Use of glutathione s-transferase in preparation of medicine for preventing and treating postharvest diseases of citrus fruits

Abstract

The application discloses application of glutathione S-transferase (GST) in preparation of a medicine for preventing and treating postharvest diseases of citrus fruits. Research results show that the GST protein can induce fruit disease resistance and effectively delay the infection process of postharvest green mold of citrus fruits. The application widens the application range of the GST protein in the field of fruit and vegetable preservation, and provides a new solution for prevention and treatment of postharvest diseases of citrus fruits.

Description

Technical Field

[0001] The present invention relates to the application of glutathione S-transferase (GST) in the field of fruit and vegetable preservation. Background Art

[0002] Citrus is a plant of the genus Citrus in the subfamily Citrus of the Rutaceae family and is one of the world's important economic fruit crops. As of 2021, global citrus production reached 158.5 million tons, with China ranking first in the world with 44.6 million tons, accounting for 28% of the global total production. For a long time, the main commercial channel for citrus fruits has been fresh sales. However, fresh citrus fruits are extremely susceptible to infection by various pathogenic fungi during post-harvest storage and transportation, causing serious industrial losses. Among them, citrus green mold, penicillium and sour rot caused by infection with Penicillium digitatum, Penicillium italicum and Geotrichum citri-aurantii are the main infectious diseases of citrus fruits during storage. At present, the industry mainly uses chemical fungicides such as prochloraz and carbendazim to control post-harvest diseases of fruits, but there are problems such as chemical fungicide residues and microbial resistance.

[0003] Proteins can induce and enhance the disease resistance of plants and fruits and vegetables. This induced disease resistance is broad-spectrum, stable, and long-lasting. They are also environmentally friendly and do not contribute to pathogen resistance, thus meeting the requirements of green agricultural development. Therefore, the research and development of protein-based compounds for the treatment of postharvest diseases of fruits and vegetables will lead the way in the development of biopesticides and hold broad prospects. Summary of the Invention

[0004] The purpose of the present invention is to provide a protein substance for use in a drug for preventing and treating post-harvest diseases of citrus fruits, so as to broaden the application scope of the protein substance in the field of fruit and vegetable preservation, and at the same time provide a new solution for the prevention and treatment of post-harvest diseases of citrus fruits.

[0005] After research, the present invention provides the following technical solutions:

[0006] Application of glutathione S-transferase in the preparation of drugs for preventing and treating postharvest diseases of citrus fruits.

[0007] Preferably, the post-harvest disease of citrus fruit is post-harvest green mold of citrus fruit.

[0008] Preferably, the amino acid sequence of the glutathione S-transferase is shown in SEQ ID No. 1. Its relative molecular mass is about 26 kDa and it is derived from Schistosoma japonicum.

[0009] The glutathione S-transferase can be obtained by heterologous expression and the like, and can be either a crude protein or a purified protein.

[0010] Preferably, the glutathione S-transferase is a crude GST protein, which is obtained by the following method: transforming the pGEX-6P-1 plasmid containing the GST encoding gene into Escherichia coli to obtain a GST protein-expressing engineered bacterium, inoculating it into LB liquid culture medium containing ampicillin, adding IPTG to induce it to express GST protein, collecting the bacteria, resuspending, lysing the cells, and centrifuging to obtain the supernatant to obtain a crude GST protein solution.

[0011] Preferably, the glutathione S-transferase is a GST purified protein, which is obtained by the following method: the pGEX-6P-1 plasmid containing the GST encoding gene is transformed into Escherichia coli to obtain a GST protein expression engineered bacterium, inoculated into LB liquid culture medium containing ampicillin, and induced to express GST protein by adding IPTG. The bacteria are collected, resuspended, and the cells are lysed. The supernatant is obtained by centrifugation and filtered with a microporous filter membrane. The filtrate is passed through a GST affinity chromatography column, eluted with a reduced glutathione solution, and the eluate is filtered with a 3K ultrafiltration tube to obtain a GST purified protein solution.

[0012] The pGEX-6P-1 plasmid is a commercially available E. coli expression vector commonly used in the art. It contains a GST-encoding gene with a nucleotide sequence as shown in SEQ ID No. 2, which encodes a glutathione S-transferase (GST) protein with an amino acid sequence as shown in SEQ ID No. 1. The crude GST protein and purified GST protein preparation methods of the present invention are simple and high in yield.

[0013] The present invention has the beneficial effect of using glutathione S-transferase in the preparation of a drug for preventing and treating postharvest diseases of citrus fruit. Research results show that glutathione S-transferase can induce disease resistance in fruit and effectively delay the infection of postharvest green mold in citrus fruit. This invention broadens the application scope of glutathione S-transferase in the field of fruit and vegetable preservation and provides a new solution for preventing and treating postharvest diseases of citrus fruit. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 is the SDS-PAGE electrophoresis diagram of GST protein; Maker is the protein molecular weight standard, supernatant is the centrifugal supernatant after bacterial cell disruption, flow-through is the effluent after the filtrate passes through the glutathione S-transferase affinity column, wash is the washing solution of the column after the filtrate passes through the glutathione S-transferase affinity column with PBS, and elutions 1 to 5 are eluents, respectively.

[0015] Figure 2Figure 3 is the control effect of GST crude protein on postharvest green mold disease in citrus fruits; A is the incidence rate, B is the lesion diameter; C is the symptom of green mold disease in citrus fruits after inoculation with Penicillium digitatum and storage at 25℃ for 7 days.

[0016] Figure 3 Figure 3 is the control effect of GST purified protein on postharvest green mold disease in citrus fruits; A is the incidence rate, B is the lesion diameter; C is the symptom of green mold disease in citrus fruits inoculated with Penicillium digitatum and stored at 25°C for 6 days. DETAILED DESCRIPTION

[0017] In order to make the objectives, technical solutions and advantages of the present invention more clear, preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0018] 1. Analysis of Glutathione S-Transferase Protein Characteristics

[0019] The molecular weight, amino acid composition, isoelectric point, instability coefficient and hydrophilicity of the glutathione S-transferase from Schistosoma japonicum, as shown in SEQ ID No. 1, were predicted using the online software Sequence Manipulation Suite.

[0020] The results showed that the molecular weight of the glutathione S-transferase was 25.498 kDa; the number of amino acid residues was 218, of which isoleucine (Leu, 12.4%), lysine (Lys, 9.6%), aspartic acid (Asp, 7.8%), glutamic acid (Glu, 7.3%), glycine (Gly, 6.4%), tyrosine (Tyr, 6.4%), alanine (Ile, 6.0%), and proline (Pro, 6.0%) were the amino acids that accounted for a relatively large proportion in the protein; its theoretical isoelectric point was 6.09, indicating an acidic protein; the hydrophilicity analysis was -0.359, indicating a hydrophilic protein; and the instability coefficient was 36.81 (less than 50), indicating a stable protein.

[0021] 2. Construction of GST protein expression engineering bacteria

[0022] Take the pGEX-6P-1 plasmid (product number: S201150B, Shanghai Dibai Biotechnology Co., Ltd.) and add it to Escherichia coli TSSeta (DE3) competent cells (product number: TSC-E04, Beijing Qingke Biotechnology Co., Ltd.), gently flick to mix, let it stand on ice for 30 minutes, water bath at 42°C for 45 seconds, and on ice for 2 minutes. Add 600 μL of antibiotic-free LB liquid medium, resuscitate at 37°C and 200 rpm for 1 hour, centrifuge at 4000 rpm for 2 minutes, discard the supernatant, and evenly spread the remaining liquid on an LB plate containing 50 μg / L ampicillin. Pick a single colony and add it to 500 μL of LB liquid medium containing 50 μg / L ampicillin. After shaking and culturing at 37°C and 200 rpm for 3 hours, detect the target fragment and use the positive clone colony with good growth as the GST protein expression engineering bacteria.

[0023] 3. Induced expression and purification of GST protein

[0024] The cultured GST protein expression engineered bacterial solution was inoculated into 10 mL of fresh LB liquid medium containing ampicillin at a volume ratio of 1:100 and cultured at 37°C and 200 rpm to OD600 = 0.4-0.7. ITPG was added to the bacterial solution to a final concentration of 1 mmol / L, cultured at 37°C and 200 rpm for 4 h, centrifuged at 4°C and 5000g for 15 min, and the cells were collected and resuspended in 10 times the volume of pH 7.0, 0.1 mol / L PBS buffer. The cells were repeatedly frozen and thawed three times with liquid nitrogen and a 25°C water bath, and ultrasonically disrupted on ice. The cells were centrifuged at 4°C and 12000g for 15 min, and the supernatant was collected to obtain a crude GST protein solution.

[0025] Gently shake the high-affinity GST purification medium (product number: L00206, Nanjing GenScript Biotechnology Co., Ltd.) to fully resuspend, draw 1.5 mL into a 10 mL chromatography column, and wash the equilibrated medium with 20 mL of pH 7.0, 0.1 mol / L PBS buffer precooled to 4°C; filter the crude GST protein solution with a 0.45 μm pore size filter membrane, add the filtrate to the chromatography column in which the medium has been equilibrated, and place it at 4°C to flow out the liquid at a flow rate of 10-15 cm / h, and collect the effluent; after all the filtrate has flowed out of the chromatography column, immediately wash the column with 20 column volumes of 1× PBS, collect the washing liquid; then use 10-15 column volumes of freshly prepared 10 mmol / L reduced glutathione solution (0.154 g reduced glutathione dissolved in 50 mL 50 mM The eluate was collected and filtered through a 3K ultrafiltration tube to remove free glutathione, thereby obtaining a GST purified protein solution.

[0026] The supernatant, effluent, washing solution and eluate collected above were respectively taken and analyzed by SDS-PAGE electrophoresis. The results are as follows: Figure 1 As shown, clear GST protein bands appeared around 25 kDa in both the supernatant and the eluate.

[0027] 4. Effect of GST crude protein on the prevention and control of postharvest green mold in citrus fruits

[0028] Select citrus fruits of uniform size and color without mechanical damage and scars, soak them in 2% sodium hypochlorite solution for 2 minutes, rinse with clean water, and dry naturally. Wipe the equatorial part of the fruit with 75% alcohol. After the alcohol is air-dried, use a 1mL sterile blue pipette tip to make a hole on each side of the equatorial part of the fruit. In the PBS control group (Control), inoculate 20μL of pH 7.0, 0.1mol / L PBS buffer per well, and inoculate 20μL of GST crude protein solution per well in the GST crude protein treatment group. After 24 hours, make another new well on the right side of the first well and inoculate a final concentration of 3×10 4 CFU / mL of Penicillium digitatum spore suspension was added. After the liquid was completely absorbed, the fruits were packaged in polyethylene film bags (170 mm × 140 mm) and stored in a room with an ambient temperature of 25°C and a relative humidity of 90-95%. The incidence rate of citrus fruits was counted and the diameter of the fruit lesions was measured.

[0029] The results are as follows Figure 2 As shown, the incidence and lesion diameter of citrus fruits in the PBS control group and the GST crude protein treatment group gradually increased with the extension of the pathogen inoculation time. However, the disease progression of citrus fruits in the GST crude protein treatment group was significantly slower than that in the PBS control group. This was demonstrated by the following: the incidence and lesion diameter of green mold disease in citrus fruits in the GST crude protein treatment group were significantly lower than those in the PBS control group (p < 0.05). On the seventh day of inoculation with Penicillium digitatum, the incidence of citrus fruits in the PBS control group exceeded 65%, while the incidence in the GST crude protein treatment group was only 24.1%. The lesion diameter of citrus fruits in the PBS control group exceeded 50 mm, while the diameter of lesions in the GST crude protein treatment group was only 22.15 mm.

[0030] 5. Effect of GST-purified protein on the prevention and control of postharvest green mold in citrus fruits

[0031] The citrus fruits with uniform size and color, no mechanical injury and scar were selected, soaked in 2% sodium hypochlorite solution for 2 min, then washed with clean water and dried naturally. The equator of the fruit was wiped with 75% alcohol, and after the alcohol was dried, 1 mL of sterile blue gun head was used to punch a hole on each opposite side of the equator of the fruit. The PBS control group was inoculated with 20 μL of PBS buffer solution with pH 7.0 and 0.1 mol / L per hole, and the GST purified protein treatment group was inoculated with 20 μL of GST purified protein solution with a final concentration of 150 μM per hole. After 24 h, a new hole was punched on the right side of the first hole, and 20 μL of Penicillium digitatum spore suspension with a final concentration of 3×10 4 CFU / mL was inoculated. After the liquid was completely absorbed, the fruit was packaged with a polyethylene film bag (170 mm×140 mm) and stored in a room with an environmental temperature of 25°C and a relative humidity of 90-95%. The incidence of citrus fruits and the lesion diameter of the fruits were measured.

[0032] The results are shown in Table 1. Figure 3 As shown in Table 1, the incidence and lesion diameter of the citrus fruits of the PBS control group and the GST purified protein group gradually increased with the extension of the inoculation time of the pathogenic bacteria. However, the incidence process of the citrus fruits of the GST purified protein group was significantly slower than that of the PBS control group, which was manifested in that the incidence and lesion diameter of the citrus fruits of the GST purified protein group were lower than those of the PBS control group, and there was a significant difference (p<0.05). On the 6th day of inoculation of Penicillium digitatum, the incidence of the citrus fruits of the PBS control group was more than 80%, while the incidence of the GST purified protein group was only 53.3%. The lesion diameter of the citrus fruits of the PBS control group was more than 50 mm, while the lesion diameter of the GST purified protein group was only 33.18 mm.

[0033] It can be seen that the GST crude protein and the GST purified protein can both induce the disease resistance of the fruits and effectively delay the invasion process of postharvest green mold of the citrus fruits, and can be used for preparing a medicine for preventing and treating postharvest diseases of the citrus fruits.

[0034] Finally, it should be pointed out that the above examples are only used to illustrate the technical solutions of the present application but not limit the present application. Although the present application has been described by referring to the preferred embodiments thereof, those skilled in the art will understand that various changes in form and detail can be made without departing from the spirit and scope of the present application as defined by the appended claims.

Claims

1. Use of glutathione S-transferase in the preparation of a medicament for preventing and treating postharvest diseases of citrus fruits, wherein the postharvest disease of citrus fruits is postharvest green mold disease of citrus fruits, and the amino acid sequence of the glutathione S-transferase is shown in SEQ ID No.

1.

2. The use according to claim 1, characterized in that The glutathione S-transferase is a crude GST protein, which is obtained by the following method: the pGEX-6P-1 plasmid containing the GST encoding gene is transformed into Escherichia coli to obtain a GST protein expression engineered bacterium, which is inoculated into LB liquid culture medium containing ampicillin, IPTG is added to induce the expression of GST protein, the bacteria are collected, resuspended, the cells are lysed, and the supernatant is obtained by centrifugation to obtain a crude GST protein solution.

3. The use according to claim 1, characterized in that The glutathione S-transferase is a GST purified protein, which is obtained by the following method: the pGEX-6P-1 plasmid containing the GST encoding gene is transformed into Escherichia coli to obtain a GST protein expression engineered bacterium, inoculated into LB liquid culture medium containing ampicillin, and induced to express the GST protein by adding IPTG. The bacteria are collected, resuspended, and the cells are lysed. The supernatant is obtained by centrifugation and filtered through a microporous filter membrane. The filtrate is passed through a GST affinity chromatography column and eluted with a reduced glutathione solution. The eluate is filtered through a 3K ultrafiltration tube to obtain a GST purified protein solution.

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