CN114480314B - Application of CRISPR/cas9 system in obtaining tomato latent She Equan urinary amino acid 3-hydroxylase mutant - Google Patents

Application of CRISPR/cas9 system in obtaining tomato latent She Equan urinary amino acid 3-hydroxylase mutant Download PDF

Info

Publication number
CN114480314B
CN114480314B CN202210127509.7A CN202210127509A CN114480314B CN 114480314 B CN114480314 B CN 114480314B CN 202210127509 A CN202210127509 A CN 202210127509A CN 114480314 B CN114480314 B CN 114480314B
Authority
CN
China
Prior art keywords
tomato
latent
amino acid
equan
crispr
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.)
Active
Application number
CN202210127509.7A
Other languages
Chinese (zh)
Other versions
CN114480314A (en
Inventor
吕志创
冀顺霞
毕思言
王晓迪
唐艳红
刘万学
张桂芬
万方浩
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.)
Institute of Plant Protection of Chinese Academy of Agricultural Sciences
Original Assignee
Institute of Plant Protection of Chinese Academy of Agricultural Sciences
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 Institute of Plant Protection of Chinese Academy of Agricultural Sciences filed Critical Institute of Plant Protection of Chinese Academy of Agricultural Sciences
Priority to CN202210127509.7A priority Critical patent/CN114480314B/en
Publication of CN114480314A publication Critical patent/CN114480314A/en
Application granted granted Critical
Publication of CN114480314B publication Critical patent/CN114480314B/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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C12N9/0073—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with NADH or NADPH as one donor, and incorporation of one atom of oxygen 1.14.13
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
    • A01K67/033—Rearing or breeding invertebrates; New breeds of invertebrates
    • A01K67/0333—Genetically modified invertebrates, e.g. transgenic, polyploid
    • A01K67/0337—Genetically modified Arthropods
    • A01K67/0339—Genetically modified insects, e.g. Drosophila melanogaster, medfly
    • 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
    • C12N15/1137—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 against enzymes
    • 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
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • 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)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
    • C12Y114/13—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with NADH or NADPH as one donor, and incorporation of one atom of oxygen (1.14.13)
    • C12Y114/13009—Kynurenine 3-monooxygenase (1.14.13.9)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2207/00—Modified animals
    • A01K2207/15—Humanized animals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/70—Invertebrates
    • A01K2227/706—Insects, e.g. Drosophila melanogaster, medfly
    • 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]
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention relates to the technical field of agricultural biology, in particular to application of a CRISPR/cas9 system in obtaining a mutant type of tomato latent She Equan urea amino acid 3-hydroxylase. The invention provides application of the tomato latent She Equan urea amino acid 3-hydroxylase gene cinnabar. The result shows that after the target gene mutation is induced by CRISPR/Cas9, the eye color of the tomato leaf miner turns red or brown, which indicates that the cinnabar gene plays a key role in canthaxanthin biosynthesis of the tomato leaf miner.

Description

Application of CRISPR/cas9 system in obtaining tomato latent She Equan urinary amino acid 3-hydroxylase mutant
Technical Field
The invention relates to the technical field of agricultural biology, in particular to application of a CRISPR/cas9 system in obtaining a mutant type of tomato latent She Equan urea amino acid 3-hydroxylase.
Background
The tomato leaf miner Tuta absorber belongs to lepidoptera, and belongs to the family of the moths, namely tomato leaf miner, tomato leaf miner and southern American tomato leaf miner, and is an important pest of tomatoes. The south america tomato leaf miner is mainly endangered by larvae, female adults like to spawn on the just-developed leaves, and once the larvae hatch, the larvae submerge into the tissues of host plants and dive into the leaves. At present, chemical pesticides are the most main measure for preventing and controlling the insects, and the chemical pesticides are continuously enhanced in drug resistance along with the continuous use of the chemical pesticides, and the chemical pesticides rapidly generate resistance to various drug targets.
CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associtdroteins) is an adaptive immune defense that organisms develop during long-term evolution, and can be used to combat invasive viruses and foreign DNA. sgRNA (singleguide RNA) with guiding effect is formed by artificial design to guide the site-directed cleavage of RNA, DNA and proteins by Cas9, cas9 can bring any fusion protein and RNA at any dsDNA sequence. The existing research results show that the CRISPR-Cas9 technology is a new method for reliably, efficiently and rapidly knocking out genes, and a plurality of mutations can be introduced at different sites. The CRISPR/Cas is utilized to carry out gene editing, so that the effect of lasting stability can be achieved, and the CRISPR/Cas is an effective genetic tool for researching gene functions. The use of functional genes with obvious phenotypic characteristics as target studies is one of the important ways to rapidly discern whether CRISPR/Cas technology is successfully constructed.
The synthesis pathway of canthaxanthin is mainly produced by tryptophan via the kynurenine pathway. Tryptophan firstly opens a pyrrole ring under the action of tryptophan oxygenase to generate formylkynurenine; then under the action of kynurenine formamidase, formylkynurenine is demethylated to generate formic acid and kynurenine; kynurenine generates 3-hydroxykynurenine under the action of kynurenine 3-hydroxylase gene cinnabar, which is a direct precursor for the synthesis of canthaxanthin. Thus, the kynurenine 3-hydroxylase gene cinnabar is a key gene in the synthesis pathway of insect canthaxanthin.
Disclosure of Invention
The invention aims to provide tomato latent She Equan urea amino acid 3-hydroxylase.
It is still another object of the present invention to provide a tomato latent She Equan urinary amino acid 3-hydroxylase gene.
It is still another object of the present invention to provide a recombinant vector comprising the tomato latent She Equan urinary amino acid 3-hydroxylase gene described above.
It is still another object of the present invention to provide the use of the above-described coding gene in the synthesis pathway of canthaxanthin of tomato leaf miner.
According to the specific embodiment of the invention, a tomato latent She Equan urinary amino acid 3-hydroxylase gene cinnabar is cloned for the first time, and the full-length nucleotide sequence of cDNA is shown as SEQ ID NO:1 is shown as follows:
ATGGCTTCACTTCAAAATGGGCATTCGAAGAAAAAATTGGATGTTGCAGTTGTGGGTGCTGGATTGGTAGGATCTTTAGAAGCTTTGTATCTAGCAAAGAGAGGTCACCGAGTTCGTCTTTACGAATACCGGGACGATATAAGACAGACCCCTCTTGTCCGCGGTCGATCTATCAATTTGGCGCTGTCTGTGCGCGGCCGCAAGGCTCTCAAAGGTGTCGGGTTAGAGGACCACATGATCAACAAACACGGTATACCGATGCGAGGGAGAATGATACACAGGCTTGACGGATCTACTTATGACATACCTTACGACGCGAGGACCAATCAGTGCATCTACTCCGTTGGAAGAAATTATCTCAATAGTTTACTTCTCGAAGAATCAGAAAAGTATGAAAATGTGGAGCGTCATTTCAATCATAAGCTACTTGAAGCTAATCTGCGTAGTGGATCTCTTACTTTTGTCAAAACCGAGAGTAACGAAACAGTTCGCGTATCAGCAGACCTGACCATAGGAGCTGATGGGGCGTTCTCCGCAGTCCGCAAAGCCATGATGAAGCAGCCACTCTTCGACTACAGCCAGAAATACATCGAACATGGATACCTGGAGCTGTGTATACCGGCGGATGAGAACGGCGGATTCCAAATGCCAGCGAACTACCTGCACATCTGGCCACGCGGCGAATTCATGATGATTGCCCTGCCGAACCAGGACTGCTCGTGGACAGTGACCCTCTTCATGCCCTTCGAGAAGTTCAAGAGTATCGAGAACGAGGAGAAACTAATCGCATTCTTCACCCAGTACTTCCCCGATTCGATCCCGTTGATCGGACGACAGAAGTTGGTTGAAGACTTCTTCGCTGGGAAACCTTCGCCTTTGGTGGCAGTGAAGTGCCGACCTTACCACGTGGAAGATAAAGCTCTAATCATAGGCGACGCTTCACACGCCGTCGTTCCGTTCTACGGGCAAGGCATGAACGCTGGCTTCGAAGACTGCACTATCTTGGACCAGCTGTTTGAAAAATATGATGATAACATAAAGGACATTCTCGAAGAGTTCTCCGCGACCAGATGGGAAGACACCTTCGCTATCAGCGATCTGGCGATGTATAATTATATTGAGATGCGTGACCTGGTGACCCGTCCATCCTACCGTCTCCGAAAAGCTGTTGACGACTTCATATTCTGGCTGGTTCCAGACTTCTGGGTTCCACTGTACAACTCCGTCACATTCACCACCATGCCTTACAGCCAGTGCGTGAGGAACAGGCAGTGGCAGAATAAGGTACTCATGTACAGCCTCCTTTTCTTTGGGTTCTCGATATTCGCTTTCCTATACGCTAAACGATAA
the coding amino acid sequence of the Tacinnabar gene of the tomato leaf miner is shown as SEQ ID NO. 2:
MASLQNGHSKKKLDVAVVGAGLVGSLEALYLAKRGHRVRLYEYRDDIRQTPLVRGRSINLALSVRGRKALKGVGLEDHMINKHGIPMRGRMIHRLDGSTYDIPYDARTNQCIYSVGRNYLNSLLLEESEKYENVERHFNHKLLEANLRSGSLTFVKTESNETVRVSADLTIGADGAFSAVRKAMMKQPLFDYSQKYIEHGYLELCIPADENGGFQMPANYLHIWPRGEFMMIALPNQDCSWTVTLFMPFEKFKSIENEEKLIAFFTQYFPDSIPLIGRQKLVEDFFAGKPSPLVAVKCRPYHVEDKALIIGDASHAVVPFYGQGMNAGFEDCTILDQLFEKYDDNIKDILEEFSATRWEDTFAISDLAMYNYIEMRDLVTRPSYRLRKAVDDFIFWLVPDFWVPLYNSVTFTTMPYSQCVRNRQWQNKVLMYSLLFFGFSIFAFLYAKR
the invention provides application of the tomato latent She Equan urea amino acid 3-hydroxylase gene in regulating and controlling eye color of tomato latent leaf moth.
The invention provides an application of the CRISPR/cas9 system of the tomato latent She Equan urea 3-hydroxylase gene in obtaining tomato latent She Equan urea 3-hydroxylase gene mutant.
The invention provides a CRISPR/cas9 system of the tomato latent She Equan urea 3-hydroxylase gene, wherein the synthetic primer of the sgRNA target site of the tomato latent She Equan urea 3-hydroxylase gene is as follows: sgRNA1-F:5'GAAATTA ATACGACTCACTATAGGAGACAGACCCCTCTTGTCCGgttttagagctagaaatagc3’；
SgRNA2-F：5’GAAATTAATACGACTCACTATAGGAATTTGGCGCTGTCTGTGCGgttttagagctagaaatagc3’；
SgRNA3-F：5’GAAATTAATACGACTCACTATAGGTGCGAGGGAGAATGATACACgttttagagctagaaatagc3’；
SgRNA4-F：5’GAAATTAATACGACTCACTATAGGATGACATACCTTACGACGCGgttttagagctagaaatagc3’；
sgRNA-R：5’AAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTgctatttctagctctaaaac3’。
The amino acid sequence has 3 transmembrane domains. The invention provides application of the tomato latent She Equan urea amino acid 3-hydroxylase gene cinnabar. The result shows that after the target gene mutation is induced by CRISPR/Cas9, the eye color of the tomato leaf miner is changed into pure black, which indicates that the cinnabar gene plays a key role in canthaxanthin biosynthesis of the tomato leaf miner. The invention induces the mutation of the tomato leaf miner cinnabar gene based on the CRISPR/Cas technology, promotes the change of the eye color of the tomato leaf miner, has important practical significance for using the gene as a marker mark, makes clear the role of the cinnabar gene in the biosynthesis of the tomato leaf miner's canthaxanthin, and provides a genetic molecular marker for researching the gene function.
Drawings
FIG. 1 shows the conserved domains of the Tacinnabar gene of tomato leaf miner;
FIG. 2 shows the phenotype of the tomato leaf miner after the Tacinnabar gene mutation.
Detailed Description
Example 1: cloning of full-length cDNA sequence of tomato leaf miner cinnabar gene
The adult tomato leaf miner is taken and put into a centrifuge tube with the volume of 1.5mL, the adult tomato leaf miner is ground into powder by a grinding rod after being frozen by liquid nitrogen, RNA is extracted, and the obtained product is preserved at the temperature of minus 80 ℃ for standby. cDNA was synthesized by reverse transcription of the extracted RNA according to the full-scale Jin Fanzhuai recording kit (One-Step gDNA Removal and cDNA Synthesis SuperMix). The cDNA is used as a template, and a primer is designed for PCR amplification. The primers used were as follows:
F：5’CGGACTCGTGTTATTTA3’
R：5’GCACTTACCAATTAGAAA3’
by using the primer sequence, the cDNA sequence of the Tacinnabar gene is obtained through PCR amplification, the total length of the cDNA sequence is 1350bp, and the obtained gene has the sequence shown in SEQ ID NO:1, which encodes 449 nucleotide sequences as set forth in SEQ ID NO:2, and a polypeptide having the amino acid sequence shown in 2. The cloned gene was analyzed for its encoded amino acid sequence and found to have 3 transmembrane domains, as shown in FIG. 1.
Example 2: effect of Tacinnabar Gene of tomato leaf miner on canthaxanthin biosynthesis
(1) In vitro synthesis of sgRNA
Determining the target site of the sgRNA, adding a T7 promoter sequence (shown in underline) before the target sequence of the F primer, and adding 20bp (gttttagagctagaaatagc) to complement the R primer after the target sequence; the R primer is the general sequence of sgRNA scafaold. The primer sequences used for the synthesis of the sgRNA transcription templates are shown in the following table.
TABLE 1
Synthesized by Shanghai Biotechnology services Inc.
Total RNA extraction and cDNA synthesis: as in example 1.sgRNA template synthesis, PCR product purification, in vitro synthesis of sgRNA and purification of sgRNA. Sgrnas were synthesized and purified using the kit, and were performed according to the kit instructions.
(2) Preparing a Cas9 protein-sgRNA mixed solution: cas9 protein (gold, nanjing) was purchased, and 10. Mu.L (Cas 9 protein (400 ng/. Mu.L), sgRNA (400 ng/. Mu.L (100 ng/. Mu.L of each sgRNA)), RNase-free water (complement)) was configured for the Cas9 protein-sgRNA injection system.
(3) Microinjection:
(1) sucking about 100 adult tomato leaf miner with a fluke tube, and laying on fresh tomato leaves for 3h;
(2) taking off the leaf with the eggs, picking the eggs under a microscope, and adhering the eggs on a double-sided adhesive tape (< 30 min);
(3) injection was performed under microscope (< 30min, with RNase-free water as control);
(4) placing the eggs into a culture dish for moisture preservation and culture, wherein the culture temperature is 25-26 ℃ and the photoperiod is 14L/10D;
(5) after injection, after hatching the larvae, transferring the newly hatched larvae to fresh tomato leaves;
(6) when the tomato leaves start to dry or the whole leaves are fed 70%, the fresh leaves are replaced, so that the food of the larvae is ensured to be sufficient;
(7) after pupation, the pupa is singly placed in a 1.5mL centrifuge tube with a small hole on the cover;
(8) after the adults emerge, observing whether the colors of the eyes of the adults change or not under a microscope.
(4) Phenotypic observation
After emergence of the adults, the color of the adult eyes was observed under a microscope to see if the eye color was changed compared to the control. The results are shown in fig. 2, and eyes exhibited a red eye phenotype (panel B in fig. 2) and a tan eye color phenotype (panel C in fig. 2) after the tomato leaf miner tacunnabar gene was mutated, as compared to the control (panel a in fig. 2). The results show that after CRISPR/Cas9 induces target gene mutation, the eye color of the tomato leaf miner is changed into a black phenotype of a homozygous line, a red eye phenotype and a tan eye color phenotype of a heterozygous line, and the induced mutation of the gene of the tomato leaf miner is used for promoting the eye color of the tomato leaf miner to change, so that the gene of the tomato leaf miner plays a key role in the canthaxanthin biosynthesis of the tomato leaf miner.
Sequence listing
<110> institute of plant protection of national academy of agricultural sciences
Application of <120> CRISPR/cas9 system in obtaining tomato latent She Equan urinary amino acid 3-hydroxylase mutant
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1350
<212> DNA
<213> tomato leaf miner (Tuta absorber)
<400> 1
atggcttcac ttcaaaatgg gcattcgaag aaaaaattgg atgttgcagt tgtgggtgct 60
ggattggtag gatctttaga agctttgtat ctagcaaaga gaggtcaccg agttcgtctt 120
tacgaatacc gggacgatat aagacagacc cctcttgtcc gcggtcgatc tatcaatttg 180
gcgctgtctg tgcgcggccg caaggctctc aaaggtgtcg ggttagagga ccacatgatc 240
aacaaacacg gtataccgat gcgagggaga atgatacaca ggcttgacgg atctacttat 300
gacatacctt acgacgcgag gaccaatcag tgcatctact ccgttggaag aaattatctc 360
aatagtttac ttctcgaaga atcagaaaag tatgaaaatg tggagcgtca tttcaatcat 420
aagctacttg aagctaatct gcgtagtgga tctcttactt ttgtcaaaac cgagagtaac 480
gaaacagttc gcgtatcagc agacctgacc ataggagctg atggggcgtt ctccgcagtc 540
cgcaaagcca tgatgaagca gccactcttc gactacagcc agaaatacat cgaacatgga 600
tacctggagc tgtgtatacc ggcggatgag aacggcggat tccaaatgcc agcgaactac 660
ctgcacatct ggccacgcgg cgaattcatg atgattgccc tgccgaacca ggactgctcg 720
tggacagtga ccctcttcat gcccttcgag aagttcaaga gtatcgagaa cgaggagaaa 780
ctaatcgcat tcttcaccca gtacttcccc gattcgatcc cgttgatcgg acgacagaag 840
ttggttgaag acttcttcgc tgggaaacct tcgcctttgg tggcagtgaa gtgccgacct 900
taccacgtgg aagataaagc tctaatcata ggcgacgctt cacacgccgt cgttccgttc 960
tacgggcaag gcatgaacgc tggcttcgaa gactgcacta tcttggacca gctgtttgaa 1020
aaatatgatg ataacataaa ggacattctc gaagagttct ccgcgaccag atgggaagac 1080
accttcgcta tcagcgatct ggcgatgtat aattatattg agatgcgtga cctggtgacc 1140
cgtccatcct accgtctccg aaaagctgtt gacgacttca tattctggct ggttccagac 1200
ttctgggttc cactgtacaa ctccgtcaca ttcaccacca tgccttacag ccagtgcgtg 1260
aggaacaggc agtggcagaa taaggtactc atgtacagcc tccttttctt tgggttctcg 1320
atattcgctt tcctatacgc taaacgataa 1350
<210> 2
<211> 449
<212> PRT
<213> tomato leaf miner (Tuta absorber)
<400> 2
Met Ala Ser Leu Gln Asn Gly His Ser Lys Lys Lys Leu Asp Val Ala
1 5 10 15
Val Val Gly Ala Gly Leu Val Gly Ser Leu Glu Ala Leu Tyr Leu Ala
20 25 30
Lys Arg Gly His Arg Val Arg Leu Tyr Glu Tyr Arg Asp Asp Ile Arg
35 40 45
Gln Thr Pro Leu Val Arg Gly Arg Ser Ile Asn Leu Ala Leu Ser Val
50 55 60
Arg Gly Arg Lys Ala Leu Lys Gly Val Gly Leu Glu Asp His Met Ile
65 70 75 80
Asn Lys His Gly Ile Pro Met Arg Gly Arg Met Ile His Arg Leu Asp
85 90 95
Gly Ser Thr Tyr Asp Ile Pro Tyr Asp Ala Arg Thr Asn Gln Cys Ile
100 105 110
Tyr Ser Val Gly Arg Asn Tyr Leu Asn Ser Leu Leu Leu Glu Glu Ser
115 120 125
Glu Lys Tyr Glu Asn Val Glu Arg His Phe Asn His Lys Leu Leu Glu
130 135 140
Ala Asn Leu Arg Ser Gly Ser Leu Thr Phe Val Lys Thr Glu Ser Asn
145 150 155 160
Glu Thr Val Arg Val Ser Ala Asp Leu Thr Ile Gly Ala Asp Gly Ala
165 170 175
Phe Ser Ala Val Arg Lys Ala Met Met Lys Gln Pro Leu Phe Asp Tyr
180 185 190
Ser Gln Lys Tyr Ile Glu His Gly Tyr Leu Glu Leu Cys Ile Pro Ala
195 200 205
Asp Glu Asn Gly Gly Phe Gln Met Pro Ala Asn Tyr Leu His Ile Trp
210 215 220
Pro Arg Gly Glu Phe Met Met Ile Ala Leu Pro Asn Gln Asp Cys Ser
225 230 235 240
Trp Thr Val Thr Leu Phe Met Pro Phe Glu Lys Phe Lys Ser Ile Glu
245 250 255
Asn Glu Glu Lys Leu Ile Ala Phe Phe Thr Gln Tyr Phe Pro Asp Ser
260 265 270
Ile Pro Leu Ile Gly Arg Gln Lys Leu Val Glu Asp Phe Phe Ala Gly
275 280 285
Lys Pro Ser Pro Leu Val Ala Val Lys Cys Arg Pro Tyr His Val Glu
290 295 300
Asp Lys Ala Leu Ile Ile Gly Asp Ala Ser His Ala Val Val Pro Phe
305 310 315 320
Tyr Gly Gln Gly Met Asn Ala Gly Phe Glu Asp Cys Thr Ile Leu Asp
325 330 335
Gln Leu Phe Glu Lys Tyr Asp Asp Asn Ile Lys Asp Ile Leu Glu Glu
340 345 350
Phe Ser Ala Thr Arg Trp Glu Asp Thr Phe Ala Ile Ser Asp Leu Ala
355 360 365
Met Tyr Asn Tyr Ile Glu Met Arg Asp Leu Val Thr Arg Pro Ser Tyr
370 375 380
Arg Leu Arg Lys Ala Val Asp Asp Phe Ile Phe Trp Leu Val Pro Asp
385 390 395 400
Phe Trp Val Pro Leu Tyr Asn Ser Val Thr Phe Thr Thr Met Pro Tyr
405 410 415
Ser Gln Cys Val Arg Asn Arg Gln Trp Gln Asn Lys Val Leu Met Tyr
420 425 430
Ser Leu Leu Phe Phe Gly Phe Ser Ile Phe Ala Phe Leu Tyr Ala Lys
435 440 445
Arg

Claims (4)

1. Tomato latent She Equan urinary amino acid 3-hydroxylase, which is characterized in that the amino acid sequence is shown in SEQ ID NO: 2.
2. A tomato latent She Equan urinary amino acid 3-hydroxylase gene encoding a tomato latent She Equan urinary amino acid 3-hydroxylase according to claim 1.
3. The tomato latent She Equan urinary amino acid 3-hydroxylase gene according to claim 2, which has a nucleotide sequence as set forth in SEQ ID NO: 1.
4. The use of the tomato latent She Equan urinary amino acid 3-hydroxylase gene according to claim 2, wherein the tomato latent She Equan urinary amino acid 3-hydroxylase gene CRISPR/cas9 system is utilized to obtain a tomato latent leaf moth eye color mutant, and the synthetic primers of the sgRNA target site of the tomato latent She Equan urinary amino acid 3-hydroxylase gene are as follows:
sgRNA1-F：5’GAAATTAATACGACTCACTATAGGAGACAGACCCCTCTTGTCCGgttttagagctagaaatagc3’；
SgRNA2-F：5’GAAATTAATACGACTCACTATAGGAATTTGGCGCTGTCTGTGCGgttttagagctagaaatagc3’；
SgRNA3-F：5’GAAATTAATACGACTCACTATAGGTGCGAGGGAGAATGATACACgttttagagctagaaatagc3’；
SgRNA4-F：5’GAAATTAATACGACTCACTATAGGATGACATACCTTACGACGCGgttttagagctagaaatagc3’；
sgRNA-R：5’AAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTgctatttctagctctaaaac3’。
CN202210127509.7A 2022-02-11 2022-02-11 Application of CRISPR/cas9 system in obtaining tomato latent She Equan urinary amino acid 3-hydroxylase mutant Active CN114480314B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202210127509.7A CN114480314B (en) 2022-02-11 2022-02-11 Application of CRISPR/cas9 system in obtaining tomato latent She Equan urinary amino acid 3-hydroxylase mutant

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202210127509.7A CN114480314B (en) 2022-02-11 2022-02-11 Application of CRISPR/cas9 system in obtaining tomato latent She Equan urinary amino acid 3-hydroxylase mutant

Publications (2)

Publication Number Publication Date
CN114480314A CN114480314A (en) 2022-05-13
CN114480314B true CN114480314B (en) 2023-11-17

Family

ID=81480620

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202210127509.7A Active CN114480314B (en) 2022-02-11 2022-02-11 Application of CRISPR/cas9 system in obtaining tomato latent She Equan urinary amino acid 3-hydroxylase mutant

Country Status (1)

Country Link
CN (1) CN114480314B (en)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2012205525A (en) * 2011-03-29 2012-10-25 National Institute Of Agrobiological Sciences Gene participating in coloration of egg and eye of silkworm and use thereof
CN113150099A (en) * 2021-02-08 2021-07-23 中国农业科学院植物保护研究所 Nanmei tomato leaf miner juvenile hormone signal pathway transcription factor Kr-h1 gene and application thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2012205525A (en) * 2011-03-29 2012-10-25 National Institute Of Agrobiological Sciences Gene participating in coloration of egg and eye of silkworm and use thereof
CN113150099A (en) * 2021-02-08 2021-07-23 中国农业科学院植物保护研究所 Nanmei tomato leaf miner juvenile hormone signal pathway transcription factor Kr-h1 gene and application thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
First Report on CRISPR/Cas9-Based Genome Editing in the Destructive Invasive Pest Tuta Absoluta (Meyrick) (Lepidoptera: Gelechiidae);Shun-Xia Ji et al;《Front Genet .》;全文 *
碱基编辑技术及其在昆虫中的应用和展望;王晓迪等;《中国生物防治学报》;第37卷(第03期);全文 *

Also Published As

Publication number Publication date
CN114480314A (en) 2022-05-13

Similar Documents

Publication Publication Date Title
CN106867977B (en) A kind of rice herbicide resistant protein and gene and its application
Takahashi et al. cDNA sequence analysis and expression of two cold-regulated genes in soybean
CN114480314B (en) Application of CRISPR/cas9 system in obtaining tomato latent She Equan urinary amino acid 3-hydroxylase mutant
CN101302523B (en) Application of physiological clock regulation protein LHY to stress-tolerant plant cultivation
CN111826364B (en) Disease and pest resistance related gene and application thereof
CN111500619B (en) Application of BXL gene or encoded protein thereof
CN108559753B (en) Application of wheat stripe rust PSTG _17694 gene in stripe rust prevention and treatment and stripe rust resistant wheat cultivation method
CN111518186A (en) Plant salt-resistant protein MsVNI1, and coding gene and application thereof
CN114231535B (en) Application of cassava MeRSZ21b gene in improving drought stress resistance of plants
CN109456983A (en) Soybean GmERF10 gene and its application
CN112513275B (en) Application of miR396 or mutant of encoding gene thereof in regulation and control of agronomic traits of plants
CN111909251B (en) Rice selenium-rich gene OsHSE2-1 and application thereof
CN111269920B (en) Wheat scab-resistant geneTaXAX1And uses thereof
WO2021047377A1 (en) Application of tpst gene in regulation of traits of plant
CN110590925B (en) Pteromalus puparum venom protein PpVPG and application thereof
CN110669790B (en) Application of CvBV12-7 gene in reducing humoral immune response of insect
CN116622666A (en) Method for regulating drought resistance of plants and application of TaMPK3 in regulating drought resistance of plants
CN110468138B (en) Gene TSG2 for controlling cold resistance of rice and application thereof
CN102899304B (en) Have kill phytopathogen activity albumen, its encoding gene and application
CN115725531A (en) Acetyl transferase OsG2 gene and application of protein coded by same in adjusting rice grain size
CN108004251B (en) Application of wheat stripe rust PSTG _11438 gene in stripe rust prevention and treatment and cultivation method of stripe rust resistant wheat
CN109182316B (en) Rice yield-related SAP30C functional protein, encoding gene, recombinant vector and application
JPH11187878A (en) New potassium channel gene derived from plants belonging to genus nicotiana
CN114041464B (en) Method for enhancing resistance of plants to bacteria
CN113173981B (en) Phyllostachys pubescens PeDREB3 gene and application thereof in plant cold resistance regulation

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