CN116064615A - Gene for encoding polygalacturonase and application thereof - Google Patents

Abstract

The invention belongs to the technical field of genetic engineering, and particularly relates to a gene for encoding polygalacturonic acid endonuclease and application thereof. A gene for encoding polygalacturonase, which has a nucleotide sequence shown in SEQ ID NO. 1. The gene for encoding the polygalacturonase can be used for rapidly preparing a large amount of the polygalacturonase or pectase, is used for pathogenicity analysis of the polygalacturonase and is beneficial to improving experimental efficiency.

Description

Gene for encoding polygalacturonase and application thereof

Technical Field

The invention relates to the technical fields of genetic engineering and enzyme engineering, in particular to a gene for encoding polygalacturonase and application thereof.

Background

Pectic enzymes are important components of cell wall degrading enzymes and play an important role in the degradation of plant cell walls. Polygalacturonase is a pectase obtained for the first time, which causes maceration of plant tissue and cell death by degrading the polygalacturonase chains, causing the structure of the cell wall to be destroyed.

The polygalacturonase is one of key enzymes for degrading pectin skeleton structures of plant cell walls, and is divided into two types of polygalacturonase endonucleases and polygalacturonase exoenzymes according to different enzyme cutting modes of the polygalacturonase acting on the galacturonic acid, wherein the polygalacturonase endonucleases are oligogalacturonate chains which randomly cut long chains of the polygalacturonase into different lengths and different sizes and have polymerization degrees of 10-14; polygalacturonic acid exonuclease cleaves the galacturonic acid residues at the non-reducing end of the oligogalacturonate chain one by one, gradually decreasing the degree of polymerization.

The polygalacturonic acid endonuclease is an extremely important cell wall degrading enzyme in the interaction of plants and microorganisms, a pathogenic bacterium colonizes a host and generates a necessary factor for pathogenicity, and the pathogenic bacterium upregulates and expresses the polygalacturonic acid endonuclease gene in the infection process, and degrades the cell wall of the plants at a certain stage to obtain nutrition for self nutrition.

Sugarcane ratoon stunting disease (Ratoon Stunting Disease, RSD) is a bacterial disease, is a worldwide sugarcane disease, and is mainly transmitted through sugarcane seeds with diseases and tools, the sugarcane after disease is characterized by dwarfing plants, reduced tillers, thinned sugarcane stems, shortened internodes, generally reduced yield by 12-37%, and reduced sucrose fraction. The pathogenic bacteria of RSD are xylem variants (Leifsonia xyli subsp. Xyli, lxx) of the species of the genus lisinia, which are parasitic to the xylem of sugarcane, have high nutrition requirements, are difficult to separate and culture in vitro, and it is very difficult to obtain a large number of polygalacturonic endonucleases for pathogenic analysis by directly separating the pathogenic bacteria. And the original sequence of the polygalacturonase gene from sugarcane ratoon stunting disease is difficult to express in escherichia coli because of containing signal peptide.

The information disclosed in this background section is only for enhancement of understanding of the general background of the invention and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person of ordinary skill in the art.

Disclosure of Invention

The invention aims to provide a gene for encoding polygalacturonic acid endonuclease and application thereof, and the gene optimizes the polygalacturonic acid endonuclease gene sequence, can rapidly obtain a large amount of polygalacturonic acid endonuclease for pathogenicity analysis, and can remarkably improve experimental efficiency.

In order to achieve the above purpose, the present invention provides the following technical solutions:

a gene for encoding polygalacturonase, wherein the nucleotide sequence of the gene is shown as SEQ ID No. 1.

The second purpose of the invention is to protect polygalacturonase, wherein the polygalacturonase is an expression product of a polygalacturonase gene, the expression product consists of 470 amino acids, the molecular weight of the polygalacturonase is 48285.4Da, and the amino acid sequence of the polygalacturonase is shown as SEQ ID No. 2.

The third object of the present invention is to protect a recombinant expression plasmid obtained by recombining an expression plasmid and the above-mentioned gene.

The fourth object of the present invention is to protect the use of the above-mentioned genes in the preparation of endopolygalacturonase or pectase.

A fifth object of the present invention is to protect the use of the polygalacturonase described above for degrading plant cell walls.

A sixth object of the present invention is to protect the use of the polygalacturonase described above for degrading polygalacturonic acid or pectin.

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

the gene for encoding the polygalacturonase can be used for rapidly preparing a large amount of the polygalacturonase or pectase, is used for pathogenicity analysis of the polygalacturonase and is beneficial to improving experimental efficiency.

Drawings

FIG. 1 is plasmid vector information;

FIG. 2 is a diagram of recombinant plasmid restriction enzyme;

FIG. 3 is a SDS-PAGE analysis of a fusion protein expression panel; wherein M is Protein Maker (Cat.No.: C600525), 1 is total Protein before induction, 2 is supernatant at 20deg.C, 3 is precipitation at 20deg.C, 4 is supernatant at 37deg.C, 5 is precipitation at 37deg.C;

FIG. 4 is a SDS-PAGE analysis of the fusion protein nickel agarose affinity chromatography purification; wherein M is Protein Maker (Cat.No.: C600525), 1 is loading, 2 is eluting, 3-4 is 20mM lmidazole eluting component, 5-6 is 50mM lmidazole eluting component, and 7 is 500mM lmidazole eluting component;

FIG. 5 is a SDS-PAGE analysis of purified proteins; m is Protein Maker (Cat.No.: C600525), 1 is fusion target Protein;

FIG. 6 is a BSA standard graph.

Detailed Description

The following description of the embodiments of the present invention will be apparent from, and is intended to provide a thorough description of, the embodiments of the present invention, and not a complete description of, the embodiments of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, are intended to fall within the scope of the present invention.

Examples

1. Construction of plasmids

1.1 according to the optimized gene sequence shown in SEQ ID NO.1, 56 primers are designed, the primer sequences are shown in SEQ ID NO.3-SEQ ID NO.58, enzyme cutting sites NdeI and XhoI are respectively added on the primers No.1 and 56, and two PCR amplifications are carried out according to overlapping PCR by two fragments.

1.1.1 amplification of the first fragment with primers 1-28, the reaction system is shown in Table 1.

PCR reaction system for amplifying first fragment by primer number 1 1-28

PCR reaction procedure: 95℃for 3min,95℃for 22s,55℃for 20s,72℃for 50s,18 cycles, 72℃for 5min.

1.1.2 amplification of the second fragment with primers 27-56, the reaction system is shown in Table 2.

PCR reaction System for amplifying second fragment by primers No. 27-56 of Table 2

PCR reaction procedure: 95℃for 3min,95℃for 22s,55℃for 20s,72℃for 50s,18 cycles, 72℃for 5min.

1.1.3 or more is the first round of PCR amplification, and the second round of PCR is performed after completion. The second round PCR reaction is shown in Table 3.

TABLE 3 second round PCR reaction System

PCR reaction procedure: 95℃for 3min,95℃for 24s,58℃for 20s,72℃for 80s,22 cycles, 72℃for 5min.

1.2 recovery and purification of the second round PCR amplification products using SanPrep column type DNA gel recovery kit, the second round PCR amplification products and plasmid vectors were digested with the endonucleases NdeI and XhoI, respectively, and the PCR product digestion system is shown in Table 4.

TABLE 4 PCR product cleavage System

Placing the mixture into a water bath kettle with constant temperature of 37 ℃ for reaction for 2 hours.

The system for enzyme digestion of the plasmid vector is shown in Table 5.

TABLE 5 plasmid vector cleavage System

Placing into a water bath kettle with constant temperature of 37 ℃ for reaction for 2 hours, wherein the information of plasmid vectors is shown in figure 1.

1.3 ligation of the digested PCR product with the plasmid vector by ligase T4 DNAliase, and ligation of ligation (8. Mu.l of the target fragment, 4. Mu.l of the digested vector pET21a, 10X T4 DNAligase Buffer 2. Mu.l, 1. Mu.l of T4 DNAliase, ddH) ₂ O up to 20 μl) was transferred into TOP10 competence, and positive clones were detected and screened for sequencing verification. Meanwhile, the recombinant plasmid is extracted by using an ion exchange type plasmid extraction kit, double digestion verification is carried out by using NdeI and XhoI enzymes, the system is the same as the digestion system of 1.2, and the digestion result is shown in figure 2.

2. Protein expression

2.1 mixing 100. Mu.L of the extracted recombinant plasmid with 100. Mu.L of competent cells BL21 (DE 3) of Escherichia coli gently, heating at 42 ℃ for 45s, adding 700. Mu.L of LB liquid medium without antibiotics, culturing for 1h at 37 ℃ with shaking (160-225 rpm), absorbing a proper amount of culture bacteria liquid, uniformly coating on a flat plate containing the corresponding antibiotic ampicillin (50. Mu.g/mL), and culturing overnight in an inverted manner.

2.2 picking up the monoclonal to culture in a liquid culture medium containing 50 mug/mL of ampicillin; when the OD value reached 0.6, 0.5mM IPTG inducer was added, the culture was continued, and the culture was continued at 20℃for 16h and 37℃for 4d, respectively, and the culture was centrifuged at 8000 Xg for 10min at 4℃with no inducer added as a negative control, and the supernatant was discarded to collect the cells.

2.3 adding PBS buffer solution of pH7.4 into the collected thalli for suspension, crushing the thalli by using an ultrasonic crusher, centrifuging at 4 ℃ and 10000 Xg for 30min, dissolving the centrifuged sediment by using a buffer solution of 8M Urea,50mM Tris-HCl,300mM NaCl,pH8.0, respectively processing the supernatant and the sediment, preparing samples, and carrying out SDS-PAGE expression detection, wherein the result is shown in figure 3.

As can be seen from FIG. 3, IPTG can induce the mass expression of the target protein at 20 ℃ and 37 ℃, obvious bands appear at the corresponding positions of the theoretical molecular weight 48.29 +/-5 kDa of the protein, and the target protein is expressed in the form of inclusion bodies in the precipitation of recombinant strains.

2.4 culturing the bacterial liquid in a culture medium containing 50 mug/mL of ampicillin, when the OD value reaches 0.6, adding 0.5mM IPTG inducer, culturing overnight at 20 ℃ for mass expression, centrifuging at 4 ℃ and 8000 Xg for 10min, and collecting cell bacterial bodies

3. Protein purification

3.1 cell lines collected were dissolved in a buffer solution of pH8.0 of 8M Urea,50mM Tris-HCl,300mM NaCl,0.1%Triton X-100, sonicated, centrifuged at 4℃and 10000 Xg for 30min, and the supernatant crude protein was collected.

3.2 taking 5mL of Ni-NAT, cleaning the balance column by using a Binding buffer with the volume of 5 times of the column bed, and the flow rate is 5mL/min; the crude protein was incubated with post-equilibration column packing for 1h.

3.3, loading the incubated product on a column, and collecting and flowing out; washing the balance column with Binding buffer, washing the column with Washing buffer, and collecting the effluent; eluting with an Elution buffer, and collecting the effluent.

3.4, the crude protein and the impurity washing effluent are respectively treated, sampled and subjected to SDS-PAGE detection, and the result is shown in FIG. 4.

As shown in FIG. 4, lmidazole has a good effect of eluting the target protein, and increasing the concentration of lmidazole can increase the purity of the target protein, and when the concentration of lmidazole is 50mM, the target protein with higher purity can be obtained.

3.5 dialyzing the components 6 and 7 with better purity into 500mM Tris,300mM NaCl,0.1%SKL,2mM DTT,pH 8.0, concentrating by using PEG20000 after the dialysis is finished, filtering by using a 0.22 mu m filter membrane, sub-packaging by 1mL/tube, and preserving at-80 ℃; and SDS-PAGE detection is carried out on the purified protein, and the result is shown in FIG. 5.

As can be seen from FIG. 5, distinct bands appear at the corresponding positions of the theoretical molecular weight 48.29.+ -.5 kDa, confirming that the fusion protein components are stored.

4. Protein concentration determination

The protein concentration was measured by using a non-interfering protein quantitative kit Cat.No. C503071, the volume of the sample to be measured was 20. Mu.L, the measurement results are shown in Table 6 below, and BSA standard curves (see FIG. 6) were drawn by taking the average value of absorption values at the wavelength of A480 as the abscissa and the protein amount of BSA as the ordinate, to obtain BSA of the target protein.

TABLE 6 results of protein concentration test

Substituting 0.847 into the equation in FIG. 6 gave a protein amount y of 7.01382 and a measured sample volume of 20. Mu.L, and thus a target protein concentration of 0.35. Mu.g/. Mu.L was finally obtained.

5. Amino acid sequence with optimized gene sequence expression

According to the optimized gene sequence, the gene is translated into an amino acid sequence by using software Vector NTI or BioXM and Expasy, and the amino acid sequence is shown as SEQ ID NO. 2.

The foregoing descriptions of specific exemplary embodiments of the present invention are presented for purposes of illustration and description. It is not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application to thereby enable one skilled in the art to make and utilize the invention in various exemplary embodiments and with various modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims and their equivalents.

Claims (6)

1. A gene for encoding polygalacturonase, which is characterized in that the nucleotide sequence of the gene is shown as SEQ ID No. 1.

2. The polygalacturonase is an expression product of a gene for encoding the polygalacturonase, and is composed of 470 amino acids, has a molecular weight of 48285.4Da and an isoelectric point of 5.63, and the amino acid sequence of the polygalacturonase is shown as SEQ ID No. 2.

3. A recombinant expression plasmid obtained by recombination of the expression plasmid and the gene according to claim 1.

4. Use of the gene according to claim 1 for the preparation of an endopolygalacturonase or pectinase.

5. Use of polygalacturonase according to claim 2 for degrading plant cell walls.

6. Use of polygalacturonase according to claim 2 for degrading polygalacturonic acid or pectin.

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