CN109593762A - Diamondback moth APN1 gene and its application are knocked out using CRISPR/Cas9 gene editing system - Google Patents
Diamondback moth APN1 gene and its application are knocked out using CRISPR/Cas9 gene editing system Download PDFInfo
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Abstract
The invention discloses a kind of knockout for realizing non-mode biology diamondback moth APN1 gene using CRISPR/Cas9 and its applications in Bt Cry1Ac insecticidal proteins resistance mechanism molecular studies.The method of the present invention designs and synthesizes diamondback moth APN1 gene specific sgRNA target sequence, microinjection is to diamondback moth front blastoderm phase ovum Posterior pole position after sgRNA is mixed with Cas9 albumen, go out to stablize the diamondback moth APN1 homozygous mutation population of heredity using CRISPR/Cas9 gene editing screening system and for Bt Cry1Ac insecticidal proteins virulence bioassay, it was demonstrated that it is resistance that diamondback moth APN1 gene mutation can cause it to generate Bt Cry1Ac insecticidal proteins.The method of the present invention is simple and efficient, time saving and energy saving, and only the further investigation of non-mode biology in vivo functionality gene does not provide technical support, is also laid a good foundation to explore new strategy of insect pest control.
Description
Technical field
The present invention relates to the knockout of genetic engineering field more particularly to diamondback moth APN1 gene and in diamondback moth Bt
Application in the research of Cry1Ac insecticidal proteins Resistance mechnism.
Background technique
Diamondback moth [Plutella xylostellaIt (L.)] is a kind of special feeding habits pest of global crop in cruciferae,
It is also one of current drug resistance pest the most serious, cross resistance spectrum is wide, resistance level is high, fast-growth.And with
Extensive cultivation of the brassicaceous vegetable in various regions, diamondback moth great prestige is caused to the production of world brassicaceous vegetable
The side of body.Bacillus thuringiensis (Bacillus thuringiensis, Bt) and Cry class insecticidal crystal protein can be generated, to a variety of elder brothers
Worm has specific toxic action, is that purposes is most wide in the world at present, the maximum microbial insecticide of yield, accounts for microbial insecticide
95% or more of agent total amount, because its to pest efficient, to safety of human and livestock, it is environmentally friendly due to worldwide obtain it is extensive
Using.Diamondback moth is to be reported in field earliest to generate Bt insecticide resistance pest, since first case comes from U.S.'s summer
Since the smooth field resistance report of prestige, the field diamondback moth in the area such as Shenzhen and Guangzhou of Japan, Malaysia and China
Different degrees of resistance is produced to Bt.Therefore, reasonable scientific method research diamondback moth Bt Resistance mechnism is found,
Fundamentally solve the problems, such as that pest resistance to insecticide is just particularly important.
Aminopeptidase N (Aminopeptidase N, APN) belongs to a kind of exopeptidase, is widely present in animals and plants
It is internal, play a significant role in multiple physiology courses.APN albumen is by glycosyl-phosphatidyl inositol (Glycosyl-
Phosphatidylinositol, GPI) it is anchored on the midgut epithelial cell film of insect, in lepidopterous insects, APN is recognized
For the functional membrane receptor for being the interaction of Bt insecticidal proteins.Experiment shows reduction and the beet armyworm of the expression quantity of APN1
(Spodoptera exigua) resistance correlation is generated to Bt Cry1C insecticidal proteins.Tiewsiri, which is equal to 2011, passes through albumen
Matter group etc. studies have shown that cabbage looper (Trichoplusia ni) in intestines APN1 and APN6 expression quantity change and its to Bt
The resistance of Cry1Ac insecticidal proteins is related, there is a kind of trans- regulatory mechanism of expression quantity, the raising of APN6 expression quantity between the two
For the reduction of APN6 expression quantity, there are a kind of complementary mechanisms to reduce fitness cost, to participate in cabbage looper to Bt
Cry1Ac insecticidal proteins it is resistance.
The short palindrome in the interval of regular cluster repeats system (Clustered regularly interspaced short
palindromic repeats；CRISPR-associated gene Cas9, CRISPR/Cas9) it is a kind of in nucleic acid
The active complex of enzyme cutting, it can recognize specific DNA sequence dna, carries out specific site cutting and causes double-strand DNA cleavage
(Double-strand breaks, DSB), the principle of the technology are artificial synthesized crRNA (CRISPR-derived first
RNA) with tracrRNA (trans- activating crRNA) two kinds of complementary pairings RNA, being formed has guiding function
SgRNA (Single guide RNA), is instructed by the sgRNA, and Cas9 cuts targeting DNA double chain, so that activation is thin
Two kinds of repair mechanisms intracellular, i.e. non-homologous end joining or homologous recombination cause incision position alkali by both repair mechanisms
Loss, insertion and the replacement of base, to obtain mutated individual.In recent years, CRISPR/Cas9 technology was as a kind of strong
Gene editing means are applied to many fields, and with Zinc finger nuclease (Zinc-finger nucleases, ZFNs) and
Activating transcription factor sample effector nuclease (Transcription activator-like effector nucleases,
TALENs) technology is compared, CRISPR-Cas9 have more rapidly, it is easy, efficiently, multidigit point, selectively targeted knock out the excellent of gene
Gesture.
Summary of the invention
The present invention is insufficient for existing diamondback moth Bt Resistance mechnism verification method, and qualification process is excessively cumbersome etc. to ask
Topic provides and a kind of knocks out diamondback moth APN1 gene and lossless using genotype using CRISPR/Cas9 gene editing system
Detection method filters out the Mutants homozygous of diamondback moth APN1 gene, passes through the biological characteristis of Bt toxin Cry1Ac, detection
The diamondback moth individual of APN1 gene mutation to the resistance level of Bt Cry1Ac insecticidal proteins, elaborate diamondback moth APN1 gene with
Resistance relationship between Bt Cry1Ac insecticidal proteins.
Present invention provide the technical scheme that a kind of knockout of the diamondback moth APN1 gene based on CRISPR/Cas9 technology
Method, this method utilize CRISPR/Cas9 gene editing system, design and synthesize diamondback moth APN1 gene specific sgRNA target
Sequence is marked, microinjection is to diamondback moth front blastoderm phase ovum Posterior pole position after sgRNA is mixed with Cas9 albumen, with four age of diamondback moth
The trace gDNA that the fresh pupa of larva is sloughed off is template, carries out PCR amplification and direct Sequencing identification G0 generation mutation using specific primer
Individual, G0 mutually mate for mutated individual and obtain G1 generation, and quantity is chosen after directly identifying with TA sequencing enough and is had and is identical dashed forward
The heterozygote individual of modification carries out selfing and obtains G2 generation, and TA sequencing identification obtains capable of stablizing hereditary G2 for homozygotic individual, with
Selfing obtains the APN1 homozygous gene saltant type population in G3 generation afterwards.
Further, the method, sgRNA target sequence design outside the 12nd of the gene coding region diamondback moth APN1
Aobvious son, nucleotide sequence is as shown in SEQ ID NO. 1；The synthetic primer of sgRNA target site are as follows: CRISPR forward primer
As shown in SEQ ID NO. 2 and CRISPR reverse primer is as shown in SEQ ID NO. 3.
The method, the final concentration of 300 ng/ μ l of final concentration of 150 ng/ μ l, the Cas9 albumen of sgRNA.
The method further can using forward primer SEQ ID NO. 4 and reverse primer SEQ ID NO. 5
With the gDNA sequence near specific amplification APN1 gene sgRNA target site.
Meanwhile the present invention also provides a kind of diamondback moth APN1 gene knockout based on CRISPR-Cas9 gene Knockout
Kit, comprising: nucleotide sequence sgRNA target sequence as shown in SEQ ID NO. 1 and Cas9 albumen.
The kit further comprises matched detection reagent, for detecting the shear effect of the gene and commenting
Estimate gene knockout efficiency.
The kit further comprises SEQ ID NO. 2 and SEQ ID NO. 3；And 4 He of SEQ ID NO.
SEQ ID NO. 5。
The present inventor designs and synthesizes diamondback moth APN1 gene specific sgRNA target sequence, utilizes CRISPR/Cas9 base
Because of editing system, microinjection passes through gene to diamondback moth front blastoderm phase ovum Posterior pole position after sgRNA is mixed with Cas9 albumen
The lossless detection method of type quickly and effectively, has other similar insect fine without destroying any diamondback moth physique structure
Reference value.What is filtered out stablizes hereditary diamondback moth APN1 homozygous mutation population and for Bt Cry1Ac desinsection
Albumen virulence bioassay, it was demonstrated that diamondback moth APN1 gene mutation can cause it to generate highly resistance to Bt Cry1Ac insecticidal proteins
Property.The present invention be the in vivo functionality research of diamondback moth APN1 gene and disclose diamondback moth APN1 gene and Bt toxin Cry1Ac it
Between relationship lay a good foundation, lay a good foundation to explore new strategy of insect pest control.
With the zinc finger endonuclease ZFN and activating transcription factor class effect nuclease TALEN for being all gene editing technology
Technology is compared, and CRISPR/Cas9 technical operation is simple, and time saving and energy saving, gene editing is high-efficient, and the mutant character of realization can be stablized
It is genetic to the next generation, so that the in vivo research of target gene becomes a kind of possibility.This finds novel efficient peace also for us
Full strategy of insect pest control provides new approaches.
Detailed description of the invention
Fig. 1 is the deoxynucleotide sequence of one section of 297-bp near target site, and wherein italic underlines
Sequence is the primer used when amplification, is forward primer (APN1-F) and reverse primer (APN1-R) respectively, arrow designates
The direction of downstream primer.
Fig. 2 is sgRNA design drawing, is labelled with the genome structure of diamondback moth APN1 gene, sgRNA design in figure in proportion
Sequence in the 12nd exon region of APN1 gene, dotted line represents the core fragment of sgRNA target sequence, underscore mark
Note is the site PAM, and black up-side down triangle represents Cas9 cleavage site.
Fig. 3 is the gDNA sloughed off using pupa as the DNA fragmentation of the 297-bp of template amplification.
Fig. 4 is mutation type of the G1 for diamondback moth individual, and asterisk is the homozygous mutation idiotype finally obtained, lower stroke
The sequence of line mark is target sequence nucleus, and black inverted triangle is Cas9 cleavage site.
Fig. 5 is successively wild type individual (DBM1Ac-S sensitive population), heterozygote individual and final acquisition from top to bottom
2-bp missing (APN1KO population) homozygous mutation individual sequencing peak figure, black underscore represents homozygous mutation population institute
The 2-bp base sequence of missing, the position of arrow meaning are the site that gene editing starts.
Specific embodiment
, diamondback moth APN1 gene sgRNA target sequence design and synthesis
1. using Cas-Designer software (http://www.rgenome.net/cas-designer/) in diamondback moth APN1
The specific regions (the 12nd exon) of gene design sgRNA target sequence (its nucleotide sequence such as 1 institute of SEQ ID NO.
Show), as shown in Fig. 2, and searching for diamondback moth DBM-DB genome database (http: // 59.79.254.1/DBM/
Index.php) and CRISPR undershooting-effect detects Cas-OFFinder software (http://www.rgenome.net/cas-
Offinder/), the potential site of missing the target of detection.
2. T7 promoter sequence is added before sgRNA core site AGGCCGTGGGTGTACTGCGC, in sgRNA core bit
The sequence complementary with crRNA/tracrRNA is added after point, forms the complete 5 ' end DNA fragmentation of sgRNA.
3. holding DNA fragmentation to pass through PCR the sgRNA 5 ' in the crRNA/tracrRNA sequence of one section of 80-bp and step 2
Denaturation, annealing extend the complete sgRNA deoxynucleotide chain of synthesis, the primer of use such as SEQ ID NO. 2 and SEQ ID
NO. shown in 3.
CRISPR forward primer (N1-CRI-F):
5′-GAAATTAATACGACTCACTATAGGAGGCCGTGGGTGTACTGCGCGTTTTAGAGCTAGAAATAGC-3′
(SEQ ID NO. 2)；
CRISPR reverse primer (CRI-R): 5 '-AAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGC
CTTATTTTAACTTGCTATTTCTAGCTCTAAAAC-3 ' (SEQ ID NO. 3).
(1) sgRNA is synthesized: using the PrimeSTAR Max Premix polymerase of TaKaRa company, carrying out 50 μ l bodies
System reacts without template PCR, specially (10 μM of 2 μ l of CRISPR-F；10 μM CRISPR-R 2 μl；PrimeSTAR
Max Premix (2×) 25 μl；ddH2O 21 μl)。
(2) PCR reaction condition are as follows: 98 °C of 2 min；38 circulation (98 °C of 10 s, 70 °C of 5 s, 72 °C
30 s)；72 °C 10 min；4 °C ∞.
(3) using the DNA clean-up kit of Beijing CoWin Bioscience Co., Ltd. to above-mentioned PCR product
It is purified, the specific steps are as follows:
The external reverse transcription of sgRNA deoxynucleotide chain obtained in step 3 is synthesized into sgRNA nucleotide sequence.
(1) sgRNA is transcribed in vitro: utilizing the MEGAshortscript T7 High Yield of Ambion company
Transcription kit carries out external reverse transcription, and corresponding reagent is added according to table 1:
Table 1
| Reagent | System |
| 10×T7 Reaction Buffer | 2 μl |
| DNA template | 1 μg |
| 10mM ATP | 2 μl |
| 10mM CTP | 2 μl |
| 10mM GTP | 2 μl |
| 10mM UTP | 2 μl |
| T7 Enzyme Mix | 2 μl |
| Nuclease-free Water | To 20 μ l |
It is gently mixed reaction reagent, 37 °C of 4 h of incubation.
1 μ l TURBO DNase is added, mixes, 37 °C of 15 min of incubation, removes DNA profiling.
(2) MEGAclear kits sgRNA nucleotide chain is used, the specific steps are as follows:
Add the DEPC water and 15 μ l ammonium acetate terminate liquids of 115 μ l, mixes.
Add 150 μ l extracts (phenol: chloroform: isoamyl alcohol=25:24:1) acutely to shake, is stored at room temperature 5 min, 4 °
C is centrifuged 15 min, takes supernatant in new pipe.
Adding 150 μ l chloroforms, acutely shakes, be stored at room temperature 1 min, 4 °C of 10 min of centrifugation take supernatant in new pipe,
Add 300 μ l ethyl alcohol, mixes gently, -20 °C of 30 min of placement.
4 °C of 10 min of centrifugation abandon supernatant, twice with 75% ethanol washing precipitating, dry.
30 μ l water are added to dissolve, -70 °C of preservations.
, Eggs of Diamondback Moth microinjection
1. microinjection previous experiments prepare:
(1) needle instrument and the EG-401 of Narishige company, Japan is drawn to grind using the P-97 of U.S. Sutter Instrument company
Needle instrument makes injection needle.
(2) diamondback moth to mate should shift to an earlier date 2 days and release, and to reach egg production peak time, when oviposition will manufacture dark surrounds,
Temperature maintains 26-28 °C in insectary.
(3) the slide forceps scissors that experiment the previous day will use in experiments Microinjection, distilled water, injection needle carry out disinfection
Sterilizing.
(4) before microinjection, fresh wild cabbage juice is squeezed, slide is immersed in 20 min, taking-up in wild cabbage juice and is dried, two
Slide overlapping, edge are sealed with sealed membrane, are placed in the diamondback moth cage of oviposition, collect fresh diamondback moth every a hour
Ovum.
(5) before microinjection, sgRNA/Cas9 mixture is prepared, sgRNA and Cas9 protein concentration are respectively 524 ng/ μ l
It is mended so that the final concentration of sgRNA and Cas9 is respectively 150 ng/ μ l and 300 ng/ μ l with sterile purified water with 1000 ng/ μ l
Enough to 5 μ l.
2. microinjection:
(1) Eggs of Diamondback Moth given birth under dark surrounds is taken, the slide for sealing up sealed membrane is unfolded, German Eppendorf is used
4 injecting systems of FemtoJet 4i and InjectMan of company carry out microinjection, and wound is small as far as possible when injection, will injure
It is preferably minimized.
(2) Eggs of Diamondback Moth after injection is put in temperature is 26 °C, in the environment that relative humidity is 65%, ovum is waited to incubate
Change, calculates hatching rate.
, mutated individual detection and screening:
(1) co-injection DBM1Ac-S strain Eggs of Diamondback Moth 211 has 117 egg hatchings in the Eggs of Diamondback Moth of G0 generation injection, incubates
Rate is 55%, has 70 to pupate in this 117 ovum, extracts the gDNA sample that fresh pupa is sloughed off, and pupa is sloughed off gDNA extraction and used
KAPA Quick Extract kit (KAPA Biosystems).
The pupa of single diamondback moth individual is sloughed off to collect respectively and is placed in 1.5 ml centrifuge tubes, stainless shot is added, is added
30 μ l extracting solutions are as shown in table 2 ground sample using electric grinding instrument.
Table 2
| Reagent | System |
| 10×KAPA Quick Extract Buffer | 3 μl |
| 1 U/μl KAPA Quick Extract Enzyme | 0.5 μl |
| PCR-grade water | 26.5 μl |
Application program (75 °C of 10 min；95 °C 5 min；4 °C of ∞) carry out PCR.
Gained PCR product is that pickles chrysalis sloughs off gDNA, is saved backup in -20 °C.
(2) specific primer (forward primer APN1-F, as shown in SEQ ID NO. 4 is utilized；Reverse primer APN1-R, such as
Shown in SEQ ID NO. 5, particular sequence is as shown in Figure 1) carry out PCR reaction amplification (table 3) sgRNA target site sequence nearby
(Fig. 3), by PCR product direct Sequencing, screening obtains 34 heterozygous mutant individuals, i.e. the mutation rate in G0 generation is about 49%.
Table 3
| Reagent | System |
| 10 μM APN1-F | 2 μl |
| 10 μM APN1-R | 2 μl |
| PrimeSTAR Max Premix (2×) | 12.5 μl |
| GDNA template | 150 ng |
| ddH2O | To 25 μ l |
(3) in 34 mutated individual phase mutual crosses for obtaining G0 generation screening, generation G1 generation, obtain 98 pupas in G1 generation, continue
After extracting the gDNA sample that fresh pupa is sloughed off, PCR product direct Sequencing, while connecting Beijing Quan Shijin Biotechnology Co., LtdpEASY- T1 cloning vector converts Escherichia coli, carries out TA sequencing, specific mutated-genotype is determined, as shown in figure 4, G1 is for miscellaneous
Closing mutated individual is 38, mutation rate 39%.
(4) it chooses G1 generation sequencing screening to obtain, quantity most (n=20) and simultaneously containing the miscellaneous of 2-bp (GC) missing
Zygote diamondback moth individual is hybridized, and G2 generation is generated.
(5) after G2 is pupated for diamondback moth, after extracting the gDNA sample that 128 fresh pupas are sloughed off, near PCR amplification target site
Sequence, by PCR product direct Sequencing, screening obtains 26 homozygous mutation individuals, and homozygous rate is about 20%.By these homozygous mutations
The G3 generation that individual generates after being hybridized is homozygous mutation population, as shown in Figure 5.
Four, diamondback moth Bt Cry1Ac insecticidal proteins virulence bioassay
(1) blade infusion process is used, using original Bt sensitive population DBM1Ac-S as negative control, by Bt Cry1Ac desinsection egg
It is white to be sequentially configured to 7 series of concentrations (DBM1Ac-S:10 mg/L, 5 mg/L, 2.5 mg/L, 1.25 mg/ by proportional diluted method
L, 0.625 mg/L, 0.3125 mg/L and 0.15625 mg/L；APN1KO:1200 mg/L, 600 mg/L, 300 mg/L, 150
Mg/L, 75 mg/L, 37.5 mg/L and 18.75 mg/L) carry out virulence bioassay.
(2) virulence is raw surveys the results show that with DBM1Ac-S groupy phase ratio, and APN1 homozygous mutation population is to Bt
The resistant multiple of Cry1Ac insecticidal proteins has reached 413 times, forms significant Cry1Ac resistant phenotype (table 4).
The virulence bioassay of 4. Populations of Diamondback Moth, Plutella Xylostella Bt Cry1Ac insecticidal proteins of table
| Population | Quantity | LC50 (95% confidence interval)a | Slope | Card side's (freedom degree) | Relevant antagonism multipleb |
| DBM1Ac-S | 210 | 0.75 (0.60-0.94) | 2.38 ± 0.28 | 1.38(5) | 1.00 |
| APN1KO | 210 | 346.96 (275.98-445.76) | 2.34 ± 0.29 | 2.09(5) | 462.61 |
aLC50Value is to kill the Cry1Ac insecticidal proteins concentration (mg/L) of 50% diamondback moth larvae, if two population LC50Value
95% confidence interval, which is not overlapped, is considered as significant difference；
bRelevant antagonism multiple=APN1KO population LC50Value/DBM1Ac-S population LC50Value.
(3) therefore, for the present invention by CRISPR/Cas9 technology, demonstrating APN1 in diamondback moth body can be used as Bt
The functional receptor of Cry1Ac insecticidal proteins, gene mutation can cause diamondback moth to produce Bt Cry1Ac insecticidal proteins
463 it is resistance.
<110>Vegetable & Flower Inst., Chinese Academy of Agriculture Science
<120>diamondback moth APN1 gene and its application are knocked out using CRISPR/Cas9 gene editing system
<160> 5
<210> 1
<211> 23
<212> DNA
<400> 1
AGGCCGTGGGTGTACTGCGCCGG
<210> 2
<211> 64
<212> DNA
<400> 2
GAAATTAATACGACTCACTATAGGAGGCCGTGGGTGTACTGCGCGTTTTAGAGCTAGAAATAGC
<210> 3
<211> 80
<212> DNA
<400> 3
AAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCT
CTAAAAC
<210> 4
<211> 25
<212> DNA
<400> 4
GCCTGTGTCTTTCAGTGGACTGTAC
<210> 5
<211> 28
<212> DNA
<400> 5
GCTCGTTGTCTTCTACTACACTATTGAG
Claims (10)
1. a kind of knockout technique of the diamondback moth APN1 gene based on CRISPR/Cas9 technology.
2. the method as described in claim 1, which is characterized in that design and synthesize diamondback moth APN1 gene specific sgRNA target
Sequence is marked, microinjection is to diamondback moth front blastoderm phase ovum Posterior pole position after sgRNA is mixed with Cas9 albumen.
3. method according to claim 2, which is characterized in that the trace gDNA sloughed off using the fresh pupa of diamondback moth four-age larva is mould
Plate carries out PCR amplification and direct Sequencing identification G0 for mutated individual using specific primer, and G0 mutually mates for mutated individual and obtains
G1 generation is obtained, directly and after TA sequencing identification the enough and heterozygote individuals with identical mutation type of quantity is chosen and carries out selfing acquisition
G2 generation, TA sequencing identification obtain to stablize the G2 of heredity for homozygotic individual, and then selfing obtains the APN1 homozygous gene in G3 generation
Saltant type population.
4. method according to claim 2, which is characterized in that the design of sgRNA target sequence is encoded in diamondback moth APN1 gene
12nd exon in area, nucleotide sequence is as shown in SEQ ID NO. 1.
5. method as claimed in claim 4, which is characterized in that the synthetic primer of sgRNA target site are as follows:
CRISPR forward primer is as shown in SEQ ID NO. 2 and CRISPR reverse primer is as shown in SEQ ID NO. 3.
6. method according to claim 2, which is characterized in that final concentration of 150 ng/ μ l, the Cas9 albumen of sgRNA
Final concentration of 300 ng/ μ l.
7. method as claimed in claim 3, which is characterized in that utilize forward primer SEQ ID NO. 4 and reverse primer SEQ
ID NO. 5 can be with the gDNA sequence near specific amplification APN1 gene sgRNA target site.
8. a kind of diamondback moth APN1 gene knockout kit based on CRISPR-Cas9 gene Knockout, comprising: nucleotides sequence
Arrange the sgRNA target sequence as shown in SEQ ID NO. 1 and Cas9 albumen.
9. kit as claimed in claim 8, which is characterized in that further comprise matched detection reagent, for detecting
State the shear effect and assessment gene knockout efficiency of gene.
10. kit as claimed in claim 8, which is characterized in that further comprise SEQ ID NO. 2 and SEQ ID NO.
The primer of sequence shown in 3；And the primer of sequence shown in SEQ ID NO. 4 and SEQ ID NO. 5.
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| CN201811642687.3A CN109593762A (en) | 2018-12-29 | 2018-12-29 | Diamondback moth APN1 gene and its application are knocked out using CRISPR/Cas9 gene editing system |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110616267A (en) * | 2019-07-26 | 2019-12-27 | 中国农业科学院蔬菜花卉研究所 | Method for detecting resistance of diamondback moth to Bt insecticidal protein Cry1Ac based on APN gene and kit thereof |
| CN110760514A (en) * | 2019-10-31 | 2020-02-07 | 福建农林大学 | Species-specific insecticides based on genome editing and DNA barcodes |
| CN111647664A (en) * | 2020-06-19 | 2020-09-11 | 河北大学 | Method for non-invasive identification of insect genotype |
| CN113528529A (en) * | 2021-08-03 | 2021-10-22 | 淮北师范大学 | Method for obtaining Period gene mutation site based on CRISPR/Cas9 technology and application |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104404036A (en) * | 2014-11-03 | 2015-03-11 | 赛业（苏州）生物科技有限公司 | Conditional gene knockout method based on CRISPR/Cas9 technology |
-
2018
- 2018-12-29 CN CN201811642687.3A patent/CN109593762A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104404036A (en) * | 2014-11-03 | 2015-03-11 | 赛业（苏州）生物科技有限公司 | Conditional gene knockout method based on CRISPR/Cas9 technology |
Non-Patent Citations (4)
| Title |
|---|
| XIN ZHANG ET AL.: "Resistance of Trichoplusia ni to Bacillus thuringiensis Toxin Cry1Ac Is Independent of Alteration of the Cadherin-Like Receptor for Cry Toxins", 《PLOS ONE》 * |
| 童晓玲等: "CRISPR/Cas9系统在昆虫中的应用", 《遗传》 * |
| 郭兆将: "小菜蛾对Bt Cry1Ac毒素抗性的分子机制研究", 《中国博士学位论文全文数据库 农业科技辑》 * |
| 龚佑静: "小菜蛾对Bt毒素Cry1Ac抗性的生化与分子机理", 《中国博士学位论文全文数据库 农业科技辑》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110616267A (en) * | 2019-07-26 | 2019-12-27 | 中国农业科学院蔬菜花卉研究所 | Method for detecting resistance of diamondback moth to Bt insecticidal protein Cry1Ac based on APN gene and kit thereof |
| CN110616267B (en) * | 2019-07-26 | 2022-08-19 | 中国农业科学院蔬菜花卉研究所 | Method for detecting resistance of diamondback moth to Bt insecticidal protein Cry1Ac based on APN gene and kit thereof |
| CN110760514A (en) * | 2019-10-31 | 2020-02-07 | 福建农林大学 | Species-specific insecticides based on genome editing and DNA barcodes |
| CN110760514B (en) * | 2019-10-31 | 2021-06-01 | 福建农林大学 | Species-specific insecticides based on genome editing and DNA barcodes |
| CN111647664A (en) * | 2020-06-19 | 2020-09-11 | 河北大学 | Method for non-invasive identification of insect genotype |
| CN113528529A (en) * | 2021-08-03 | 2021-10-22 | 淮北师范大学 | Method for obtaining Period gene mutation site based on CRISPR/Cas9 technology and application |
| CN113528529B (en) * | 2021-08-03 | 2023-01-24 | 淮北师范大学 | Method for obtaining Period gene mutation site based on CRISPR/Cas9 technology and application |
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