50, carrying out shake culture at 37 ℃ and 200-₆₀₀Rapidly placing on ice for 5-10min when the temperature reaches 0.3-0.6, and standing at 15 deg.C for 40-50 min; then adding inducer IPTG with final concentration of 0.3-0.5mmol/L, culturing at 15 deg.C and 200-.

(3.2) purification of Pear PbrPME protein

After the induction of expression, at 4 degrees C under 12000rpm centrifugal 10min after abandoning the supernatant, collecting the precipitate, to the precipitation adding lysate (150mM sodium chloride, 50mM three (hydroxymethyl) aminomethane-hydrochloric acid, 1mM ethylene diamine tetraacetic acid, 0.1% sodium dodecyl sulfate (w/v), 0.1% Triton X-100(v/v), 50X EDTA-free protease inhibitor cocktails III (Germany Merck Mich), pH 7.3) heavy suspension precipitation; and (3) carrying out ultrasonic crushing on the resuspended bacterial liquid with the power of 240W under the conditions: turn on for 3s, stop for 7s, and total 27 min. After the ultrasonication is finished, centrifuging at 12000rpm for 10min, collecting supernatant, filtering the supernatant by a 0.45-micron filter membrane, purifying the pear PbrPME protein by using a nickel column affinity chromatography method, and balancing a chromatography medium by using 10 times of column volume of a balancing buffer solution (500mM sodium chloride, 20mM tris (hydroxymethyl) aminomethane, 5mM imidazole, pH 7.3) before purifying the protein by using the nickel column affinity chromatography medium; the purified protein was collected, concentrated and desalted at 8000rpm at 4 ℃ using an ultrafiltration tube with a cut-off of 30 kDa. Then 50 μ l of the purified sample is taken, 50 μ l of 2 xSDS-PAGE sample buffer is added, 10 μ l of the sample is taken for SDS-PAGE electrophoresis, the purification condition of the pear PbrPME protein is determined after Coomassie brilliant blue staining, the purified protein is collected, and finally the pear PbrPME protein is stored at-80 ℃ for standby.

(4) Pear PbrPME protein processing pear pollen tube

And (3) concentrating and desalting the purified protein, and then determining the protein concentration with a Nanodrop2000 instrument. The pear pollen was pre-cultured for 1-2 hours using 200. mu.l of pollen liquid medium (0.55mM calcium nitrate, 1.60mM boric acid, 1.60mM magnesium sulfate, 1.00mM potassium nitrate, 440.00mM sucrose and 5.00mM2- (N-morpholino) ethanesulfonic acid, pH 6.0-6.2). Adding 5-8 μ g pear PbrPME protein after 1-2h, treating for 1-2h, taking 10-20 μ l sample, observing under optical microscope, and counting pollen tube length with Image-Pro Plus 6.0 software.

Compared with the prior art, the invention has the beneficial effects that:

the pear PbrPME recombinant protein in-vitro expression method disclosed by the invention has the optimum expression temperature of 15 ℃, fully ensures the activity of the PbrPME recombinant protein, and has low cost and high yield. The pear PbrPME recombinant protein purified by the invention has single strip and high purity, can meet the requirements of pear pollen related experiments, and can be used for extracting leaf protoplast and the like.

Drawings

FIG. 1 is a diagram of the prediction of the transmembrane domain of the pear PbrPME gene sequence.

FIG. 2 is a diagram of agarose gel electrophoresis of PCR amplified pear PbrPME gene;

wherein, 1 is the DNA standard molecular mass; 2 is PCR amplification product.

FIG. 3 is a SDS-PAGE diagram of the induced expression and purification of recombinant pear PbrPME protein;

wherein M is a protein standard molecular weight; 1 is induced thallus total protein without IPTG; 2 is total protein of the thallus after induction by 0.5mmol/L IPTG; and 3, the purified recombinant pear PbrPME protein.

FIG. 4 is a pictorial view of a recombinant PbrPME protein treated pear pollen tube;

wherein, -PbrPME is a control without adding recombinant pear PbrPME protein; + PbrPME was added to the recombinant pear PbrPME protein treated group.

FIG. 5 is a statistical representation of pollen tube length after recombinant PbrPME protein treatment of pear pollen tubes;

wherein, the abscissa is corresponding processing; the ordinate is the pollen tube length; -PbrPME is PbrPME protein control without added recombinant pear; + PbrPME was added to the recombinant pear PbrPME protein treated group.

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 PbrPME-pCold-TF recombinant plasmid

Taking fresh Dangshan pear pollen, extracting the total RNA of the flower column by using a plant total RNA extraction kit, and performing reverse transcription by using a reverse transcription kit to obtain total cDNA; as shown in FIG. 1, the sequence of the transmembrane domain of the pear PbrPME gene was analyzed and removed, primers were designed, and PCR amplification was performed using cDNA as a template.

Design forward primer (PbrPME-pCold-Xho I-Lp) as 5' -CCCTCGAGATGAAAAACAAAGGAGGAGGAG-3 '(Xho I cleavage site underlined, start codon in bold) and 5' -GC for the reverse primer (PbrPME-pCold-Xba I-Rp)TCTAGAAACGGAAACCATGCCAGC-3' (Xba I cleavage site underlined);

the PCR reaction system (total reaction system 50. mu.l) was: ddH₂O31. mu.l, 2.5. mu.l each of the upstream and downstream primers, 1. mu.l of cDNA, 10. mu.l of 5 XPisuion GC Buffer, 1. mu.l of 10mM dNTPs, 50mM MgCl₂solution 1μl,Phusion DNAPolymerase 1μl。

The PCR reaction conditions are as follows: pre-denaturation at 98 ℃ for 30min, then at 98 ℃ for 10s, 60 ℃ for 30s, and 72 ℃ for 2min for 30 cycles, and finally extension at 72 ℃ for 10 min. The PCR product was identified by electrophoresis on a 1% agarose gel. As can be seen from FIG. 2, there is a clear bright band around 2000bp, which matches the expected size.

The method comprises the steps of recovering PCR products by referring to instructions of a kit for recovering Gel extraction kit fast agarose Gel DNA of century corporation, inserting the recovered PCR products into a pCold-TF expression vector subjected to the same double enzyme digestion through T4-DNA ligase after Xho I and Xba I double enzyme digestion, connecting for 18h at 4 ℃, constructing PbrPME-pCold-TF recombinant plasmids, transforming the recombinant plasmids into an Escherichia coli strain DH5 α, culturing on an LB solid plate (containing 100 mu g/ml ampicillin) at 37 ℃, selecting single colonies after enzyme digestion identification is correct, and verifying the sequence correctness by DNA sequencing of Suzhou Jinzhi Nizhi Biotechnology Limited company.

Example 2: expression of pear PbrPME in Escherichia coli

1. Obtaining recombinant expression strain for expressing pear PbrPME

Selecting bacteria with successful sequencing, inoculating the bacteria into 4mL LB liquid culture medium containing ampicillin (100 mug/mL), shaking and culturing at 37 ℃ at 250rpm for overnight, extracting PbrPME-pCold-TF recombinant expression vector according to a quick Plasmid small extraction kit of QuickPure Plasmid Mini kit Kangji corporation, taking 1ng of recombinant expression vector PbrPME-pCold-TF to chemically transform escherichia coli Rosetta (DE3), coating the transformed escherichia coli Rosetta (DE3) on an LB plate containing 100 mug/mL ampicillin to screen recombinants, shaking and culturing at 37 ℃ and 250rpm for overnight to obtain the recombinants which are genetic engineering bacteria for expressing pear PbrPME, randomly selecting 1 single colony for streak culture, inoculating a small amount of grown streaking bacteria into 1mL LB liquid culture medium (containing 100 mug/mL penicillin) for overnight shaking and culturing at 37 ℃ and 250rpm, then adding 300 mul of sterilized 50% (v/v) glycerol into 700 mul of the bacterial liquid, mixing uniformly, using liquid nitrogen to quickly freeze and storing in a refrigerator at minus 80 ℃ to obtain the recombinant expression strain for expressing the pear PbrPME.

2. Expression of pear PbrPME recombinant protein

The pear PbrPME recombinant expression strain obtained above was inoculated into 100ml of LB liquid medium (containing 100. mu.g/ml ampicillin) at a volume ratio of 1:50, cultured overnight at 37 ℃ with shaking at 250rpm, and the recombinant expression strain was activated. The activated recombinant expression strain was transferred to 300mL LB liquid medium (containing 100. mu.g/mL ampicillin) at a volume ratio of 1:50 and cultured under conditions of 37 ℃ and 200rpm with shaking to OD₆₀₀Taking 5ml of bacterial liquid as a negative control when the concentration is 0.4-0.6, rapidly placing the rest bacterial liquid on ice for 10min, then placing the bacterial liquid in a shaking table at 15 ℃ for standing for 40min, finally adding IPTG with the final concentration of 0.5mmol/L, performing shake culture at 15 ℃ and 240rpm, and performing induced expression for 24 h. After the expression is finished, 5ml of bacterial liquid is taken for centrifugation, and thalli are collectedPrecipitating, adding 200 μ l 10% SDS (w/v, g/100ml) into the precipitate and negative control, mixing, water-bathing with boiling water at 100 deg.C for 10min, cooling in ice for 2min, centrifuging at 12000rpm at 4 deg.C for 10min, collecting 50 μ l supernatant, adding 50 μ l 2 × protein loading buffer, collecting 10 μ l, performing 12% conventional SDS-PAGE, staining with Coomassie brilliant blue, decolorizing, and detecting recombinant protein expression, the results are shown in

lanes

1 and 2 in FIG. 3.

Example 3: separation, purification and identification of recombinant pear PbrPME protein

The pear PbrPME recombinant expression strain is inoculated into 100ml LB liquid culture medium (containing 100 mug/ml ampicillin) according to the ratio of 1:50, and is cultured overnight at 37 ℃ and 250rpm with shaking, and the recombinant expression strain is activated. The activated recombinant expression strain was transferred to 300mL LB medium (containing 100. mu.g/mL ampicillin) at a ratio of 1:50 and cultured at 37 ℃ with shaking at 200rpm until OD₆₀₀After the concentration is 0.4-0.6, quickly placing on ice for 10min, then placing in a shaking table at 15 ℃ for standing for 40min, finally adding IPTG with the final concentration of 0.5mmol/L, and carrying out shake culture at 15 ℃ and 240rpm for induced expression for 24 h. After completion of the expression, the supernatant was centrifuged at 12000rpm for 10min at 4 ℃ to collect pellet of the cells, and the pellet was sonicated and disrupted with a lysis solution (150mM NaCl, 50mM Tris (hydroxymethyl) aminomethane-HCl, 1mM EDTA, 0.1% sodium dodecyl sulfate (W/v), 0.1% Triton X-100(v/v), 50 × EDTA-free protease inhibitor cocktail III (Merck Miclebo, Germany) at pH 7.3) and then resuspended in the cell, under the condition of 240W: starting for 3s, stopping for 7s, and crushing for 27min until the solution is clear. After the ultrasonication was completed, the mixture was centrifuged at 12000rpm at 4 ℃ for 10min, and the supernatant was collected and filtered through a 0.45 μm filter to remove impurities. The Ni-NTA agarose affinity chromatography filler of Germany Mercury corporation is used for purifying the pear PbrPME recombinant protein, and the specific operation is as follows: column volume 10 times of equilibration buffer (500mM sodium chloride, 20mM tris (hydroxymethyl) aminomethane, 5mM imidazole, pH 7.3) equilibrates the column with a controlled flow rate of 1 ml/min; passing 20ml of protein supernatant filtered by the filter membrane through a purification column, and controlling the flow rate to be 1 ml/min; a20 column volume wash containing 30mM imidazole (500mM sodium chloride, 20mM tris (hydroxymethyl) aminomethane, 30mM imidazole, pH 7.3) washed the column at a flow rate of 1 ml/min; 10 times column volumeEluting with 80mM imidazole-containing eluate (500mM sodium chloride, 20mM tris (hydroxymethyl) aminomethane, 80mM imidazole, pH 7.3) to obtain purified protein at flow rate of 1ml/min, collecting eluate to obtain purified protein, concentrating with ultrafiltration tube (cut-off of 30kDa) at 8000rpm and 4 deg.C, desalting, and storing at-80 deg.C. A small amount of protein was analyzed for SDS-PAGE purity and identified by Western Blotting, and the results are shown in lane 3 of FIG. 3.

Example 4: activity detection of recombinant pear PbrPME protein

The concentration of recombinant pear PbrPME protein was determined on a Nanodrop2000 instrument. Pear pollen was pre-cultured for 2 hours using 200. mu.l of pollen liquid medium (0.55mM calcium nitrate, 1.60mM boric acid, 1.60mM magnesium sulfate, 1.00mM potassium nitrate, 440.00mM sucrose and 5.00mM2- (N-morpholino) ethanesulfonic acid, pH 6.0-6.2). After 2h, a total of 5. mu.g of pear PbrPME protein was added for 1h, after which 10. mu.l of the sample was observed under a light microscope and the pollen tube length was counted using Image-Pro Plus 6.0 software, the results are shown in FIGS. 4 and 5.

Sequence listing

<110> Nanjing university of agriculture

<120> in vitro expression method of recombinant pectin methylesterase PbrPME, and coding gene and application thereof

<160>4

<170>SIPOSequenceListing 1.0

<210>1

<211>1737

<212>DNA

<213> Pear (Pyrus spp)

<400>1

atgaacgctg aacataataa gaagaagaag ttcgccatca tcggcgtttc atctgtgatt 60

ctagtagcca tggtggttgc cgtaacggtt ggggtaaccg caaaaaacaa aggaggagga 120

gggcacaaaa actcggacgg cgggcatgtc accacgtcga caaaagcaat aaaggctata 180

tgccagccga cggattataa ggaaacctgc gagaggagtc tggaatcggc tgctggcaac 240

agcacggagc cgaaggacct catcaaggca ggtttcaacg ttaccatgga caaacttcgg 300

gccatcataa aaaactccac cacattgcaa gaactggcca aggacgaaag tacaaaccaa 360

gctctagaaa attgcaagga gctcttggag tatgccatcg acgatatcac gacatctttc 420

caaaaattgg gtcctttcga catcagcaag atcgacgatt acgcggaaga tctcaaggtc 480

tggctcagcg cggctattac ttaccaacaa acatgcttgg acgggtttca gaacaccaaa 540

ggtgacgcag gcgaaaagat gaagcatttc ttgaacacca cgcaacagct caccagcaac 600

ggacttgcca tggtgaccga aattacgtcc gttcttgggt ctctgaattt gaaagcaaac 660

cggcgtcgtt tgctggcagg gggagggggt ggaagtaatg cgaagaatgc tccaaaggtt 720

attcctacgt ggatcaacaa taagcgtagc ctcgacgtgt ctaccgtgac tccacagtcg 780

attaaaccag acgcggtggt ggctaaggat gggagcggac agtacgagac tattggtgaa 840

gctgtgaaga tgatcccgaa gaacaatgcg gacaagacgt ttgtgattta tgtgaaggag 900

ggagtgtaca gtgagtatgt catgattgac aagtatatga ccaacgtcat gttgattggg 960

gatggcgcta ctaagaccaa ggtcaccggt agcaaaaact tcgctgcggg agtccaaact 1020

tttcagactg caacagttgt tgtagttggg gactacttca ttgctaagga catagggatt 1080

gagaactcag caggagccga agggcaccaa gccgtggctc tgcgagtgca gtcggacttg 1140

tccatcttct accgctgcca aatggacggg taccaagaca ctctctacac tcaaacccac 1200

cggcaattct accgcgactg caccatcagc ggcacaatcg actttatctt tggtgacgct 1260

gccgtagtgt tccagaactg caagatgatc gtaagaaaac caatggacaa ccaagcttgt 1320

atggtcacag cccaagggag aattgaccgg cgtcagccct cgggtatcat cctccaaaac 1380

tgcaccatct ccggggaccc ggagtacatt cccgtgaagg acaagaacaa gtcgtacctg 1440

gggaggccgt ggaagaacct cgctaggacc gtcgttatgc agtcgcagat tgatggcatc 1500

attgctccgg aagggtggat ggagtggacg ggttcgaaca atcatcaaag ttgctggttt 1560

ggcgaataca gaaacagagg gcctggctcc gacatgagca agagggtgaa gtggagcggt 1620

atcaagaatc ttggtgccga ccaggctgtc gctttcacct ccggtaaatt tgttgagggt 1680

gataagtgga tcaagcccag cggtgtgcct tacgttgctg gcatggtttc cgtttag 1737

<210>2

<211>578

<212>PRT

<213> Pear (Pyrus spp)

<400>2

Met Asn Ala Glu His Asn Lys Lys Lys Lys Phe Ala Ile Ile Gly Val

1 5 10 15

Ser Ser Val Ile Leu Val Ala Met Val Val Ala Val Thr Val Gly Val

20 25 30

Thr Ala Lys Asn Lys Gly Gly Gly Gly His Lys Asn Ser Asp Gly Gly

35 40 45

His Val Thr Thr Ser Thr Lys Ala Ile Lys Ala Ile Cys Gln Pro Thr

50 55 60

Asp Tyr Lys Glu Thr Cys Glu Arg Ser Leu Glu Ser Ala Ala Gly Asn

65 70 75 80

Ser Thr Glu Pro Lys Asp Leu Ile Lys Ala Gly Phe Asn Val Thr Met

85 90 95

Asp Lys Leu Arg Ala Ile Ile Lys Asn Ser Thr Thr Leu Gln Glu Leu

100 105 110

Ala Lys Asp Glu Ser Thr Asn Gln Ala Leu Glu Asn Cys Lys Glu Leu

115 120 125

Leu Glu Tyr Ala Ile Asp Asp Ile Thr Thr Ser Phe Gln Lys Leu Gly

130 135 140

Pro Phe Asp Ile Ser Lys Ile Asp Asp Tyr Ala Glu Asp Leu Lys Val

145 150 155 160

Trp Leu Ser Ala Ala Ile Thr Tyr Gln Gln Thr Cys Leu Asp Gly Phe

165 170 175

Gln Asn Thr Lys Gly Asp Ala Gly Glu Lys Met Lys His Phe Leu Asn

180 185 190

Thr Thr Gln Gln Leu Thr Ser Asn Gly Leu Ala Met Val Thr Glu Ile

195 200 205

Thr Ser Val Leu Gly Ser Leu Asn Leu Lys Ala Asn Arg Arg Arg Leu

210 215 220

Leu Ala Gly Gly Gly Gly Gly Ser Asn Ala Lys Asn Ala Pro Lys Val

225 230 235 240

Ile Pro Thr Trp Ile Asn Asn Lys Arg Ser Leu Asp Val Ser Thr Val

245 250 255

Thr Pro Gln Ser Ile Lys Pro Asp Ala Val Val Ala Lys Asp Gly Ser

260 265 270

Gly Gln Tyr Glu Thr Ile Gly Glu Ala Val Lys Met Ile Pro Lys Asn

275 280 285

Asn Ala Asp Lys Thr Phe Val Ile Tyr Val Lys Glu Gly Val Tyr Ser

290 295 300

Glu Tyr Val Met Ile Asp Lys Tyr Met Thr Asn Val Met Leu Ile Gly

305 310 315 320

Asp Gly Ala Thr Lys Thr Lys Val Thr Gly Ser Lys Asn Phe Ala Ala

325 330 335

Gly Val Gln Thr Phe Gln Thr Ala Thr Val Val Val Val Gly Asp Tyr

340 345 350

Phe Ile Ala Lys Asp Ile Gly Ile Glu Asn Ser Ala Gly Ala Glu Gly

355 360 365

His Gln Ala Val Ala Leu Arg Val Gln Ser Asp Leu Ser Ile Phe Tyr

370 375 380

Arg Cys Gln Met Asp Gly Tyr Gln Asp Thr Leu Tyr Thr Gln Thr His

385 390 395 400

Arg Gln Phe Tyr Arg Asp Cys Thr Ile Ser Gly Thr Ile Asp Phe Ile

405 410 415

Phe Gly Asp Ala Ala Val Val Phe Gln Asn Cys Lys Met Ile Val Arg

420 425 430

Lys Pro Met Asp Asn Gln Ala Cys Met Val Thr Ala Gln Gly Arg Ile

435 440 445

Asp Arg Arg Gln Pro Ser Gly Ile Ile Leu Gln Asn Cys Thr Ile Ser

450 455 460

Gly Asp Pro Glu Tyr Ile Pro Val Lys Asp Lys Asn Lys Ser Tyr Leu

465 470 475 480

Gly Arg Pro Trp Lys Asn Leu Ala Arg Thr Val Val Met Gln Ser Gln

485 490 495

Ile Asp Gly Ile Ile Ala Pro Glu Gly Trp Met Glu Trp Thr Gly Ser

500 505 510

Asn Asn His Gln Ser Cys Trp Phe Gly Glu Tyr Arg Asn Arg Gly Pro

515 520 525

Gly Ser Asp Met Ser Lys Arg Val Lys Trp Ser Gly Ile Lys Asn Leu

530 535 540

Gly Ala Asp Gln Ala Val Ala Phe Thr Ser Gly Lys Phe Val Glu Gly

545 550 555 560

Asp Lys Trp Ile Lys Pro Ser Gly Val Pro Tyr Val Ala Gly Met Val

565 570 575

Ser Val

<210>3

<211>1635

<212>DNA

<213> Pear (Pyrus spp)

<400>3

atgaaaaaca aaggaggagg agggcacaaa aactcggacg gcgggcatgt caccacgtcg 60

acaaaagcaa taaaggctat atgccagccg acggattata aggaaacctg cgagaggagt 120

ctggaatcgg ctgctggcaa cagcacggag ccgaaggacc tcatcaaggc aggtttcaac 180

gttaccatgg acaaacttcg ggccatcata aaaaactcca ccacattgca agaactggcc 240

aaggacgaaa gtacaaacca agctctagaa aattgcaagg agctcttgga gtatgccatc 300

gacgatatca cgacatcttt ccaaaaattg ggtcctttcg acatcagcaa gatcgacgat 360

tacgcggaag atctcaaggt ctggctcagc gcggctatta cttaccaaca aacatgcttg 420

gacgggtttc agaacaccaa aggtgacgca ggcgaaaaga tgaagcattt cttgaacacc 480

acgcaacagc tcaccagcaa cggacttgcc atggtgaccg aaattacgtc cgttcttggg 540

tctctgaatt tgaaagcaaa ccggcgtcgt ttgctggcag ggggaggggg tggaagtaat 600

gcgaagaatg ctccaaaggt tattcctacg tggatcaaca ataagcgtag cctcgacgtg 660

tctaccgtga ctccacagtc gattaaacca gacgcggtgg tggctaagga tgggagcgga 720

cagtacgaga ctattggtga agctgtgaag atgatcccga agaacaatgc ggacaagacg 780

tttgtgattt atgtgaagga gggagtgtac agtgagtatg tcatgattga caagtatatg 840

accaacgtca tgttgattgg ggatggcgct actaagacca aggtcaccgg tagcaaaaac 900

ttcgctgcgg gagtccaaac ttttcagact gcaacagttg ttgtagttgg ggactacttc 960

attgctaagg acatagggat tgagaactca gcaggagccg aagggcacca agccgtggct 1020

ctgcgagtgc agtcggactt gtccatcttc taccgctgcc aaatggacgg gtaccaagac 1080

actctctaca ctcaaaccca ccggcaattc taccgcgact gcaccatcag cggcacaatc 1140

gactttatct ttggtgacgc tgccgtagtg ttccagaact gcaagatgat cgtaagaaaa 1200

ccaatggaca accaagcttg tatggtcaca gcccaaggga gaattgaccg gcgtcagccc 1260

tcgggtatca tcctccaaaa ctgcaccatc tccggggacc cggagtacat tcccgtgaag 1320

gacaagaaca agtcgtacct ggggaggccg tggaagaacc tcgctaggac cgtcgttatg 1380

cagtcgcaga ttgatggcat cattgctccg gaagggtgga tggagtggac gggttcgaac 1440

aatcatcaaa gttgctggtt tggcgaatac agaaacagag ggcctggctc cgacatgagc 1500

aagagggtga agtggagcgg tatcaagaat cttggtgccg accaggctgt cgctttcacc 1560

tccggtaaat ttgttgaggg tgataagtgg atcaagccca gcggtgtgcc ttacgttgct 1620

ggcatggttt ccgtt 1635

<210>4

<211>545

<212>PRT

<213> Pear (Pyrus spp)

<400>4

Met Lys Asn Lys Gly Gly Gly Gly His Lys Asn Ser Asp Gly Gly His

1 5 10 15

Val Thr Thr Ser Thr Lys Ala Ile Lys Ala Ile Cys Gln Pro Thr Asp

20 25 30

Tyr Lys Glu Thr Cys Glu Arg Ser Leu Glu Ser Ala Ala Gly Asn Ser

35 40 45

Thr Glu Pro Lys Asp Leu Ile Lys Ala Gly Phe Asn Val Thr Met Asp

50 55 60

Lys Leu Arg Ala Ile Ile Lys Asn Ser Thr Thr Leu Gln Glu Leu Ala

65 70 75 80

Lys Asp Glu Ser Thr Asn Gln Ala Leu Glu Asn Cys Lys Glu Leu Leu

85 90 95

Glu Tyr Ala Ile Asp Asp Ile Thr Thr Ser Phe Gln Lys Leu Gly Pro

100 105 110

Phe Asp Ile Ser Lys Ile Asp Asp Tyr Ala Glu Asp Leu Lys Val Trp

115 120 125

Leu Ser Ala Ala Ile Thr Tyr Gln Gln Thr Cys Leu Asp Gly Phe Gln

130 135 140

Asn Thr Lys Gly Asp Ala Gly Glu Lys Met Lys His Phe Leu Asn Thr

145 150 155 160

Thr Gln Gln Leu Thr Ser Asn Gly Leu Ala Met Val Thr Glu Ile Thr

165 170 175

Ser Val Leu Gly Ser Leu Asn Leu Lys Ala Asn Arg Arg Arg Leu Leu

180 185 190

Ala Gly Gly Gly Gly Gly Ser Asn Ala Lys Asn Ala Pro Lys Val Ile

195 200 205

Pro Thr Trp Ile Asn Asn Lys Arg Ser Leu Asp Val Ser Thr Val Thr

210 215 220

Pro Gln Ser Ile Lys Pro Asp Ala Val Val Ala Lys Asp Gly Ser Gly

225 230 235 240

Gln Tyr Glu Thr Ile Gly Glu Ala Val Lys Met Ile Pro Lys Asn Asn

245 250 255

Ala Asp Lys Thr Phe Val Ile Tyr Val Lys Glu Gly Val Tyr Ser Glu

260 265 270

Tyr Val Met Ile Asp Lys Tyr Met Thr Asn Val Met Leu Ile Gly Asp

275 280 285

Gly Ala Thr Lys Thr Lys Val Thr Gly Ser Lys Asn Phe Ala Ala Gly

290 295 300

Val Gln Thr Phe Gln Thr Ala Thr Val Val Val Val Gly Asp Tyr Phe

305 310 315 320

Ile Ala Lys Asp Ile Gly Ile Glu Asn Ser Ala Gly Ala Glu Gly His

325 330 335

Gln Ala Val Ala Leu Arg Val Gln Ser Asp Leu Ser Ile Phe Tyr Arg

340 345 350

Cys Gln Met Asp Gly Tyr Gln Asp Thr Leu Tyr Thr Gln Thr His Arg

355 360 365

Gln Phe Tyr Arg Asp Cys Thr Ile Ser Gly Thr Ile Asp Phe Ile Phe

370 375 380

Gly Asp Ala Ala Val Val Phe Gln Asn Cys Lys Met Ile Val Arg Lys

385 390 395 400

Pro Met Asp Asn Gln Ala Cys Met Val Thr Ala Gln Gly Arg Ile Asp

405 410 415

Arg Arg Gln Pro Ser Gly Ile Ile Leu Gln Asn Cys Thr Ile Ser Gly

420 425 430

Asp Pro Glu Tyr Ile Pro Val Lys Asp Lys Asn Lys Ser Tyr Leu Gly

435 440 445

Arg Pro Trp Lys Asn Leu Ala Arg Thr Val Val Met Gln Ser Gln Ile

450 455 460

Asp Gly Ile Ile Ala Pro Glu Gly Trp Met Glu Trp Thr Gly Ser Asn

465 470 475 480

Asn His Gln Ser Cys Trp Phe Gly Glu Tyr Arg Asn Arg Gly Pro Gly

485 490 495

Ser Asp Met Ser Lys Arg Val Lys Trp Ser Gly Ile Lys Asn Leu Gly

500 505 510

Ala Asp Gln Ala Val Ala Phe Thr Ser Gly Lys Phe Val Glu Gly Asp

515 520 525

Lys Trp Ile Lys Pro Ser Gly Val Pro Tyr Val Ala Gly Met Val Ser

530 535 540

Val

545

Claims

1. A method for in vitro expression of pear PbrPME protein is characterized by comprising the following steps:

e. purifying the pear PbrPME protein for induction expression.

2. The method for in vitro expression of pear PbrPME protein according to claim 1, wherein in step a, the forward primer used for PCR amplification of pear PbrPME gene is 5'-CCCTCGAGATGAAAAACAAAGGAGGAGGAG-3', and the reverse primer is 5'-GCTCTAGAAACGGAAACCATGCCAGC-3'.

3. The method for in vitro expression of pear PbrPME protein according to claim 1 or 2, wherein in the step a, the PCR reaction system for PCR amplification of pear PbrPME gene is: ddH₂O31. mu.l, 2.5. mu.l each of the upstream and downstream primers, 1. mu.l of cDNA, 10. mu.l of 5 XPisuion GC Buffer, 1. mu.l of 10mM dNTPs, 50mM MgCl₂solution 1. mu.l, Phusion DNApolymerase 1. mu.l; the PCR reaction conditions are as follows: pre-denaturation at 98 ℃ for 30min, then at 98 ℃ for 10s, 60 ℃ for 30s, and 72 ℃ for 2min for 30 cycles, and finally extension at 72 ℃ for 10 min.

4. The method for in vitro expression of pear PbrPME protein according to claim 1, wherein in step b, when constructing prokaryotic recombinant expression vector for expressing pear PbrPME protein, the prokaryotic expression vector used is Escherichia coli expression vector pCold-TF, and the insertion site for cloning pear PbrPME gene is between Xho I and Xba I restriction enzyme sites.

5. The method for in vitro expression of pear PbrPME protein according to claim 4, wherein the PCR product is analyzed by 1% agarose gel electrophoresis and recovered by cutting gel, and the recovered product is double-digested with Xho I and Xba I restriction enzymes and then ligated to Xho I and Xba I sites of the same double-digested Escherichia coli expression vector pCold-TF.

6. The method for in vitro expression of pear PbrPME protein according to claim 1, wherein in step d, said low temperature treatment is ice-cooling for 5-10min, then standing at 10-15 ℃ for 40-50min, the final concentration of said inducer IPTG is 0.3-0.5mmol/L, and said condition for inducing expression is 15 ℃ for 22-24 h.

7. The method for in vitro expression of pear PbrPME protein according to claim 1 or 6, wherein the detailed process of inducing expression of pear PbrPME protein in step d is: the constructed recombinant expression strain of PbrPME was inoculated into 100ml of LB liquid containing 100. mu.g/ml ampicillin at a ratio of 1:50Culturing overnight in a culture medium at 37 ℃ and 200-250rpm with shaking, then transferring into 300ml LB liquid culture medium containing 100 mu g/ml ampicillin according to the proportion of 1:50, and culturing at 37 ℃ and 200-250rpm with shaking until the OD of bacterial liquid₆₀₀Rapidly placing on ice for 5-10min when the temperature reaches 0.3-0.6, and standing at 15 deg.C for 40-50 min; then adding inducer IPTG with final concentration of 0.3-0.5mmol/L, carrying out shake culture at 15 ℃ and 200-250rpm, and carrying out induced expression for 22-24 h;

the detailed process of purifying the pear PbrPME protein for induction expression in the step e is as follows: after the induction expression is finished, centrifuging at 12000rpm for 10min at 4 ℃, then abandoning the supernatant, collecting the precipitate, and adding a lysate into the precipitate to resuspend the precipitate; ultrasonically crushing the resuspended bacterial liquid, centrifuging at 12000rpm for 10min after the ultrasonic crushing is finished, collecting supernatant, filtering the supernatant by a 0.45-micron filter membrane, purifying the pear PbrPME protein by using a nickel column affinity chromatography method, and balancing the chromatography medium by using a balance buffer solution with 10 times of column volume before purifying the protein by using the nickel column affinity chromatography medium; collecting purified protein, concentrating and desalting at 8000rpm at 4 deg.C by use of 30kDa ultrafiltration tube, and storing at-80 deg.C;

the formula of the lysis solution is as follows: 150mM sodium chloride, 50mM tris (hydroxymethyl) aminomethane-hydrochloric acid, 1mM ethylenediaminetetraacetic acid, 0.1% sodium dodecyl sulfate (w/v), 0.1% Triton X-100(v/v), 50 × EDTA-free protease inhibitor cocktail III (Merck Michelia, Germany), pH 7.3;

8. The method for in vitro expression of pear PbrPME protein according to claim 1, wherein the pear PbrPME protein has 6 histidine tags (His-Tag) at the N-terminus.