CN115466318B - Pichia glabra secretory protein PgAsp1 and application thereof - Google Patents

Pichia glabra secretory protein PgAsp1 and application thereof Download PDF

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CN115466318B
CN115466318B CN202210725229.6A CN202210725229A CN115466318B CN 115466318 B CN115466318 B CN 115466318B CN 202210725229 A CN202210725229 A CN 202210725229A CN 115466318 B CN115466318 B CN 115466318B
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pgasp1
pichia
glabra
secretory protein
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CN115466318A (en
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曾凯芳
陈鸥
姚世响
易兰花
朱容
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Southwest University
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Abstract

The invention discloses a Pichia glabra secretory protein PgAsp1 with an amino acid sequence shown as SEQ ID No.1 and application thereof in preparation of antistaling agent for inducing disease resistance of citrus fruits, and research results prove that the genetic transformation mediated by agrobacterium is used for transiently expressing the secretory protein PgAsp1 on the citrus fruits, so that the onset of citrus fruit green mold and the increase of lesion diameter can be inhibited to a certain extent; the secretory protein PgAsp1 with the function of inducing the disease resistance of the citrus fruits is extracted from the Pichia glabra, so that the mechanism of inducing the disease resistance of the citrus fruits by the Pichia glabra is enriched, the application mode of antagonistic yeast in the field of fruit preservation is widened, and a novel solution is provided for preventing and treating the post-harvest green mold of the citrus fruits.

Description

Pichia glabra secretory protein PgAsp1 and application thereof
Technical Field
The invention belongs to the technical field of genetic engineering technology and biological control of fruit postharvest diseases, and relates to pichia glabra (Pichia galeiformis) secretion protein PgAsp1 and application thereof.
Background
Antagonistic yeast is a biological control antagonistic bacterium commonly used in the control of fruit postharvest diseases. A great number of documents report that antagonistic yeast can induce the disease resistance of fruits, and relates to the expression of various disease resistance genes of fruits, hormone synthesis and signal transduction pathways, synthesis of disease resistance related substances and the like. However, the effect of antagonizing yeast secreted proteins on fruit disease resistance is not yet known.
Citrus is a plant of the citrus subfamily of the family rutaceae, the most productive fruit in the world and china. Fresh citrus fruit is extremely easy to be infected by microorganisms in the processes of transportation, storage and the like after picking, and the loss can reach more than 30% of the total yield. The green mold caused by the fungus penicillium digitatum (Penicillium digitatum) is a major invasive disease during storage of citrus fruits. At present, chemical bactericides such as prochloraz and the like are mainly adopted in industry to control postharvest diseases of fruits, but the problems of residual bactericides, drug resistance caused by microorganisms and the like exist.
Disclosure of Invention
The invention aims to explore and clearly antagonize the role of yeast secreted proteins in inducing fruit disease resistance so as to provide a new solution for preventing and treating fruit postharvest diseases.
Through researches, the invention provides the following technical scheme:
1. pichia glabra secretory protein PgAsp1 has an amino acid sequence shown in SEQ ID No. 1.
2. A coding gene of Pichia glabra secretory protein PgAsp1.
Further, the nucleotide sequence of the coding gene is shown as SEQ ID No. 2.
3. Recombinant expression vector containing Pichia glabra secretory protein PgAsp1 encoding gene.
Furthermore, the recombinant expression vector is obtained by cloning a Pichia helmet-shaped secretory protein PgAsp1 coding gene with a nucleotide sequence shown as SEQ ID No.2 into a plant expression vector pCAMBIA2300 between multiple cloning sites KpnI and PstI.
4. Engineering bacteria containing the recombinant expression vector.
Furthermore, the engineering bacteria are obtained by transferring a recombinant expression vector containing a Pichia glabra secretory protein PgAsp1 encoding gene into agrobacterium GV 3101.
5. Application of Pichia glabra secretory protein PgAsp1 in preparing antistaling agent for inducing disease resistance of citrus fruit is provided.
Further, the induction of the disease resistance of the citrus fruit is the induction of the disease resistance of the green mold of the citrus fruit.
6. The application of the engineering bacteria in preparing the antistaling agent for inducing the disease resistance of citrus fruits.
Further, the induction of the disease resistance of the citrus fruit is the induction of the disease resistance of the green mold of the citrus fruit.
The invention has the beneficial effects that: the invention provides Pichia glabra secretory protein PgAsp1 and application thereof in preparation of antistaling agent for inducing disease resistance of citrus fruits, and research results prove that agrobacterium-mediated genetic transformation can instantaneously express the secretory protein PgAsp1 on the citrus fruits, and can inhibit onset of citrus fruit green mold and increase of lesion diameter to a certain extent. The secretory protein PgAsp1 with the function of inducing the disease resistance of the citrus fruits is extracted from the Pichia glabra, so that the mechanism of inducing the disease resistance of the citrus fruits by the Pichia glabra is enriched, the application mode of antagonistic yeast in the field of fruit preservation is widened, and a novel solution is provided for preventing and treating the post-harvest green mold of the citrus fruits.
Drawings
FIG. 1 is an electrophoretogram of the gene encoding Pichia glabra secretory protein PgAsp1.
FIG. 2 is an electrophoretogram of recombinant vector pCAMBIA2300-PgAsp1.
FIG. 3 shows the electrophoresis pattern of the engineering bacterium of Agrobacterium containing the PgAsp1 encoding gene.
FIG. 4 shows the induction effect of Pichia glabra secretory protein PgAsp1 on post-harvest green mold of citrus fruits; a is the incidence, B is the lesion diameter, which indicates a significant difference (P < 0.05) compared to the Control group (Control); c is the symptom of onset of green mold after 6 days of storage of citrus fruits at 25℃after inoculation with Penicillium digitatum.
Detailed Description
In order to make the objects, technical solutions and advantageous effects of the present invention more apparent, preferred embodiments of the present invention are described in detail below.
Pichia glabra used in this example was isolated from the surface of lemon fruit in citrus orchards.
1. Primer design
The following primers were designed based on the gene sequence of Pichia glabra and the multiple cloning sites KpnI and PstI on the pCAMBIA2300 vector, and were commissioned for synthesis by the division of biological engineering (Shanghai).
PgAsp1-F：caatttactattctagggtaccATGAATTTCAAATCGTCGGTTCTG(SEQ ID No.3)
PgAsp1-R：gtatgggtatctagactgcagTTAGATAGCCTTTGCTAATCCGAC(SEQ ID No.4)
2. Pichia glabra total RNA extraction and cDNA synthesis
The total RNA of Pichia helmet-shaped is extracted by using a fungus RNA extraction kit (RE 781-50T) of Beijing Cool Leucocalm technology Co., ltd, and the specific steps are carried out according to the instruction book of the kit.
PrimeScript for the total RNA of Pichia glabra TM The RT regent Kit (Takara) is used for reverse transcription to synthesize cDNA, the specific steps are carried out according to the instruction book of the Kit, the concentration and the quality of the obtained cDNA are detected by an enzyme-labeled instrument Take3, and the cDNA is preserved at-20 ℃ for standby.
3. Cloning of Pichia helmet-shaped secretory protein PgAsp1 coding gene sequence
The cDNA synthesized by reverse transcription is used as a template, primers PgAsp1-F and PgAsp1-R are used, a Norpran high-fidelity enzyme P505 kit is used, and the PCR amplification of the PgAsp1 coding gene sequence with the nucleotide sequence shown as SEQ ID No.2 is carried out according to the specification of the kit.
The PCR products were subjected to 1% agarose gel electrophoresis (150V, 30 min) and the result was shown in FIG. 1, the target strips were cut off with a sterile knife, recovered with a gel recovery kit, and the specific steps were performed according to the kit instructions, and the recovered products were stored at-20℃for further use.
4. KpnI and PstI double cleavage of plasmid pCAMBIA2300
Plasmid pCAMBIA2300 was subjected to KpnI and PstI double cleavage, and the cleavage reaction solution was placed in a metal bath at 37℃for 30min.
The enzyme-cut product is subjected to agarose gel electrophoresis, a target strip is cut off by a sterile blade, and is recovered by a gel recovery kit, the specific steps are carried out according to the specification of the kit, and the recovered product is preserved at the temperature of minus 20 ℃ for later use.
5. Construction of recombinant vector pCAMBIA2300-PgAsp1
The recovered linearized plasmid pCAMBIA2300 and PgAsp1 coding gene sequences were ligated using a nonizane homologous recombinase, the specific steps were performed according to the reagent instructions, and the ligation reaction was placed in a metal bath at 37℃for 30min.
The ligation product transformed E.coli competent cells. And (5) performing bacterial liquid PCR verification on the transformant by using a plasmid sequencing primer. Transformants with positive results were commissioned for sequencing by the biological engineering (Shanghai) Co., ltd. And comparing the sequencing result with the correct recombinant plasmid to obtain the recombinant vector pCAMBIA2300-PgAsp1 which is successfully constructed.
6. Construction of agrobacteria engineering bacteria
Extracting recombinant vector pCAMBIA2300-PgAsp1 (the electrophoresis result is shown in figure 2), adding into competent cells of Agrobacterium GV3101, flicking, mixing, placing on ice for 5min, liquid nitrogen for 5min at 37deg.C for 5min, and placing on ice for 5min; adding 600 mu L of antibiotic-free LB liquid medium, culturing at 28 ℃ for 2-3 h at 200rpm, centrifuging at 4000rpm for 2min, discarding the supernatant, and uniformly coating the residual liquid on an LB plate (containing 25 mu g/L rifampicin and 50 mu g/L kanamycin); single colonies were picked up into 500. Mu.L LB liquid medium (containing 25. Mu.g/L rifampicin and 50. Mu.g/L kanamycin), cultured at 28℃under shaking at 200rpm for 16 hours, and then the target fragments were detected. The result is shown in figure 3, and the agrobacterium containing the PgAsp1 coding gene is the successfully constructed PgAsp1 agrobacterium engineering bacteria.
7. Effect of agrobacterium engineering bacteria on instantaneously expressing secretory protein on citrus fruit to induce disease resistance of citrus fruit green mold
Culturing engineering bacteria of Agrobacterium of PgAsp1 in LB medium (containing 25 μg/L rifampicin and 50 μg/L kanamycin) overnight, and collecting bacterial liquid OD 600 About 1, the cells were centrifuged at 4000rpm for 5min, and the cells were incubated with an incubation liquid (1L of the incubation liquid contained 5g glucose, 1.0663g MES (using a concentration of 5 mM), 0.760g Na 3 PO 4 ·12H 2 O (2 mM), 0.785mL 127.4mM AS mother solution (0.1 mM), and adjusting OD600 = 0.8, mixing gene silencing inhibitor P19 in equal amount, and incubating at 28deg.C in dark for 2-3 h to obtain agrobacterium engineering bacteria solution; selecting citrus fruit with uniform size, uniform color and no mechanical injury or scar, soaking in 2% sodium hypochlorite for 2min, washing with clear water, naturally air drying, wiping equatorial part of the fruit with 75% alcohol, air drying with alcohol, punching a hole on opposite side of equatorial part of the fruit with 1mL sterile gun head, and injecting into the hole with 1mL syringe (without needle head)About 0.5mL of agrobacteria engineering bacteria liquid is injected, and agrobacteria containing pCAMBIA2300 plasmid are used as control; after inoculating the engineering bacteria of Agrobacterium for 1d, a hole was punched 1cm to the right of each hole, and 10. Mu.L of 1X 10 was inoculated with a pipetting gun 4 CFU/mL of the penicillium digitatum (Penicillium digitatum) spore suspension, after bacterial liquid absorption, single fruit bagging, and placing in an environment of 25 ℃ in a dark place, and counting the morbidity and the lesion diameter from day 3.
As shown in FIG. 4, the agrobacterium engineering bacteria containing the PgAsp1 coding gene transiently express the secretion protein PgAsp1 (the amino acid sequence is shown as SEQ ID No. 1) on citrus fruits, and can inhibit the onset of citrus fruit green mold and the increase of the lesion diameter to a certain extent.
Finally, it is noted that the above-mentioned preferred embodiments are only intended to illustrate rather than limit the invention, and that, although the invention has been described in detail by means of the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention as defined by the appended claims.
Sequence listing
<110> university of southwest
<120> Pichia helmet secretory protein PgAsp1 and application thereof
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 412
<212> PRT
<213> Pichia helmet shape (Pichia galeiformis)
<400> 1
Met Asn Phe Lys Ser Ser Val Leu Thr Ser Leu Val Leu Met Leu Ala
1 5 10 15
Ser Leu His Gly Ala Asp Ala Lys Val His Ser Ala Lys Ile His Lys
20 25 30
His Pro Leu Glu Glu Ser Leu Lys Asp Val Ser Phe Ile Asp Tyr Val
35 40 45
Asp Ser Val Lys Ser Lys Tyr Leu Lys Thr Phe Val Ser Ser Val Asn
50 55 60
Ala Pro Tyr Val Pro Phe Val Glu Thr Val Leu Asp Glu Glu Leu Ala
65 70 75 80
Ser Ile Glu Lys Thr His Asp Leu Pro Leu Thr Asn Tyr Leu Asn Ala
85 90 95
Gln Tyr Phe Thr Glu Ile Gln Leu Gly Thr Pro Gly Gln Thr Phe Lys
100 105 110
Val Ile Leu Asp Thr Gly Ser Ser Asn Leu Trp Val Pro Gly Thr Glu
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Cys Gly Ser Leu Ala Cys Tyr Leu His Glu Lys Tyr Asp His Asp Ser
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Ser Ser Thr Tyr Lys Lys Asn Gly Thr Ser Phe Ala Ile Lys Tyr Gly
145 150 155 160
Ser Gly Ser Leu Glu Gly Tyr Val Ser Gln Asp Leu Leu Thr Phe Gly
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Asp Leu Val Ile Pro Asn Gln Asp Phe Ala Glu Ala Thr Ser Glu Pro
180 185 190
Gly Leu Ala Phe Ala Phe Gly Lys Phe Asp Gly Ile Leu Gly Leu Ala
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Tyr Asp Thr Ile Ser Val Asp Lys Ile Val Pro Pro Ile Tyr Asn Ala
210 215 220
Ile Ser Gln Gly Leu Leu Asp Ala Pro Gln Phe Ala Phe Tyr Leu Gly
225 230 235 240
Asp Thr Ala Lys Ser Glu Glu Asp Gly Gly Val Ala Ser Leu Gly Gly
245 250 255
Tyr Asp Lys Ser Lys Phe Glu Gly Glu Ile Thr Trp Leu Pro Val Arg
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Arg Lys Ala Tyr Trp Glu Val Lys Phe Asp Gly Ile Gly Leu Gly Asp
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Glu Tyr Ala Val Leu Glu Gly His Gly Ala Ala Ile Asp Thr Gly Thr
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Ser Leu Ile Ala Leu Pro Ser Gln Leu Ala Glu Ile Leu Asn Ala Gln
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Ile Gly Ala Glu Lys Ser Trp Asn Gly Gln Tyr Thr Val Asp Cys Asn
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Ala Arg Glu Lys Leu Pro Glu Leu Thr Phe Thr Phe Asp Gly Tyr Asn
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Phe Thr Leu Ser Ala Tyr Asp Tyr Thr Leu Glu Val Ser Gly Ser Cys
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Ile Ser Ala Phe Thr Pro Met Asp Phe Pro Asp Pro Ile Gly Pro Leu
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Ala Ile Ile Gly Asp Ala Phe Leu Arg Arg Tyr Tyr Ser Ile Tyr Asp
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Leu Gly Lys Asp Ala Val Gly Leu Ala Lys Ala Ile
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<210> 2
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<213> Pichia helmet shape (Pichia galeiformis)
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atgaatttca aatcgtcggt tctgacctcg ttggtcttga tgcttgcatc cctccatggc 60
gcagacgcca aggtgcactc agccaagatc cacaagcatc ctctcgaaga gagcttgaag 120
gacgtctcct tcatcgacta cgtcgactcc gtcaagagca agtacttgaa gacgtttgtc 180
agcagcgtca acgctccata cgtgcctttc gtcgagacgg tccttgacga agagctggcc 240
agcatcgaaa agacacacga cttgccattg accaactatt tgaacgcaca gtacttcaca 300
gagatccagc ttggtactcc aggccagacg ttcaaggtca tcctcgacac aggctcgtcc 360
aacttgtggg tcccaggcac agagtgcggc tcgttggcgt gctatctcca cgagaagtac 420
gaccacgact cttcttccac ctataagaag aacggcacca gcttcgccat caagtacggt 480
tccggctccc tcgaaggata cgtctcccag gatttgctca cctttggtga cttggtcatc 540
ccaaaccagg actttgcaga ggctaccagc gaaccaggct tggctttcgc ctttgggaaa 600
ttcgacggta tcttgggatt ggcctacgac accatttcag tcgacaagat tgttcctcca 660
atctataacg caataagtca gggcctattg gacgctcctc aattcgcctt ctacctaggc 720
gataccgcaa agtcggaaga ggacggcggc gtcgcctccc tcggtggata cgacaagtcc 780
aagttcgaag gcgaaatcac gtggttgcct gtcagaagaa aggcttactg ggaagtcaag 840
ttcgacggta tcggattggg cgatgagtac gcagtcttgg aaggccatgg tgcagcaatc 900
gacaccggta catccttgat tgcccttcct tcccaattgg ccgagatctt gaacgcccaa 960
atcggcgcag aaaaatcttg gaacggccag tacaccgtcg actgtaacgc cagagaaaaa 1020
ttaccagagt taaccttcac ctttgacggc tacaacttca ccctttctgc ctatgactac 1080
actttggagg tctccggaag ctgcatttcc gcattcaccc caatggactt cccagaccca 1140
atcggtcctt tggctatcat cggtgatgcc ttccttagaa gatactactc tatctacgat 1200
ctaggaaagg acgctgtcgg attagcaaag gctatctaa 1239
<210> 3
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<213> Artificial sequence (Artificial Sequence)
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caatttacta ttctagggta ccatgaattt caaatcgtcg gttctg 46
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<212> DNA
<213> Artificial sequence (Artificial Sequence)
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gtatgggtat ctagactgca gttagatagc ctttgctaat ccgac 45

Claims (8)

1. The Pichia glabra secretory protein PgAsp1 is characterized in that the amino acid sequence is shown as SEQ ID No. 1.
2. A gene encoding Pichia helminth secretory protein PgAsp1 as claimed in claim 1.
3. The coding gene of claim 2, wherein the nucleotide sequence is shown in SEQ ID No. 2.
4. A recombinant expression vector comprising the coding gene of claim 2.
5. The recombinant expression vector of claim 4, wherein: is obtained by cloning the coding gene of Pichia glabra secretory protein PgAsp1 with the nucleotide sequence shown as SEQ ID No.2 into the multiple cloning sites KpnI and PstI of a plant expression vector pCAMBIA 2300.
6. An engineered bacterium comprising the recombinant expression vector of claim 4.
7. The engineered bacterium of claim 6, wherein said recombinant expression vector is transformed into agrobacterium GV 3101.
8. The use of pichia glabra secretory protein PgAsp1 according to claim 1 or the engineering bacterium according to claim 6 in the manufacture of a preservative for inducing disease resistance of citrus fruit green mold.
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CN103667274B (en) * 2013-12-10 2016-05-04 中国科学院微生物研究所 A kind of multiple-shaped nuohan inferior yeast genetic manipulation strategy and application thereof
WO2018141872A1 (en) * 2017-02-02 2018-08-09 Lallemand Hungary Liquidity Management Llc Heterologous protease expression for improving alcoholic fermentation
CN114521585A (en) * 2022-01-25 2022-05-24 西南大学 Method for preventing and treating postharvest diseases of jujube fruits by using antagonistic yeast to remold epiphytic microbial communities

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