CN108018222A - For suppressing the preparation GS115/Ac-AMP2 of pear fruit Postharvest Penicillium - Google Patents
For suppressing the preparation GS115/Ac-AMP2 of pear fruit Postharvest Penicillium Download PDFInfo
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- CN108018222A CN108018222A CN201711289272.8A CN201711289272A CN108018222A CN 108018222 A CN108018222 A CN 108018222A CN 201711289272 A CN201711289272 A CN 201711289272A CN 108018222 A CN108018222 A CN 108018222A
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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 kind of preparation GS115/Ac AMP2 for being used to suppress pear fruit Postharvest Penicillium, to import the recombination yeast GS115/Ac AMP2 after Ac AMP2 antibacterial peptides.The construction method of recombination yeast GS115/Ac AMP2 is to comprise the following steps：Ac AMP2 genes after optimization are building up on pPICZ α A carriers by Xho I and Not I double digestions and T4 ligases, after the pPICZ α A/Ac AMP2 recombinant expression carrier linearized enzyme digestions of formation, in electricity conversion GS115 competent cells, picking single bacterium colony PCR and sequence verification in containing Zeocin resistant panels, obtain recombinant yeast pichia pastoris GS115/Ac AMP2 bacterial strains.The present invention can be used to suppress the fresh-keeping of postharvest diseases of fruit.
Description
Technical field
The present invention relates to postharvest diseases of fruit Prevention Technique field, and more particularly one kind is in Pichia pastoris GS115 transfer
Change and be overexpressed Ac-AMP2 antibacterial peptides and improve its Prevention Technique to fruit disease.
Background technology
Disease after fruit vegetable is mainly prevented using the method for chemical bactericide at this stage, but excessively uses chemical sterilization
The problems such as agent can be detrimental to health, cause environmental pollution and develop immunity to drugs.Currently, European Union has forbidden chemical bactericide to exist
Application in kernel approaches fruit, therefore safe and non-toxic, environmental-friendly and Efficient antibacterial the chemical bactericide substitute of searching becomes
Active demand at this stage.
The biological control that antagonistic microbe is used for postharvest diseases of fruit is considered as most possibly substituting chemical bactericide
One of method, antagonism yeast have the advantages that safe and non-toxic, easy to use and inheritance stability make its commercialization use become can
Energy.But the biological control effect of current antagonistic microbe is still unable to reach or close to chemical bactericide, therefore to further improve
The biocontrol effect of antagonism yeast, researchers have carried out a variety of trials, including environment stress processing, training systern, adverse circumstance
Condition separates antagonism strain and expression alien gene etc., wherein with the high speed development of molecular engineering, expression product is had
Direct inhibitory effect and the foreign genes such as antagonistic microbe resistance orientation conversion antagonism yeast can be improved it is increasingly becoming biology
The research hotspot of prevention and control field.Pichia pastoris is with genetic manipulation is simple, foreign gene expression levels are high and stablize, expression product
It can process and be subject to researcher to favor the features such as modification, at present existing a variety of antibacterial peptides successful expression in Pichia pastoris.
The small molecule polypeptide that antibacterial peptide is produced as a kind of organism specific gene coding, has efficiency light to bacterium
The killing ability of spectrum, while also have to disease fungus, virus, protozoon etc. and acted on compared with high inhibition.
Ac-AMP2 antibacterial peptides come from amaranthus caudatus (Amaranthus caudatus) seed, and have strong heat endurance
It is still active between 2~11 with PH stability, boiling water bath 10min and PH；It is notable to disease fungus inhibition at the same time,
Wherein to the 503nhibiting concentration (IC of alternaric bacteria (Alternaria brassicola)50) as low as 4 μ g/mL, to Botrytis cinerea
503nhibiting concentration (the IC of (Botrytis cinerea)50) as low as 8 μ g/mL.To neuron like cells and human skin flesh
Meat fiber cell does not have toxic action.
Ac-AMP2 antibacterial peptides target gene (that is, original Ac-AMP2 genes) is in Antimicrobial peptides
from Amaranthus caudatus seeds with sequence homology to the cysteine/
It is disclosed in glycine-rich domain of chitin-binding proteins, its sequence is：
ATGGTGAACATGAAGAGTGTTGCATTGATAGTTATAGTTATGATGGCGTTTATGATGGTGGATCCATCAATGGGAGT
GGGAGAATGTGTGAGAGGACGTTGCCCAAGTGGGATGTGTTGCAGTCAGTTTGGGTACTGTGGTAAAGGCCCAAAGT
ACTGTGGCCGTGCCAGTACTACTGTGGATCACCAAGCTGATGTTGCTGCCACCAAAACTGCCAAGAATCCTACCGAT
GCTAAACTTGCTGGTGCTGGTAGTCCA。
The content of the invention
The technical problem to be solved in the present invention is to provide one kind not by chemical bactericide on the premise of, using importing Ac-
Recombinant yeast pichia pastoris GS115/Ac-AMP2 after AMP2 antibacterial peptide genes suppresses the preservation technology of postharvest diseases of fruit.
In order to solve the above technical problem, the present invention provides a kind of preparation for being used to suppress pear fruit Postharvest Penicillium
GS115/Ac-AMP2, to import the recombination yeast GS115/Ac- after Ac-AMP2 (Ac-AMP2 after optimization) antibacterial peptide
AMP2。
As the improvement for being used to suppress the preparation GS115/Ac-AMP2 of pear fruit Postharvest Penicillium of the present invention, ferment is recombinated
The construction method of female GS115/Ac-AMP2 is to comprise the following steps：
1), Ac-AMP2 antibacterial peptide target gene is optimized according to the Preference of Pichia pastoris codon, is optimized
Ac-AMP2 genes afterwards；
2) the Ac-AMP2 genes after optimization, are building up to pPICZ by Xho I and Not I double digestions and T4 ligases
On α A carriers, pPICZ α A/Ac-AMP2 recombinant expression carriers are formed；
Xho I and Not I restriction enzyme sites are added in Ac-AMP2 target gene front and back end i.e., after optimization respectively, with double enzymes
After cutting pPICZ α A carriers earnestly, target gene fragment is connected on carrier pPICZ α A, forms pPICZ α A/Ac-AMP2
Recombinant expression carrier；
3), by after pPICZ α A/Ac-AMP2 recombinant expression carrier linearized enzyme digestions, electricity converts GS115 competent cells
In, picking single bacterium colony PCR and sequence verification in containing Zeocin resistant panels, obtain recombinant yeast pichia pastoris GS115/Ac-
AMP2 bacterial strains.
Remarks：The sequence verification is the sequence judged whether with the Ac-AMP2 genes after optimization, i.e. is led to verify
Enter the gene in GS115 yeast whether be optimization after Ac-AMP2 genes.
The further of the preparation GS115/Ac-AMP2 for being used to suppress pear fruit Postharvest Penicillium as the present invention is improved,
Ac-AMP2 genes after optimization are following nucleotide sequence (SEQ ID NO:1)：
CTCGAGAAAAGAATGGTGAACATGAAGTCCGTGGCATTGATCGTCATTGTCATGATGGCATTTATGATGGTTGACCC
ATCCATGGGAGTTGGATAATGCGTTAGGGGACGTTGCCCATCTGGTATGTGTTGTTCTCAGTTCGGGTACTGTGGTA
AGGGCCCTAAGTATTGTGGTAGAGCCAGTACGACCGTAGACCATCAAGCTGATGTTGCTGCTACTAAGACTGCTAAA
AATCCTACAGATGCCAAACTTGCCGGTGCTGGTTCACCCGCGGCCGC。
It is Xho I restriction enzyme sites, Not I restriction enzyme sites respectively that underscore is corresponding at above-mentioned 2.
The present invention in actual use, is adjustable to thalline suspension as 1 × 106~1 × 108Cell/mL, then coated on pears
The surface of fruit.
The present invention is when carrying out the prevention effect experiment of restructuring GS115/Ac-AMP2 yeast bios, by recombinant yeast pichia pastoris
It is 1 × 10 that GS115/Ac-AMP2 (that is, GS115/Ac-AMP2 recombinant bacterial strains), which is adjusted to thalline suspension,6~1 × 108Cell/mL,
Then it is inoculated with pear fruit wound.
The present invention relates to a kind of conversion and overexpression antibacterial peptide to improve prevention of the antagonism yeast to pear fruit postharvest disease
Method.The present invention is experiments verify that the recombinant yeast pichia pastoris GS115/Ac-AMP2 bacterial strains of successful expression Ac-AMP2 are given birth to as fruit
Thing antistaling agent, fruit wound surface is acted on by the bio-preservative, can effectively suppress the generation of fruit penicilliosis.
It is an advantage of the invention that：
(1) the Ac-AMP2 antibacterial peptides that the present invention is used derive from amaranthus caudatus seed, and it is verified that to people's umbilical blood vessels
Endothelial cell and the effect of human skin meat fiber cytotoxic evil, thus its security is protected；The antibacterial peptide pair at the same time
A variety of disease fungus micromole levels play inhibitory action, thus can effectively reduce rotting for fruit, extend the fresh-keeping of fruits and vegetables
Phase.
(2) after Ac-AMP2 antibacterial peptides being imported GS115 and expression, the biological and ecological methods to prevent plant disease, pests, and erosion effect of recombinant yeast pichia pastoris is significantly carried
Rise, can effectively reduce the use of chemical bactericide, while reduce the food-safe and harm of environment and chemical bactericide can
The problems such as resistance to the action of a drug that can be produced.
(3) using the method for eukaryotic system expression Ac-AMP2 antibacterial peptides, it can effectively reduce and directly be extracted with amaranthus caudatus seed
Cost of the Ac-AMP2 antibacterial peptides as antibacterial substance.
(4) compared with the heterologous expression system of other antibacterial peptides of forefathers, which expresses the production of Ac-AMP2 antibacterial peptides
Amount (210 μ g/mL) has more advantage.
Such as：Kuddus etc. (2016) is reported its Snakin-1 after antibacterial peptide gene Snakin-1 conversion Pichia pastoris
Expression quantity is 40 μ g/mL；Its expression quantity is 14.247 μ after antibacterial peptide Cecropin A are converted Pichia pastoris by Ren etc. (2011)
g/mL。
In conclusion on the premise of the present invention can effectively reduce chemical bactericide use, it is effective to suppress pear fruit mould
The generation of disease, large-scale production and antibacterial peptide to the antibacterial peptide provide base in the utilization for suppressing postharvest diseases of fruit
Plinth.
Brief description of the drawings
The PCR that Fig. 1 is recombinant plasmid pPICZ alpha A/Ac-AMP2 is identified；
Wherein M is 5000bp DNA Marker ladder；1 is not connected with the PCR results of Ac-AMP2 for pPICZ α A；2 are
PPICZ α A/Ac-AMP2 plasmid PCR qualification results；
Fig. 2 is the PCR qualification results of recombination yeast (recombinant bacterial strain) GS115/Ac-AMP2；
Wherein M is 5000bp DNA Marker ladder；1 is the PCR results of GS115；2 be yeast strain GS115/
The PCR results of pPICZ α A；3 be the PCR qualification results of recombination yeast GS115/Ac-AMP2；
Fig. 3 is the protein content of recombination yeast GS115/Ac-AMP2；
The Western-blot that Fig. 4 is recombinant bacterial strain GS115/Ac-AMP2 is identified；
Wherein M is 6.5~270kDa protein ladder；1 is the Western-blot results of GS115；2 be yeast
The Western-blot results of bacterial strain GS115/pPICZ α A；3 reflect for the Western-blot of recombination yeast GS115/Ac-AMP2
Determine result；
Fig. 5 is prevention effects of the recombination yeast GS115/Ac-AMP2 to pears penicilliosis；
It is incidence wherein to scheme (A), and figure (B) is lesion diameter；
The different lowercases of column diagram are represented analyzes significant difference (p by Duncan multiple range<0.05)；Error bar table
Show the standard error of three parallel tests.
Embodiment
With reference to specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in
This：
Embodiment 1, recombinant expression carrier structure
1st, experiment material：
Restriction enzyme：Xho I and Not I, and T4 ligases；
The DNA gel QIAquick Gel Extraction Kit of Axygen；
2×Phanta Max Master Mix；
Competent escherichia coli cell DH5 α；
Plasmid pPICZ α A.
2nd, handle：
(1) by the plasmid pPICZ α A and target gene (Ac-AMP2 after optimization containing Xho I, Not I restriction enzyme sites
Gene, SEQ ID NO:Described in 1) after double digestion, double digestion product is detected with 1% agarose gel electrophoresis and returned with DNA gel
Purpose fragment and expression vector after kit recycling optimization are received, the purpose fragment after recycling is through 4 DEG C of connection 16h of T4 ligases.
Double digestion system is：
After the expression vector for recycling purpose fragment and linearisation, the reaction system connected overnight is：
(2) by reaction overnight system (obtained by step (1)) conversion competent escherichia coli cell DH5 α, it is coated on less salt
LB+Zeocin tablets (25 μ g/mL of Zeocin concentration), 37 DEG C are incubated overnight to formation single bacterium colony.
(3) picking single bacterium colony carries out PCR detections and follow-up sequencing identification respectively.PCR primer is
5′AOX1:5 '-GACTGGTTCCAATTGACAAGC-3 ',
3′AOX1:5 '-GCAAATGGCATTCTGACATCC-3 ',
Template is single bacterium colony lysate.
PCR reaction systems are：
PCR response procedures are：95 DEG C of pre-degeneration 30s；95 DEG C of denaturation 15s；60 DEG C of annealing 15s；72 DEG C of extension 30s；Circulation
35 times；72 DEG C of last extension 5min.
3rd, result：
As shown in Figure 1, in 3 bacterial strains of picking, there are 2 single bacterium colonies the band 739bp consistent with theoretical value occur, its
In 1 single bacterium colony pPICZ α A carrier do not cut completely through, the 2 single bacterium colony PCR results consistent with theoretical value are further surveyed
Sequence verifies that sequencing result shows that Ac-AMP2 has correctly been building up on pPICZ α A expression vectors, obtains pPICZ α A/Ac-AMP2
Recombinant expression carrier.
Remarks explanation：Do not connect Ac-AMP2, after PCR band be 589bp.
Embodiment 2, the conversion and identification for recombinating GS115/Ac-AMP2 yeast
1st, experiment material：
Recombinant expression carrier pPICZ α A/Ac-AMP2
Plasmid pPICZ α A
Pichia pastoris GS115
Restriction enzyme Sac I
2nd, handle：
(1) above-mentioned verification is built into successful recombinant expression carrier pPICZ α A/Ac-AMP2 and does not import the sky of antibacterial peptide
Carrier pPICZ α A are handled with Sac I digestions.
(2) the GS115 competent cells that the carrier 10 μ L and 80 μ L of linearisation are prepared are taken to mix and be transferred to electric revolving cup
In, the program that is set according to Bio-Rad electroporations is electroporated in competence yeast cells, the rapid 1M sorbs for adding 1mL
Alcohol, 28 DEG C of standing 1h, takes the 200 electroporated liquid of μ l to be spread evenly across YPD+Zeocin resistant panels (100 μ g/ of Zeocin concentration
ML), and under 28 DEG C of constant temperatures cultivate to formation single bacterium colony.
(3) picking single bacterium colony carries out the extraction of Yeast genome respectively, and carries out PCR and sequencing identification.
PCR primer, PCR response procedures are equal to embodiment 1；
PCR reaction systems, except following corresponding change：Template is changed to the Yeast genome of extraction by bacterium colony lysate；
Remaining is equal to embodiment 1.
3rd, result：
As shown in Fig. 2, Pichia pastoris GS115, import empty plasmid after GS115 and import recombinant expression carrier after
GS115 can detect band in 2000bp, illustrate that the AOX1 and 3 ' of universal primer 5 ' AOX1 used can identify GS115 genomes
Other sites, but only import empty plasmid and import antibacterial peptide recombinant plasmid GS115 Pichia pastoris in occur it is of different sizes,
Two band (589bp, 739bp) in line, illustrates empty carrier pPICZ α A and recombinant expression carrier pPICZ α A/Ac-
AMP2 is already connected in GS115 genomes, further correct through sequence verification result.
When sequencing result is consistent with the Ac-AMP2 sequences after optimization, it can confirm to obtain recombinant yeast pichia pastoris GS115/Ac-
AMP2。
The expression and identification of embodiment 3, Ac-AMP2 in yeast
First, the Ac-AMP2 expressions of recombination yeast GS115/Ac-AMP2
1st, experiment material：
Recombination yeast GS115/Ac-AMP2
Bradford determination of protein concentration kits；
2nd, handle：
(1) picking has been integrated into the recombinant bacterial strain GS115/Ac-AMP2 and sky of Yeast genome by verifying purpose gene
Plasmid control bacterial strain GS115/pPICZ α A single bacterium colonies, carry out induced expression.Held every 24h addition methanol to final concentration of 1%
Continuous induction 108h；Take 1mL Sample storages measure peptide expression horizontal every 12h.
(2) sample 8000g is centrifuged into 10min, collects supernatant and precipitate respectively, each sampling time point takes 50uL supernatants
Liquid carries out the protein concentration in Braford methods detection sample.
3rd, result：
As shown in figure 3, with the extension of induction time, recombinant bacterial strain GS115/Ac-AMP2 histone concentration constantly increases,
After inducing 60h, protein concentration reaches 210 μ g/mL of maximum, and control group is then constantly in reduced levels.
2nd, the Western-blot identifications of recombinant bacterial strain GS115/Ac-AMP2
1st, experiment material：
Recombination yeast GS115/Ac-AMP2
TCA (trichloroacetic acid), acetone
Primary antibody c-Myc Tag Monoclonal Antibody, secondary antibody HRP-labeled Goat Anti-Mouse IgG
(H+L)。
2nd, handle：
(1) supernatant obtained after sample is centrifuged is with TCA according to 9:1 ratio adds 100% TCA, mixes postposition
In precipitates overnight on ice.The acetone vibration that precooling is added after the centrifugation of overnight precipitation sample is resuspended, and centrifugation again adds SDS
Sample Buffer are resuspended, boiling water bath processing 10min.
(2) 10 μ L samples are taken to carry out Tricine-SDS-PAGE electrophoresis, the gel after electrophoresis carries out transferring film processing, Ran Houjia
Enter Western confining liquids slowly to shake, room temperature closing 1h, carries out the incubation of primary antibody and secondary antibody, finally carry out egg after the completion of closing
White detection.
3rd, result：
As shown in figure 4, GS115 Pichia pastoris and import empty plasmid GS115/pPICZ α A Pichia pastoris in colloid not
There are band, and then there are a molecular mass to be about for the restructuring GS115/Ac-AMP2 Pichia pastoris imported after antibacterial peptide
The band that the expected results of 11.9kDa are consistent, the experimental result further prove that antibacterial peptide successfully imports Pichia pastoris GS115
In, and being capable of successful expression.
Embodiment 4, restructuring GS115/Ac-AMP2 yeast bio prevention effects
1st, experiment material
Fruit is pears, and kind is Shuijing Pear
Pathogen：Penicillium expansum (Penicillium expansum), 25 DEG C activation 7 days it is spare.
Recombination yeast GS115/Ac-AMP2, is adjusted to 1 × 10 using sterile water7Cell/mL, obtains GS115/Ac-AMP2 bacterium
Liquid suspension.
2nd, handle：
(1) fruit pre-processes：The fruit that equal in magnitude, maturity is identical, surface has no mechanical damage is chosen, first uses tap water
Cleaning, then immerses in 0.1% liquor natrii hypochloritis and sterilizes 2 minutes again, takes out, then is rinsed well with tap water, dries in the air naturally
It is dry spare.
(2) with the card punch of aseptic process, position manufactures five unified sizes to each fruit under the line (5mm wide, 5mm are deep)
Wound, respectively toward add in wound 50 μ L sterile water (Control), 1 × 107The GS115 thallus suspension liquids of cell/mL, 1
×107The GS115/pPICZ α A thallus suspension liquids of cell/mL, 1 × 107The GS115/Ac-AMP2 thallus suspension liquids of cell/mL,
1×107The original Ac-AMP2 thallus suspension liquids of GS115/ of cell/mL.After naturally dry 2h, each wound be inoculated with 30 μ l 1 ×
104The mould Spores suspension of spore/mL.Choose 9 pear fruits to be placed in feed frame as a processing, use preservative film
Sealing keeps 90% humidity environment, starts to observe after 25 DEG C of constant temperature storage 48h and records the incidence and lesion diameter of fruit.
Each experimental setup 3 is parallel, and experiment is repeated twice, until subject to identical result.
Remarks explanation：The construction method of the above-mentioned original Ac-AMP2 of GS115/ is：Substituted with " original Ac-AMP2 genes "
The original Ac-AMP2 of recombinant yeast pichia pastoris GS115/ of " the Ac-AMP2 genes after optimization " structure gained of the present invention.
3rd, result：
As shown in Fig. 5 (A), GS115/Ac-AMP2 recombinant yeast pichia pastoris can effectively inhibit the generation of penicilliosis, through weight
Pears experimental group after group yeast GS115/Ac-AMP2 suspension processing, the incidence of penicilliosis are substantially less than control group, are reduced to
41.7%, and sterile water, GS115, the GS115/pPICZ α A containing empty plasmid and import original Ac-AMP2 GS115/ it is original
Without significant changes after the yeast bacterium suspension processing of Ac-AMP2, incidence is respectively 83.3%, 75%, 83.3% and 79%.Pears
Fruit lesion diameter also has same trend, and as shown in Fig. 5 (B), the experimental group of recombination yeast GS115/Ac-AMP2 processing is averaged
Lesion diameter is minimum, is only 2.92mm, compared to sterile water, GS115, imports empty plasmid GS115/pPICZ α A and importing original
The control group of the original Ac-AMP2 processing of the GS115/ of beginning Ac-AMP2, average lesion diameter of the invention declines 65.5% respectively,
52%th, 60.6% and 56.7%, the lesion diameter of aforementioned four control group be respectively 8.46mm, 6.08mm, 7.42mm and
6.7mm.After comprehensive penicilliosis incidence and average lesion diameter explanation importing optimization after cecropin A c-AMP2, can significantly it carry
The fungistatic effect of high GS115.
Finally, it should also be noted that it is listed above be only the present invention several specific embodiments.Obviously, this hair
It is bright to be not limited to above example, there can also be many deformations.Those of ordinary skill in the art can be from present disclosure
All deformations for directly exporting or associating, are considered as protection scope of the present invention.
Sequence table
<110>Zhejiang University
<120>For suppressing the preparation GS115/Ac-AMP2 of pear fruit Postharvest Penicillium
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 278
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
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 (3)
1. the preparation GS115/Ac-AMP2 for suppressing pear fruit Postharvest Penicillium, it is characterized in that：To import Ac-AMP2 antibacterials
Recombination yeast GS115/Ac-AMP2 after peptide.
2. the preparation GS115/Ac-AMP2 according to claim 1 for being used to suppress pear fruit Postharvest Penicillium, its feature
It is that the construction method of recombination yeast GS115/Ac-AMP2 is to comprise the following steps：
1), Ac-AMP2 antibacterial peptide target gene is optimized according to the Preference of Pichia pastoris codon, after being optimized
Ac-AMP2 genes；
2) the Ac-AMP2 genes after optimization, are building up to pPICZ α A by Xho I and Not I double digestions and T4 ligases to carry
On body, pPICZ α A/Ac-AMP2 recombinant expression carriers are formed；
3), by after pPICZ α A/Ac-AMP2 recombinant expression carrier linearized enzyme digestions, in electricity conversion GS115 competent cells, containing
There are picking single bacterium colony PCR and sequence verification in Zeocin resistant panels, obtain recombinant yeast pichia pastoris GS115/Ac-AMP2 bacterial strains.
3. the preparation GS115/Ac-AMP2 according to claim 2 for being used to suppress pear fruit Postharvest Penicillium, its feature
It is that the Ac-AMP2 genes after optimization are following nucleotide sequence：
CTCGAGAAAAGAATGGTGAACATGAAGTCCGTGGCATTGATCGTCATTGTCATGATGGCATTTATGATGGTTG
ACCCATCCATGGGAGTTGGATAATGCGTTAGGGGACGTTGCCCATCTGGTATGTGTTGTTCTCAGTTCGGGTACTGT
GGTAAGGGCCCTAAGTATTGTGGTAGAGCCAGTACGACCGTAGACCATCAAGCTGATGTTGCTGCTACTAAGACTGC
TAAAAATCCTACAGATGCCAAACTTGCCGGTGCTGGTTCACCCGCGGCCGC。
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| CN201711289272.8A CN108018222B (en) | 2017-12-07 | 2017-12-07 | Preparation GS115/Ac-AMP2 for inhibiting penicilliosis of pear fruit after harvest |
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