CN112852806A - sgRNA of female specific E3 exon of targeted spodoptera frugiperda Doublesex gene and application thereof - Google Patents
sgRNA of female specific E3 exon of targeted spodoptera frugiperda Doublesex gene and application thereof Download PDFInfo
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- CN112852806A CN112852806A CN202110055205.XA CN202110055205A CN112852806A CN 112852806 A CN112852806 A CN 112852806A CN 202110055205 A CN202110055205 A CN 202110055205A CN 112852806 A CN112852806 A CN 112852806A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—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
-
- 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
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
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- 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
- C12N15/89—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microinjection
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- 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
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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]
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- 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
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
- Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants
Abstract
The invention relates to the field of pest genetic control, in particular to sgRNA of a female specific E3 exon of a targeted Spodoptera frugiperda Doublesex gene and application thereof. The nucleotide sequences of two targets of sgRNA of E3 exon of the targeted Spodoptera frugiperda Doublesex gene on female specific E3 exon of the Spodoptera frugiperda Doublesex gene are shown as SEQ ID No.1 and SEQ ID No. 2. The method for genetically controlling spodoptera frugiperda by using the CRISPR/Cas9 system comprises the following steps: 1) obtaining a Spodoptera frugiperda female specific E3 exon sequence; 2) designing sgRNA of E3 exon of spodoptera frugiperda Doublesex gene; 3) mixing mRNA or protein of Cas9 with the sgRNA designed in the step 2), and introducing the mixture into fertilized eggs of spodoptera frugiperda to obtain female-specific sterile spodoptera frugiperda. The method realizes the efficient and accurate knockout of the spodoptera frugiperda dsx female specific exon by using the CRISPR/Cas9 technology, and determines the important application value of the dsx gene as a target in spodoptera frugiperda genetic control.
Description
Technical Field
The invention relates to the field of pest genetic control, in particular to sgRNA of a female specific E3 exon of a targeted Spodoptera frugiperda Doublesex gene and application thereof.
Background
At present, the crop pest control in China mainly depends on chemical control measures, and a series of problems are brought while the pest control is carried out: pesticide residue, environmental pollution, increase of pest drug resistance and reduction of variety of beneficial species. The pest genetic prevention and control can not only obviously reduce the use amount of chemical pesticides and avoid pesticide residues in agricultural products, but also greatly improve the quality safety level of the agricultural products and protect the ecological environment. The genetic modification-based insect genetic sterility technology (SIT) comprises a female sterility technology and a male sterility technology, is used as a species-specific, environment-friendly, scientific and efficient pest genetic prevention and control technology, and has great application prospects in pest prevention and control.
Spodoptera frugiperda (J.E. Smith) is an important agricultural pest which issues early warning to the world in 2018 United nations grain and crop organizations, and is also an important foreign invasive species in China, the Spodoptera frugiperda has the advantages of extremely wide host, extremely wide growth range, extremely strong reproductive capacity, extremely high migration diffusion speed, serious sudden hazard and great prevention and control difficulty, the traditional pest control means cannot realize accurate prevention and control of Spodoptera frugiperda, and the research and development of genetic prevention and control technology of Spodoptera frugiperda is urgent.
Disclosure of Invention
The invention aims to overcome the defects in the prior art, provides sgRNA of E3 exon specific to spodoptera frugiperda Doublesex (dsx) gene female and application thereof, and further utilizes the important regulation and control function of spodoptera frugiperda Doublesex gene in reproductive development to perform spodoptera frugiperda genetic control.
The specific technical scheme of the invention is as follows:
the sgRNA of E3 exon of the targeted Spodoptera frugiperda Doublesex gene has nucleotide sequences of two targets on a female specific E3 exon of the Spodoptera frugiperda Doublesex gene as shown in SEQ ID No.1 and SEQ ID No. 2.
The invention also provides application of the sgRNA in genetic control of spodoptera frugiperda.
The invention also provides a method for genetically controlling spodoptera frugiperda by using the CRISPR/Cas9 system, which comprises the following steps:
1) obtaining a Spodoptera frugiperda female specific E3 exon sequence;
2) designing sgRNA of a spodoptera frugiperda Doublesex gene female specific E3 exon;
3) mixing mRNA or protein of Cas9 with the sgRNA designed in the step 2), introducing the mixture into fertilized eggs of spodoptera frugiperda, knocking out E3 exon of the Doublesex gene, and obtaining female-specific sterile spodoptera frugiperda.
According to the method, preferably, the nucleotide sequences of two targets of the sgRNA on the female specific E3 exon of spodoptera frugiperda Doublesex gene in the method are shown as SEQ ID NO.1 and SEQ ID NO. 2.
Further, according to the method of the present invention, preferably, step 3) introduces the mixture into a fertilized egg of spodoptera frugiperda by microinjection.
The invention also provides a kit for genetically controlling spodoptera frugiperda, which comprises sgRNA of a female specific E3 exon of spodoptera frugiperda Doublesex gene and mRNA or protein of Cas 9.
Further preferably, the nucleotide sequences of two targets of the sgRNA on the female specific E3 exon of spodoptera frugiperda Doublesex gene in the kit are shown as SEQ ID No.1 and SEQ ID No. 2.
The invention obtains the cd sequence of spodoptera frugiperda dsx genes through spodoptera frugiperda whole genome sequencing and lepidoptera insect Doublesex gene (dsx) homologous comparison. And (3) respectively amplifying dsx gene transcripts of different sexes of males and females, and comparing sequence differences of dsx transcripts of different sexes to obtain an exon E3 specific to female sex. Specific targeted sgRNA is designed aiming at Spodoptera frugiperda E3 sequence, and female specific exon E3 knockout is realized by using CRISPR/Cas9 technology. By testing phenotype and genetic performance of E3 exon knockout strains, the developmental deformity of female external genitalia organs is found after the dsx gene E3 exon is knocked out, and the reproductive system of dissected female is found that the E3 exon knockout homozygous individual fertilized sac is absent and the ovary is malformed; after the heterozygous individual copulates, the heterozygous individual does not lay eggs, and the eggs at the position of the ovarian duct close to the lateral oviduct are arranged in a plurality of rows. Preliminarily determining the application value of the dsx gene as an important target for genetic control of Spodoptera frugiperda.
Drawings
FIG. 1 is a schematic diagram of a Cas9 protein cleavage site of exon E3 of a Doublesex gene in an embodiment of the present invention;
FIG. 2 is a drawing illustrating the genotype test of the deletion of exon E3 of the Doublesex gene in the example of the present invention, wherein the wild type amplified fragment (WT) is 511bp, and the deleted fragment (KO) is 472 bp;
FIG. 3 is a schematic diagram of E3 exon knockout of the Doublesex gene in which sgRNA target sites are underlined, according to an embodiment of the present invention;
FIG. 4 is a diagram of pupal germ-line malformation following exon knockout of dsx-E3 in accordance with embodiments of the present invention;
FIG. 5 is a graph showing anogenital dysplasia in female adults following exon knockout of dsx-E3 in accordance with embodiments of the present invention;
FIG. 6 is a schematic representation of female adult ovarian developmental malformation following exon knock-out of dsx-E3 in accordance with embodiments of the present invention.
Detailed Description
The technical solution of the present invention will be described in detail with reference to examples. The reagents and biomaterials used below were all commercial products unless otherwise specified. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The molecular biological experiments, which are not specifically described in the examples, were carried out according to the methods specified in molecular cloning, A laboratory Manual (fourth edition) J. SammBruke, or according to the kit and product instructions.
Example 1 PCR amplification of different transcripts of the Doublesex Gene
Obtaining a dsx gene candidate sequence through Spodoptera frugiperda whole genome Blast, and designing a primer sequence as follows:
F:ATGGTGTCCGTGGGCTCGTG(SEQ ID NO.3)
R:CGCCGCCTGCGCACTTGTAG(SEQ ID NO.4)
taking 3 male and female adults of Spodoptera frugiperda, respectively mixing the male and female adults with the male and female adults, respectively extracting RNA of the male and female adults, carrying out reverse transcription to obtain cDNA of the male and female adults, and carrying out amplification by using the cDNA as a template and the primers, wherein an amplification system and a program are as follows:
2×Phanta Master Mix：25uL
and (3) primer F: 1uL
And (3) primer R: 1uL
cDNA：1uL
Water: 22uL
The procedure is as follows: 95 ℃/3 min; 95 ℃/10sec, 64 ℃/10sec, 72 ℃/90 sec; amplification was performed for 34 cycles at 72 deg.C/5 min.
Recovering the TA clone of the above band and sequencing to obtain the coding region sequences of different male and female transcripts of dsx gene as follows:
sfDSX-M (Male) (SEQ ID NO.5)
sfDSX-F1 (female) (SEQ ID NO.6)
sfDSX-F2 (female) (SEQ ID NO.7)
sfDSX-F3 (female) (SEQ ID NO.8)
sfDSX-F4 (female) (SEQ ID NO.9)
Example 2 female-specific exon E3 knockout of Doublesex Gene and genotyping
As shown in figure 1, sgRNA target site scanning is carried out on dsx gene E3 exon by using sgRNA 9 design software, off-target risk assessment is carried out by taking the whole genome as a reference sequence, and an N18NGG sequence (shown in figure 1 by underlining, wherein the Cas9 protein cleavage site is represented by ^ a) with the highest score and no off-target risk is selected, and the site 1 sequence is as follows: GTTCGACGGGTTAGAGCTGAGG (PAM sequence in bold) (SEQ ID NO.1), site 2 sequence: GATAATCAACGAATACGCGCGG (PAM sequence in bold) (SEQ ID NO. 2); 2 pairs of specific primers were designed based on the above sequence:
E3-1F:TAATACGACTCACTATAGGATAATCAACGAATACGCG(SEQ ID NO.10)
E3-1R:TTCTAGCTCTAAAACCGCGTATTCGTTGATTATCC(SEQ ID NO.11)
E3-4F:TAATACGACTCACTATAGGTTCGACGGGTTAGAGCTG(SEQ ID NO.12)
E3-4R:TTCTAGCTCTAAAACCAGCTCTAACCCGTCGAACC(SEQ ID NO.13)
the sgRNA was transcribed in vitro using Precision gRNA Synthesis Kit (Invitrogen, A29377) Kit. Introducing the sgRNA and the Cas9 protein into Spodoptera frugiperda fertilized eggs in a microinjection mode, normally feeding, and taking 5-instar terminal molt for genotype detection, wherein detection primers are as follows:
E3F511：CGTAAACGACGATGTGCTGT(SEQ ID NO.14)
E3R511：GAGAAATGTCTCTCCCGCCTT(SEQ ID NO.15)
the amplification system and procedure were as follows:
DreamTaq Green PCR Master Mix(2X)：12.5uL
and (3) primer F: 1uL
And (3) primer R: 1uL
DNA：4uL
Water: 6.5uL
The procedure is as follows: 95 ℃/3 min; 95 ℃/30sec, 58 ℃/30sec, 72 ℃/30 sec; amplification was performed for 34 cycles at 72 deg.C/5 min.
The results of the detection are shown in FIG. 2. Wherein, the wild type amplified fragment (WT) is 511bp, and the knockout fragment (KO) is 472bp, which shows that the knockout is successful. The fragments were sequenced, and the result is shown in fig. 3, the expected target fragment deletion near the Cas9 protein cleavage site, and the underlined sequence in the sequencing peak shows the residual sequence of sgRNA target site Cas9 protein cleavage.
Example 3 phenotypic analysis of Doublesex Gene female-specific exon E3 knockout lines
The development of external genital organs in and out of dsx knockout female individuals was observed and recorded by photography, and the results are shown in FIGS. 4-6. In FIG. 4, pupal germ-line malformations following exon knockout of dsx-E3 can be seen. In FIG. 5, genitalia-external developmental malformations of female adults following knockout of the dsx-E3 exon can be seen. In FIG. 6, malformation of female adult ovarian development following knockout of the dsx-E3 exon can be seen.
Example 4 determination of genetic Properties of female-specific exon E3 knockout individuals of the Doublesex Gene
And (3) hybridizing and pairing the female sterile line heterozygous individuals with the males of the wild type individuals (1:2), detecting mating cyst changes by observing mating behaviors and dissecting, determining the mating condition of the female individuals, continuously feeding the mated adults for 7 days, and counting the spawning condition. The results show that no matter whether the genotype of the dsx-E3 KO female is heterozygous or homozygous, the mating behavior is not obviously abnormal, and the female can successfully mate with the male, but the mated female does not lay eggs.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and are not limited. Although the present invention has been described in detail with reference to the embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the spirit and scope of the invention as defined in the appended claims.
Sequence listing
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gacgtggacg aggcatcacg gaaaatagac gaaggaaaga tgataatcaa cgaatacgcg 660
cggaagaaca atctgaacgt gttcgacggg ttagagctga ggaactcgac acgccagaaa 720
atggcggaaa ttaataatat aagtggtgta ctatcgtcgt cgatgaagtt attttgcgaa 780
tga 783
<210> 8
<211> 747
<212> DNA
<213> Spodoptera frugiperda (Spodoptera frugiperda)
<400> 8
atggtgtccg tgggctcgtg gaggcgccgc acgcccgacg actgcgagga gcgctccgag 60
cccggcgcct ccagctccgc cgtgccgcgc gcgccgccga actgcgcgcg ctgtcggaac 120
caccggctca agatcgagct gaagggccac aagcggtact gcaagtaccg caactgtact 180
tgcgagaagt gtatactgac ggctgaccgg cagcgagtga tggcacaaca gactgctatg 240
cggcgtgccc aggcgcagga cgaggcgcgc gcgcgggccg ggatgcaggc gctgggcgtg 300
gagctggagc agccggagcc gccggtggtg aaggcgccgc gcagccccgc ggtgccgccg 360
ccgcgctcgc tgggctcggc cagctgcgac tcggtcccgg gctcgcccgg agtgtccccg 420
tacgcgccgc tgccgccctc cataccgctg ccgcaagcga tgccgcctct gctgccgcca 480
caacagccgg cggtttcctt agaaaactta gtggataact gtaacaagct gctggagaag 540
ttccactact cctgggagat gatgcctctg gtcctggtca tcctgaatta cgcaggcagt 600
gacgtggacg aggcatcacg gaaaatagac gaaggaaaga tgataatcaa cgaatacgcg 660
cggaagaaca atctgaacgt gttcgacggg ttagagctga ggaactcgac acgccatgac 720
cggacgaagg aaaatggcgg aaattaa 747
<210> 9
<211> 759
<212> DNA
<213> Spodoptera frugiperda (Spodoptera frugiperda)
<400> 9
atggtgtccg tgggctcgtg gaggcgccgc acgcccgacg actgcgagga gcgctccgag 60
cccggcgcct ccagctccgc cgtgccgcgc gcgccgccga actgcgcgcg ctgtcggaac 120
caccggctca agatcgagct gaagggccac aagcggtact gcaagtaccg caactgtact 180
tgcgagaagt gtatactgac ggctgaccgg cagcgagtga tggcacaaca gactgctatg 240
cggcgtgccc aggcgcagga cgaggcgcgc gcgcgggccg ggatgcaggc gctgggcgtg 300
gagctggagc agccggagcc gccggtggtg aaggcgccgc gcagccccgc ggtgccgccg 360
ccgcgctcgc tgggctcgac cagctgcgac tcggtcccgg gctcgcccgg agtgtccccg 420
tacgcgccgc tgccgccttc cataccgctg ccgcaagcga tgccgcctct gctgccgcca 480
caacagccgg cggtttcctt agaaaactta gtggataact gtaacaaact gctggagaag 540
ttccactact cctgggagat gatgcctctg gtcctggtca tcctgaatta cgcaggcagt 600
gacgtggacg aggcatcacg gaaaatagac gaaggaaaga tgataatcaa cgaatacgcg 660
cggaagaaca atctgaacgt gttcgacggg ttagagctga ggaactcgac acgccatgac 720
cggacgaagg tggtgaaatt cgaaatgcaa cctcagtga 759
<210> 10
<211> 37
<212> DNA
<213> Artificial
<400> 10
taatacgact cactatagga taatcaacga atacgcg 37
<210> 11
<211> 35
<212> DNA
<213> Artificial
<400> 11
ttctagctct aaaaccgcgt attcgttgat tatcc 35
<210> 12
<211> 37
<212> DNA
<213> Artificial
<400> 12
taatacgact cactataggt tcgacgggtt agagctg 37
<210> 13
<211> 35
<212> DNA
<213> Artificial
<400> 13
ttctagctct aaaaccagct ctaacccgtc gaacc 35
<210> 14
<211> 20
<212> DNA
<213> Artificial
<400> 14
cgtaaacgac gatgtgctgt 20
<210> 15
<211> 21
<212> DNA
<213> Artificial
<400> 15
gagaaatgtc tctcccgcct t 21
Claims (7)
1. A sgRNA targeting a spodoptera frugiperda Doublesex gene female specific E3 exon is characterized in that nucleotide sequences of two targets of the sgRNA on the spodoptera frugiperda Doublesex gene female specific E3 exon are shown as SEQ ID No.1 and SEQ ID No. 2.
2. The sgRNA of claim 1, used for genetic control of Spodoptera frugiperda.
3. A method for genetically controlling spodoptera frugiperda using a CRISPR/Cas9 system, comprising the steps of:
1) obtaining a Spodoptera frugiperda female specific E3 exon sequence;
2) designing sgRNA of female specific E3 exon of spodoptera frugiperda Doublesex gene;
3) mixing mRNA or protein of Cas9 with the sgRNA designed in the step 2), introducing the mixture into fertilized eggs of spodoptera frugiperda, knocking out E3 exon of the Doublesex gene, and obtaining female-specific sterile spodoptera frugiperda.
4. The method of claim 3, wherein the sgRNA has nucleotide sequences for two targets on exons of Spodoptera frugiperda Doublesex gene female-specific E3 as shown in SEQ ID No.1 and SEQ ID No. 2.
5. The method according to claim 3 or 4, wherein step 3) the mixture is introduced into fertilized eggs of Spodoptera frugiperda by microinjection.
6. A kit for genetically controlling spodoptera frugiperda, comprising sgRNA of the spodoptera frugiperda douublesex gene female-specific E3 exon and mRNA or protein of Cas 9.
7. The kit of claim 6, wherein the sgRNA has nucleotide sequences of two targets on exons specific to Spodoptera frugiperda Doublesex gene female E3, as shown in SEQ ID No.1 and SEQ ID No. 2.
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| CN117210582A (en) * | 2023-11-09 | 2023-12-12 | 广东省农业科学院植物保护研究所 | Specific molecular marker for early sex identification of litchi rough shin cui chi moth and detection method |
| CN117210582B (en) * | 2023-11-09 | 2024-02-27 | 广东省农业科学院植物保护研究所 | Specific molecular marker for early sex identification of litchi rough shin cui chi moth and detection method |
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