CN110938634B - Apoptosis gene fragment for reducing egg laying amount of small brown planthopper and application thereof - Google Patents
Apoptosis gene fragment for reducing egg laying amount of small brown planthopper and application thereof Download PDFInfo
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- CN110938634B CN110938634B CN201911316663.3A CN201911316663A CN110938634B CN 110938634 B CN110938634 B CN 110938634B CN 201911316663 A CN201911316663 A CN 201911316663A CN 110938634 B CN110938634 B CN 110938634B
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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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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N57/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic phosphorus compounds
- A01N57/10—Biocides, pest repellants or attractants, or plant growth regulators containing organic phosphorus compounds having phosphorus-to-oxygen bonds or phosphorus-to-sulfur bonds
- A01N57/16—Biocides, pest repellants or attractants, or plant growth regulators containing organic phosphorus compounds having phosphorus-to-oxygen bonds or phosphorus-to-sulfur bonds containing heterocyclic radicals
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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
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1096—Processes for the isolation, preparation or purification of DNA or RNA cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
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- 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
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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
- C12N2310/14—Type of nucleic acid interfering N.A.
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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
- C12N2330/00—Production
- C12N2330/30—Production chemically synthesised
Abstract
The invention discloses an apoptosis gene fragment for reducing the egg laying amount of Laodelphax striatellus and application thereof, wherein the gene sequence is shown as SEQ ID NO.1, and the amino acid sequence is shown as SEQ ID NO. 2. In the invention, dsRNA of the gene fragment for apoptosis of the laodelphax striatellus is synthesized by a molecular biology technology, the egg laying amount of the laodelphax striatellus can be obviously reduced by using a feeding method, and a gene which can be used as an effective target spot for biological control of the laodelphax striatellus is found; the dsRNA sequence of the synthetic Laodelphax striatellus apoptosis gene segment is shown as SEQ ID NO.5, can effectively silence genes, and well resist the degradation of RNA enzyme; meanwhile, the synthesis cost is low, and the large-scale popularization and application are facilitated; the dsRNA of the laodelphax striatellus apoptosis gene has obvious effect of reducing the egg laying amount of the laodelphax striatellus through an RNAi technology, provides a new way for establishing a pest control method by utilizing the RNAi technology, and reduces the use of chemical pesticides.
Description
Technical Field
The invention relates to the technical field of agricultural science, in particular to an apoptosis gene fragment for reducing the egg laying amount of Laodelphax striatellus and application thereof.
Background
The Laodelphax striatellus is used as a main pest in food areas in China, can directly eat and damage food crops, can transmit various crop viruses to indirectly damage the crops, and causes the yield of the food crops to be reduced by 30-50 percent every year or even the food crops are completely harvested. In China, chemical pesticides are important and main means for controlling Laodelphax striatellus. However, the continuous single use of chemical pesticides has long led to the development of varying degrees of resistance to a variety of pesticides by the laodelphax striatellus. In order to achieve satisfactory control effect, farmers continuously increase the pesticide consumption, but the control effect is not ideal and the environmental pollution is aggravated, thus forming a vicious circle. Therefore, in agricultural production practice, the development of green, safe, and sustainable biological control methods and means for laodelphax striatellus has been necessary.
The primary objective of pest control is to control pest population numbers below economic thresholds (to prevent pest occurrence above economic damage levels when control measures should be taken). RNA interference (RNAi) technology developed in recent years has been widely used in research on gene function and pest control. Theoretically, if the RNAi technology is used to interfere the expression of important reproductive genes in agricultural pests, the reproductive capacity of the pests is reduced, and the population number is reduced, so that the purpose of controlling the pests is achieved.
The Chinese invention patent CN106191003B discloses a drug-resistant carboxylesterase gene LSCE12 of laodelphax striatellus, a gene fragment for reducing the drug resistance of the laodelphax striatellus and application thereof, and provides a drug-resistant carboxylesterase gene LSCE12 of the laodelphax striatellus, wherein the gene fragment (namely dsRNA) for reducing the drug resistance of the laodelphax striatellus can be obtained by utilizing the gene, RNAi is fed, and the drug resistance of the laodelphax striatellus is further reduced, so that the laodelphax striatellus is more easily killed by an insecticide, and the using amount of chemical pesticides is reduced. Although the method effectively reduces the using amount of chemical pesticide, the chemical pesticide still needs to be used, and the offspring of the laodelphax striatellus with high drug resistance also has higher drug resistance, thus being not beneficial to the long-term control of the laodelphax striatellus.
In view of the above, there is an urgent need to improve the existing methods for controlling laodelphax striatellus, reduce the use of chemical pesticides and facilitate long-term control.
Disclosure of Invention
The invention aims to solve the technical problem that the existing method for preventing and controlling the Laodelphax striatellus needs to use chemical pesticides.
In order to solve the technical problems, the technical scheme adopted by the invention is to provide the apoptosis gene fragment for reducing the egg laying amount of the laodelphax striatellus, wherein the gene sequence is shown as SEQ ID NO.1, and the amino acid sequence is shown as SEQ ID NO. 2.
In the scheme, the method for cloning the apoptosis gene fragment for reducing the egg laying amount of the laodelphax striatellus comprises the following steps:
step one, extracting total RNA of female adult Laodelphax striatellus, and synthesizing a first strand cDNA by taking the total RNA of the Laodelphax striatellus as a template;
step two, taking the first chain of the laodelphax striatellus cDNA synthesized in the step one as a template, and carrying out PCR amplification on a target gene through an upstream primer P1 with a sequence of SEQ ID NO.3 and a downstream primer P2 with a sequence of SEQ ID NO. 4;
separating the obtained PCR product by agarose gel electrophoresis, purifying the product, and sequencing by using a full-automatic sequencer to obtain a sequence, namely the apoptosis gene;
step four, designing a primer containing a T7 promoter for the apoptosis gene sequence obtained by sequencing the step three, synthesizing dsRNA of an apoptosis gene fragment in vitro and storing for later use.
In the scheme, the synthesis method of the apoptosis gene fragment for reducing the egg laying amount of the laodelphax striatellus comprises the steps of taking a first strand of a cDNA of the laodelphax striatellus as a template, taking an upstream primer P3 with a sequence of SEQ ID NO.6 and a downstream primer P4 with a sequence of SEQ ID NO.7, and carrying out PCR amplification to obtain a dsRNA of the apoptosis gene of the laodelphax striatellus.
In the scheme, the method for synthesizing the apoptosis gene fragment for reducing the egg laying amount of the laodelphax striatellus comprises the following specific steps:
extracting total RNA of female adult laodelphax striatellus, and synthesizing a cDNA first strand by using the total RNA of the laodelphax striatellus as a template;
step two, taking the first strand of the cDNA of the laodelphax striatellus synthesized in the step one as a template, and carrying out PCR amplification by using an upstream primer P3 sequence containing a T7 promoter as SEQ ID NO.6 and a downstream primer P4 sequence containing a T7 promoter as SEQ ID NO. 7;
step three, separating the products obtained by PCR in the step two through agarose gel electrophoresis, and recovering a target DNA band;
step four, connecting the target DNA fragment recovered in the step three to a pEASY-T1 vector by using T4 ligase, introducing the target DNA fragment into escherichia coli DH5 alpha through transformation, coating the escherichia coli DH5 alpha on an LB solid culture medium containing ampicillin, and culturing the ampicillin overnight at 37 ℃;
fifthly, selecting the bacterial colony in the fourth step, and carrying out PCR identification and sequencing on the bacterial colony;
step six, performing amplification culture on the positive recombinants by using an LB liquid culture medium containing ampicillin, and extracting clone plasmids;
step seven, taking the plasmid in the step six as a template, and carrying out PCR amplification on a target gene by using an upstream primer P3 sequence containing a T7 promoter as SEQ ID NO.6 and a downstream primer P4 sequence containing a T7 promoter as SEQ ID NO. 7;
and step eight, purifying the PCR product of the target gene amplified in the step seven, and amplifying by taking the PCR product as a template to obtain the dsRNA of the gene for reducing the oviposition amount of the laodelphax striatellus.
In the scheme, the dsRNA is uniformly mixed with feed and then fed to the laodelphax striatellus.
In the scheme, the concentration of the dsRNA of the apoptosis gene segment for reducing the egg laying amount of the Laodelphax striatellus is 3000-5000 ng/mu L.
Compared with the prior art, the invention has the following advantages:
1. synthesizing dsRNA of a small brown planthopper apoptosis gene fragment by a molecular biology technology, obviously reducing the egg laying amount of the small brown planthopper by a feeding method, and finding out a gene which can be used as an effective target for biological control of the small brown planthopper;
2. the dsRNA synthesized into the gene fragment for apoptosis of the laodelphax striatellus can effectively silence the gene and well resist the degradation of RNA enzyme; meanwhile, the synthesis cost is low, and large-scale popularization and application are facilitated;
3. the dsRNA of the laodelphax striatellus apoptosis gene has obvious effect of reducing the egg laying amount of the laodelphax striatellus through an RNAi technology, provides a new way for establishing a pest control method by utilizing the RNAi technology, and avoids using chemical pesticides.
Drawings
FIG. 1 is a DNA band of a fragment of the apoptosis gene Lsbcl-2;
FIG. 2 shows a dsRNA band of a segment of the apoptosis gene Lsbcl-2.
Detailed Description
The invention provides an apoptosis gene fragment for reducing the egg laying amount of small brown planthoppers and application thereof, wherein the small brown planthoppers are effectively prevented and treated by reducing the egg laying amount of the small brown planthoppers. The invention is described in detail below with reference to the drawings and the detailed description.
Example 1, method for cloning Lsbcl-2 fragment of apoptosis gene of Laodelphax striatellus.
The method comprises the following specific steps:
step one, taking 40-50 female adult Laodelphax striatellus ovaries, and extracting total RNA by a TRizoL method;
step two, taking the total RNA as a template, and synthesizing a cDNA first chain by reverse transcription;
step three, acquiring a gene fragment sequence from the gene group of the Laodelphax striatellus, predicting a Laodelphax striatellus apoptosis gene Lsbcl-2 after homology comparison in an NCBI database (http:// www.ncbi.nlm.nih.gov /), designing an upstream Primer P1(SEQ ID NO.3) and a downstream Primer P2(SEQ ID NO.4) by using Primer-Blast (https:// www.ncbi.nlm.nih.gov/tools/Primer-Blast /) in the NCBI database, and carrying out PCR amplification;
upstream primer P1(SEQ ID NO. 3): 5'-CGTCTGCTGAAGTGGTGGAT-3'
Downstream primer P2(SEQ ID NO. 4): 5'-TCAGGAAACACCTCTCGCAC-3'
The PCR amplification system is as follows: mu.L of 5 Xreaction Buffer, 2. mu.L of dNTP, 1. mu.L of cDNA template in step (1), 2. mu.L of upstream primer P1 with sequence of SEQ ID NO.3, 2. mu.L of downstream primer P2 with sequence of SEQ ID NO.4, 0.5. mu.L of Taq enzyme, 8.5. mu.L of dd H 2 O, 20 mu L in total; the PCR amplification reaction procedure was as follows: denaturation at 95 deg.C for 2 min; 35 cycles of 95 ℃ 20sec, 55 ℃ 20sec, 72 ℃ 1 min; stretching at 72 ℃ for 10 min; storing at 10 deg.C;
and step four, separating the obtained PCR product through agarose gel electrophoresis, purifying the product, and sequencing the product by using a full-automatic sequencer to obtain the sequence, namely the apoptosis gene, namely the gene fragment shown in SEQ ID NO. 1.
Example 2 dsRNA synthesis and recovery of Laodelphax striatellus apoptosis gene Lsbcl-2.
The method comprises the following specific steps:
step one, according to the verified gene sequence, an upstream Primer P3(SEQ ID NO.6) and a downstream Primer P4(SEQ ID NO.7) are designed by using Primer-Blast (https:// www.ncbi.nlm.nih.gov/tools/Primer-Blast /) in NCBI database
Upstream primer P3(SEQ ID NO. 6):
5′-taatacgactcactatagggGTGAGCGATTTGCCTTTGGG-3′
downstream primer P4(SEQ ID NO. 7):
5′-taatacgactcactatagggGTAGACCGCCATCCGATTGT-3′
control GFP gene design upstream primer P5(SEQ ID NO.8) and downstream primer P6(SEQ ID NO.9)
Upstream primer P5(SEQ ID NO. 8):
5′-taatacgactcactatagggAGAATGAGTAAAGGAGAAGAACTTTTC-3′
downstream primer P6(SEQ ID NO. 9):
5′-taatacgactcactatagggAGATTTGTATAGTTCATCCATGCCATGT-3′
the PCR amplification system is as follows: 10 μ L of 2 × reaction Buffer, 2 μ L of forward primer P3(SEQ ID NO.8), 2 μ L of reverse primer P4(SEQ ID NO.9), 2 μ L of cDNA template, 4 μ L of dd H 2 O, 20 mu L in total; the PCR amplification reaction procedure was as follows: denaturation at 95 deg.C for 3 min; 30sec at 95 ℃, 30sec at 60 ℃, 1min at 72 ℃ and 35 cycles; extending at 72 ℃ for 7 min;
step two, separating the PCR amplification product by agarose gel electrophoresis with the concentration of 1.2 percent, cutting the gel under an ultraviolet lamp, and recovering by adopting an AxyPrep DNA gel recovery kit of Aisijin biotechnology company;
the specific operation is as follows:
1. cutting agarose gel containing purposeful DNA under an ultraviolet lamp, putting the agarose gel into a 1.5 mL-weighed microcentrifuge tube, and weighing again to obtain the weight of the cut gel;
2. adding 3 gel volumes of Buffer DE-A (100mg to 100 mu L volume) according to the weight of the obtained gel, mixing uniformly, then carrying out water bath at 75 ℃, and intermittently and uniformly mixing until the gel is completely melted;
3. adding 0.5 Buffer DE-A volume of Buffer DE-B and uniformly mixing;
4. transferring the mixed solution to a DNA preparation tube, centrifuging at 12,000 Xg for 1min, and discarding the filtrate;
5. add 500. mu.L Buffer W1 to the DNA preparation tube, centrifuge at 12,000 Xg for 30s, discard the filtrate;
6. add 700. mu.L Buffer W2 to the DNA preparation tube, centrifuge at 12,000 Xg for 30s, discard the filtrate;
7. add 700. mu.L Buffer W2 again to the DNA preparation tube, centrifuge at 12,000 Xg for 1min, discard the filtrate;
8. without adding liquid, placing the DNA preparation tube in a centrifuge tube, and centrifuging for 1min at 12,000 Xg;
9. transferring the DNA preparation tube to a new 1.5mL microcentrifuge tube, adding 30 μ L of nucleic-Free Water, standing at room temperature for 1min, and centrifuging at 12,000 Xg for 1 min;
10. the DNA product of interest was collected and stored at-20 ℃.
Step three, connecting the recovered PCR amplification product to a pEASY-T1 vector under the action of T4 ligase, transforming the recombinant plasmid into escherichia coli DH5 alpha, coating the escherichia coli DH5 alpha on an LB solid culture medium containing 50ng/mL of ampicillin, and culturing overnight at 37 ℃;
picking colonies from an LB solid culture medium containing ampicillin, carrying out amplification culture on the colonies in an LB liquid culture medium containing ampicillin, and extracting clone plasmids;
and step five, performing PCR amplification again by taking the plasmid as a DNA template, wherein the system is as follows: 50 μ L of 2 × reaction Buffer, 4 μ L of forward primer P3(SEQ ID NO.8), 4 μ L of reverse primer P4(SEQ ID NO.9), 8 μ L of cDNA template, 34 μ L of dd H 2 O, 100 mu L in total; the PCR amplification reaction procedure was as follows: denaturation at 95 deg.C for 3 min; 30sec at 95 ℃, 30sec at 60 ℃, 1min at 72 ℃ and 35 cycles; extension was carried out at 72 ℃ for 7 min. Purifying PCR amplification products by using a recovery kit;
and step six, obtaining dsRNA of the Laodelphax striatellus apoptosis gene Lsbcl-2 by using a PCR product as a template for amplification. The amplification system was as follows: mu.L 10 × reaction buffer, 2. mu.L ATP, 2. mu.L GTP, 2. mu.L UTP, 2. mu.L CTP, 1. mu.g purified PCR product as template, 2. mu. L T7 RNA Polymerase Mix, plus dd H 2 O, 20 mu L in total; the reaction procedure is as follows: at 37 ℃ for 6 h; dsRNA of the gene was obtained.
Example 3, laodelphax striatellus feeding dsRNA experiment.
The method comprises the following specific steps:
uniformly mixing feed for Laodelphax striatellus and dsRNA (the concentration is 4000 ng/mu L) of an apoptosis gene;
step two, preparing a plurality of double-pass glass tubes, sealing one ends of the double-pass glass tubes by using gauze and rubber bands, and sucking 2-3-year-old Laodelphax striatellus nymphs by using an electric trematode device to be added into the double-pass glass tubes;
sealing the other end of the double-way glass tube by using a Parafilm sealing film, sucking 100 mu L of dsRNA mixed solution (treatment group) of the feed for the laodelphax striatellus and the apoptosis gene or 100 mu L of dsGFP mixed solution (control group) of the feed for the laodelphax striatellus and the control GFP gene by using a liquid transfer gun, dripping the dsGFP mixed solution (control group) of the feed for the laodelphax striatellus and the control GFP gene at the center of the sealing film, and sealing the mixed solution of the feed for the laodelphax striatellus and the apoptosis dsRNA mixed solution between two layers of sealing films by using the other sealing film;
and step four, placing the double-pass glass tube added with the mixed solution of the laodelphax striatellus feed and the cell apoptosis dsRNA at the temperature of 25 +/-1 ℃, the relative humidity of 60% and the illumination period of 16L-8D. Feeding the plant hoppers by using phototaxis habits of the plant hoppers, and changing the mixed solution of the plant hopper feed and cell apoptosis dsRNA (double strand ribonucleic acid) every 24 hours or the dsGFP mixed solution of the plant hopper feed and a control GFP gene;
and step five, continuously feeding the laodelphax striatellus until the laodelphax striatellus emerges into adults. And (3) independently picking out the female adults, placing the female adults in test tubes with rice seedlings, matching, and checking the egg laying amount every 24h until the female adults die. During the period, if the male adult dies, the male adult is added again. The results are shown in table 1, and the dsRNA mixed solution of the feed for feeding the laodelphax striatellus and the apoptosis gene Lsbcl-2 has obvious effect of reducing the egg laying amount of the laodelphax striatellus from the table 1.
TABLE 1
The invention has the following advantages:
1. synthesizing dsRNA of a laodelphax striatellus apoptosis gene Lsbcl-2 by a molecular biology technology, and utilizing a feeding method to obviously reduce the egg laying amount of the laodelphax striatellus so as to find a gene which can be used as an effective target for biological prevention and control of the laodelphax striatellus;
2. the dsRNA synthesized into the Laodelphax striatellus apoptosis gene Lsbcl-2 can effectively silence the gene and well resist the degradation of RNA enzyme; meanwhile, the synthesis cost is low, and the large-scale popularization and application are facilitated;
3. the dsRNA of the laodelphax striatellus apoptosis gene has obvious effect of reducing the egg laying amount of the laodelphax striatellus through an RNAi technology, provides a new way for establishing a pest control method by utilizing the RNAi technology, and reduces the use of chemical pesticides.
The present invention is not limited to the above preferred embodiments, and any structural changes made under the teaching of the present invention shall fall within the protection scope of the present invention.
Sequence listing
<110> Zhongshan university
<120> apoptosis gene fragment for reducing egg laying amount of Laodelphax striatellus and use thereof
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<400> 5
gacggcucag caacgucagc gacgccgucu cucggaaguu cuccaacaca aucggauggc 60
ggucuaccau uccagucgau cacaucgugc aacagggacg gacgcucugc ggacaguacg 120
uccgcagucg ccugaaacgg uccggucucu ucagccgaaa gugcggacug cagcggcugc 180
ggagugcggc uaguuugccu ggcgguuacg uagugcgaga gguguuuccu gagauguugg 240
gcgugggagu ggagcuggag cgcaugcauc cgaagcugua cgccggggug gccagacagg 300
cgaguugcag ccccggcg 318
<210> 6
<211> 40
<212> DNA
<213> Artificial sequence (2 Ambystoma latex x Ambystoma texanum)
<400> 6
taatacgact cactataggg gtgagcgatt tgcctttggg 40
<210> 7
<211> 40
<212> DNA
<213> Artificial sequence (2 Ambystoma latex x Ambystoma texanum)
<400> 7
taatacgact cactataggg gtagaccgcc atccgattgt 40
<210> 8
<211> 47
<212> DNA
<213> Artificial sequence (2 Ambystoma latex x Ambystoma texanum)
<400> 8
taatacgact cactataggg agaatgagta aaggagaaga acttttc 47
<210> 9
<211> 48
<212> DNA
<213> Artificial sequence (2 Ambystoma latex x Ambystoma texanum)
<400> 9
taatacgact cactataggg agatttgtat agttcatcca tgccatgt 48
Claims (6)
1. The apoptosis gene segment for reducing the egg laying amount of the Laodelphax striatellus is characterized in that the gene sequence is shown as SEQ ID NO.1, and the amino acid sequence is shown as SEQ ID NO. 2.
2. The method for cloning the apoptosis gene fragment capable of reducing the egg laying amount of the laodelphax striatellus according to claim 1, comprising the following steps:
step one, extracting total RNA of female adult Laodelphax striatellus, and synthesizing a first strand cDNA by taking the total RNA of the Laodelphax striatellus as a template;
step two, taking the first strand of the cDNA of the laodelphax striatellus synthesized in the step one as a template, and carrying out PCR amplification on a target gene through an upstream primer P1 with a sequence of SEQ ID NO.3 and a downstream primer P2 with a sequence of SEQ ID NO. 4;
separating the obtained PCR product by agarose gel electrophoresis, purifying the product, and sequencing by using a full-automatic sequencer to obtain a sequence, namely the apoptosis gene;
step four, designing a primer containing a T7 promoter for the apoptosis gene sequence obtained by sequencing the step three, synthesizing dsRNA of an apoptosis gene fragment in vitro and storing for later use.
3. The method for synthesizing the apoptosis gene fragment capable of reducing the egg laying amount of the laodelphax striatellus according to claim 1, wherein the first strand of the cDNA of the laodelphax striatellus is used as a template, and the dsRNA of the apoptosis gene of the laodelphax striatellus is obtained by performing PCR amplification on an upstream primer P3 with the sequence of SEQ ID No.6 and a downstream primer P4 with the sequence of SEQ ID No. 7.
4. The method for synthesizing the apoptosis gene fragment capable of reducing the egg laying amount of the laodelphax striatellus according to claim 3, comprising the following steps:
extracting total RNA of female adult laodelphax striatellus, and synthesizing a cDNA first strand by using the total RNA of the laodelphax striatellus as a template;
step two, taking the first chain of the laodelphax striatellus cDNA synthesized in the step one as a template, and performing PCR amplification through an upstream primer P3 sequence containing a T7 promoter as SEQ ID NO.6 and a downstream primer P4 sequence containing a T7 promoter as SEQ ID NO. 7;
step three, separating the products obtained by PCR in the step two through agarose gel electrophoresis, and recovering a target DNA band;
step four, connecting the target DNA fragment recovered in the step three to a pEASY-T1 vector by using T4 ligase, introducing the target DNA fragment into escherichia coli DH5 alpha through transformation, coating the escherichia coli DH5 alpha on an LB solid culture medium containing ampicillin, and culturing the ampicillin overnight at 37 ℃;
fifthly, selecting the bacterial colony in the fourth step, and carrying out PCR identification and sequencing on the bacterial colony;
step six, performing amplification culture on the positive recombinants by using an LB liquid culture medium containing ampicillin, and extracting clone plasmids;
step seven, taking the plasmid in the step six as a template, and carrying out PCR amplification on a target gene by using an upstream primer P3 sequence containing a T7 promoter as SEQ ID NO.6 and a downstream primer P4 sequence containing a T7 promoter as SEQ ID NO. 7;
and step eight, purifying the PCR product of the target gene amplified in the step seven, and amplifying by taking the PCR product as a template to obtain the dsRNA of the gene for reducing the oviposition amount of the laodelphax striatellus.
5. Use of the apoptotic gene fragment for reducing oviposition of laodelphax striatellus according to claim 1, wherein for reducing oviposition of laodelphax striatellus, dsRNA obtained by the cloning method of the apoptotic gene fragment for reducing oviposition of laodelphax striatellus according to claim 2 or the synthesis method of the apoptotic gene fragment for reducing oviposition of laodelphax striatellus according to any one of claims 3 to 4 is fed to laodelphax striatellus after being uniformly mixed with feed.
6. The use of the apoptotic gene fragment for reducing oviposition of laodelphax striatellus according to claim 5, wherein the dsRNA is present at a concentration of 4000ng/μ L.
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| CN106191003A (en) * | 2016-07-18 | 2016-12-07 | 江苏省农业科学院 | Small brown rice planthopper Drug resistance carboxylesterase gene LSCE12, the reduction drug-fast genetic fragment of small brown rice planthopper and application thereof |
| WO2016205445A1 (en) * | 2015-06-16 | 2016-12-22 | Pioneer Hi-Bred International, Inc. | Compositions and methods to control insect pests |
| CN110066798A (en) * | 2019-04-16 | 2019-07-30 | 杭州师范大学 | Application of the brown paddy plant hopper NlInR gene as target spot in preparation prevention and treatment brown paddy plant hopper drug |
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| WO2016205445A1 (en) * | 2015-06-16 | 2016-12-22 | Pioneer Hi-Bred International, Inc. | Compositions and methods to control insect pests |
| CN106191003A (en) * | 2016-07-18 | 2016-12-07 | 江苏省农业科学院 | Small brown rice planthopper Drug resistance carboxylesterase gene LSCE12, the reduction drug-fast genetic fragment of small brown rice planthopper and application thereof |
| CN110066798A (en) * | 2019-04-16 | 2019-07-30 | 杭州师范大学 | Application of the brown paddy plant hopper NlInR gene as target spot in preparation prevention and treatment brown paddy plant hopper drug |
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