CN109837283B - Preparation and application of citrus natural bacteriostatic protein CsLTP1 - Google Patents

Abstract

The invention discloses preparation and application of a citrus natural bacteriostatic protein CsLTP1, and particularly discloses a codon-optimized non-specific lipid transfer protein CsLTP1 gene in Rutaceae plants, which is characterized by comprising a sequence shown as SEQ ID NO. 2. According to the invention, the rCsLTP1 protein obtained by codon optimization of the bacteriostatic protein CsLTP1, construction of a prokaryotic expression vector, and recombinant expression, purification and identification of the rCsLTP1 has better thermal stability and acid-base stability, and has an obvious inhibiting effect on penicillium digitatum of pathogenic bacteria of the postharvest green mold of citrus under the living condition.

Description

Preparation and application of citrus natural bacteriostatic protein CsLTP1

Technical Field

The invention belongs to the field of prevention and control of postharvest green mildew of citrus, and particularly relates to preparation and application of a citrus natural bacteriostatic protein CsLTP1, which can be applied to prevention and control of postharvest green mildew of rutaceae plant fruits (such as citrus), namely a method for preparing the citrus natural bacteriostatic protein CsLTP1 by using genetic engineering and application of an extract prepared by using the method as a plant source bacteriostatic agent in prevention and control of postharvest disease green mildew of citrus.

Background

Citrus is the first fruit in the world, and during the period of harvesting, transportation and storage, the fruit is damaged due to improper measures, and the fruit is easy to be infected by various pathogenic bacteria to generate postharvest diseases, so that the fruit is rotten and goes bad, and huge economic loss is brought to the citrus industry [1]. Citrus green mold and blue mold caused by Penicillium Digitatum (Penicillium Digitatum) and Penicillium Italicum (Penicillium Italicum) are two major diseases after the harvest of citrus, resulting in a rot rate of 70% -80% [2-3], with the rot caused by Penicillium Digitatum being particularly severe [4]. The disease of the picked citrus is mainly characterized in that the diseased part of the citrus is softened and water-soaked, a white mildew (mycelium of pathogenic bacteria) is generated after 2 to 3 days, a green powdery mildew layer (fruiting body of pathogenic bacteria) appears in the center of the disease, the citrus has stuffy aroma when smelling, the citrus is quickly rotten, and the citrus pulp is bitter and can not be eaten. At present, the method for controlling postharvest diseases and storing and refreshing citrus mainly adopts chemical bactericides (such as benzimidazoles, imidazoles, imazalil, biguanide salts and the like) and physical storage technologies (such as low temperature, heat treatment and the like) to treat fruits [5]. Although the chemical bactericide has a certain control effect on postharvest diseases of citrus, the chemical bactericide has high toxicity and serious environmental pollution, and brings serious threat to human health, and the use of a plurality of chemical bactericides is limited in many countries. Moreover, the long-term use of the agents causes pathogenic bacteria to generate drug resistance, so that the postharvest disease control method is more complicated, and the fresh-keeping effect is influenced. Therefore, the finding of a plant natural active substance with the activity of inhibiting citrus pathogenic bacteria to control citrus postharvest diseases has great significance for the development of the citrus industry.

Antimicrobial peptides (AMPs) have broad-spectrum antibacterial activity, and high-efficiency antifungal, viral and protozoal activities; the thermal stability is good; the antibacterial agent has the characteristics of unique antibacterial mechanism, difficult generation of drug resistance and the like, is attracting attention of people, and is expected to become one of products for replacing antibiotics. The research finds that the antibacterial peptide is widely present in prokaryotes, plants and animals, and plays an important role in the natural defense system of organisms. Antibacterial peptides include antibacterial polypeptides and antifungal polypeptides [6]. At present, relatively few researches on plant-derived antibacterial peptides are made in China. Non-specific lipid transporter proteins (nsLTPs) have been reported to have a broad antifungal activity [7]. However, research and development application reports of directly adopting antibacterial peptides such as non-specific lipid transporters as plant-derived bacteriostats in the aspect of disease prevention and control after fruit and vegetable harvest are not found. Reference documents:

[1]Brown G E,Chambers M.Evaluation of biological products for the control of postharvest diseases of Florida citrus[C]//Proc.Fla.State Hort.Soc.1996,109:278-282.

[2]Demirci F.Effects of'Pseudomonas fluorescens'and'Candida famata'on Blue Mould of Citrus Caused by'Penicillium Italicum'[J].Australian Journal of Crop Science,2011,5(3):344.

[3] henfu, mandarin [ M ]. Beijing, agricultural Press of China, 1997.

[4]Macarisin D,Cohen L,Eick A,et al.Penicillium digitatum suppresses production of hydrogen peroxide in host tissue during infection of citrus fruit[J].Phytopathology,2007,97(11):1491-1500.

[5]Sayago J E,

R M,Kovacevich L N,et al.Antifungal activity of extracts of extremophile plants from the Argentine Puna to control citrus postharvest pathogens and green mold[J].Postharvest biology and technology,2012,67:19-24.

[6]de Souza

E,Sousa D A,Viana J C,et al.The use of versatile plant antimicrobial peptides in agribusiness and human health[J].Peptides,2014,55:65-78.

[7]Kirubakaran S I,Begum S M,Ulaganathan K,et al.Characterization of a new antifungal lipid transfer protein from wheat[J].Plant physiology and Biochemistry,2008,46(10):918-927.

Disclosure of Invention

Aiming at the defects or improvement requirements of the prior art, the invention aims to provide a preparation method and application of a citrus natural bacteriostatic protein CsLTP1, wherein the bacteriostatic protein CsLTP1 can be used as a plant source bacteriostatic agent for preventing and treating postharvest green mold of rutaceae plant fruits (such as citrus), the codon optimization of the bacteriostatic protein CsLTP1, the construction of a prokaryotic expression vector, the recombinant expression, purification and identification of the CsLTP1 are carried out, and the obtained rCsLTP1 protein has good thermal stability and acid-base stability, and has an obvious inhibiting effect on Penicillium digitatum (Penicillium digitatum) pathogenic bacteria of postharvest green mold of citrus under the living condition. Compared with the prior art, the plant-derived bacteriostatic protein CsLTP1 is a natural bacteriostatic agent capable of being produced in a large scale, can obviously reduce the occurrence of the postharvest green mold of the citrus fruits, reduces the harm caused by chemical bactericides and has a good application prospect in the field of preservative and fresh-keeping of the citrus fruits.

To achieve the above objects, according to one aspect of the present invention, there is provided a codon-optimized non-specific lipid transfer protein CsLTP1 gene of a rutaceae plant, comprising a sequence shown in SEQ ID No. 2.

According to still another aspect of the present invention, the present invention provides a primer for constructing a prokaryotic expression vector of a CsLTP1 gene, comprising a forward primer and a reverse primer, wherein,

the forward primer comprises a sequence shown as SEQ ID NO. 3;

the reverse primer comprises a sequence shown as SEQ ID NO. 4.

According to still another aspect of the present invention, there is provided a recombinant protein of rCsLTP1, characterized in that the protein is:

i. 1, a protein consisting of an amino acid sequence shown in SEQ ID NO;

a protein having from 80% to 100% homology with the amino acid sequence defined in SEQ ID No.1, which encodes an amino acid sequence of a protein having the same function;

or iii, a derivative protein having equivalent activity by adding, deleting or substituting one or more amino acids in the amino acid sequence shown in SEQ ID No. 1.

According to still another aspect of the present invention, there is provided a method for inducible expression of a recombinant protein rCsLTP1, comprising the steps of: adding IPTG (isopropyl-beta-D-thiogalactoside) with the final concentration of 0.01-1 mmol/L into the recombinant strain in the logarithmic growth phase, and inducing at 12-37 ℃ for 4-24 h to generate rCsLTP1 recombinant protein; preferably, the recombinant strain is added with IPTG (isopropyl thiogalactoside) with the final concentration of 0.1mmol/L in the logarithmic growth phase and induced at the temperature of 20 ℃ for 16h to generate rCsLTP1 recombinant protein;

wherein the recombinant bacteria are escherichia coli transferred into a CsLTP1 gene prokaryotic expression vector; the CsLTP1 gene prokaryotic expression vector is specifically obtained by taking the non-specific lipid transfer protein CsLTP1 gene in the rutaceae plant optimized by the codon as claimed in claim 1 as a template and utilizing the primer for constructing the CsLTP1 gene prokaryotic expression vector as claimed in claim 2 to construct through PCR reaction.

As a further preferred aspect of the invention, the generated rCsLTP1 recombinant protein is further subjected to purification treatment, wherein the purification treatment specifically comprises the steps of collecting the induced recombinant bacteria, then performing PBS washing, centrifugation, resuspension in an ice-precooled lysis buffer solution and ultrasonic bacteria breaking treatment on the recombinant bacteria, then centrifuging to collect a supernatant, then filtering the supernatant with a filter membrane, purifying and eluting the obtained filtrate with an HIS nickel column purification system, and then collecting an eluent to obtain the rCsLTP1 recombinant protein.

According to another aspect of the invention, the invention provides a method for identifying rCsLTP1 recombinant protein, which is characterized in that a protein to be identified and CsLTP1 protein are identified by MALDI-TOF/TOF mass spectrum, if 4 effective specific peptide sections which can be matched with the CsLTP1 protein are identified from the protein to be identified, the protein to be identified belongs to the rCsLTP1 recombinant protein; otherwise, the protein to be identified does not belong to the rCsLTP1 recombinant protein;

wherein the CsLTP1 protein has a sequence shown as SEQ ID NO. 1.

According to still another aspect of the present invention, there is provided a method for detecting the thermal stability or acid-base stability of the above rCsLTP1 recombinant protein, characterized in that,

placing the rCsLTP1 recombinant protein at the temperature of not more than 120 ℃ for 0-15 min, preferably placing the rCsLTP1 recombinant protein at the temperature of-80-120 ℃ for 0-15 min, and detecting the thermal stability of the rCsLTP1 recombinant protein by observing whether the protein is degraded or not, preferably, the protein is not degraded;

or, the rCsLTP1 recombinant protein is placed under the pH condition of 4-9, and whether the protein is degraded or not is observed to detect the acid-base stability of the rCsLTP1 recombinant protein, and preferably, the protein is not degraded.

According to another aspect of the invention, the invention provides a drug for preventing and treating postharvest green mold of rutaceae plant fruits, which is characterized in that the drug contains the rCsLTP1 recombinant protein; preferably, the medicament is in a composition containing 10 ³ -10 ⁸ CFU/ml suspension of Penicillium digitatum spores is mixed with rCsLTP1 recombinant protein; preferably, the penicillium digitatum spores in the suspension satisfy 10 ⁶ CFU/ml; the addition amount of the rCsLTP1 recombinant protein in the suspension is 0.1-1mg/ml, and preferably 500 mug/ml.

According to still another aspect of the present invention, there is provided a method for detecting the inhibitory effect of the recombinant protein rCsLTP1 against penicillium digitatum, a pathogen of post-harvest chlorophyceae of Rutaceae plants, comprising inoculating the rCsLTP1 recombinant protein as defined in claim 3, mixed at 0.1-1mg/ml, containing 10 mg/ml, with the recombinant protein rCsLTP1 ³ -10 ⁸ A fruit of a Rutaceae plant, inoculated with an unmixed rCsLTP1 recombinant protein according to claim 3, but containing 10, of a suspension of spores of Penicillium digitatum in CFU/ml ³ -10 ⁸ Comparison of decay rates of two Rutaceae plant fruits of CFU/ml Penicillium digitatum spore suspension, and detection of antimicrobial peptide rCsLTP1 recombinant protein for post-harvest chlorophycemia pathogen of Rutaceae plant fruitsInhibitory effects of penicillium digitatum;

preferably, the inoculum is mixed with 0.1-1mg/ml rCsLTP1 recombinant protein and contains 10 ³ -10 ⁸ The decay rate of Rutaceae plant fruits of CFU/ml of Penicillium digitatum spore suspension is reduced;

preferably, 0.1-1mg/ml of the rCsLTP1 recombinant protein is preferably 500 μ g/ml; the penicillium digitatum spores in the suspension all meet 10 ⁶ CFU/ml；

Preferably, the rutaceae plant fruit is citrus.

According to another aspect of the present invention, the present invention provides the non-specific lipid transfer protein CsLTP1 gene in the codon-optimized rutaceae plant, the primer for constructing the prokaryotic expression vector of CsLTP1 gene, the recombinant protein rCsLTP1 as a plant-derived bacteriostatic agent, the method for inducing expression of recombinant protein rCsLTP1, the method for identifying recombinant protein rCsLTP1, the method for detecting the thermal stability or acid-base stability of recombinant protein rCsLTP1, the drug for preventing and treating the postharvest chlorophyllasis of rutaceae plant fruits, or the application of the method for detecting the inhibitory effect of the antimicrobial peptide rCsLTP1 recombinant protein on the postharvest chlorophyllasis pathogenic bacteria penicillium digitatum of rutaceae plant fruits.

Compared with the prior art, the method for preventing and treating the postharvest penicillium notatum of the rutaceae plant fruits is provided through the codon-optimized non-specific lipid transfer protein CsLTP1 gene in the rutaceae plant, a primer for constructing a prokaryotic expression vector of the CsLTP1 gene, rCsLTP1 recombinant protein, a method for inducing and expressing the rCsLTP1 recombinant protein, a method for identifying the rCsLTP1 recombinant protein, a method for detecting the thermal stability or the acid-base stability of the rCsLTP1 recombinant protein, a medicine for preventing and treating the postharvest penicillium notatum of the rutaceae plant fruits or a method for detecting the inhibiting effect of the antibacterial peptide rCsLTP1 recombinant protein on the postharvest penicillium notatum of the rutaceae plant fruits, and the like; in addition, a non-specific lipid transfer protein CsLTP1 gene in the codon-optimized Rutaceae plant, a primer for constructing a prokaryotic expression vector of the CsLTP1 gene, rCsLTP1 recombinant protein, a method for inducing and expressing the rCsLTP1 recombinant protein, a method for identifying the rCsLTP1 recombinant protein and the like can be applied to preparation of a medicament or preservative for preventing and treating postharvest green mold of Rutaceae plant fruits; the rCsLTP1 recombinant protein can be used as antibacterial peptide and a plant source bacteriostatic agent for preventing and treating postharvest green mold of citrus. The plant-derived bacteriostatic protein CsLTP1 obtained by the invention is a natural bacteriostatic agent capable of being produced in a large scale, can obviously reduce the occurrence of green mold of the picked citrus fruits and harm caused by chemical bactericides, and has a good application prospect in the field of preservative and fresh-keeping of the citrus fruits.

The invention particularly optimizes the process conditions and parameters of the steps of induction expression and purification of rCsLTP1 recombinant protein, adds the recombinant bacteria with the final concentration of 0.01-1 mmol/L IPTG in the logarithmic growth phase, and induces the recombinant bacteria for 4-24 h at 12-37 ℃ (particularly, adds the recombinant bacteria with the final concentration of 0.1mmol/L IPTG in the logarithmic growth phase and induces the recombinant bacteria for 16h at 20 ℃), thus inducing and generating the rCsLTP1 recombinant protein; and for the generated rCsLTP1 recombinant protein, the invention also utilizes a purification process to process, namely, firstly, the induced recombinant bacteria are collected, then PBS washing, centrifugation, resuspension in an ice-precooled lysis buffer solution and ultrasonic bacteria breaking treatment are carried out on the recombinant bacteria, then, the supernatant is centrifugally collected, then, the supernatant is filtered by a filter membrane, the obtained filtrate is purified and eluted by an HIS nickel column purification system, and then, the eluent is collected to obtain the rCsLTP1 recombinant protein. The invention controls the whole process flow adopted by the steps of the induced expression and the purification of the recombinant protein and the specific process conditions and parameters (such as the types and the proportion of reagents, the processing temperature and time, the centrifugal processing time and the centrifugal force and the like) related to each step, so that the rCsLTP1 recombinant protein has high yield and high purity.

The nucleotide sequence comprises a non-specific lipid transfer protein CsLTP1 gene in a rutaceae plant with optimized codon of a sequence shown as SEQ ID NO. 2, namely the nucleotide sequence with optimized codon of the CsLTP1 gene, and is particularly suitable for escherichia coli expression; when the rCsLTP1 protein is induced and expressed, escherichia coli transferred with a CsLTP1 gene prokaryotic expression vector is used as a recombinant bacterium (the CsLTP1 gene prokaryotic expression vector is specifically obtained by taking the non-specific lipid transfer protein CsLTP1 gene in the rutaceae plant optimized by the codon as a template and utilizing the primer for constructing the CsLTP1 gene prokaryotic expression vector to construct through PCR reaction, wherein the introduction is based on the non-specific lipid transfer protein CsLTP1 gene in the rutaceae plant such as citrus), so that the rCsLTP1 can be expressed in a large amount and in a soluble way; the bacteria after induction expression can be purified to obtain recombinant protein, and the purity of the purified protein is high (the molecular weight of the protein is about 27kDa, for example, the protein can obtain 4 specific effective peptide sections through MALDI-TOF/TOF mass spectrum identification).

The rCsLTP1 recombinant protein of the present invention can be used as an antimicrobial peptide, for example, the rCsLTP1 protein can be mixed with a commonly used suspension of Penicillium digitatum spores (e.g., containing 10. Sup. F) ⁶ CFU/ml suspension of penicillium digitatum spores) so that the concentration of the antimicrobial peptide rCsLTP1 in the final suspension is, for example, 500 μ g/ml, and then inoculating, for example, citrus fruits, so that the rate of decay of the citrus fruits can be reduced; the present invention also allows the detection of the inhibitory effect of the antimicrobial peptide rCsLTP1 on penicillium digitatum, a pathogen such as the postharvest phytophthora parasitica of citrus under in vivo conditions (by comparing it with a rutaceae plant fruit inoculated with only a suspension of penicillium digitatum spores, the decay rate of citrus fruit is significantly reduced by using a drug containing rCsLTP1 recombinant protein, in particular by inoculating a mixed suspension of a specific rCsLTP1 recombinant protein, with the spore content of penicillium digitatum). In addition, the rCsLTP1 protein has good thermal stability, can endure high-temperature treatment at 120 ℃ and is not degraded; the acid-base stability is good, and the pH value can be tolerated by the condition treatment of 4-9.

In conclusion, the method for preparing the citrus natural bacteriostatic protein CsLTP1 and the application of the extract prepared by the method as a plant source bacteriostatic agent in prevention and treatment of citrus postharvest disease green mold are provided. The method comprises the steps of constructing a prokaryotic expression system of an antibacterial peptide allergen CsLTP1 by optimizing a CsLTP1 gene prokaryotic expression sequence and gene synthesis, optimizing a method for expressing and purifying rCsLTP1 protein, identifying the recombinant protein by mass spectrometry, detecting the thermal stability and the acid-base stability of the rCsLTP1 protein, and detecting the inhibiting effect of the antibacterial peptide rCsLTP1 on penicillium digitatum of pathogenic bacteria of citrus postharvest green mold under a living condition. Compared with the prior art, the antibacterial peptide rCsLTP1 provided by the invention has an obvious inhibition effect on penicillium digitatum of pathogenic bacteria of post-harvest green mold of citrus, and the plant-derived antibacterial protein CsLTP1 adopted by the invention is a plant-derived antibacterial agent capable of being produced in a large scale, can reduce harm caused by chemical bactericides, and has important significance on fruit preservation and fresh-keeping of citrus and food safety.

Drawings

FIG. 1 is a technical flow chart of antigen expression, protein purification, thermal stability and acid-base stability detection of citrus antimicrobial protein CsLTP1 and application thereof.

FIG. 2 is a diagram of prokaryotic expression vector construction. (a) a schematic diagram of the constructed prokaryotic expression vector. (b), lane M: DNA Ladder; lane 1: the constructed vector pET-32a (+) -CsLTP1, namely pET-32a (+)/Trx-His-CsLTP 1; lane 2, pET-32a (+)/Trx-His-CsLTP 1 plasmid double cut (EcoRI, xhoI).

FIG. 3 shows the expression and purification of Trx-His-CsLTP1 protein. M, protein molecular mass standard; 1, the recombinant bacterium is not induced; 2, inducing the recombinant bacteria by IPTG; 3, cracking the supernatant of the recombinant bacteria after IPTG induction; 4, purified Trx-His-CsLTP1 protein.

FIG. 4 shows the identification of CsLTP1 protein by MALDI-TOF/TOF. The effectively identified peptide fragments are shown in bold.

FIG. 5 shows the stability assay of rCsLTP1 protein. (a) Detecting the thermal stability of the rCsLTP1 protein, and (b) detecting the acid-base stability of the rCsLTP1 protein.

FIG. 6 shows the onset of citrus fruit on day 3 after rCsLTP1 protein treatment inoculated with Penicillium digitatum.

FIG. 7 is a statistical analysis of the lesion area of citrus fruit on day 3 after rCsLTP1 protein treatment inoculated with Penicillium digitatum.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and do not limit the invention. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

Example 1: retrieval, codon optimization and whole-gene synthesis of citrus natural bacteriostatic protein CsLTP1 gene

1.1 Citrus Natural bacteriostatic protein CsLTP1 Gene retrieval

The amino acid sequence of non-specific lipotransferase protein (GenBank XP-420069565.1) in the sweet orange genome (http:// Citrus. Hzau. Edu. Cn/orange /) was searched for the corresponding Citrus non-specific lipotransferase protein by sequence homology and the gene sequences were obtained as shown in Table 1 by published articles (e.g., jinlong Wu, lin Chen, dingbo Lin, zaocheng, and Xiuxin Deng. Development and Application of a multiple Real-Time PCR Assay as an Indicator of Potential allergy in Citrus fruit juice, journal of agricultural and food chemistry,2016,64 (47): 9089-9098.).

TABLE 1 Citrus non-specific lipid transfer protein CsLTP1 Gene information

1.2 allergen Gene CsLTP1 sequence codon optimization and Total Gene Synthesis

The usage frequency of synonymous codons is different between species and species, and there is codon bias. Under the condition that the usage frequency of the synonymous codon of the exogenous gene is matched with that of an expression host, the expression level of the exogenous gene can be obviously improved. The allergen gene CsLTP1 of the present invention can be codon optimized using JCat tool (http:// www.jcat. De /), and the optimized amino acid and nucleotide sequences are shown in Table 2. The amino acid sequence encoded by the CsLTP1 gene after codon optimization is shown in Table 2. The whole gene synthesis is entrusted to Wuhan Kingkurui bioengineering GmbH to complete.

TABLE 2 codon-optimized nucleotide sequences

Note: csLTP1 ^＊ Is an amino acid sequence, csLTP1 is an original nucleotide sequence, csLTP1 ^# For the optimized nucleotide sequence

Example 2 prokaryotic expression vector construction

2.1 primer design

The primers of the present invention were designed using Primer 5.0 software, and the nucleotide sequences of the primers are shown in Table 3. All primers were synthesized by Biotechnology Ltd of the New Engineers of Ongkogaku, beijing.

TABLE 3 primers

Note: csLTP1-F and CsLTP1-R in the table are forward and reverse primers. The bold text 15bp indicates the linker used for vector construction. The 5 'EcoRI and 3' XhoI cleavage sites are indicated in italics.

2.2 vector construction

The invention uses a method of a homologous recombination One-Step Cloning Kit (Clonexpress II One Step Cloning Kit, provided by Nanjing Novowed Biotechnology Co., ltd.) to construct a prokaryotic expression vector pET-32a (+)/Trx-His-CsLTP 1. Specifically, a PCR product is obtained by PCR reaction using a synthesized CsLTP1 gene as a template, csLTP1-F and CsLTP1-R as primers. PCR systems are shown in Table 4, and procedures are shown in Table 5, PCR Master Mix (2X) was purchased from Thermo Fisher Scientific (cat # K0171). Meanwhile, the pET-32a (+) vector was digested with EcoRI and XhoI restriction enzymes. The endonuclease system was referenced to Invitrogen. The PCR product was mixed with the vector cleavage product according to the method of the homologous recombination one-step cloning kit, and the mixture was treated at 37 ℃ for 30 minutes, immediately followed by incubation on ice for 5 minutes. According to the method of Beijing Quanji Biotechnology Limited, the obtained mixture is transformed into E.coli DH5 alpha, and under appropriate culture conditions, positive recombinants are screened and sent to Beijing Optimalaceae New Biotechnology Limited for DNA sequence determination. Taking the clone with the complete correct sequence, using

Recombinant plasmids were extracted with AxyPrep plasmid DNA minikit and verified by EcoRI and XhoI double digestion (FIG. 2). The enzyme cutting fragment is 282bp through agarose electrophoresis, so the invention successfully constructs the prokaryotic expression vector pET-32a (+)/Trx-His-CsLTP 1.

TABLE 4 PCR System

TABLE 5 PCR reaction conditions

Example 3 recombinant CsLTP1 protein expression, protein purification, mass Spectrometry identification

3.1 Inducible expression of rCsLTP1 protein

Coli BL21 (DE 3) competent cells were transformed with the correctly sequenced positive plasmid, inoculated into 5mL LB medium containing 100. Mu.g/mL ampicillin, and cultured overnight with shaking at 37 ℃. Transferring the culture solution into 50mL of the culture solution at a ratio of 1: 100 the next day, culturing at 37 deg.C to logarithmic phase (A600 = 0.4-0.6), adding IPTG with final concentration of 0.1mmol/L, inducing at 20 deg.C for 16h, centrifuging at 10000g for 5min, and collecting bacteria; washed with PBS, centrifuged, resuspended in ice-cold lysis buffer (50 mmol/L NaH) ₂ PO ₄ 300mmol/L NaCl, 0.1g/L lysozyme and 1mmol/L PMSF, pH 8.0), ultrasonic bacteria breaking, 10000g centrifugation for 10min, collecting supernatant, and performing SDS-PAGE analysis (figure 3). According to the result analysis of the gel chart, the rCsLTP1 protein is expressed in the supernatant and is expressed in a soluble way.

3.2 rCsLTP1 protein purification

And (3) centrifuging 1L of bacteria liquid subjected to induced expression at 6000r/min to obtain thalli, adding 40mL of lysis buffer solution to fully resuspend the thalli, adding 1mmol/L protease inhibitor phenylmethylsulfonyl fluoride (PMSF) at the final concentration, and then carrying out ultrasonic lysis under the ice bath condition. The lysate is centrifuged at 12000r/min at 4 ℃ for 50min, and the supernatant is collected and filtered through a 0.22 μm filter for use. The HIS-labeled nickel-ion protein purification column was equilibrated with 5 column volumes of lysis buffer (50mM Tris,100mM NaCl,5% glycerol), added with lysis buffer, incubated with shaking at 4 ℃ for 4h, and the affinity medium was able to specifically adsorb the target protein, followed by elution of the target protein using imidazole eluent (50mM Tris,100mM NaCl and 5% glycerol,100-200mM imidazole). The eluate was collected and analyzed by SDS-PAGE. The fourth lane in fig. 3 is the corresponding purified rCsLTP1 protein. The detection result shows that 1L of bacteria can produce 20.4mg of recombinant protein. The obtained protein was subjected to buffer exchange using an ultrafiltration tube, and the original Tris buffer was replaced with PBS phosphate buffered saline at pH 7.2.

3.3 rCsLTP1 protein mass spectrometric identification

The gel strip corresponding to the rCsLTP1 protein in the fourth lane of FIG. 3 was removed, and the gel strip was sent to Nippon Hippocampus Biotech Limited for mass spectrometry, with a molecular weight of about 27 kDa. And carrying out protein identification on the rCsLTP1 protein by using a MALDI-TOF/TOF protein identification technology. As shown in FIG. 4, four specific peptides were identified.

Example 4 detection of the thermal stability and acid-base stability of the bacteriostatic protein CsLTP1

4.1 rCsLTP1 protein thermal stability assay

The rCsLTP1 protein is dissolved in PBS phosphate buffer solution, treated for 15min at 40 ℃, 50 ℃, 60 ℃, 70 ℃, 80 ℃, 90 ℃,100 ℃ and 120 ℃, and analyzed by SDS-PAGE to determine the thermal stability. As shown in FIG. 5a, the rCsLTP1 protein has good thermal stability and can endure high temperature treatment at 120 ℃ without degradation.

4.2 rCsLTP1 protein acid-base stability detection

Dissolving rCsLTP1 protein in PBS phosphate buffer solution, and adjusting pH to 3, 4, 5, 6 and 7 with 1mol/L HCl; and adjusting the pH value of the rCsLTP1 protein solution to 8, 9 and 10 by using 1mol/L NaOH, and determining the acid-base stability of the rCsLTP1 protein solution through SDS-PAGE detection analysis. As shown in FIG. 5b, rCsLTP1 protein has good acid-base stability and can tolerate the condition of PH 4-9.

Example 5 inhibitory Effect of the antimicrobial peptide rCsLTP1 on the action of Penicillium digitatum which is a pathogen of post-harvest Chloromyces fragilis in Citrus

5.1 sample preparation

Two citrus varieties, namely red summer orange and fused kumquat, are used in the invention. The red summer orange is picked at the national orange breeding center of the university of agriculture in Huazhong, and the Jingan is purchased from the market. The penicillium digitatum is separated from a biotechnology laboratory (the laboratory) and stored after being picked by gardening and forestry academy of Huazhong agriculture university.

5.2 Effect of CsLTP1 protein treatment on diameter of lesion of Penicillium digitatum inoculated on citrus fruits

First, the CsLTP1 protein used in this experiment was the product prepared in example 3. First, PBS solutions containing penicillium digitatum spores to which CsLTP1 protein was added and to which CsLTP1 protein was not added were prepared, wherein the control was CsLTP1 protein-not added. Then, the resultant was inoculated onto red summer orange and jingle cumquat fruits (8 to 10 fruits), and the occurrence of the test material was observed to detect an inhibitory activity against penicillium digitatum containing CsLTP1 protein. The method comprises the following specific steps: (1) selecting spores of Penicillium digitatum in sterile PBS solution to obtain a solution with a concentration of 10 ⁶ CFU/ml spore suspension, preparing a bacterial solution added with CsLTP1 protein and without the CsLTP1 protein by using the spore suspension, wherein the concentration of the added CsLTP1 protein is 500 mu g/ml; (2) treating fruits, cleaning the surfaces of the fruits by using distilled water, soaking the fruits in 2% sodium hypochlorite for 2min, cleaning the fruits twice by using the distilled water, and airing at room temperature; (3) pricking holes on the citrus peel with sterilized inoculating needles (10 needles bundled together), small holes with diameter of 2.5mm and depth of 1.5mm, and two holes on one fruit; (4) inoculating 20 μ l of bacteria liquid to each hole by using a liquid transfer gun, transferring the fruits to a preservation box with wet tissue after the bacteria liquid is completely absorbed, culturing in a constant-temperature incubator at 25 ℃, and observing the morbidity of the fruits in the culturing process.

The lesion diameter on day 3 was measured, and the size of the lesion was calculated as the average width (cm) of the lesion spread in two directions perpendicular to the direction. The susceptibility to citrus storage is shown in fig. 5, and the CsLTP 1-treated red summer orange and Pongan cuneata fruits have smaller lesion area significance than the control group 3 days after the penicillium digitatum inoculation (fig. 6). The data show that the CsLTP1 protein has obvious inhibition effect on the penicillium notatum of the pathogenic bacterium of citrus green mold when being treated as a bacteriostatic agent.

In conclusion, the invention provides a method for preparing a natural bacteriostatic protein CsLTP1 of citrus and application of an extract prepared by the method as a plant-derived bacteriostatic agent in prevention and treatment of citrus postharvest diseases green mold, and fills a blank of plant-derived antimicrobial peptide in prevention and treatment of citrus postharvest diseases. The invention not only searches the non-specific lipocalin CsLTP1 gene in the sweet orange genome, but also optimizes codons in order to improve the expression level of exogenous genes, synthesizes nucleotide sequences suitable for escherichia coli expression, is particularly suitable for rutaceae plants, establishes a prokaryotic expression system of the CsLTP1 protein, and can rapidly obtain the rCsLTP1 protein in a large amount. Meanwhile, the rCsLTP1 protein is found to have better thermal stability and acid-base stability. In addition, under the condition of living bodies, the antibacterial peptide rCsLTP1 provided by the invention has an obvious inhibition effect on penicillium digitatum which is a pathogenic bacterium of postharvest green mold of citrus. The plant-derived bacteriostatic protein CsLTP1 provided by the invention is a plant-derived bacteriostatic agent which can be produced in a large scale, can reduce harm caused by a chemical bactericide, and has important significance for the preservation and freshness preservation of citrus fruits and food safety.

The pET-32a (+) carrier adopted by the invention is provided with an HIS label, is a commonly used affinity label and does not influence the function and immunogenicity of target protein. In order to improve the purification efficiency, the HIS tag of the vector is fully utilized, the termination codon of the target gene is deleted when a downstream primer is designed, and the T7 terminator of the vector is selected. The recombinant protein is produced by using the escherichia coli, and the method has the advantages of short period, easiness in purification and the like. The invention constructs a prokaryotic expression vector pET-32a (+) -CsLTP1, successfully induces and expresses protein with molecular weight of 27kDa after E.coli BL21 (DE 3) bacteria is transformed, and the protein mainly exists in a soluble form. In the protein purification process, the HIS-labeled nickel ion protein purification column is used for purifying the protein. The HIS nickel column purification system utilizes the principle that nickel chloride in a Ni column can be combined with HIs (histone) labeled protein and the HIS nickel column is purified by imidazole elution. The purified CsLTP1 protein has high purity through SDS-PAGE identification.

The rutaceae fruits in the invention comprise citrus plants (such as oranges, mandarins, pomelos, oranges, lemons and the like) and other common citrus plants (such as trifoliate orange, kumquat and the like). The plant source bacteriostatic agent CsLTP1 provided by the invention is suitable for almost all citrus varieties to prevent and control the postharvest green mold diseases of citrus.

It will be understood by those skilled in the art that the foregoing is only a preferred embodiment of the present invention, and is not intended to limit the invention, and that any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the scope of the present invention.

Sequence listing

<110> university of agriculture in Huazhong

<120> preparation and application of citrus natural bacteriostatic protein CsLTP1

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Claims (4)

1. An application of a non-specific lipid transfer protein CsLTP1 gene in a codon-optimized Rutaceae plant in preventing and treating postharvest green mold of Rutaceae plant fruits is characterized in that the nucleotide sequence of the gene is shown as SEQ ID NO. 2.

2. The application of the method for inducing expression of rCsLTP1 recombinant protein in prevention and control of postharvest green mold of rutaceae plant fruits is characterized by comprising the following steps: adding IPTG (isopropyl-beta-D-thiogalactoside) with the final concentration of 0.01-1 mmol/L into the recombinant strain in the logarithmic growth phase, and inducing at 12-37 ℃ for 4-24 h to generate rCsLTP1 recombinant protein;

wherein the recombinant bacterium is escherichia coli transferred into a CsLTP1 gene prokaryotic expression vector; the CsLTP1 gene prokaryotic expression vector is specifically constructed by taking the non-specific lipid transfer protein CsLTP1 gene in the rutaceae plant optimized by the codon as claimed in claim 1 as a template through PCR reaction.

3. The application of the method for inducing and expressing the rCsLTP1 recombinant protein in the prevention and treatment of postharvest green mold of Rutaceae plant fruits as claimed in claim 2, wherein the recombinant strain is added with IPTG (0.1 mmol/L) at the final concentration in the logarithmic growth phase, and the rCsLTP1 recombinant protein is induced and generated after the induction at 20 ℃ for 16 h.

4. The application of the method for inducible expression of the rCsLTP1 recombinant protein in prevention and treatment of postharvest green mildew of Rutaceae plant fruits as claimed in claim 2, wherein the generated rCsLTP1 recombinant protein is further subjected to purification treatment, wherein the purification treatment specifically comprises the steps of collecting the induced recombinant bacteria, then washing the recombinant bacteria with PBS, centrifuging, resuspending in an ice-precooled lysis buffer solution, ultrasonically disrupting the bacteria, then centrifuging to collect a supernatant, then filtering the supernatant with a filter membrane, purifying and eluting the obtained filtrate with an HIS nickel column purification system, and collecting an eluent to obtain the rCsLTP1 recombinant protein.

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