CN108588077A - The application of CYP6AB9 genes and the dsRNA and the two of interference bollworm gossypol metabolism in preventing bollworm - Google Patents
The application of CYP6AB9 genes and the dsRNA and the two of interference bollworm gossypol metabolism in preventing bollworm Download PDFInfo
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- CN108588077A CN108588077A CN201810424082.0A CN201810424082A CN108588077A CN 108588077 A CN108588077 A CN 108588077A CN 201810424082 A CN201810424082 A CN 201810424082A CN 108588077 A CN108588077 A CN 108588077A
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- bollworm
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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
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- A01N57/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic phosphorus compounds
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Abstract
The present invention relates to biotechnologies, specifically, provide a kind of application of the dsRNA and the two of CYP6AB9 genes and interference bollworm gossypol metabolism in preventing bollworm, the dsRNA that will be designed according to bollworm CYP6AB9 genes, bollworm is imported again, and actually the expression of CYP6AB9 genes is interfered using RNAi technology.The results show that dsRNA, which enters the intracellular of bollworm, has occurred RNAi cascade reactions, the expression of target gene receives interference.In the presence of gossypol, the weight reduction more apparent than control group of the bollworm of dsRNA is injected, dsRNA provided by the invention can actually interfere the gossypol metabolic detoxification process of bollworm；But when not having gossypol stress, the bollworm of the dsRNA normal growth and development as control group is injected, eliminates the possibility that CYP6AB9 genes itself participate in adjusting cotton bollworm larvae growth and development.
Description
Technical field
The present invention relates to biotechnologies, in particular to a kind of CYP6AB9 genes and interference bollworm gossypol generation
Applications of the dsRNA and the two thanked in preventing bollworm.
Background technology
Gossypol is the distinctive Secondary metabolites of cotton, has certain resist to the pest of many cottons
Property.Free gossypol has bigger toxicity, it is not only a kind of natural insecticide or a kind of Substance.Gossypol
Containing can interact with the amino acid of protein there are two the aldehyde radical of high activity and to form schiff base structure protein is made to hand over
Connection reaction, and then protein is made to lose activity.Bollworm is the important agricultural insect using cotton as main host plants, in Helicoverpa armigera
Cytochrome P450 (CYP) participate in gossypol metabolic detoxification approach, therefore, bollworm to gossypol have certain resistance.
Bollworm belongs to Lepidoptera, Noctuidae, is a kind of worldwide pest.Because its host range it is wide, it is adaptable,
It is also easy to produce the features such as drug resistance also has both migrating property so that bollworm becomes a kind of omnivorousness agriculture endangering serious and more difficult prevention
Industry pest.The means of prevention of early stage mainly prevents bollworm by chemical pesticide, with indiscriminate use of pesticide, field
There is drug resistance in bollworm, and control effect is also worse and worse while polluting environment.Bt insecticidal protein genes are transferred to by the later stage
Cotton obtains the transgenic cotton of expression Bt insecticidal proteins, can reduce making for pesticide with lepidoptera pests such as high resistance helicoverpa armigeras
Dosage and frequency, this largely alleviates the resistance problem of bollworm.But with the extensive plantation of Bt cottons, bollworm pair
Bt cottons also produce a degree of drug resistance, present need exist for combined use chemical pesticide and come to preventing bollworm.Therefore,
It is extremely urgent to explore new medicament target for the problem of being increasingly difficult in face of cotton bollworm control.
It refers to that external source or endogenous double-stranded RNA (dsRNA) specifically cause silenced gene expression that RNA, which interferes (RNAi),
Phenomenon.DsRNA enters after cell the microRNA i.e. siRNA that 21-23bp can be cut by Dicer digestions, and then RNA is being induced
Under the action of silencing complex (RNA-induced silencing complex, RISC) siRNA by uncoiling at single-stranded, instead
Adopted chain is specifically combined with homology targets mRNA, and silenced gene expression is eventually led to.Since RNAi specificity is higher, it is fabricated to
The features such as this is relatively low, has been used as a kind of new method to be applied in agricultural insect pests control.
In view of this, special propose the present invention.
Invention content
The first object of the present invention is to provide a kind of dsRNA, can induce bollworm CYP6AB9 silenced gene expressions,
And then bollworm is to the resistance of gossypol；
The second object of the present invention is to provide the preparation method of above-mentioned dsRNA, provides a kind of prepare and prevent bollworm production
The preparation method of product；
The third object of the present invention is to provide the application of above-mentioned dsRNA, and CYP6AB9 genes is utilized to participate in bollworm gossypol
It is metabolized this function, bollworm is prevented by RNAi technology；
The fourth object of the present invention is to provide the product for preventing bollworm, to alleviate bollworm pair in the prior art
The resistance of pesticide and Bt cottons is more and more stronger, the more and more difficult problem of the preventing and controlling of bollworm；
The fifth object of the present invention is to provide the product that CYP6AB9 genes prevent bollworm in prevention bollworm or preparation
In application, utilize CYP6AB9 genes participate in metabolic pathway reduce bollworm gossypol resistance；
The sixth object of the present invention is to provide expression cassette, recombinant vector, recombinant bacterial strain containing SEQ ID No.2 sequences
Or transgenosis system cell line, a variety of products that can quickly prepare prevention bollworm dsRNA are provided；
The seventh object of the present invention is to provide a kind of method of prevention bollworm, to alleviate bollworm pair in the prior art
The resistance of pesticide and Bt cottons is more and more stronger, the more and more difficult problem of the preventing and controlling of bollworm.
In order to realize that the above-mentioned purpose of the present invention, spy use following technical scheme：
The present invention provides a kind of dsRNA, for it is following a) or b)：
A) double-stranded RNA that the nucleotide sequence shown in SEQ ID No.1 and its reverse complementary sequence form；
B) double-stranded RNA and a) limited has 80% or more homology and double-stranded RNA with the same function.
Further, the DNA sequence dna of dsRNA is nucleotide sequence shown in SEQ ID No.2 in encoding a)；
Preferably, the sense primer that amplification obtains the DNA sequence dna of dsRNA in the coding a) is shown in SEQ ID No.3
Nucleotide sequence, amplification obtain the DNA sequences of dsRNA in the coding a) downstream primer be SEQ ID No.4 shown in
Nucleotide sequence.
The present invention provides the preparation method of above-mentioned dsRNA, and the preparation method includes：According to the sequence of CYP6AB9 genes
Designated rna interferes primer, and amplification bollworm genomic templates obtain the DNA sequence dna of coding dsRNA, and then synthesize and obtain dsRNA.
Further, the sense primer of the RNA interference primer is nucleotide sequence shown in SEQ ID No.3, described
It is nucleotide sequence shown in SEQ ID No.4 that RNA, which interferes the downstream primer of primer,；
Preferably, the DNA sequence dna of the coding dsRNA is nucleotide sequence shown in SEQ ID No.2, the dsRNA
In positive-sense strand be SEQ ID No.1 shown in nucleotide sequence.
The present invention provides applications of the above-mentioned dsRNA in following any one of (a)-(e)：
(a) reducing survival rate or preparation of the bollworm in the presence of gossypol reduces survival rate of bollworm in the presence of gossypol
Product；
(b) growth or preparation of the bollworm in the presence of gossypol is inhibited to inhibit the production of growth of bollworm in the presence of gossypol
Product；
(c) inhibit the metabolism of bollworm gossypol or prepare the product for inhibiting the metabolism of bollworm gossypol；
(d) bollworm CYP6AB9 gene expression amounts are reduced or prepare the production for reducing bollworm CYP6AB9 gene expression amounts
Product；
(e) it prevents bollworm or prepares the product of prevention bollworm.
The present invention is provided to prevent the product of bollworm, active constituent includes above-mentioned dsRNA.
The present invention provides application of the CYP6AB9 genes in prevention bollworm or in preparing the product for preventing bollworm, described
The sequence of CYP6AB9 genes is nucleotide sequence shown in SEQ ID No.5.
The present invention provides the expression cassette of DNA sequence dna containing above-mentioned coding dsRNA, recombinant vector, recombinant bacterial strain or turns base
Because being cell line.
The present invention provides a kind of method of prevention bollworm, by inhibiting the expression of CYP6AB9 genes to the cotton of bollworm
Phenol metabolism is interfered, to realize prevention bollworm.
Further, inhibit CYP6AB9 gene expressions by the way that above-mentioned dsRNA is imported bollworm；
Preferably, the mode of the importing includes injection or feeding；
Preferably, described feed is：Direct-fed dsRNA, the bacterial strain for expressing dsRNA or the feed containing dsRNA；
Preferably, described inject is：By drawing the capillary that needle instrument pulls out that dsRNA is injected into the abdomen inverse the of larva
Between two and third podomere；
Preferably, when the larva is 2-3 instar larvaes, the injection volume of dsRNA is 2-3 μ g.
Compared with prior art, beneficial effects of the present invention are：
A kind of dsRNA of present invention offer is designed using RNAi technology according to bollworm CYP6AB9 genes
DsRNA, then dsRNA is imported into bollworm, actually the expression of CYP6AB9 genes is interfered using RNAi technology.Knot
Fruit shows, the bollworm CYP6AB9 genes from mRNA level in-site after detection injection dsRNA are significantly reduced, illustrate dsRNA into
Enter the intracellular of bollworm and RNAi cascade reactions have occurred, the expression of target gene CYP6AB9 genes receives interference.From form
From the point of view of learning angle, in the presence of gossypol, the weight drop more apparent than control group of the bollworm of injection CYP6AB9 genes dsRNA
Low, bollworm gene C YP6AB9 genes take part in the metabolic detoxification process of gossypol really, and dsRNA provided by the invention really can
Enough interfere the gossypol metabolic detoxification process of bollworm；But when there is no gossypol stress, the cotton of injection CYP6AB9 genes dsRNA
Earworm normal growth and development as control group eliminates CYP6AB9 genes itself and participates in adjusting cotton bollworm larvae growth and development
Possibility.This shows that CYP6AB9 genes dsRNA can be used for the prevention of bollworm, and the present invention is to utilize RNAi technology in next step
The transgenic cotton for creating new varieties is laid a good foundation, and the later stage can utilize CYP6AB9 genes or dsRNA provided by the present application to prepare
Transgenic cotton realizes the species specific green prevention and control of bollworm object in conjunction with the Bt Insect Resistant Cottons of existing plantation, slows down target pest resistance
Generation.
Description of the drawings
Experimental group during Fig. 1 measures for bollworm feeding gossypol feed body weight increase after the injection dsRNA of the embodiment of the present invention 4
With changes of weight amount result schematic diagram of the control group under 0.1% gossypol stress after 72h；
Fig. 2 is experimental group in the silence efficiency detection of the embodiment of the present invention 4 and control group respectively when for 24 hours with 72h in body
CYP6AB9 genes expression amount on rna level changes result schematic diagram；
Fig. 3 is that experimental group and control group exist in the influence that the CYP6AB9 gene pairs insect growths of the embodiment of the present invention 4 are developed
The changes of weight amount result schematic diagram after 72h is raised under normal condition.
Specific implementation mode
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.It is not specified in embodiment specific
Condition person carries out according to conventional conditions or manufacturer's recommended conditions.
The present invention provides a kind of dsRNA, for it is following a) or b)：
A) double-stranded RNA that the nucleotide sequence shown in SEQ ID No.1 and its reverse complementary sequence form；
B) double-stranded RNA and a) limited has 80% or more homology and double-stranded RNA with the same function.
The dsRNA can cause the expression silencing of bollworm CYP6AB9 genes, and then inhibit metabolism of the bollworm to gossypol
Approach reduces resistance of the bollworm to gossypol, to reduce the survival rate of bollworm, achievees the purpose that prevent bollworm.
In an embodiment of the invention, the DNA sequence dna of dsRNA is core shown in SEQ ID No.2 in encoding a)
Nucleotide sequence.
In an embodiment of the invention, the sense primer of the DNA sequence dna of dsRNA is during amplification obtains coding a)
Nucleotide sequence shown in SEQ ID No.3, downstream primer are nucleotide sequence shown in SEQ ID No.4.Above-mentioned primer pair
Specificity is good, and amplification efficiency is high.
In an embodiment of the invention, the expression cassette of the DNA sequence dna containing above-mentioned coding dsRNA, recombinant vector, again
Group bacterial strain or transgenosis system cell line all belong to the scope of the present invention.Pass through expression cassette, recombinant vector, recombinant bacterial strain or transgenosis
Being cell line independently can quickly obtain above-mentioned dsRNA, or independently can be used for carrier mediated RNAi technology,
Or it independently can be used for preparing the transgenic cotton of bollworm resisting.
The present invention provides the preparation method of above-mentioned dsRAN again, and above-mentioned dsRNA is obtained by following methods：It is interfered using RNA
Technology interferes primer according to the sequence design RNA of CYP6AB9 genes, primer amplification bollworm genomic templates is interfered using RNA
The DNA sequence dna of coding dsRNA is obtained, and then synthesizes and obtains dsRNA.This method is prepared for the sequence of CYP6AB9 genes
DsRNA, the dsRAN specificity being prepared is high, can efficiently inhibit the expression of CYP6AB9 genes, induce CYP6AB9 genes
Silence.
In an embodiment of the invention, it is core shown in SEQ ID No.3 that RNA, which interferes the sense primer of primer,
Nucleotide sequence, it is nucleotide sequence shown in SEQ ID No.4 that RNA, which interferes the downstream primer of primer,.The primer specificity is good, expands
Increasing Efficiency is high.
In an embodiment of the invention, the DNA sequence dna that RNA interferes primer amplification to obtain is SEQ ID No.2 institutes
The nucleotide sequence shown, the positive-sense strand in the dsRNA that DNA sequence dna is prepared are nucleotide sequence shown in SEQ ID No.1.
The present invention provides applications of the above-mentioned dsRNA in following any one of (a)-(e) again：
(a) reducing survival rate or preparation of the bollworm in the presence of gossypol reduces survival rate of bollworm in the presence of gossypol
Product；
(b) growth or preparation of the bollworm in the presence of gossypol is inhibited to inhibit the production of growth of bollworm in the presence of gossypol
Product；
(c) inhibit the metabolism of bollworm gossypol or prepare the product for inhibiting the metabolism of bollworm gossypol；
(d) bollworm CYP6AB9 gene expression amounts are reduced or prepare the production for reducing bollworm CYP6AB9 gene expression amounts
Product；
(e) it prevents bollworm or prepares the product of prevention bollworm.
Gossypol is the distinctive Secondary metabolites of cotton, has certain resist to the pest of many cottons
Property.And bollworm is the important agricultural insect using cotton as main host plants, can by internal metabolic detoxification enzyme to gossypol into
Row metabolism, wherein Cytochrome P450 (CYP) plays an important role wherein.Therefore, pass through the table of interference key CYP genes
It reaches, so that bollworm is significantly reduced the resistance of gossypol, can achieve the purpose that prevention and control bollworm.The dsRNA of the present invention is directed to
CYP6AB9 genes are prepared, and can expeditiously inhibit the expression of bollworm CYP6AB9 genes with characteristic, are coerced in gossypol
Under the conditions of, the gossypol metabolic pathway of bollworm receives inhibition, and the gossypol resistance of bollworm reduces, and then the life of bollworm always
It is long, the survival rate of bollworm is reduced, achievees the purpose that prevent bollworm.
The present invention provides the product for preventing bollworm again, and active constituent includes above-mentioned dsRNA.
The present invention provides bollworm CYP6AB9 genes answering in the product of prevention bollworm or preparation prevention bollworm
With, wherein the sequence of CYP6AB9 genes is nucleotide sequence shown in SEQ ID No.5.The present invention is obtained by experimental verification
Bollworm CYP6AB9 genes are not the essential genes needed for bollworm normal growth, but participate in the gossypol metabolism way of bollworm
Diameter can be inhibited the metabolism of bollworm gossypol, reach prevention bollworm using this function by inducing CYP6AB9 gene silencings
Purpose.CYP6AB9 genes participation bollworm gossypol is metabolized this function and can be applied in the work of cotton bollworm resisting.
The present invention finally provides a kind of method of prevention bollworm, by inhibiting the expression of CYP6AB9 genes to bollworm
Gossypol metabolism interfered, to realize prevention bollworm.
In an embodiment of the invention, above-mentioned dsRNA is imported into bollworm to inhibit CYP6AB9 gene expressions.
In an embodiment of the invention, the mode of importing includes injection or feeding.
In an embodiment of the invention, it feeds and is：Direct-fed dsRNA, the bacterial strain for expressing dsRNA contain
The feed of dsRNA.Feeding method is easy to operate and simple, is influenced on bollworm small.
In an embodiment of the invention, it injects and is：DsRNA is injected by the capillary for drawing needle instrument to pull out
The abdomen of larva is second from the bottom between third podomere.Injection method is efficient, can shorten and import the period.
In an embodiment of the invention, when larva is 2-3 instar larvaes, the injection volume of dsRNA is 2-3 μ g.With
The increase in worm age, corresponding dsRNA import volumes will also increase, when jamming effectiveness reaches 40%-50% or more for interference at
Work(.
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.
Bioinformatics technique is utilized in the present invention, analyzes this laboratory bollworm feeding cotton after measured, big
Transcript profile under the conditions of beans, corn and the different host plants of four kinds of capsicum, in conjunction with programs such as NCBI Blast, it is determined that cotton boll
Worm CYP6AB9 genes.
Strain (96S) is raised in bollworm room initially to acquire from Chinese Academy of Agricultural Sciences's Xinxiang Experimental Base in 1996.
Man-made feeds are raised so far, and rearing conditions are 27 ± 2 DEG C of temperature, relative humidity 75% ± 10%, photoperiod 14hL：10hD, at
Worm feeds 10% syrup of preparation after sprouting wings.
The extraction of embodiment 1RNA
(a) it by the cotton bollworm larvae of collection, pupa and adult, is ground with liquid nitrogen, 50-100mg powder is taken to be transferred to
In 1.5ml RNase-free centrifuge tubes, 1ml Trizol are added and mix well.It is incubated at room temperature 5min, in favor of in homogenised sample
Ribosome is kept completely separate.
(b) 200 μ l chloroforms are added, shakes 15s, is stored at room temperature 5min, is then centrifuged under the conditions of 12000rpm, 4 DEG C
15min。
(c) it takes 400 μ l of supernatant in new centrifuge tube, isometric isopropanol is then added, turn upside down to fully mixed
It is even, it is stored at room temperature 10min, 10min is centrifuged under the conditions of 12000rpm, 4 DEG C.
(d) it abandons supernatant, 75% ethyl alcohol of 1ml is added and gently washs, subsequent 12000rpm, centrifuge 5min under the conditions of 4 DEG C.
(e) supernatant is removed, after drying at room temperature precipitates, 50-150 μ l nuclease free waters is added and fully dissolve.
The synthesis of embodiment 2cDNA
Use reverse transcription reagent box (TransScript One-step gDNA Removal and cDNA Synthesis
SuperMix), cDNA is synthesized to the total serum IgE reverse transcription extracted in embodiment 1 according to its operating instruction.Concrete operations are as follows：
Configuration scheme such as following table：
Gently mixing is incubated 15min at 42 DEG C；85 DEG C of heating 5s inactivate reverse transcriptase.After completion of the reaction, super in -80 DEG C
Low temperature refrigerator preserves.
The preparation of embodiment 3dsRNA
GFP genes (green fluorescent protein) gene as a contrast is chosen, the gene is in insect bodies and is not present, and a large amount of
Research is all made of GFP gene chemical synthesis dsRNA as a contrast.
1) design of CYP6AB9 gene primers：
According to the sequence of target gene CYP6AB9 genes (SEQ ID No.5), using primer-design software Primer
Primer of the designs of Premier 5 with T7 promoters send to Shanghai life work and carries out primer synthesis.Primer sequence is as follows：
2) preparation of target fragment PCR amplification and dsRNA
Using bollworm cDNA as template, with FastPfu enzymatic amplification genes.Reaction system is as follows：
After the above reaction solution mixing, then of short duration centrifugation is reacted in PCR instrument by following procedure：
After the completion of PCR amplification, returned according to Wizard SV Gel and PCR Clean-Up System kit specifications
Receive PCR product.Then, with reference to the T7High Yield RNA Transcription Kit operation instructions of Nanjing Nuo Weizan companies
The sequence of positive-sense strand is shown in SEQ ID No.1 in book synthesis dsRNA, dsRNA：
Liquid-transfering gun gently mixing each component, of short duration centrifugation, 37 DEG C of incubation 4h.The DNase of 1 μ l is added in the reaction system
I, 37 DEG C of incubation 15min, digests the DNA profiling of transcription.The dsRNA electrophoretic analysis of synthesis saves backup after purification.
Embodiment 4 dsRNA injections
Uniform 3 instar larvae of build on the same day is selected, is placed on ice.By drawing the capillary that needle instrument pulls out by 2.5 μ g's
DsRNA in embodiment 3 be injected into larva abdomen it is second from the bottom between third podomere be used as experimental group, injection dsGFP make
For control group.
1) bollworm feeding gossypol feed body weight increase measures after injecting dsRNA
Larva single head after injection, which is placed in 24 orifice plates equipped with chow diet, to be restored for 24 hours, and experiment is then randomly selected
Group and each 8 of control group bollworm sample weigh weight record M1, are transferred in the gossypol feed containing 0.1%, claim after 72h
Record M2, experiment is taken to be repeated 3 times.The result collect statistics that 3 times are tested, shown in result figure 1, it can be seen that injection CYP6AB9
The larval weight growth of gene dsRNA is obviously suppressed (* expressions p compared with control group (dsGFP)<0.05).Illustrate in cotton
Under phenol stress conditions, CYP6AB9 genes dsRNA can cause the growth of bollworm significantly to be inhibited.
2) silence efficiency detects
Experimental group and control group inject respectively dsRNA and dsGFP for 24 hours and 72h, prepare sample simultaneously utilize qPCR technologies
The silence efficiency of CYP6AB9 genes is detected, reference gene selects β-actin.All detections include that 3 biology repeat, each
It repeats to include 5 samples, significance difference analysis is carried out using the independent sample t test of SPSS softwares.The results are shown in Figure 2,
DsRNA provided by the invention obviously inhibits the expression of CYP6AB9 genes, for 24 hours when inhibition it is apparent, as a result illustrate in life
The expression for surveying CYP6AB9 genes in the time is repressed with obvious effects, because dsRNA has degradation in vivo after 72h, inhibits
Effect is declined.
Wherein, the primer of reference gene β-actin is：β-actin-F：5’-cctggtattgctgaccgtatgc-3’
(SEQ ID No.8), β-actin-R：5’-ctgttggaaggtggagagggaa-3’(SEQ ID No.9).
3) influence of CYP6AB9 gene pairs insect growth development
The sample of experimental group and control group is fed into chow diet together, experimental group is chosen and control group bollworm sample is each
8, experiment 3 times is repeated, observes changes of weight, the results are shown in Figure 3, finds two kinds of body weight increases without significant difference, row after 72h
In addition to CYP6AB9 genes itself participate in adjusting the possibility of cotton bollworm larvae growth and development.
The above results show that bollworm CYP6AB9 genes take part in the metabolic detoxification process of gossypol.
Although illustrate and describing the present invention with specific embodiment, it will be appreciated that without departing substantially from the present invention's
Many other change and modification can be made in the case of spirit and scope.It is, therefore, intended that in the following claims
Including belonging to all such changes and modifications in the scope of the invention.
SEQUENCE LISTING
<110>Shenzhen agricultural Joint Genome Institute of the Chinese Academy of Agricultural Sciences
<120>The application of CYP6AB9 genes and the dsRNA and the two of interference bollworm gossypol metabolism in preventing bollworm
<160> 9
<170> PatentIn version 3.5
<210> 1
<211> 469
<212> RNA
<213>Artificial sequence
<400> 1
accaagaauu ucaaauacug ggagaaaaga ggaaucaaac augacaagcc gcucccgcuc 60
uucggaaaca auauaaaggg auauuuuuuc uauucgagua ugacacaagu agccgacgag 120
uuguacugga aguaucccaa cgaacgaguc gugggcuucu uccgugccuc agaaccugaa 180
cuccugauaa gagacccaga aaucgcgaag cgacuuuuaa gcaaugacuu uucucauuuc 240
uuucaacgag gauuuacucc uaacaagacc guguuugaac caaugaugca aaaucucuuc 300
uuugcugaag gagaucucug gaagcuacug cgacagagaa ugacuccugc auuuacuuca 360
gggaagcuga aggcuauguu ccccuugaua guggagagag cagagaggcu gaagacucga 420
gcgcucgccg cagccgccga uggcaagucu uuggacgcuc gugaucuga 469
<210> 2
<211> 469
<212> DNA
<213>Artificial sequence
<400> 2
accaagaatt tcaaatactg ggagaaaaga ggaatcaaac atgacaagcc gctcccgctc 60
ttcggaaaca atataaaggg atattttttc tattcgagta tgacacaagt agccgacgag 120
ttgtactgga agtatcccaa cgaacgagtc gtgggcttct tccgtgcctc agaacctgaa 180
ctcctgataa gagacccaga aatcgcgaag cgacttttaa gcaatgactt ttctcatttc 240
tttcaacgag gatttactcc taacaagacc gtgtttgaac caatgatgca aaatctcttc 300
tttgctgaag gagatctctg gaagctactg cgacagagaa tgactcctgc atttacttca 360
gggaagctga aggctatgtt ccccttgata gtggagagag cagagaggct gaagactcga 420
gcgctcgccg cagccgccga tggcaagtct ttggacgctc gtgatctga 469
<210> 3
<211> 43
<212> DNA
<213>Artificial sequence
<400> 3
taatacgact cactataggg accaagaatt tcaaatactg gga 43
<210> 4
<211> 41
<212> DNA
<213>Artificial sequence
<400> 4
taatacgact cactataggg tcagatcacg agcgtccaaa g 41
<210> 5
<211> 1542
<212> DNA
<213>Bollworm
<400> 5
atgattaccc tcgccataat agtggtcctg ttgatctcct tgtacctcta cggtaccaag 60
aatttcaaat actgggagaa aagaggaatc aaacatgaca agccgctccc gctcttcgga 120
aacaatataa agggatattt tttctattcg agtatgacac aagtagccga cgagttgtac 180
tggaagtatc ccaacgaacg agtcgtgggc ttcttccgtg cctcagaacc tgaactcctg 240
ataagagacc cagaaatcgc gaagcgactt ttaagcaatg acttttctca tttctttcaa 300
cgaggattta ctcctaacaa gaccgtgttt gaaccaatga tgcaaaatct cttctttgct 360
gaaggagatc tctggaagct actgcgacag agaatgactc ctgcatttac ttcagggaag 420
ctgaaggcta tgttcccctt gatagtggag agagcagaga ggctgaagac tcgagcgctc 480
gccgcagccg ccgatggcaa gtctttggac gctcgtgatc tgatggccag gtacactacc 540
gacttcatag gagcctgcgg gtttggtcta gacgcagaca ctctcaacga tgaaaactct 600
ccatttagac aacttggtat caacattttc aaactaaaac ctatcgagct agcgaaaatt 660
gttgtaaagg aaatgtttcc ggacctgtgc cagaacgtga agatttggac tcgtgtggag 720
aaagacatca atgagttggt cggagagata atggcgaaaa gaaacaataa gcctagtgga 780
agaggagact tcattgattt gatgttggag tgcaagatga aaggcacaat ggtcggcgag 840
tctattgagt cgaggaagcc tgacggatcc cctgaaacag ccagcttgga gttcaacgac 900
ggattaatag cagctcaagt gtttgtattc ttcgcagctg ggtttgagac gtcatcatca 960
gctactagct tcactctcca ccagttagcg taccatccag aggttcagaa gaaagctcaa 1020
gaggaagtgg accgcatttt agccaaacat gacggcaagc tgtcgtatga ctccatcaaa 1080
gagatgaact acctcgagat ggcgttcaag gaagggttac gaatgtttcc ttcgctgggc 1140
ttcttactgc gtcagtgcac tcgaccctac accttcccag agttcaatat gaccatcgat 1200
gaaacttgca agattctgat tccgctgcag tctctgcata acgaccccaa gtacttccct 1260
aaccctgagg tgttccggcc ggagagattt tctccagaag agttcgattc caataataag 1320
ttcgtctatt tgccgtttgg actaggacca agagcttgca taggtgagcg gctaggtctg 1380
atgcagtctc tagcaggcct ggcagcagtg ctgtccgaat tcacagtgga gccagcgccc 1440
gagaccctac ggtaccctgt ggtagaccct cagtccagca tagtgcagag tgttcaagga 1500
ggcctacccc ttatgttccg ccctaggaag aagcttgctt ga 1542
<210> 6
<211> 38
<212> DNA
<213>Artificial sequence
<400> 6
taatacgact cactataggg cgatttcaag gaggacgg 38
<210> 7
<211> 39
<212> DNA
<213>Artificial sequence
<400> 7
taatacgact cactataggg ccatgccatg tgtaatccc 39
<210> 8
<211> 22
<212> DNA
<213>Artificial sequence
<400> 8
cctggtattg ctgaccgtat gc 22
<210> 9
<211> 22
<212> DNA
<213>Artificial sequence
<400> 9
ctgttggaag gtggagaggg aa 22
Claims (10)
1. a kind of dsRNA, which is characterized in that for it is following a) or b)：
A) double-stranded RNA that the nucleotide sequence shown in SEQ ID No.1 and its reverse complementary sequence form；
B) double-stranded RNA and a) limited has 80% or more homology and double-stranded RNA with the same function.
2. dsRNA according to claim 1, which is characterized in that the DNA sequence dna of dsRNA is SEQ ID No.2 in encoding a)
Shown in nucleotide sequence；
Preferably, the sense primer that amplification obtains the DNA sequence dna of dsRNA in the coding a) is core shown in SEQ ID No.3
Nucleotide sequence, the downstream primer that amplification obtains the DNA sequence dna of dsRNA in the coding a) are nucleosides shown in SEQ ID No.4
Acid sequence.
3. the preparation method of dsRNA as claimed in claim 1 or 2, which is characterized in that the preparation method includes：According to
The sequence design RNA of CYP6AB9 genes interferes primer, amplification bollworm genomic templates to obtain the DNA sequence dna of coding dsRNA,
And then it synthesizes and obtains dsRNA.
4. preparation method according to claim 3, which is characterized in that the sense primer of the RNA interference primer is SEQ
The downstream primer of nucleotide sequence shown in ID No.3, the RNA interference primer is nucleotides sequence shown in SEQ ID No.4
Row；
Preferably, the DNA sequence dna of the coding dsRNA is nucleotide sequence shown in SEQ ID No.2, in the dsRNA
Positive-sense strand is nucleotide sequence shown in SEQ ID No.1.
5. applications of the dsRNA as claimed in claim 1 or 2 in following any one of (a)-(e)：
(a) reducing survival rate or preparation of the bollworm in the presence of gossypol reduces the production of survival rate of bollworm in the presence of gossypol
Product；
(b) growth or preparation of the bollworm in the presence of gossypol is inhibited to inhibit the product of growth of bollworm in the presence of gossypol；
(c) inhibit the metabolism of bollworm gossypol or prepare the product for inhibiting the metabolism of bollworm gossypol；
(d) bollworm CYP6AB9 gene expression amounts are reduced or prepare the product for reducing bollworm CYP6AB9 gene expression amounts；
(e) it prevents bollworm or prepares the product of prevention bollworm.
6. for preventing the product of bollworm, which is characterized in that its active constituent includes that right wants dsRNA described in 1 or 2.
Application of the 7.CYP6AB9 genes in prevention bollworm or in preparing the product for preventing bollworm, which is characterized in that described
The sequence of CYP6AB9 genes is nucleotide sequence shown in SEQ ID No.5.
8. expression cassette, recombinant vector, recombinant bacterial strain or the transgenosis of the DNA sequence dna containing the dsRNA described in coding claim 2
It is cell line.
9. a kind of method of prevention bollworm, which is characterized in that by inhibiting the expression of CYP6AB9 genes to the gossypol of bollworm
Metabolism is interfered, to realize prevention bollworm.
10. according to the method described in claim 9, it is characterized in that, by the way that dsRNA described in claims 1 or 2 is imported cotton boll
Worm inhibits CYP6AB9 gene expressions；
Preferably, the mode of the importing includes injection or feeding；
Preferably, described feed is：Direct-fed dsRNA, the bacterial strain for expressing dsRNA or the feed containing dsRNA；
Preferably, described inject is：By draw the capillary that needle instrument pulls out dsRNA is injected into larva abdomen it is second from the bottom with
Between third podomere；
Preferably, when the larva is 2-3 instar larvaes, the injection volume of dsRNA is 2-3 μ g.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109762839A (en) * | 2019-01-24 | 2019-05-17 | 中国农业科学院深圳农业基因组研究所 | CCE001a protein expression and/or active substance is inhibited to reduce the application in tolerance of the bollworm to gossypol |
| CN113519547A (en) * | 2021-07-05 | 2021-10-22 | 广东省科学院动物研究所 | Application of AtCYP303A1 inhibitor in prevention and treatment of bee parasites |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107406849A (en) * | 2015-02-27 | 2017-11-28 | 先锋国际良种公司 | To prevent and treat the composition of insect pest and method |
-
2018
- 2018-05-03 CN CN201810424082.0A patent/CN108588077B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107406849A (en) * | 2015-02-27 | 2017-11-28 | 先锋国际良种公司 | To prevent and treat the composition of insect pest and method |
Non-Patent Citations (2)
| Title |
|---|
| JIN M等: "《Adaptive regulation of detoxification enzymes in Helicoverpa armigera to different host plants》", 《INSECT MOLECULAR BIOLOGY》 * |
| 靳明辉: "《棉铃虫对棉酚的代谢机制研究》", 《中国博士学位论文全文数据库 农业科技辑》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109762839A (en) * | 2019-01-24 | 2019-05-17 | 中国农业科学院深圳农业基因组研究所 | CCE001a protein expression and/or active substance is inhibited to reduce the application in tolerance of the bollworm to gossypol |
| CN113519547A (en) * | 2021-07-05 | 2021-10-22 | 广东省科学院动物研究所 | Application of AtCYP303A1 inhibitor in prevention and treatment of bee parasites |
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