CN110004173A - A method of it obtaining non-transgenic shelf-stable and eats fructus lycii raw - Google Patents

A method of it obtaining non-transgenic shelf-stable and eats fructus lycii raw Download PDF

Info

Publication number
CN110004173A
CN110004173A CN201910276123.0A CN201910276123A CN110004173A CN 110004173 A CN110004173 A CN 110004173A CN 201910276123 A CN201910276123 A CN 201910276123A CN 110004173 A CN110004173 A CN 110004173A
Authority
CN
China
Prior art keywords
sequence
leu
fructus lycii
lys
sgrna
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN201910276123.0A
Other languages
Chinese (zh)
Other versions
CN110004173B (en
Inventor
张康
高崑
冉毅东
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Ji Nuowo Bio Tech Ltd Tianjin
Original Assignee
Ji Nuowo Bio Tech Ltd Tianjin
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Ji Nuowo Bio Tech Ltd Tianjin filed Critical Ji Nuowo Bio Tech Ltd Tianjin
Priority to CN201910276123.0A priority Critical patent/CN110004173B/en
Publication of CN110004173A publication Critical patent/CN110004173A/en
Application granted granted Critical
Publication of CN110004173B publication Critical patent/CN110004173B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • 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
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8213—Targeted insertion of genes into the plant genome by homologous recombination
    • 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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • 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)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • 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
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • 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
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
    • C12Y301/01011—Pectinesterase (3.1.1.11)
    • 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)
    • C12Y302/01015—Polygalacturonase (3.2.1.15)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y402/00—Carbon-oxygen lyases (4.2)
    • C12Y402/02—Carbon-oxygen lyases (4.2) acting on polysaccharides (4.2.2)
    • C12Y402/02002—Pectate lyase (4.2.2.2)
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
    • 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
    • C12N2810/00—Vectors comprising a targeting moiety
    • C12N2810/10—Vectors comprising a non-peptidic targeting moiety

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Cell Biology (AREA)
  • Mycology (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)

Abstract

The invention discloses a kind of methods of acquisition non-transgenic shelf-stable fresh food fructus lycii.The present invention provides a kind of methods of cultivation shelf-stable fresh food fructus lycii, include the following steps: the expression for inhibiting pectin lyase enzyme gene in purpose fructus lycii, to make fructus lycii shelf-stable；Or inhibit the content or activity of pectin lyase in purpose fructus lycii, to make fructus lycii shelf-stable.The nutritive value of medlar fresh fruit is significantly higher than dry fruit, but it is the main reason for limiting fresh fruit sale that shelf life is short.This method can rapidly obtain the new varieties of shelf-stable compared to traditional breeding technology.

Description

A method of it obtaining non-transgenic shelf-stable and eats fructus lycii raw
Technical field
The invention belongs to biotechnology breeding fields, are related to a kind of method of acquisition non-transgenic shelf-stable fresh food fructus lycii, In particular to a kind of to utilize genome editing technique rite-directed mutagenesis lycium barbarum pectin lyase related gene, obtain the resistance to storage of fructus lycii The method for depositing breeding material.
Background technique
Fructus lycii (Lycium barbarumL.) is the important resources of medicinal plant in China.Containing polysaccharides, glycine betaine, Chinese holly Qi pigment etc., modern medicine prove that fructus lycii has extensive medical value.Furthermore fructus lycii is drought-resistant, is suitable for sand ground growth, therefore It can be used as the shrub of water and soil conservation, and due to its saline-alkali tolerant, become salt-soda soil and open tree pioneer.
The various nutritional ingredients of medlar fresh fruit are higher than dry fruit, have been increasingly becoming by the favorite seasonal fruit of consumer. It is few sweet in flavor that fresh food fructus lycii has big fruit, thin skin meat thickness, seed.But medlar fresh fruit is berry, and moisture content is up to 80%, and is rich in Sugar, the speed to go bad after fruit harvesting is fast, easily goes rotten, and fruit supplies the phase and shelf life is very short, it is difficult to meet the market demand. Though thin skin meat is thick to improve mouthfeel, the difficulty of storage and transportational process is increased.So that the selling cost of medlar fresh fruit is very high, Dangerous.
Summary of the invention
In order to obtain non-transgenic shelf-stable fresh food fructus lycii, the present invention provides the following technical scheme that
The present invention provides a kind of methods of cultivation shelf-stable fresh food fructus lycii, include the following steps:
The expression for inhibiting pectin lyase enzyme gene in purpose fructus lycii, to make fructus lycii shelf-stable；
Or inhibit the content or activity of pectin lyase in purpose fructus lycii, to make fructus lycii shelf-stable.
In the above method, the pectin lyase is following (a1) or (a2) or (a3):
(a1) protein shown in the sequence 3 of sequence table；
(a2) (a1) by the substitution and/or deletion and/or addition of one or several amino acid residues and is had identical The protein of function；
(a3) with (a1) have 99% or more, 95% or more, 90% or more, 85% or more or 80% or more identity and Protein with the same function.
In the above method, the pectin lyase enzyme gene is any in following (b1)-(b4):
(b1) code area DNA molecular as shown in the sequence 1 of sequence table；
(b2) code area DNA molecular as shown in the sequence 2 of sequence table；
(b3) hybridize and encode DNA points of pectin lyase with (b1) or (b2) DNA molecular limited under strict conditions Son；
(b 4) and (b1) or (b2) has 99% or more, 95% or more, 90% or more, 85% or more or 80% or more Identity and the DNA molecular for encoding pectin lyase.
It is described to inhibit the expression of pectin lyase enzyme gene in fructus lycii or inhibit pectin lyase in fructus lycii in the above method Content or activity are realized by CRISPR/Cas9 system.
In the above method, the CRISPR/Cas9 system includes sgRNA and Cas9 albumen；
Or the CRISPR/Cas9 system includes the plasmid for expressing sgRNA and Cas9 albumen；
The target sequence of the sgRNA is following (c1) or (c2):
(c1) nucleic acid molecules shown in the sequence 7 of sequence table；
(c2) there is 99% or more, 95% or more, 90% or more, 85% or more or 80% or more identity with (c1) Nucleic acid molecules.
Or described it is embodied as the sgRNA and the Cas9 albumen importing purpose fructus lycii by CRISPR/Cas9 system In.
The method that the inhereditary material or the non-inhereditary material are imported into the cell or tissue of the fructus lycii is gene Marksmanship, Agrobacterium infestation method, PEG induction protoplasm body or other any introduction methods.
In the above method, the nucleotides sequence of the sgRNA is classified as the RNA or sequence 10 that sequence 4 encodes in sequence table；
The sgRNA is following (d1) or (d2) or (d3):
(d1) RNA that the sequence 4 of sequence table encodes；
(d2) nucleic acid molecules shown in the sequence 10 of sequence table；
(d3) same with 99% or more, 95% or more, 90% or more, 85% or more or 80% or more with (d1) or (d2) The nucleic acid molecules of one property.
Or the amino acid sequence of the Cas9 albumen is sequence 11.
Another object of the present invention is to provide a kind of method for preparing shelf-stable fresh food fructus lycii.
Method provided by the invention, include the following steps: in the cell or tissue of purpose fructus lycii import Cas9 albumen and Special sgRNA obtains the fresh food fructus lycii of shelf-stable；
The target sequence of the sgRNA is following (c1) or (c2):
(c1) nucleic acid molecules shown in the sequence 7 of sequence table；
(c2) there is 99% or more, 95% or more, 90% or more, 85% or more or 80% or more identity with (c1) Nucleic acid molecules.
In the above method, the sgRNA is following (d1) or (d2) or (d3):
(d1) RNA that the sequence 4 of sequence table encodes；
(d2) nucleic acid molecules shown in the sequence 10 of sequence table；
(d3) same with 99% or more, 95% or more, 90% or more, 85% or more or 80% or more with (d1) or (d2) The nucleic acid molecules of one property.
3rd purpose of the invention is to provide a kind of sgRNA or expresses its plasmid.
SgRNA provided by the invention, target sequence are following (c1) or (c2):
(c1) nucleic acid molecules shown in the sequence 7 of sequence table；
(c2) there is 99% or more, 95% or more, 90% or more, 85% or more or 80% or more identity with (c1) Nucleic acid molecules.
The present invention also provides a kind of albumen, for as follows (a1) or (a2) or (a3):
(a1) protein shown in the sequence 3 of sequence table；
(a2) (a1) by the substitution and/or deletion and/or addition of one or several amino acid residues and is had identical The protein of function；
(a3) with (a1) have 99% or more, 95% or more, 90% or more, 85% or more or 80% or more identity and Protein with the same function.
Among the above, the sgRNA is following (d1) or (d2) or (d3):
(d1) RNA that the sequence 4 of sequence table encodes；
(d2) nucleic acid molecules shown in the sequence 10 of sequence table；
(d3) same with 99% or more, 95% or more, 90% or more, 85% or more or 80% or more with (d1) or (d2) The nucleic acid molecules of one property.
The cell is any cell that can be simultaneously regenerated as intact plant by tissue cultures as importing receptor；It is described Tissue is any tissue that can be simultaneously regenerated as intact plant by tissue cultures as importing receptor；Specifically, the cell For protoplasm somatocyte or suspension cell；The tissue is leaf dish, stem section or cotyledon.
The nutritive value of medlar fresh fruit is significantly higher than dry fruit, but it is the main reason for limiting fresh fruit sale that shelf life is short.This Method can rapidly obtain the new varieties of shelf-stable compared to traditional breeding technology.In safety, this method is special using sequence The instant expression method of different nuclease can make artificial nuclease gene unconformity to acceptor gene group, while obtain without transgenosis The genetic stocks that the fixed point of trace knocks out.Fixed point knocks out compared with traditional transgenic technology, considerably reduces due to the machine transplanting of rice Enter and integrate the security risks that brought non-premixed flame generates.Therefore, the technology turns than traditional from technological layer Gene technology safety with higher.In cost, this method considerably reduces the population sample quantity for needing to screen, and only needs The breeding cycle for taking 2-3, the breeding cycle than transgenosis 8-10 years significantly shorten.In policy, based on the consideration in safety, This method can obtain the examination & approval of policy with being more rapidly also easier to.
Detailed description of the invention
Fig. 1 is the figure of fructus lycii LbPL gene target site to be knocked out.
Fig. 2 is wild peaceful Qi 7 and picks the gentle contrast effect figure for putting 15 days of rear chamber with pl mutant fresh fruit.
Fig. 3 is PHSE401-LbPL1 structure chart.
Specific embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
The determination of embodiment 1, the clone of fructus lycii LbPL gene and target sequence
One, the clone of fructus lycii LbPL gene
The cDNA obtained using fructus lycii blade total serum IgE reverse transcription expands fructus lycii pectin lyase as template, using RT-PCR method The full-length cDNA of enzyme LbPL gene.Conserved sequence design primer based on plant of Solanaceae tomato PL gene
F:ATGGG(G/C)CACTTCCTCTGTTTT
R:TT(T/C)AGCAACGAGAACCCTTTTTAC
It is expanded with primer combination F/R, it is complete that RT-PCR result, which obtains the segment (Fig. 2) of 1212bp through sequencing, Opening code-reading frame encodes 403 amino acid.It is compared by Blast, is LbPL according to the unnamed gene that homology is represented.
The sequence of the genomic DNA of LbPL gene is sequence 1, and cDNA sequence is sequence 2；The albumen LbPL of gene coding Amino acid sequence be sequence 3.
Two, the selection of fructus lycii LbPL gene target site to be knocked out
LbPL gene locus number is Solyc03g111690, is located at the 3rd article of chromosome of fructus lycii, comprising 4 exons, 3 Introne, encodes 403 amino acid altogether, and the target site of building knockout carrier selection is located in exon 2 (Fig. 1).
The sequence of target site sequence and its sgRNA can be following 3 kinds:
The coded sequence of sgRNA-1 are as follows: GGGTATTTATCGTGTGGTACCGG (sequence 4), target sequence 5 '- GGGTATTTATCGTGTGGTACCGG-3 ' (sequence 7)；
The coded sequence of sgRNA-2 are as follows: CGGTTAGCTGACTGTGCAATTGG (sequence 5), target sequence 5 '- CGGTTAGCTGACTGTGCAATTGG-3 (sequence 8)；
The coded sequence of sgRNA-3 are as follows: ACTGTGGATTATTTTCGCGAGGG (sequence 6), target sequence 5 '- ACTGTGGATTATTTTCGCGAGGG-3 (sequence 9).
The mutation efficiency highest of sgRNA-1 is analyzed, therefore sgRNA-1 is selected to carry out follow-up work.
The knockout of embodiment 2, LbPL gene
One, the sgRNA of the preparation of Cas9 albumen and in-vitro transcription
1, the preparation of Cas9 albumen
It presents and is obtained containing purified Cas9 albumen (ammonia from Inst. of Genetics and Development Biology, CAS's Developmental Biology research Base acid sequence is sequence 11).
2, the in-vitro transcription of target site sgRNA
The target sequence of sgRNA-1 is Target-LbPL1:5 '-GGGTATTTATCGTGTGGTACCGG-3 ' (sequence 7)
1), the building of recombinant plasmid
Synthesize following single-stranded primers with cohesive end (underscore part): LbPL1F:5 '- ATTGGGGTATTTATCGTGTGGTAC-3'；LbPL1R:5 '-AAACGTACCACACGATAAATACCC-3 ' is moved back by primer Fiery program is formed with the double-stranded DNA of cohesive end；
The double-stranded DNA of toughness end pHSE401 carrier is inserted into again (to record in the following literature: A CRISPR/ Cas9toolkit for multiplex genome editing in plants.Xing HL,Dong L,Wang ZP, Zhang HY,Han CY,Liu B,Wang XC,Chen QJ.BMC Plant Biol.2014Nov 29；14 (1): 327) To get the plasmid containing target-LbPL1 between two BsaI restriction enzyme sites.
Plasmid has sequence 4 (the corresponding non-coding DNA molecules of sgRNA) institute through sequence verification positive plasmid, the positive plasmid The DNA molecular shown, the sgRNA that expressed sequence 4 encodes, the positive plasmid are named as PHSE401-LbPL1 (Fig. 3).
2, the in-vitro transcription of the sgRNA of the target site containing LbPL
Synthetic primer:
T7-LbPL-sgRNA-F:TAATACGACTCACTATAGGGGGTATTTATCGTGTGGTAC,
GRNA-Sc-R:AAAAGCACCGACTCGGTGCCA；
PCR amplification is carried out by template of PHSE401-LbPL1, obtained PCR product is purified with PCR purification kit (Quan Shijin EP101-02), PCR product are the DNA molecular containing T7 promoter and LbPL target spot sgRNA1, utilize Quan Shijin's It is synthesized the RNA (sgRNA-LbPL1) containing T7 promoter and LbPL target spot sgRNA1 by T7 in-vitro transcription kit in vitro: ggguauuuaucgugugguacguuuuagagc uagaaauagc aaguuaaaau aaggcuaguc cguuaucaac Uugaaaaagu ggcaccgagu cggugcuuuu uuu (sequence 10), and add PolyA tail in the 3 ' ends of sgRNA, with Improve the stability of mRNA.
SgRNA-LbPL1 exists as a solution, solute sgRNA-LbPL1, and solvent is water, and concentration is 250ng/ μ L。
Two, fructus lycii LbPL gene is carried out using the Cas9 albumen of Bombardment-Mediated Transformation purifying and the sgRNA of in-vitro transcription Fixed point editor
The Cas9 albumen and sgRNA-LbPL1 that above-mentioned one is obtained are peaceful by the method importing lycium barbarum of via Particle Bombardment Transformation In Qi 7.Using peaceful Qi 7 leaf dishes as transformation receptor, complete regenerated plant is obtained by tissue cultures after conversion, carries out molecule Detection, filters out mutant, specific as follows:
1, silica loads Cas9 albumen and sgRNA-LbPL1
Choosing the silica Au-MSN that aperture is 10nm is medium, and the Au-MSN of 20mg is added to the phosphorus of 5mL pH7.4 It is ultrasonically treated in phthalate buffer (PBS), then adds the purified Cas9 albumen of 7mg.By this mixture at 22 DEG C Stirring 24 hours, then be centrifuged with 12000rpm, supernatant is abandoned, precipitating is spun up with PBS buffer solution again, obtains Cas9 albumen-Au- MSN (10 μ g/ μ L) medium.The sgRNA-LbPL1 (250ng/ μ L) of the in-vitro transcription of 4 μ L is added to 10 μ L Cas9 albumen- In Au-MSN (10 μ g/ μ L) medium, the CaCl of 12.5 μ L 2.5M is added2With the spermidine of 5 μ L 0.1M, then 5000rpm from Heart 15s abandons supernatant and uses, then twice, then with 100% alcohol of 5 μ L has hanged the load that mRNA is wrapped up with 100% ethanol wash precipitating There is the Au-MSN of Cas9 albumen, forms sgRNA-Cas9-Au-MSN complex.
2, via Particle Bombardment Transformation fructus lycii leaf dish
1) it takes the seed of peaceful Qi 7 to be placed on after 3%NaClO sterilizes on MS culture medium to germinate, then clip every two weeks The stem section of 2cm expands in cuttage to 1/2MS culture medium numerous.
2) two layers of neutral filter paper, clip Chinese holly are covered on induced medium (MS+1.0mg/L NAA+0.5mg/L 6-BA) Qi aseptic blade, distal shaft end upward, are placed in induced medium, and preculture 1 day.
3) blade is bombarded with Bio-Rad PDS-1000/He particle gun, by the sgRNA-Cas9-Au-MSN of 5 μ L Complex is loaded on load sample film；It is bombarded using particle gun, the target distance of each bombardment is 6cm, bombarding pressure For 1100psi, bombardment diameter is 2cm.
4) blade after bombarding is placed into 25 DEG C of illumination cultivations 2 days, is then cut into 9mm2Square, be seeded in induction respectively On culture medium, 25 DEG C illumination cultivation 2-4 weeks, subculture is primary every two weeks.
5) by the callus subculture induced to differential medium (MS+0.1mg/L NAA+0.5mg/L 6-BA), Induce seedling.
It 6) will be on the seedling subculture of 2cm or more to root media (MS+0.2mg/L NAA+0.01mg/L 6-BA).14- After 28 days, T0 is obtained for fructus lycii plant after gene editing.
Three, Molecular Detection
T0 is extracted into genomic DNA for fructus lycii plant shoots after gene editing, using the DNA as template, carries out PCR/RE (Polymerase Chain Reaction/Restriction digestion) experimental analysis.Wild type fructus lycii is set simultaneously DNA as control.PCR/RE analysis method is with reference to document Shan, Qi.et al.Rapid and efficient gene Modification in rice and Brachypodium using TALENs.Molecular Plant (2013), due to There are the identification sequences (5 '-GGTACC-3 ') of restriction enzyme on the target fragments of fructus lycii endogenous gene LbPL, thus test It is middle that polymerase chain reaction-restriction endonuclease analysis (PCR/RE) experiment is carried out using restriction enzyme (PstI).
Wherein, PCR amplification the primer are as follows:
LbPL-F:5 '-CCTCCTTGCCTCCTCTAATCC-3 '；
LbPL-R:5 '-ACAGTTCTCCACCCGTAATGC-3 '.
Because target spot sgRNA-1 is selected at pstI restriction enzyme site, therefore if running adhesive tape band is two, display PCR expands Increase band to be cut open, for hint Plant Genome there is no variation at the target spot, plant is not just knockout mutations body；If run Adhesive tape band is one, and display PCR amplification band is not cut open, and implies that Plant Genome Plant Genome occurs at the target spot Variation, plant is Mutants homozygous；If running adhesive tape band is three, display PCR amplification band one is cut open, and one is not cut It opens, implies that Plant Genome chain at the target spot morphs, for a chain there is no variation, plant is heterozygous mutant Body.
PCR/RE experiment analysis results show that the LbPL gene target site after gene editing in fructus lycii plant shoots has occurred Mutation.Band in recycling, sequencer map, sequencing result show that base insertion/deletion all has occurred in the target site of LbPL gene The mutation of type；
Here is a mutation as a result, deleting a base according to first site:
AATTTAGGGTATTTATCGTGTGGTACCGGAAATCC WT
AATTTAGGGTATTTATCGTGTG.TACCGGAAATCC pl。
Plant with the mutation is named as T0 for fructus lycii mutant plants pl.
506 leaf dishes have been bombarded, 3662 fructus lycii plant are obtained, have all carried out PCR/RE experiment detection, discovery is wherein 52 mutant, wherein 36 are Mutants homozygous.
Four, the fructescence identification of pl Mutants homozygous
It is pure for fructus lycii to obtain T0 for Mutants homozygous is selected in fructus lycii mutant plants pl by the T0 that above-mentioned three screening is obtained Close mutant plants pl.
T0 is planted with the peaceful Qi of same size wild type 7 in peaceful Xia Zhongning's Newcastle town for fructus lycii Mutants homozygous plant pl The big Tanaka in the village Song Ying, all plants are divided into two groups, every group each 100 plants.T0 is for fructus lycii Mutants homozygous plant pl and wild type 7 It is number consistent in fertilising, watering, trimming and other farmland management modes, the 3rd year, fresh fruit was harvested in the same time.By comparing fresh Fruit single fruit weight, single plant yield, color, soluble solid, polysaccharides, fructus lycii glycine betaine are found without significant difference.
But in identical environment item after T0 is picked for fructus lycii Mutants homozygous plant pl with wild type 7 (WT) fresh fruits Part: 24 degree, place 15d in the incubator that relative humidity is 20% after, wild type is obviously bigger than the degree that addles of mutant (as schemed 2)。
Sequence table
<110>Tianjin Ji Nuowo Biotechnology Co., Ltd
<120>a kind of method for obtaining non-transgenic shelf-stable fresh food fructus lycii
<160> 11
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2152
<212> DNA
<213> Artificial sequence
<400> 1
tatataactt aaacgcgttt aattggacgt atgctagagt agtaatcata tgctaacaag 60
agatagatac tgttacttga acagacaaaa actaccgtag tcttcttctc tctatcatct 120
aggttagagg aatctccgta aaacatgtaa tcacgaagga acaagggacc catccctttt 180
gtttaattac gctaatttct ctactttttc cttctttata aactcctcca tttccctctc 240
ttttcttcat aacccaaaat cctctgtttt taaagctaat ttataaaaca aacaatgggc 300
acttcctctg tttttctact attccttctt tcttttcttc tccttctccc gtccctcctt 360
gcctcctcta atcctcaaca agttgtcgat gaagtacaca ggtacgtgca tttttaattt 420
tattttaatt caacattgca tatttgaaaa gggacagagt cagataggaa caaggagttc 480
attcgtatta aaataattgc atatggtaat actcatgtgt acattattat tataataatg 540
caggagtata aatggttcaa ggaggaattt agggtattta tcgtgtggta ccggaaatcc 600
aatcgacgat tgttggcgtt gtgatccgaa ttgggagaaa aaccgtcagc ggttagctga 660
ctgtgcaatt ggatttggga aaaacgctat tggtggtaga gacggtaaaa tctacgtggt 720
gaccgattcg ggtgacgaca atgccgtcac tccgaagccg gggactctcc ggcacgcagt 780
gattcagact gagccactgt ggattatttt cgcgagggat atggttatac agttgaaaga 840
ggaattaatt atgaattcgt ttaaaacgat agacggaagg ggcgctagcg tacacatagc 900
gggtggtccg tgtataacaa tacagtacgt aacgaatatt attatacacg gaattcatat 960
acatgactgt aaacaaggtg gaaatgctat ggtgcggagc agtccatcgc attacgggtg 1020
gagaactgtt tcggacggtg atggagtgtc catattcggt gggagccatg tttgggtgga 1080
tcattgttct ttgtccaact gtaaggacgg tctgattgat gcgattatgg ggtctacagc 1140
aataaccatt tcaaataatt acatgacaca tcatgataaa gttatgctct tgggacatag 1200
tgatacatat actcaagata aaaacatgca agtaactata gcttttaatc actttggtga 1260
aggtcttgtc caaagaatgc caaggtatga gcctgagtta tttaactttt atatactgac 1320
aattttgaca atgtgaattt tatgaattat tatttggaat aaatgcagat gtagacatgg 1380
ttacttccat gtggtgaaca atgactacac acattgggaa atgtatgcta ttggtggaag 1440
tgctgatccc actatcaata gtcaagggaa ccgatttctt gcccctgata tcaggtttag 1500
caaagaggtt agttagttat ttcacttaca tttttctatc tagtctgtta atgagttaac 1560
gggtaatttt attatattta tgtaggttac aaagcacgag gatgcaccgg agagtgaatg 1620
gaagaattgg aattggagga ccgatgggga cctaatgttg aatggtgcat tttttacgcg 1680
atcaggagtt agaactggtt catcaagtta tgctaaagct tcgagtttga gtgcaaggcc 1740
gtcttcatta gtagccaatc ttgtgtctag ttctggtgca ctcaactgta aaaagggttc 1800
tcgttgctaa ttattattat tatttcctaa aaaagacaca agaaccaaag caaaagggga 1860
ttaattattt aacttggttt tgaaaaaaga aaaaggatgt aatttgaaga aaaaaaccat 1920
ctagtgaggg tgaaaaagta agggacaaga agtttagggt tgtgctttta atatcatttt 1980
ttcactccct ttatttttat ttttactttt taagtttttt ttgacttgat tgtcaaatac 2040
aacctcggcc tgttattctc agccattggg acttttagtt cttgttggtt ttagagagag 2100
gcaagctaag aagtgttgat atatacatca atatatatgt atttattttt ga 2152
<210> 2
<211> 1212
<212> DNA
<213> Artificial sequence
<400> 2
atgggcactt cctctgtttt tctactattc cttctttctt ttcttctcct tctcccgtcc 60
ctccttgcct cctctaatcc tcaacaagtt gtcgatgaag tacacaggag tataaatggt 120
tcaaggagga atttagggta tttatcgtgt ggtaccggaa atccaatcga cgattgttgg 180
cgttgtgatc cgaattggga gaaaaaccgt cagcggttag ctgactgtgc aattggattt 240
gggaaaaacg ctattggtgg tagagacggt aaaatctacg tggtgaccga ttcgggtgac 300
gacaatgccg tcactccgaa gccggggact ctccggcacg cagtgattca gactgagcca 360
ctgtggatta ttttcgcgag ggatatggtt atacagttga aagaggaatt aattatgaat 420
tcgtttaaaa cgatagacgg aaggggcgct agcgtacaca tagcgggtgg tccgtgtata 480
acaatacagt acgtaacgaa tattattata cacggaattc atatacatga ctgtaaacaa 540
ggtggaaatg ctatggtgcg gagcagtcca tcgcattacg ggtggagaac tgtttcggac 600
ggtgatggag tgtccatatt cggtgggagc catgtttggg tggatcattg ttctttgtcc 660
aactgtaagg acggtctgat tgatgcgatt atggggtcta cagcaataac catttcaaat 720
aattacatga cacatcatga taaagttatg ctcttgggac atagtgatac atatactcaa 780
gataaaaaca tgcaagtaac tatagctttt aatcactttg gtgaaggtct tgtccaaaga 840
atgccaagat gtagacatgg ttacttccat gtggtgaaca atgactacac acattgggaa 900
atgtatgcta ttggtggaag tgctgatccc actatcaata gtcaagggaa ccgatttctt 960
gcccctgata tcaggtttag caaagaggtt acaaagcacg aggatgcacc ggagagtgaa 1020
tggaagaatt ggaattggag gaccgatggg gacctaatgt tgaatggtgc attttttacg 1080
cgatcaggag ttagaactgg ttcatcaagt tatgctaaag cttcgagttt gagtgcaagg 1140
ccgtcttcat tagtagccaa tcttgtgtct agttctggtg cactcaactg taaaaagggt 1200
tctcgttgct aa 1212
<210> 3
<211> 403
<212> PRT
<213> Artificial sequence
<400> 3
Met Gly Thr Ser Ser Val Phe Leu Leu Phe Leu Leu Ser Phe Leu Leu
1 5 10 15
Leu Leu Pro Ser Leu Leu Ala Ser Ser Asn Pro Gln Gln Val Val Asp
20 25 30
Glu Val His Arg Ser Ile Asn Gly Ser Arg Arg Asn Leu Gly Tyr Leu
35 40 45
Ser Cys Gly Thr Gly Asn Pro Ile Asp Asp Cys Trp Arg Cys Asp Pro
50 55 60
Asn Trp Glu Lys Asn Arg Gln Arg Leu Ala Asp Cys Ala Ile Gly Phe
65 70 75 80
Gly Lys Asn Ala Ile Gly Gly Arg Asp Gly Lys Ile Tyr Val Val Thr
85 90 95
Asp Ser Gly Asp Asp Asn Ala Val Thr Pro Lys Pro Gly Thr Leu Arg
100 105 110
His Ala Val Ile Gln Thr Glu Pro Leu Trp Ile Ile Phe Ala Arg Asp
115 120 125
Met Val Ile Gln Leu Lys Glu Glu Leu Ile Met Asn Ser Phe Lys Thr
130 135 140
Ile Asp Gly Arg Gly Ala Ser Val His Ile Ala Gly Gly Pro Cys Ile
145 150 155 160
Thr Ile Gln Tyr Val Thr Asn Ile Ile Ile His Gly Ile His Ile His
165 170 175
Asp Cys Lys Gln Gly Gly Asn Ala Met Val Arg Ser Ser Pro Ser His
180 185 190
Tyr Gly Trp Arg Thr Val Ser Asp Gly Asp Gly Val Ser Ile Phe Gly
195 200 205
Gly Ser His Val Trp Val Asp His Cys Ser Leu Ser Asn Cys Lys Asp
210 215 220
Gly Leu Ile Asp Ala Ile Met Gly Ser Thr Ala Ile Thr Ile Ser Asn
225 230 235 240
Asn Tyr Met Thr His His Asp Lys Val Met Leu Leu Gly His Ser Asp
245 250 255
Thr Tyr Thr Gln Asp Lys Asn Met Gln Val Thr Ile Ala Phe Asn His
260 265 270
Phe Gly Glu Gly Leu Val Gln Arg Met Pro Arg Cys Arg His Gly Tyr
275 280 285
Phe His Val Val Asn Asn Asp Tyr Thr His Trp Glu Met Tyr Ala Ile
290 295 300
Gly Gly Ser Ala Asp Pro Thr Ile Asn Ser Gln Gly Asn Arg Phe Leu
305 310 315 320
Ala Pro Asp Ile Arg Phe Ser Lys Glu Val Thr Lys His Glu Asp Ala
325 330 335
Pro Glu Ser Glu Trp Lys Asn Trp Asn Trp Arg Thr Asp Gly Asp Leu
340 345 350
Met Leu Asn Gly Ala Phe Phe Thr Arg Ser Gly Val Arg Thr Gly Ser
355 360 365
Ser Ser Tyr Ala Lys Ala Ser Ser Leu Ser Ala Arg Pro Ser Ser Leu
370 375 380
Val Ala Asn Leu Val Ser Ser Ser Gly Ala Leu Asn Cys Lys Lys Gly
385 390 395 400
Ser Arg Cys
<210> 4
<211> 23
<212> DNA
<213> Artificial sequence
<400> 4
gggtatttat cgtgtggtac cgg 23
<210> 5
<211> 23
<212> DNA
<213> Artificial sequence
<400> 5
cggttagctg actgtgcaat tgg 23
<210> 6
<211> 23
<212> DNA
<213> Artificial sequence
<400> 6
actgtggatt attttcgcga ggg 23
<210> 7
<211> 23
<212> DNA
<213> Artificial sequence
<400> 7
gggtatttat cgtgtggtac cgg 23
<210> 8
<211> 23
<212> DNA
<213> Artificial sequence
<400> 8
cggttagctg actgtgcaat tgg 23
<210> 9
<211> 23
<212> DNA
<213> Artificial sequence
<400> 9
actgtggatt attttcgcga ggg 23
<210> 10
<211> 103
<212> RNA
<213> Artificial sequence
<400> 10
ggguauuuau cgugugguac guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu uuu 103
<210> 11
<211> 1368
<212> PRT
<213> Artificial sequence
<400> 11
Met Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val
1 5 10 15
Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe
20 25 30
Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile
35 40 45
Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu
50 55 60
Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys
65 70 75 80
Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser
85 90 95
Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys
100 105 110
His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr
115 120 125
His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp
130 135 140
Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His
145 150 155 160
Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro
165 170 175
Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr
180 185 190
Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala
195 200 205
Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn
210 215 220
Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn
225 230 235 240
Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe
245 250 255
Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp
260 265 270
Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp
275 280 285
Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp
290 295 300
Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser
305 310 315 320
Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys
325 330 335
Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe
340 345 350
Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser
355 360 365
Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp
370 375 380
Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg
385 390 395 400
Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu
405 410 415
Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe
420 425 430
Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile
435 440 445
Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp
450 455 460
Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu
465 470 475 480
Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr
485 490 495
Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser
500 505 510
Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys
515 520 525
Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln
530 535 540
Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr
545 550 555 560
Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp
565 570 575
Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly
580 585 590
Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp
595 600 605
Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr
610 615 620
Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala
625 630 635 640
His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr
645 650 655
Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp
660 665 670
Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe
675 680 685
Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe
690 695 700
Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu
705 710 715 720
His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly
725 730 735
Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly
740 745 750
Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln
755 760 765
Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile
770 775 780
Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro
785 790 795 800
Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu
805 810 815
Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg
820 825 830
Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys
835 840 845
Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg
850 855 860
Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys
865 870 875 880
Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys
885 890 895
Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp
900 905 910
Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr
915 920 925
Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp
930 935 940
Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser
945 950 955 960
Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg
965 970 975
Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val
980 985 990
Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe
995 1000 1005
Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys
1010 1015 1020
Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser
1025 1030 1035 1040
Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu
1045 1050 1055
Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile
1060 1065 1070
Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser
1075 1080 1085
Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly
1090 1095 1100
Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile
1105 1110 1115 1120
Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser
1125 1130 1135
Pro Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly
1140 1145 1150
Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile
1155 1160 1165
Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala
1170 1175 1180
Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys
1185 1190 1195 1200
Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser
1205 1210 1215
Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr
1220 1225 1230
Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser
1235 1240 1245
Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His
1250 1255 1260
Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val
1265 1270 1275 1280
Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys
1285 1290 1295
His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu
1300 1305 1310
Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp
1315 1320 1325
Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp
1330 1335 1340
Ala Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile
1345 1350 1355 1360
Asp Leu Ser Gln Leu Gly Gly Asp
1365

Claims (10)

1. a kind of method for cultivating shelf-stable fresh food fructus lycii, includes the following steps:
The expression for inhibiting pectin lyase enzyme gene in purpose fructus lycii, to make fructus lycii shelf-stable；
Or inhibit the content or activity of pectin lyase in purpose fructus lycii, to make fructus lycii shelf-stable.
2. according to the method described in claim 1, it is characterized by: the pectin lyase be following (a1) or (a2) or (a3):
(a1) protein shown in the sequence 3 of sequence table；
(a2) (a1) by the substitution and/or deletion and/or addition of one or several amino acid residues and had into identical function Protein；
(a3) there is 99% or more, 95% or more, 90% or more, 85% or more or 80% or more identity with (a1) and have The protein of identical function.
3. according to the method described in claim 2, it is characterized by: the pectin lyase enzyme gene is in following (b1)-(b4) It is any:
(b1) code area DNA molecular as shown in the sequence 1 of sequence table；
(b2) code area DNA molecular as shown in the sequence 2 of sequence table；
(b3) hybridize under strict conditions with (b1) or (b2) DNA molecular limited and encode the DNA molecular of pectin lyase；
(b4) there is 99% or more, 95% or more, 90% or more, 85% or more or 80% or more identity with (b1) or (b2) And encode the DNA molecular of pectin lyase.
4. method according to claim 1 to 3, it is characterised in that: pectin lyase enzyme gene in the inhibition fructus lycii Expression or inhibit fructus lycii in pectin lyase content or activity be to be realized by CRISPR/Cas9 system.
5. according to the method described in claim 4, it is characterized by:
The CRISPR/Cas9 system includes sgRNA and Cas9 albumen；
Or the CRISPR/Cas9 system includes the plasmid for expressing sgRNA and Cas9 albumen；
The target sequence of the sgRNA is following (c1) or (c2):
(c1) nucleic acid molecules shown in the sequence 7 of sequence table；
(c2) there is the nucleic acid of 99% or more, 95% or more, 90% or more, 85% or more or 80% or more identity with (c1) Molecule；
Or described it is embodied as importing the sgRNA and the Cas9 albumen in purpose fructus lycii by CRISPR/Cas9 system.
6. according to the method described in claim 5, it is characterized by: the sgRNA is following (d1) or (d2) or (d3):
(d1) RNA that the sequence 4 of sequence table encodes；
(d2) nucleic acid molecules shown in the sequence 10 of sequence table；
(d3) there is 99% or more, 95% or more, 90% or more, 85% or more or 80% or more identity with (d1) or (d2) Nucleic acid molecules.
7. a kind of method for preparing shelf-stable fresh food fructus lycii, includes the following steps: to import in the cell or tissue of purpose fructus lycii Cas9 albumen and special sgRNA obtain the fresh food fructus lycii of shelf-stable；
The target sequence of the sgRNA is following (c1) or (c2):
(c1) nucleic acid molecules shown in the sequence 7 of sequence table；
(c2) there is the nucleic acid of 99% or more, 95% or more, 90% or more, 85% or more or 80% or more identity with (c1) Molecule.
8. method according to claim 7, it is characterised in that:
The sgRNA is following (d1) or (d2) or (d3):
(d1) RNA that the sequence 4 of sequence table encodes；
(d2) nucleic acid molecules shown in the sequence 10 of sequence table；
(d3) there is 99% or more, 95% or more, 90% or more, 85% or more or 80% or more identity with (d1) or (d2) Nucleic acid molecules.
9. a kind of sgRNA or the plasmid for expressing it；The target sequence of the sgRNA is following (c1) or (c2):
(c1) nucleic acid molecules shown in the sequence 7 of sequence table；
(c2) there is the nucleic acid of 99% or more, 95% or more, 90% or more, 85% or more or 80% or more identity with (c1) Molecule；
Or, a kind of albumen, for as follows (a1) or (a2) or (a3):
(a1) protein shown in the sequence 3 of sequence table；
(a2) (a1) by the substitution and/or deletion and/or addition of one or several amino acid residues and had into identical function Protein；
(a3) there is 99% or more, 95% or more, 90% or more, 85% or more or 80% or more identity with (a1) and have The protein of identical function.
10. sgRNA according to claim 9, it is characterised in that: the sgRNA is following (d1) or (d2) or (d3):
(d1) RNA that the sequence 4 of sequence table encodes；
(d2) nucleic acid molecules shown in the sequence 10 of sequence table；
(d3) there is 99% or more, 95% or more, 90% or more, 85% or more or 80% or more identity with (d1) or (d2) Nucleic acid molecules.
CN201910276123.0A 2019-04-08 2019-04-08 Method for obtaining non-transgenic storable fresh-eating Chinese wolfberry Active CN110004173B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201910276123.0A CN110004173B (en) 2019-04-08 2019-04-08 Method for obtaining non-transgenic storable fresh-eating Chinese wolfberry

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201910276123.0A CN110004173B (en) 2019-04-08 2019-04-08 Method for obtaining non-transgenic storable fresh-eating Chinese wolfberry

Publications (2)

Publication Number Publication Date
CN110004173A true CN110004173A (en) 2019-07-12
CN110004173B CN110004173B (en) 2023-03-14

Family

ID=67170269

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201910276123.0A Active CN110004173B (en) 2019-04-08 2019-04-08 Method for obtaining non-transgenic storable fresh-eating Chinese wolfberry

Country Status (1)

Country Link
CN (1) CN110004173B (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114214336A (en) * 2022-02-23 2022-03-22 中国科学院华南植物园 Lycium ruthenicum LrNOR gene and application of protein thereof
CN114470188A (en) * 2022-03-28 2022-05-13 扬州大学 Preparation method and application of lycium barbarum polysaccharide ultra-large mesoporous silica nano adjuvant

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2000253888A (en) * 1999-03-11 2000-09-19 Kao Corp Pecinic acid lyase gene
CN106755067A (en) * 2016-12-05 2017-05-31 天津吉诺沃生物科技有限公司 To plant gene pinpoint the method for insertion and the Transient expression systems of acquisition, tissue and mutant plant by transient expression
CN108610408A (en) * 2012-09-07 2018-10-02 美国陶氏益农公司 FAD2 performances locus and the target site specificity binding protein that targeted disruption can correspondingly be induced

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2000253888A (en) * 1999-03-11 2000-09-19 Kao Corp Pecinic acid lyase gene
CN108610408A (en) * 2012-09-07 2018-10-02 美国陶氏益农公司 FAD2 performances locus and the target site specificity binding protein that targeted disruption can correspondingly be induced
CN106755067A (en) * 2016-12-05 2017-05-31 天津吉诺沃生物科技有限公司 To plant gene pinpoint the method for insertion and the Transient expression systems of acquisition, tissue and mutant plant by transient expression

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
GENBANK: "NCBI Reference Sequence: NM_001365026.1", 《GENBANK》 *
张德有等: "香蕉果胶裂解酶基因的克隆", 《植物生理与分子生物学学报》 *
申艳红等: "番木瓜果肉果胶裂解酶基因克隆及反义植物表达载体的构建", 《热带作物学报》 *
黄少勇等: "CRISPR/Cas9技术在园艺作物中的研究进展", 《新疆农业科学》 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114214336A (en) * 2022-02-23 2022-03-22 中国科学院华南植物园 Lycium ruthenicum LrNOR gene and application of protein thereof
CN114214336B (en) * 2022-02-23 2022-05-03 中国科学院华南植物园 Lycium ruthenicum LrNOR gene and application of protein thereof
CN114470188A (en) * 2022-03-28 2022-05-13 扬州大学 Preparation method and application of lycium barbarum polysaccharide ultra-large mesoporous silica nano adjuvant
CN114470188B (en) * 2022-03-28 2024-01-23 扬州大学 Preparation method and application of medlar polysaccharide ultra-large mesoporous silica nanoadjuvant

Also Published As

Publication number Publication date
CN110004173B (en) 2023-03-14

Similar Documents

Publication Publication Date Title
CN101743314A (en) Transgenic plants with increased stress tolerance and yield
CN106470544A (en) The melon plant that fruit yield improves
CN103732615B (en) Jaz5A is used to improve the purposes of drought resistance in plants
CN108728420A (en) It is a kind of regulation and control crop downgrade and its yield gene and its application
CN108603197A (en) The method for improving plant nitrogen use efficiency
WO2023065966A1 (en) Application of bfne gene in tomato plant type improvement and biological yield increase
CN104611359B (en) The application of ZmSPL1 albumen and its encoding gene in regulation and control Maize Kernel Development
CN110872598A (en) Cotton drought-resistant related gene GhDT1 and application thereof
CN109609527A (en) CDPK18L gene is improving the application in tomato bacterial leaf spot resistance and high temperature resistance as negative regulatory factor
CN110358772B (en) OsEBP89 gene for improving abiotic stress resistance of rice, and preparation method and application thereof
CN110004173A (en) A method of it obtaining non-transgenic shelf-stable and eats fructus lycii raw
CN102757487A (en) Plant dwarfing related protein GA2ox, and encoding gene and application thereof
CN107022563A (en) Genetically modified plants
CN112210566B (en) Application of rice OsS6K1 gene or OsS6K2 gene in improving rice yield and/or drought resistance
CN113501867A (en) Corn drought-resistant gene ZmMYBR38 and application thereof
CN113151307A (en) Gene related to tobacco ethylene response transcription factor and application thereof
CN113684225A (en) Application of tomato SlHMGA3 gene in cultivation of tomato with delayed fruit ripening
CN108048481A (en) Application of the RLI1 albumen in adjusting and controlling rice leaf angle
CN103570805A (en) Active polypeptide and application of active polypeptide in plant root growth
CN112980876B (en) Application of GhGPAT12 protein and GhGPAT25 protein in regulation and control of cotton male reproductive development
CN111073896B (en) Gene for controlling corn grain filling, encoding product, primer, carrier and application
CN108456683A (en) The function of one adjusting and controlling rice Heading date gene SID1 and application
CN111748560B (en) Application of rice OsNRT2.1 gene in improving manganese content in rice grains
CN106811448B (en) Cotton tyrosine phosphatase GhPTP1 and its encoding gene and application
CN104962564B (en) One clone for regulating and controlling grass Plant height gene INDETERMINATE1 and application

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant