CN108018222B - Preparation GS115/Ac-AMP2 for inhibiting penicilliosis of pear fruit after harvest - Google Patents
Preparation GS115/Ac-AMP2 for inhibiting penicilliosis of pear fruit after harvest Download PDFInfo
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- CN108018222B CN108018222B CN201711289272.8A CN201711289272A CN108018222B CN 108018222 B CN108018222 B CN 108018222B CN 201711289272 A CN201711289272 A CN 201711289272A CN 108018222 B CN108018222 B CN 108018222B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
- A23B7/00—Preservation or chemical ripening of fruit or vegetables
- A23B7/14—Preserving or ripening with chemicals not covered by groups A23B7/08 or A23B7/10
- A23B7/153—Preserving or ripening with chemicals not covered by groups A23B7/08 or A23B7/10 in the form of liquids or solids
- A23B7/154—Organic compounds; Microorganisms; Enzymes
- A23B7/155—Microorganisms; Enzymes; Antibiotics
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
- C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
Abstract
The invention discloses a preparation GS115/Ac-AMP2 for inhibiting penicilliosis after pear fruit harvest, which is recombinant yeast GS115/Ac-AMP2 after Ac-AMP2 antibacterial peptide is introduced. The construction method of the recombinant yeast GS115/Ac-AMP2 comprises the following steps: constructing the optimized Ac-AMP2 gene on a pPICZ alpha A vector through Xho I and Not I double enzyme digestion and T4 ligase, performing linearization enzyme digestion on the formed pPICZ alpha A/Ac-AMP2 recombinant expression vector, performing electric transformation on GS115 competent cells, selecting a single colony in a Zeocin resistance plate, performing PCR (polymerase chain reaction) and sequencing verification to obtain the recombinant Pichia pastoris GS115/Ac-AMP2 strain. The invention can be used for inhibiting the postharvest diseases of fruits for fresh keeping.
Description
Technical Field
The invention relates to the technical field of fruit postharvest disease control, in particular to a technology for improving fruit disease control by transforming and over-expressing Ac-AMP2 antibacterial peptide in pichia pastoris GS 115.
Background
At present, the disease after fruit and vegetable is mainly prevented and treated by adopting a chemical bactericide method, but the problems of harm to human health, environmental pollution, generation of drug resistance and the like caused by excessive use of the chemical bactericide are solved. Currently, the application of chemical bactericides in stone fruits is prohibited by the European Union, so that the search for safe, nontoxic, environment-friendly and efficient antibacterial chemical bactericide substitutes is an urgent need at the present stage.
The antagonistic microbe is used for biological control of fruit postharvest diseases, which is regarded as one of the most possible methods for replacing chemical bactericides, and the antagonistic yeast has the advantages of safety, no toxicity, convenient use, genetic stability and the like, so that the commercial application of the antagonistic yeast becomes possible. However, the biological control effect of antagonistic microorganisms cannot reach or approach chemical bactericides at present, so researchers have made various attempts to further improve the biocontrol effect of antagonistic yeast, including stress treatment, culture condition optimization, antagonistic strain separation under stress conditions, exogenous gene expression and the like, wherein with the rapid development of molecular technology, the directional transformation of exogenous genes, such as expression products with direct bacteriostatic efficacy and capability of improving the antagonistic properties of antagonistic microorganisms, into antagonistic yeast gradually becomes a research hotspot in the field of biological control. Pichia pastoris has the characteristics of simple genetic operation, high and stable exogenous gene expression level, processable and modified expression products and the like, is favored by researchers, and various antibacterial peptides are successfully expressed in the Pichia pastoris at present.
The antibacterial peptide is a micromolecular polypeptide generated by a specific gene code of an organism, has high-efficiency spectral killing capability on bacteria, and also has strong inhibiting effect on pathogenic fungi, viruses, protozoa and the like.
The Ac-AMP2 antibacterial peptide is from Amaranthus caudatus (Amaranthus caudatus) seeds, has strong thermal stability and pH stability, and still has activity in boiling water bath for 10min and at a pH of 2-11; has remarkable inhibitory effect on pathogenic fungi, wherein the Inhibitory Concentration (IC) of Alternaria brassicola is half inhibitory concentration50) Semi Inhibitory Concentration (IC) as low as 4. mu.g/mL against Botrytis cinerea50) As low as 8. mu.g/mL. Has no toxic effect on human umbilical vascular endothelial cells and human skin muscle fiber cells.
The Ac-AMP2 Antimicrobial peptide target gene (i.e., the original Ac-AMP2 gene) is disclosed in the Antimicrobial peptides from Amaranthus caudatus seeds with sequence homology to the cysteine/glycine-rich domain of peptide-binding proteins, having the sequence:
ATGGTGAACATGAAGAGTGTTGCATTGATAGTTATAGTTATGATGGCGTTTATGATGGTGGATCCATCAATGGGAGTGGGAGAATGTGTGAGAGGACGTTGCCCAAGTGGGATGTGTTGCAGTCAGTTTGGGTACTGTGGTAAAGGCCCAAAGTACTGTGGCCGTGCCAGTACTACTGTGGATCACCAAGCTGATGTTGCTGCCACCAAAACTGCCAAGAATCCTACCGATGCTAAACTTGCTGGTGCTGGTAGTCCA。
disclosure of Invention
The technical problem to be solved by the invention is to provide a fresh-keeping technology for inhibiting postharvest diseases of fruits by using the recombinant pichia pastoris GS115/Ac-AMP2 into which the Ac-AMP2 antibacterial peptide gene is introduced without the help of a chemical bactericide.
In order to solve the technical problems, the invention provides a preparation GS115/Ac-AMP2 for inhibiting penicilliosis after pear fruit harvest, which is recombinant yeast GS115/Ac-AMP2 after Ac-AMP2 (optimized Ac-AMP2) antibacterial peptide is introduced.
As the improvement of the preparation GS115/Ac-AMP2 for inhibiting the penicilliosis of the pear fruit after harvest, the construction method of the recombinant yeast GS115/Ac-AMP2 comprises the following steps:
1) optimizing the target gene of the Ac-AMP2 antibacterial peptide according to the preference of a pichia pastoris codon to obtain an optimized Ac-AMP2 gene;
2) constructing the optimized Ac-AMP2 gene on a pPICZ alpha A vector through Xho I and Not I double enzyme digestion and T4 ligase to form a pPICZ alpha A/Ac-AMP2 recombinant expression vector;
respectively adding Xho I and Not I enzyme cutting sites at the front end and the rear end of an optimized Ac-AMP2 target gene, cutting a pPICZ alpha A vector by double enzyme cutting, and connecting a target gene fragment to the vector pPICZ alpha A to form a pPICZ alpha A/Ac-AMP2 recombinant expression vector;
3) after the pPICZ alpha A/Ac-AMP2 recombinant expression vector is subjected to linearization enzyme digestion, a GS115 competent cell is electrically transformed, a single colony is selected from a Zeocin resistance-containing plate for PCR and sequencing verification, and the recombinant Pichia pastoris GS115/Ac-AMP2 strain is obtained.
Remarking: the sequencing was performed to determine whether the sequence of the optimized Ac-AMP2 gene was identical, i.e., to determine whether the gene introduced into GS115 yeast was the optimized Ac-AMP2 gene.
As a further improvement of the preparation GS115/Ac-AMP2 for inhibiting the post-harvest penicilliosis of the pear fruit, the optimized Ac-AMP2 gene is the following nucleotide sequence (SEQ ID NO: 1):
CTCGAGAAAAGAATGGTGAACATGAAGTCCGTGGCATTGATCGTCATTGTCATGATGGCATTTATGATGGTTGACCCATCCATGGGAGTTGGATAATGCGTTAGGGGACGTTGCCCATCTGGTATGTGTTGTTCTCAGTTCGGGTACTGTGGTAAGGGCCCTAAGTATTGTGGTAGAGCCAGTACGACCGTAGACCATCAAGCTGATGTTGCTGCTACTAAGACTGCTAAAAATCCTACAGATGCCAAACTTGCCGGTGCTGGTTCACCCGCGGCCGC。
the Xho I site and the Not I site correspond to the underlined positions in the above 2.
When the invention is practically used, the suspension of the thalli can be adjusted to be 1 multiplied by 106～1×108cells/mL, then applied to the surface of the pear fruit.
In the invention, when the biological control effect experiment of the recombinant GS115/Ac-AMP2 yeast is carried out, the recombinant Pichia pastoris GS115/Ac-AMP2 (namely, GS115/Ac-AMP2 recombinant strain) is regulated to the thallus suspension of 1 × 106～1×108cells/mL, then the pear fruit wounds were inoculated.
The invention relates to a method for preventing and treating pear fruit postharvest diseases by transforming and overexpressing antibacterial peptide to improve antagonistic yeast. Experiments prove that the recombinant Pichia pastoris GS115/Ac-AMP2 strain successfully expressing Ac-AMP2 is used as a fruit biological preservative, and the biological preservative acts on the surface of a fruit wound and can effectively inhibit the occurrence of fruit penicilliosis.
The invention has the advantages that:
(1) the Ac-AMP2 antibacterial peptide applied by the invention is derived from Amaranthus caudatus seeds, and has no toxic and harmful effects on human umbilical vascular endothelial cells and human skin muscle fiber cells, so that the safety of the Ac-AMP2 antibacterial peptide is ensured; meanwhile, the antimicrobial peptide has an inhibiting effect on micromolar levels of various pathogenic fungi, so that the rot of fruits can be effectively reduced, and the fresh-keeping period of fruits and vegetables can be prolonged.
(2) After the Ac-AMP2 antibacterial peptide is introduced into GS115 and expressed, the biocontrol effect of the recombinant Pichia pastoris is remarkably improved, the use of a chemical bactericide can be effectively reduced, and the problems of harm to food safety and environment, possible drug resistance of the chemical bactericide and the like are solved.
(3) The method for expressing the Ac-AMP2 antibacterial peptide by adopting a eukaryotic system can effectively reduce the cost of taking the Ac-AMP2 antibacterial peptide directly extracted from the Amaranthus caudatus seeds as a bacteriostatic substance.
(4) Compared with the heterologous expression system of other antibacterial peptides of the foreigners, the eukaryotic system has more advantages in the yield (210 mu g/mL) of the Ac-AMP2 antibacterial peptide.
For example: kuddus et al (2016) report that the expression quantity of Snakin-1 of an antibacterial peptide gene Snakin-1 is 40 mug/mL after the antibacterial peptide gene Snakin-1 is converted into pichia pastoris; ren et al (2011) expressed 14.247 μ g/mL after transforming the Cecropin A into Pichia pastoris.
In conclusion, the invention can effectively inhibit the occurrence of the penicilliosis of the pear fruits on the premise of effectively reducing the use of the chemical bactericide, and provides a foundation for the large-scale production of the antibacterial peptide and the development and utilization of the antibacterial peptide in the inhibition of the postharvest diseases of the fruits.
Drawings
FIG. 1 is a PCR identification of recombinant plasmid pPICZ α A/Ac-AMP 2;
wherein M is 5000bp DNA Marker ladder; 1 is the PCR result of pPICZ α A not linked with Ac-AMP 2; 2 is the PCR identification result of pPICZ alpha A/Ac-AMP2 plasmid;
FIG. 2 shows the results of PCR identification of recombinant yeast (recombinant strain) GS115/Ac-AMP 2;
wherein M is 5000bp DNA Marker ladder; 1 is the PCR result of GS 115; 2 is the PCR result of yeast strain GS115/pPICZ alpha A; 3 is the PCR identification result of the recombinant yeast GS115/Ac-AMP 2;
FIG. 3 is the protein content of recombinant yeast GS115/Ac-AMP 2;
FIG. 4 shows Western blot identification of recombinant strain GS115/Ac-AMP 2;
wherein M is a protein ladder of 6.5-270 kDa; 1 is a Western-blot result of GS 115; 2 is a Western-blot result of the yeast strain GS115/pPICZ alpha A; 3 is a Western-blot identification result of the recombinant yeast GS115/Ac-AMP 2;
FIG. 5 shows the control effect of recombinant yeast GS115/Ac-AMP2 on Penicillium piricolum disease;
wherein panel (A) is the incidence and panel (B) is the lesion diameter;
the different lower case letters of the bar graph represent significant differences by duncan multiple range analysis (p < 0.05); error bars represent the standard error of three parallel experiments.
Detailed Description
The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:
example 1 recombinant expression vector construction
1. Experimental materials:
restriction enzymes: xho I and Not I, and T4 ligase;
axygen's DNA gel recovery kit;
2×Phanta Max Master Mix；
e.coli competent cell DH5 α;
plasmid pPICZ α A.
2. And (3) treatment:
(1) after plasmid pPICZ alpha A and a target gene (optimized Ac-AMP2 gene, SEQ ID NO:1) containing Xho I and Not I enzyme cutting sites are subjected to double enzyme cutting, a double enzyme cutting product is detected by using 1% agarose gel electrophoresis, an optimized target fragment and an expression vector are recovered by using a DNA gel recovery kit, and the recovered target fragment is connected for 16h at 4 ℃ by T4 ligase.
The double enzyme cutting system is as follows:
after recovery of the desired fragment and linearized expression vector, the overnight ligation reaction system was:
(2) the overnight reaction system (obtained in step (1)) was transformed into E.coli competent cells DH 5. alpha. and plated on LB + Zeocin plates (Zeocin concentration 25. mu.g/mL) with low salt content and cultured overnight at 37 ℃ until single colonies were formed.
(3) And respectively picking single colonies for PCR detection and subsequent sequencing identification. The PCR primer is
5′AOX1:5′-GACTGGTTCCAATTGACAAGC-3′，
3′AOX1:5′-GCAAATGGCATTCTGACATCC-3′，
The template is single colony lysate.
The PCR reaction system is as follows:
the PCR reaction program is: pre-denaturation at 95 ℃ for 30 s; denaturation at 95 ℃ for 15 s; annealing at 60 ℃ for 15 s; extension at 72 ℃ for 30 s; circulating for 35 times; final extension at 72 ℃ for 5 min.
3. As a result:
as shown in figure 1, 2 single colonies of the picked 3 strains have a 739bp band consistent with a theoretical value, wherein 1 single colony pPICZ alpha A vector is not completely cut, 2 single colony PCR results consistent with the theoretical value are further sequenced and verified, and the sequencing result shows that Ac-AMP2 is correctly constructed on the pPICZ alpha A expression vector, so that the pPICZ alpha A/Ac-AMP2 recombinant expression vector is obtained.
Remarks explanation: the band after PCR, to which Ac-AMP2 was not ligated, was 589 bp.
Example 2 transformation and characterization of recombinant GS115/Ac-AMP2 Yeast
1. Experimental materials:
recombinant expression vector pPICZ alpha A/Ac-AMP2
Plasmid pPICZ alpha A
Pichia pastoris GS115
Restriction enzyme Sac I
2. And (3) treatment:
(1) the successfully constructed recombinant expression vector pPICZ alpha A/Ac-AMP2 and the empty vector pPICZ alpha A without the antibacterial peptide are subjected to enzyme digestion treatment by Sac I.
(2) Uniformly mixing 10 mu L of linearized vector and 80 mu L of prepared GS115 competent cells, transferring the mixture into an electrotransfer cup, performing electric shock transformation into the competent yeast cells according to a program set by a Bio-Rad electrotransfer instrument, quickly adding 1mL of 1M sorbitol, standing for 1h at 28 ℃, uniformly coating 200 mu L of electric shock transformation liquid on a YPD + Zeocin resistance plate (the concentration of Zeocin is 100 mu g/mL), and culturing at the constant temperature of 28 ℃ until a single colony is formed.
(3) And respectively picking single colonies for yeast genome extraction, and performing PCR and sequencing identification.
PCR primers, PCR reaction procedure and the like were the same as in example 1;
PCR reaction system except for the following corresponding changes: changing the bacterial colony lysate into the extracted yeast genome by the template; the rest is equivalent to embodiment 1.
3. As a result:
as shown in FIG. 2, the Pichia pastoris GS115, the GS115 after the introduction of the empty plasmid and the GS115 after the introduction of the recombinant expression vector can detect a band at 2000bp, which indicates that the used universal primers 5 'AOX 1 and 3' AOX1 can identify other sites of the GS115 genome, but only two bands (589bp and 739bp) which are different in size and are consistent with the expectation appear in the GS115 Pichia pastoris after the introduction of the empty plasmid and the introduction of the antibacterial peptide recombinant plasmid, which indicates that the empty vector pPICZ alpha A and the recombinant expression vector pPICZ alpha A/Ac-AMP2 are connected to the GS115 genome, and the result is correct after the sequencing verification.
When the sequencing result is consistent with the optimized Ac-AMP2 sequence, the recombinant Pichia pastoris GS115/Ac-AMP2 can be obtained.
Example 3 expression and characterization of Ac-AMP2 in Yeast
First, Ac-AMP2 expression level of recombinant yeast GS115/Ac-AMP2
1. Experimental materials:
recombinant yeast GS115/Ac-AMP2
A Bradford protein concentration assay kit;
2. and (3) treatment:
(1) single colonies of a recombinant strain GS115/Ac-AMP2 and an empty plasmid control strain GS115/pPICZ alpha A, which are verified that the target gene is integrated into the yeast genome, are picked for induced expression. Adding methanol every 24h until the final concentration is 1%, and continuously inducing for 108 h; and taking 1mL of samples every 12h for storage to determine the expression level of the antibacterial peptide.
(2) Samples 8000g were centrifuged for 10min, and the supernatant and pellet were collected separately, and 50uL of the supernatant was taken at each sampling time point for testing the protein concentration in the samples by the Braford method.
3. As a result:
as shown in FIG. 3, the histone concentration of the recombinant strain GS115/Ac-AMP2 was increased with the increase of the induction time, and after 60 hours of induction, the maximum protein concentration was 210. mu.g/mL, while the control group was at a lower level.
II, Western-blot identification of the recombinant strain GS115/Ac-AMP2
1. Experimental materials:
recombinant yeast GS115/Ac-AMP2
TCA (trichloroacetic acid), acetone
Primary Anti-c-Myc Tag Monoclonal Antibody, secondary Anti-HRP-labeled Goat Anti-Mouse IgG (H + L).
2. And (3) treatment:
(1) adding 100% TCA into the supernatant obtained after the sample centrifugation and TCA according to the proportion of 9:1, mixing uniformly, and then placing on ice for precipitation overnight. After the overnight precipitation Sample is centrifuged, precooled acetone is added for shaking and resuspension, and SDS Sample Buffer is added for resuspension after the centrifugation again, and the mixture is treated for 10min in boiling water bath.
(2) Taking 10 mu L of sample to carry out Tricine-SDS-PAGE electrophoresis, carrying out membrane transfer treatment on gel after electrophoresis, then adding Western confining liquid to slowly shake, carrying out room temperature sealing for 1h, carrying out primary antibody and secondary antibody incubation after sealing is finished, and finally carrying out protein detection.
3. As a result:
as shown in FIG. 4, there is no band in the gel between GS115 Pichia pastoris and GS 115/pPICZ. alpha.A Pichia pastoris into which the empty plasmid is introduced, while there is a band with the expected result that the molecular mass of the recombinant GS115/Ac-AMP2 Pichia pastoris is about 11.9kDa after the antibacterial peptide is introduced, and the experimental result further proves that the antibacterial peptide is successfully introduced into the Pichia pastoris GS115 and can be successfully expressed.
Example 4 biocontrol effects of recombinant GS115/Ac-AMP2 Yeast
1. Experimental Material
The fruit is pear, the variety is crystal pear
Pathogenic bacteria: penicillium expansum (Penicillium expansum), activated at 25 ℃ for 7 days for use.
Recombinant yeast GS115/Ac-AMP2, adjusted to 1X 10 with sterile water7The cells/mL to obtain GS115/Ac-AMP2 cell suspension.
2. And (3) treatment:
(1) fruit pretreatment: selecting fruits with the same size, the same maturity and no mechanical damage on the surfaces, cleaning the fruits by using tap water, then soaking the fruits into a 0.1% sodium hypochlorite solution for disinfection for 2 minutes, taking the fruits out, then washing the fruits by using the tap water, and naturally airing the fruits for later use.
(2) Each fruit was prepared into five wounds of uniform size (5mm wide and 5mm deep) at the equatorial region using a sterile punch, and 50. mu.L of sterile water (Control) and 1X 10 were added to each wound7GS115 cell suspension/mL, 1X 107GS115/pPICZ alpha A cell suspension/mL, 1X 107GS115/Ac-AMP2 cell suspension at cell/mL, 1X 107cells/mL GS 115/original Ac-AMP2 cell suspension. After air drying for 2h, each wound was inoculated with 30. mu.l of 1X 104spores/mL of the Penicillium pathogen spore suspension. Selecting 9 pear fruits as a treatment, placing the pear fruits in a feed frame, sealing the pear fruits by using a preservative film, keeping a 90% humidity environment, and observing and recording the morbidity and the lesion diameter of the fruits after storing the pear fruits for 48 hours at a constant temperature of 25 ℃. Each experiment was set up in 3 replicates and the experiments were repeated twice until the same results were obtained.
Remarks explanation: the construction method of the GS 115/original Ac-AMP2 comprises the following steps: the 'original Ac-AMP2 gene' is used for replacing the 'optimized Ac-AMP2 gene' of the invention to construct the obtained recombinant Pichia pastoris GS 115/original Ac-AMP 2.
3. As a result:
as shown in figure 5(A), the recombinant Pichia pastoris GS115/Ac-AMP2 can effectively inhibit the occurrence of penicilliosis, the incidence of penicilliosis of a pear experimental group treated by the recombinant yeast GS115/Ac-AMP2 suspension is obviously lower than that of a control group and is reduced to 41.7%, and the incidence of penicilliosis is respectively 83.3%, 75%, 83.3% and 79% after the treatment of sterile water, GS115/pPICZ alpha A containing empty plasmids and a yeast suspension of GS 115/original Ac-AMP2 introduced into original Ac-AMP 2. The pear lesion diameters also have the same trend, as shown in FIG. 5(B), the average lesion diameter of the experimental group treated by the recombinant yeast GS115/Ac-AMP2 is the smallest and is only 2.92mm, compared with the control group treated by sterile water, GS115, introduced empty plasmid GS115/pPICZ alpha A and introduced original Ac-AMP2 GS 115/original Ac-AMP2, the average lesion diameter of the present invention is respectively reduced by 65.5%, 52%, 60.6% and 56.7%, and the lesion diameters of the four control groups are respectively 8.46mm, 6.08mm, 7.42mm and 6.7 mm. The incidence rate of the penicilliosis and the average lesion diameter are combined, so that the antibacterial effect of the GS115 can be obviously improved after the optimized antibacterial peptide Ac-AMP2 is introduced.
Finally, it is also noted that the above-mentioned lists merely illustrate a few specific embodiments of the invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.
Sequence listing
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<120> preparation GS115/Ac-AMP2 for inhibiting penicilliosis after pear fruit harvest
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<170> SIPOSequenceListing 1.0
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ctcgagaaaa gaatggtgaa catgaagtcc gtggcattga tcgtcattgt catgatggca 60
tttatgatgg ttgacccatc catgggagtt ggataatgcg ttaggggacg ttgcccatct 120
ggtatgtgtt gttctcagtt cgggtactgt ggtaagggcc ctaagtattg tggtagagcc 180
agtacgaccg tagaccatca agctgatgtt gctgctacta agactgctaa aaatcctaca 240
gatgccaaac ttgccggtgc tggttcaccc gcggccgc 278
Claims (2)
1. Preparation GS115/Ac-AMP2 inhibits Penicillium expansum (c: (A))Penicillium expansum) The application in (1) is characterized in that: the preparation is recombinant pichia pastoris GS115/Ac-AMP2 after Ac-AMP2 antibacterial peptide is introduced;
the Ac-AMP2 is optimized Ac-AMP2, and the nucleotide sequence of the Ac-AMP2 is shown in SEQ ID NO. 1.
2. The application of the preparation GS115/Ac-AMP2 in inhibiting penicillium expansum according to claim 1, wherein the construction method of the recombinant pichia pastoris GS115/Ac-AMP2 comprises the following steps:
1) optimizing the target gene of the Ac-AMP2 antibacterial peptide according to the preference of a pichia pastoris codon to obtain an optimized Ac-AMP2 gene;
2) constructing the optimized Ac-AMP2 gene on a pPICZ alpha A vector through Xho I and Not I double enzyme digestion and T4 ligase to form a pPICZ alpha A/Ac-AMP2 recombinant expression vector;
3) after the pPICZ alpha A/Ac-AMP2 recombinant expression vector is subjected to linearization enzyme digestion, a GS115 competent cell is electrically transformed, a single colony is selected from a Zeocin resistance-containing plate for PCR and sequencing verification, and the recombinant Pichia pastoris GS115/Ac-AMP2 strain is obtained.
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