CN110317740A - One plant of recombinant yeast pichia pastoris, its expression product and its application - Google Patents
One plant of recombinant yeast pichia pastoris, its expression product and its application Download PDFInfo
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- CN110317740A CN110317740A CN201810297225.6A CN201810297225A CN110317740A CN 110317740 A CN110317740 A CN 110317740A CN 201810297225 A CN201810297225 A CN 201810297225A CN 110317740 A CN110317740 A CN 110317740A
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- Prior art keywords
- sweet potato
- chitinase
- ibchia
- expression
- pichia pastoris
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Links
- 240000004808 Saccharomyces cerevisiae Species 0.000 title claims abstract description 19
- 241000235058 Komagataella pastoris Species 0.000 title claims abstract description 15
- 108010022172 Chitinases Proteins 0.000 claims abstract description 39
- 102000012286 Chitinases Human genes 0.000 claims abstract description 34
- 244000017020 Ipomoea batatas Species 0.000 claims abstract description 29
- 235000002678 Ipomoea batatas Nutrition 0.000 claims abstract description 29
- 229920002101 Chitin Polymers 0.000 claims abstract description 21
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 21
- 238000000034 method Methods 0.000 claims abstract description 14
- 230000001939 inductive effect Effects 0.000 claims abstract description 8
- 241000196324 Embryophyta Species 0.000 claims abstract description 7
- 244000000004 fungal plant pathogen Species 0.000 claims abstract description 4
- 239000013589 supplement Substances 0.000 claims abstract description 3
- 238000006243 chemical reaction Methods 0.000 claims description 7
- 230000007062 hydrolysis Effects 0.000 claims description 4
- 238000006460 hydrolysis reaction Methods 0.000 claims description 4
- 230000002401 inhibitory effect Effects 0.000 claims description 4
- 239000013598 vector Substances 0.000 claims description 3
- 239000002773 nucleotide Substances 0.000 claims 6
- 125000003729 nucleotide group Chemical group 0.000 claims 6
- 108700028369 Alleles Proteins 0.000 claims 2
- 102000004190 Enzymes Human genes 0.000 abstract description 11
- 108090000790 Enzymes Proteins 0.000 abstract description 11
- 230000000694 effects Effects 0.000 abstract description 11
- 241000233866 Fungi Species 0.000 abstract description 10
- 241000894006 Bacteria Species 0.000 abstract description 8
- 235000008331 Pinus X rigitaeda Nutrition 0.000 abstract description 7
- 241000018646 Pinus brutia Species 0.000 abstract description 7
- 235000011613 Pinus brutia Nutrition 0.000 abstract description 7
- 244000052769 pathogen Species 0.000 abstract description 5
- 230000001717 pathogenic effect Effects 0.000 abstract description 5
- 238000002360 preparation method Methods 0.000 abstract description 5
- 239000000047 product Substances 0.000 abstract description 5
- 238000003259 recombinant expression Methods 0.000 abstract description 5
- 238000010353 genetic engineering Methods 0.000 abstract description 4
- 239000013604 expression vector Substances 0.000 abstract description 3
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- 239000000411 inducer Substances 0.000 abstract description 2
- 230000005764 inhibitory process Effects 0.000 abstract description 2
- 239000000758 substrate Substances 0.000 abstract description 2
- 244000061176 Nicotiana tabacum Species 0.000 abstract 1
- 235000002637 Nicotiana tabacum Nutrition 0.000 abstract 1
- 239000000416 hydrocolloid Substances 0.000 abstract 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 17
- 210000004027 cell Anatomy 0.000 description 11
- 239000006228 supernatant Substances 0.000 description 10
- 230000009182 swimming Effects 0.000 description 10
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- 230000029087 digestion Effects 0.000 description 6
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- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 239000012153 distilled water Substances 0.000 description 5
- 239000013613 expression plasmid Substances 0.000 description 5
- 238000000855 fermentation Methods 0.000 description 5
- 230000004151 fermentation Effects 0.000 description 5
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 4
- 238000009835 boiling Methods 0.000 description 4
- 238000001816 cooling Methods 0.000 description 4
- 229920001542 oligosaccharide Polymers 0.000 description 4
- 150000002482 oligosaccharides Polymers 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 239000000600 sorbitol Substances 0.000 description 4
- 102100036826 Aldehyde oxidase Human genes 0.000 description 3
- 241000223600 Alternaria Species 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Natural products CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 101000928314 Homo sapiens Aldehyde oxidase Proteins 0.000 description 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 3
- 239000001888 Peptone Substances 0.000 description 3
- 108010080698 Peptones Proteins 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 235000019319 peptone Nutrition 0.000 description 3
- 239000013612 plasmid Substances 0.000 description 3
- 241000607479 Yersinia pestis Species 0.000 description 2
- 230000003385 bacteriostatic effect Effects 0.000 description 2
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- 238000005119 centrifugation Methods 0.000 description 2
- 238000013461 design Methods 0.000 description 2
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- 238000001962 electrophoresis Methods 0.000 description 2
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- 235000019441 ethanol Nutrition 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- YGSDEFSMJLZEOE-UHFFFAOYSA-N salicylic acid Chemical compound OC(=O)C1=CC=CC=C1O YGSDEFSMJLZEOE-UHFFFAOYSA-N 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- GEHJYWRUCIMESM-UHFFFAOYSA-L sodium sulfite Chemical compound [Na+].[Na+].[O-]S([O-])=O GEHJYWRUCIMESM-UHFFFAOYSA-L 0.000 description 2
- 239000008223 sterile water Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 238000001262 western blot Methods 0.000 description 2
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 244000052909 Dioscorea esculenta Species 0.000 description 1
- 235000006536 Dioscorea esculenta Nutrition 0.000 description 1
- 102000002322 Egg Proteins Human genes 0.000 description 1
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- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
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- 208000031888 Mycoses Diseases 0.000 description 1
- OVRNDRQMDRJTHS-UHFFFAOYSA-N N-acelyl-D-glucosamine Natural products CC(=O)NC1C(O)OC(CO)C(O)C1O OVRNDRQMDRJTHS-UHFFFAOYSA-N 0.000 description 1
- OVRNDRQMDRJTHS-FMDGEEDCSA-N N-acetyl-beta-D-glucosamine Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-FMDGEEDCSA-N 0.000 description 1
- MBLBDJOUHNCFQT-LXGUWJNJSA-N N-acetylglucosamine Natural products CC(=O)N[C@@H](C=O)[C@@H](O)[C@H](O)[C@H](O)CO MBLBDJOUHNCFQT-LXGUWJNJSA-N 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 244000061456 Solanum tuberosum Species 0.000 description 1
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- 238000009825 accumulation Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- -1 alpha-mercapto ethyl Chemical group 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 229940121375 antifungal agent Drugs 0.000 description 1
- 239000003429 antifungal agent Substances 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
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- 239000000084 colloidal system Substances 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 244000000037 crop pathogen Species 0.000 description 1
- 230000008260 defense mechanism Effects 0.000 description 1
- 230000003413 degradative effect Effects 0.000 description 1
- 239000008121 dextrose Substances 0.000 description 1
- 230000006806 disease prevention Effects 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 235000014103 egg white Nutrition 0.000 description 1
- 210000000969 egg white Anatomy 0.000 description 1
- 230000005611 electricity Effects 0.000 description 1
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- 235000013305 food Nutrition 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- ZJYYHGLJYGJLLN-UHFFFAOYSA-N guanidinium thiocyanate Chemical compound SC#N.NC(N)=N ZJYYHGLJYGJLLN-UHFFFAOYSA-N 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 239000004570 mortar (masonry) Substances 0.000 description 1
- 229950006780 n-acetylglucosamine Drugs 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- FJKROLUGYXJWQN-UHFFFAOYSA-N papa-hydroxy-benzoic acid Natural products OC(=O)C1=CC=C(O)C=C1 FJKROLUGYXJWQN-UHFFFAOYSA-N 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- VZOPRCCTKLAGPN-ZFJVMAEJSA-L potassium;sodium;(2r,3r)-2,3-dihydroxybutanedioate;tetrahydrate Chemical compound O.O.O.O.[Na+].[K+].[O-]C(=O)[C@H](O)[C@@H](O)C([O-])=O VZOPRCCTKLAGPN-ZFJVMAEJSA-L 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 238000003672 processing method Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 229960004889 salicylic acid Drugs 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 229940074446 sodium potassium tartrate tetrahydrate Drugs 0.000 description 1
- 235000010265 sodium sulphite Nutrition 0.000 description 1
- 230000004763 spore germination Effects 0.000 description 1
- 230000003068 static effect Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 210000005253 yeast cell Anatomy 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
-
- 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
-
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
- C12N9/2442—Chitinase (3.2.1.14)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01014—Chitinase (3.2.1.14)
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Plant Pathology (AREA)
- Mycology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Medicinal Chemistry (AREA)
- Agronomy & Crop Science (AREA)
- Pest Control & Pesticides (AREA)
- Virology (AREA)
- Dentistry (AREA)
- Environmental Sciences (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
One plant of recombinant yeast pichia pastoris, its expression product and its application are operably connected with expression vector using the sweet potato chitinase gene IbChiA of clone, obtain the recombinant expression carrier that can express the albumen.By importing above-mentioned recombinant expression carrier in appropriate host cell, the genetic engineering bacterium of expression IbChiA is obtained.It is to obtain chitinase IbChiA through inducing expression by cultivating pPICZ α+IbChiA-X33 the present invention also provides a kind of method for preparing above-mentioned IbChiA.The inductive condition is inducer methanol of every 24 hours supplements, until methanol final concentration of 1.0%.The present invention also provides application of the above-mentioned sweet potato chitinase in hydrocolloid chitin and inhibition plant pathogenic fungi.The sweet potato chitinase of preparation all has very strong fungistatic effect to sweet potato black rot pathogen and pine root fungus；Restructured Pichia pastoris in expression sweet potato chitinase method provided by the invention, exocytosis amount is up to 403mg/L, and using tobacco brown spot pathogen as substrate, enzyme activity reaches 20U/ml.
Description
Technical field
The present invention relates to molecular biology, genetic engineering field, produce more particularly to one plant of recombinant yeast pichia pastoris, its expression
Object and its application.
Background technique
Chitin is distributed widely in animal, in microbial body, is the second largest renewable resource for being only second to cellulose, Nian Sheng
Object synthetic quantity is up to 10,000,000,000 tons.The catabolite chitin oligo saccharide of chitin is that a kind of functionality with unique physiological activity is oligomeric
Sugar all has a wide range of applications in each field such as agricultural, biomedicine, food, cosmetics, but chitin biology money at present
The exploitation in source chemically based on, nearly all to use strong acid and strong base, environment is caused greatly to pollute.Utilize chitin
Enzyme hydrolysis chitin, which prepares chitin oligo saccharide, has working condition mildly controllable, the few advantage of environmental pollution, is substitution chemistry side
The desirable technique of method.
In crop pathogens and the field of biological control of pest, chitinase is made of degradative fungi chitin
Cell wall ruptures its plasmalemma, to inhibit the spore germination and mycelia growth of disease fungus, while catabolite chitin
Oligosaccharides is used as the defense mechanism of exciton activated plant again, induces the accumulation of chitinase and other antibacterial materials, reaches anti-true
The effect of bacterium and bacterium；In terms of pest-resistant effect, chitinase has synergistic effect to sporeine preparation, improves larva
The death rate；In terms of the fungal disease prevention and treatment of the mankind, chitinase is equally safe and effective antifungal agent, is possessed important
And wide medical application prospect.
Therefore it studies and using chitinase by with great economic value and theoretical value, with to chitinase
Increase in demand will be inexorable trend with the chitinase that outstanding genetic engineering bacterium produces a large amount of high activity.
Summary of the invention
First mesh of the invention is to provide comprising above-mentioned recombinant expression carrier Pichi strain；Of the invention second
Purpose is to provide a kind of method for preparing above-mentioned chitinase；Third object of the present invention is to provide sweet potato chitinases to press down
Application in plant pathogenic fungi processed；Fourth object of the present invention is to provide sweet potato chitinase answering in hydrolysis chitin
With.
In order to realize that the above goal of the invention, the present invention utilize the sweet potato chitinase gene IbChiA and expression vector cloned
It is operably connected, obtains the recombinant expression carrier that can express the albumen.
Above-mentioned recombinant expression carrier is imported in appropriate host cell, obtains the genetic engineering bacterium of expression IbChiA.
It is by cultivating pPICZ α+IbChiA-X33, warp the present invention also provides a kind of method for preparing above-mentioned IbChiA
Inducing expression obtains chitinase IbChiA.The condition of culture is 28 DEG C, and initial pH is that 7,200rpm is cultivated 144 hours.Institute
Stating inductive condition is inducer methanol of every 24 hours supplements, until methanol final concentration of 1.0%.
The present invention also provides application of the above-mentioned sweet potato chitinase in hydrolysis chitin and inhibition plant pathogenic fungi.
Beneficial effects of the present invention: sweet potato chitinase prepared by the present invention all has sweet potato black rot pathogen and pine root fungus
There is very strong fungistatic effect, the application for the enzyme in agricultural and industry is laid a good foundation；Recombinant yeast pichia pastoris provided by the invention
Sweet potato chitinase method is expressed, exocytosis amount is up to 403mg/L, and using chitin as substrate, enzyme activity reaches 20U/ml.
Detailed description of the invention
Fig. 1 is that expression plasmid constructs flow chart；
Fig. 2 is expression plasmid double digestion electrophoretogram, wherein swimming lane 1:DNA standard molecular weight；Swimming lane 2: empty plasmid pPICZ α
Result after EcoR I and Kpn I double digestion；Swimming lane 3: expression plasmid pPICZ α+IbChiA is through the bis- enzymes of EcoR I and Kpn I
Result after cutting；
Fig. 3 is the destination protein in SDS-PAGE and Western-blot detection pPICZ α+IbChiA-X33 fermentation supernatant
Result, wherein swimming lane 1: protein standard molecular weight；Swimming lane 2:pPICZ α+IbChiA-X33 fermented supernatant fluid；
Fig. 4 is inhibiting effect experimental result of the sweet potato chitinase IbChiA to alternaria and pine root fungus, wherein A:
Alternaria；B: pine root fungus；0: distilled water；1, the fermentation supernatant of 2,3:IbChiA.
Specific embodiment:
Project implementation scheme provided by the invention is not limited to research range of the invention for illustrating the present invention.
If be not specifically stated, the technological means in present invention implementation is the common technology of this research field, and raw materials used is market
It is purchased.Unless otherwise specified, the conventional means that technological means used in embodiment is well known to those skilled in the art.
Embodiment 1
(1) design of primers
Following pair of primers has been synthesized according to the design of the sequence (SEQ ID No.1) of IbChiA gene:
FW:5 '-CCGGAATTCATGAGTGTGGGGTCCATTGT -3 '
REV:5 '-CGGGGTACCGTGGAGCCAAAAGGCCT -3 '
The both ends FW, REV are respectively designed with EcoR I and Kpn I restriction enzyme site,
(2) PCR amplification of IbChiA gene
Surpass fidelity PCR Master Mix (New England BaiLabs), FW, REV primer, to contain with Phusion
The T plasmid of IbChiA is template, and PCR reaction system is 25ul:
Reaction condition are as follows: 98 DEG C of 3min；98 DEG C, 30s；58 DEG C, 30s；72 DEG C, 50s；72 DEG C, 10min；Circulation 34 times；4
DEG C keep, electrophoresis detection result.
(3) target gene is connected to pichia pastoris yeast expression vector pPICZ α
As shown in Figure 1, being carried out respectively to the PCR product of IbChiA and pPICZ α with restriction enzyme EcoRI and Kpn I
Then double digestion recycles target fragment, connected with T4 ligase, and connection product is converted bacillus coli DH 5 alpha
In, extra large JaRa company sequencing is served in PCR detection.Double digestion detection figure is as shown in Figure 2, wherein swimming lane 1:DNA standard molecular weight
(bp)；Swimming lane 2: result of the empty plasmid pPICZ α after EcoR I and Kpn I double digestion；Swimming lane 3: expression plasmid pPICZ α+
Result of the IbChiA after EcoR I and Kpn I double digestion.
2 recombinant vector pPICZ α+IbChiA electrotransformation of embodiment is to the secreting, expressing in pichia pastoris yeast X-33
(1) preparation of pichia pastoris yeast X-33 (purchase of Invitrogen company) Electroporation-competent cells and its electricity
Hit conversion
The fresh single bacterium of S11, picking is fallen in 5ml YPD fluid nutrient medium, and in 30 DEG C, 250rpm is cultivated 12-14 hours；
S12, it is inoculated into 0.1% inoculum concentration in the 2L triangular flask of the culture medium of YPD containing 500ml, in 30 DEG C, 250rpm
Culture 12-14 hours, makes its OD600=1.3-1.5；
S13,1500rpm is centrifuged 5 minutes at 4 DEG C, collects cell；
S14, with 500-250ml ice be pre-chilled sterile water washing cell twice；
S15, to wash cell primary for the 1M sorbitol solution being pre-chilled with 20ml ice；
S16, cell is resuspended with the 1M sorbitol solution that 1ml ice is pre-chilled, until final volume is 1.5ml or so, with 80 μ l packing
In small centrifuge tube.
(2) pichia pastoris yeast yeast cells is electroporated
S21, by the about 10 μ l of the expression plasmid containing target gene of ready about 100 μ g/ μ l, with 80 μ l competent yeasts
Cell mixes, and places on ice about 5 minutes；
S22, the electric revolving cup for the competent cell for being mixed with DNA being transferred to the 0.2cm that ice is pre-chilled；In 1.5 kilovolts of voltage
Lower conversion；
S23, the 1M sorbitol solution that the pre-cooling of 1ml ice is then added at once mix cell, turn in transformed cell
Enter the small centrifuge tube of 1.5ml, 30 DEG C of static 1-2h.
S24,50-200ul is taken to be coated on containing 100ug/ml YPDS plate (yeast extract 1%, peptone
2%, dextrose 2%, Sorbitol 1M, agar 2%), in 30 DEG C of cultures, 2 to 3 days observation results.
(3) screening of the recombinant bacterial strain of the gene containing IbChiA
Yeast colony PCR method identifies the transformant correctly integrated, and chooses positive bacterium colony, on plate with 5 ' AOX1,3 '
AOX1 is primer, further verifies the transformant correctly integrated using the method for yeast colony PCR.
Primer sequence are as follows: 5 ' AOX1:5 '-GACTGGTTCCAATTGACAAGC-3 '
3 ' AOX1:5 '-GCAAATGGCATTCTGACATCC-3 '
The processing method of template:
It is S31, a little with sterile suction nozzle picking colony, it is dissolved into the D2-Buffer (1L: guanidinium isothiocyanate of 50ul
472.64g, 1mol/L pH8.0TrisHCl buffer 50ml, alpha-mercapto ethyl alcohol 7ml) in mix；
S32, by mixed liquor in 100 DEG C of boiling water bath 5min；
S33,12000rpm are centrifuged 30s, abandon supernatant；
S34, it is precipitated 2 times with sterile water washing；
S35, the ddH2O, 95 DEG C of effect 5min that precipitating is dissolved in 20ul；
It is template that supernatant is obtained after S36, centrifugation.
PCR reaction system:
Reaction condition: 95 DEG C of 5min；95 DEG C of 40s, 60 DEG C of 40s, 72 DEG C of 1min 30s, 30cycles；72℃10min.
(4) in pichia pastoris yeast purpose chitinase expression
1. chitinase activity measures
The preparation of chitin: the powdered chitin of 5g, which is put into mortar in grinding, is added dense HCl 200ml pre-cooling hydrochloric acid,
It is placed in beaker, magnetic stirrer while is slowly added into 1000ml50% ethyl alcohol, makes chitin Precipitation.Centrifugation,
It abandons supernatant and collects cotton-shaped chitin, with distilled water repeated flushing to neutrality, 4 DEG C of refrigerators are saved.
The preparation of DNS solution: sodium potassium tartrate tetrahydrate 182g is dissolved in 500ml distilled water, and 3,5- dinitro is added in 45 DEG C of water-baths
Base salicylic acid 6.3g, sodium hydroxide 21g, phenol 5g, sodium sulfite 5g, it is cooling after to be dissolved, it is settled to 1000ml.
500ul enzyme solution and 1% chitin is taken to mix, 42 DEG C of water-baths keep the temperature 60min, and boiling water bath 6min is added
1mlDNS, boiling water bath 5min, cooling, 10000rpm is centrifuged 1min, measures 0D540, before 42 DEG C of water-bath heat preservations of blank control group first
Boiling water bath 5min, other operations are consistent with the above.
Enzyme activity definition: under the determination condition, 1umol N-acetylglucosamine is generated to decompose colloid per hour
Enzyme amount is an enzyme activity unit (U/ml).
2. the expression of the chitinase of mesh
Picking single colonie is inoculated in 25ml BMGY culture medium (1% yeast powder, 2% peptone, 1% glycerol), in 28 DEG C,
200rpm shaking table culture is 4.0-8.0 or so to OD600, is transferred to equipped with 50ml BMMY culture medium (1% yeast powder, 2% egg
White peptone, 1.0% methanol, 100mmol/l phosphate buffer, pH 7.0) 500ml triangular flask in, same culture conditions continue to train
It supports, adds within every 24 hours 100% methanol in culture medium to final concentration of 1.0%, inducing expression 6 days.It is every to take at regular intervals
Sample measures the enzyme activity of its cell density (OD600) and extracellular chitinase.Testing result shows that initial pH is 7, culture temperature
Degree is 28 DEG C, and 144 hours under conditions of BMMY culture media shaking vase culture ,+IbChiA-X33 the enzyme activity of α containing pPICZ reaches highest
21U/ml.Simultaneously with the destination protein in SDS-PAGE and Western-blot detection pPICZ α+IbChiA-X33 fermentation supernatant
Matter, as a result as shown in Figure 3, wherein protein standard molecular weight is from top to bottom successively are as follows: 160kDa, 120kDa, 100kDa,
70kDa,50kDa,40kDa,30kDa,25kDa,14kDakDa；Swimming lane 1,2 is pPICZ α+IbChiA-X33 fermentation supernatant
Liquid detects the purpose band (swimming lane 1,2) of IbChiA between 25-30kDa, with Compute pI/Mw tool (http: //
Web.expasy.org/compute_pi/ the molecular weight of IbChiA) is predicted are as follows: 26.8kDa.
The bacteriostatic activity test of the sweet potato chitinase of 3 mesh of embodiment
Using sweet potato black rot pathogen and pine root fungus as strains tested, PDA culture medium is configured, is inoculated with sweet potato black rot pathogen and sweet
The bacteria cake of potato pine root fungus is uniformly placed in Oxford cup at distance center radius 2.5cm, often when mycelia covers with radius 1cm
The fermented liquid supernatant of 100ul pPICZ α+IbChiA-X33 is added in a Oxford cup, and distilled water is control, continues to cultivate, and observation should
Chitinase bacteriostatic activity, as a result as shown in Figure 4, wherein A: alternaria；B: pine root fungus；0: distilled water；1,2,3:
The fermentation supernatant of IbChiA, it is seen that the sweet potato chitinase has apparent inhibiting effect to two kinds of germs, is that it is inhibiting to plant
Application in object disease fungus is laid a good foundation.
Implement 4
Using being used to hydrolyze chitin for above-mentioned sweet potato chitinase to prepare chitin oligo saccharide, this generating mode produces item
Part is mildly controllable, the few advantage of environmental pollution, alternative existing chemical method.
Sequence table
<110>Jiangsu Normal University
<120>one plants of recombinant yeast pichia pastoris, its expression product and its application
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 804
<212> DNA
<213>sweet potato (Dioscorea esculenta Lour. Burkill)
<400> 1
atgagggtta ttctgggatt gggtttggtt tgtgttgccc tgtctgtcct gacagggaca 60
ataatggcgc agggtgtggg gtccattgtg acaaaaccgt tgtttgatga gatgcttaag 120
catcgtaacg acgcaaactg tgcgagtggg ttttacacct acgaagcttt cattcaggcg 180
gctaactcct tcgccgcttt tgggaccgcc ggcgacgttg atactcgtaa gagagagatt 240
gctgcctttt tggctcaaac ttctcacgaa actactggtg gatgggcaac tgcaccagat 300
ggaccatatg catggggata ctgcttcaag caagaacaag gcaacccacc agattactgt 360
caagcaagcc aagagtggcc ttgtgctccc ggcaagaagt attttggtcg tggtcctatc 420
caaatttcct acaacttcaa ctatggtcca gctgggaaag ccataggatc ggatctacta 480
aacaacccgg atttggtggc taccgacccg gtgatatcct tcaagacggc cttctggttc 540
tggatgacac cccaatctcc caagccgtcg gcccacgccg tcatgaccgg cggatggact 600
ccgtcggcgg cagacaccgc cgccggccgc gtccccggct acggtgtggt caccaacatt 660
atcaacggcg ggattgagtg cgggaaggga tctaacccgc agatggagga taggattggg 720
ttttacaaga gatattgtga cattcttgga gttggatatg gcaataattt ggattgcgcc 780
aaccaaaggc cttttggctc ctaa 804
Claims (6)
1. one plant of recombinant yeast pichia pastoris, for the host cell of recombinant vector conversion, which is characterized in that the recombinant vector contains
The nucleotides sequence of sweet potato chitinase gene, the sweet potato chitinase gene is classified as one of following sequence:
(1) nucleotide sequence as shown in SEQ ID No.1；
(2) nucleotide sequence is added as shown in SEQ ID No.1, replace, be inserted into or lack one or several nucleotide and
At homologous sequence；
(3) nucleotides sequence of the allele of the nucleotide sequence as shown in SEQ ID No.1 or the allele derivative
Column.
2. a kind of method for preparing sweet potato chitinase, which is characterized in that comprising steps of
S1: recombinant yeast pichia pastoris described in culture claim 1；
S2: inducing the sweet potato chitinase gene in the recombinant yeast pichia pastoris to express, and obtains sweet potato chitinase.
3. a kind of method for preparing sweet potato chitinase as described in right 2, which is characterized in that the culture item in the step S1
Part is 28 DEG C, and initial pH=7,200rpm are cultivated 144 hours；Inductive condition in the step S2 is that every 24 hours supplements are primary
Methanol, inducing expression 6 days.
4. sweet potato chitinase prepared by a kind of method for preparing sweet potato chitinase described in right 2 or 3.
5. sweet potato chitinase as claimed in claim 4 is inhibiting the application in plant pathogenic fungi.
6. application of the sweet potato chitinase as claimed in claim 4 in hydrolysis chitin.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110358781A (en) * | 2019-07-31 | 2019-10-22 | 湖北大学 | A kind of acidic mammalian chitinase encoding gene and application |
| CN111286464A (en) * | 2020-04-06 | 2020-06-16 | 湖北大学 | Engineering bacteria for efficiently expressing chitinase and application of engineering bacteria in plant growth promotion |
| CN111763665A (en) * | 2019-04-01 | 2020-10-13 | 江苏师范大学 | Preparation method of high-activity sweet potato chitinase and application of high-activity sweet potato chitinase in preparation of plant pathogenic fungus antibacterial agent |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101307319A (en) * | 2008-06-06 | 2008-11-19 | 浙江工商大学 | Recombined endo-chitinase gene order and recombinant vector threreof |
| CN103305539A (en) * | 2012-03-07 | 2013-09-18 | 青岛农业大学 | Trichoderma asperellum chitinase gene and method thereof for expressing trichoderma asperellum chitinase |
| CN107318839A (en) * | 2016-04-28 | 2017-11-07 | 闫合 | A kind of bactericide and its application |
| CN109897861A (en) * | 2019-02-20 | 2019-06-18 | 中国水产科学研究院黄海水产研究所 | A kind of Portunus trituberculatus Miers chitinase gene and its recombinant expression protein and application |
-
2018
- 2018-03-30 CN CN201810297225.6A patent/CN110317740B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101307319A (en) * | 2008-06-06 | 2008-11-19 | 浙江工商大学 | Recombined endo-chitinase gene order and recombinant vector threreof |
| CN103305539A (en) * | 2012-03-07 | 2013-09-18 | 青岛农业大学 | Trichoderma asperellum chitinase gene and method thereof for expressing trichoderma asperellum chitinase |
| CN107318839A (en) * | 2016-04-28 | 2017-11-07 | 闫合 | A kind of bactericide and its application |
| CN109897861A (en) * | 2019-02-20 | 2019-06-18 | 中国水产科学研究院黄海水产研究所 | A kind of Portunus trituberculatus Miers chitinase gene and its recombinant expression protein and application |
Non-Patent Citations (6)
| Title |
|---|
| NCBI: ""PREDICTED:Ipomoea nil basic endochitinase-like(LOC109177747),mRNA"", 《GENBANK DATABASE》 * |
| WEN-CHI HOU等: ""Chitinase activity of sweet potato(Ipomoes babatas[L.]Lam var.Tainong 57"", 《BOT.BULL.ACAD.SIN》 * |
| YANHUA FAN等: ""Expression of a Beauveria bassiana chitinase(Bbchit1) in Escherichia coli and Pichia pastoris"", 《PROTEIN EXPRESSION AND PURIFICATION》 * |
| 左豫虎等: ""β-1,3-葡聚糖酶和几丁质酶活性与大豆对疫霉根腐病抗性的关系"", 《植物病理学报》 * |
| 张健等: ""甘薯几丁质酶的性质研究"", 《江苏农业科学》 * |
| 朱刘影等: ""核黄素和脱乙酰几丁质对甘薯几丁质酶的诱导作用"", 《江苏农业科学》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111763665A (en) * | 2019-04-01 | 2020-10-13 | 江苏师范大学 | Preparation method of high-activity sweet potato chitinase and application of high-activity sweet potato chitinase in preparation of plant pathogenic fungus antibacterial agent |
| CN110358781A (en) * | 2019-07-31 | 2019-10-22 | 湖北大学 | A kind of acidic mammalian chitinase encoding gene and application |
| CN111286464A (en) * | 2020-04-06 | 2020-06-16 | 湖北大学 | Engineering bacteria for efficiently expressing chitinase and application of engineering bacteria in plant growth promotion |
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