CN109456977B - Gypsy moth BURS gene, encoding protein thereof and application of dsRNA thereof in pest control - Google Patents
Gypsy moth BURS gene, encoding protein thereof and application of dsRNA thereof in pest control Download PDFInfo
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Abstract
Gypsy moth BURS gene, coding protein thereof and application of dsRNA thereof in pest control relate to the field of molecular biology, in particular to gypsy moth BURS gene, coding protein thereof and application thereof. The invention aims to solve the problems of drug resistance and environmental pollution of the existing chemical control of gypsy moth. The nucleotide sequence of the gypsy moth BURS gene is shown as SEQ ID NO: 1 is shown. The amino acid sequence of the CDS coding protein of the coding region is shown as SEQ ID NO: 3, respectively. The gypsy moth BURS gene dsRNA can obviously inhibit the expression of BURS gene, so that the phenomenon that the female and male adult wings of gypsy moth cannot be normally stretched and the wings are deformed. The invention is used in the field of gypsy moth pest control.
Description
Technical Field
The invention relates to the field of molecular biology, in particular to a gypsy moth BURS gene, and a coding protein and application thereof.
Background
Gypsy moth (Lymantria dispar Linnaeus) is an important leaf-eating pest for agriculture and forestry, and has the characteristics of wide distribution range, multiple feeding plant types, high propagation speed, large propagation quantity, periodic outbreak and the like. According to domestic reports, gypsy moth can harm about 500 kinds of plants, which not only causes huge economic loss to forestry, but also damages the ecological benefit. At present, although natural enemy microorganisms, insects or physical methods are applied to the control of gypsy moth pests, the control effect on the explosive pests is not obvious, and chemical pesticides still occupy a main control means in the control of gypsy moth pests due to the efficient and quick disinfestation characteristics of the chemical pesticides. The long-time, high-frequency and continuous increase of the chemical pesticide causes the pests to have the problems of drug resistance, increase of the pests and the environment pollution such as the pesticide residue and the like. The development of the defects after the chemical pesticide is used promotes the development of the new generation of pest control research. The rapid development of molecular biology technology, and the application of RNA interference technology in the physiological mechanism research and prevention and control of pests, wherein the RNA interference technology becomes an important means for plant protectors to achieve pest control by means of the inhibition of normal development of pests caused by silencing functional genes by virtue of the characteristics of high-efficiency silencing, strong specificity, simple and convenient operation, time saving, small experimental scale and capability of hindering the normal physiological mechanism operation of insects. In the research of pest control through RNAi, effective gene fragments in important target genes are the key points of success.
Therefore, in order to solve the problems of drug resistance and environmental pollution of the existing chemical control of gypsy moth, the research of pest control by adopting a biological means is very important.
Disclosure of Invention
The invention aims to solve the problems of drug resistance and environmental pollution of the existing chemical control of gypsy moth, and provides a gypsy moth BURS gene, a coding protein thereof and application of dsRNA thereof in pest control.
The nucleotide sequence of the BURS gene of the gypsy moth is shown as SEQ ID NO: 1 is shown. The nucleotide sequence of the coding region CDS is shown as SEQ ID NO: 2, respectively.
The amino acid sequence of CDS coding protein of the coding region of the gypsy moth BURS gene is shown as SEQ ID NO: 3, respectively.
The dsRNA sequence of the buds gene of the gypsy moth is shown as SEQ ID NO: 4, respectively.
The invention takes gypsy moth BURS gene fragment as a template, designs a specific dsRNA primer pair, and synthesizes BURS gene dsRNA through a MEGAscript RNAi kit (Ambion).
The dsRNA of the buds gene of the gypsy moth is applied to pest control.
The specific method comprises the following steps: and injecting dsRNA of the gypsy moth BURS gene into 6-instar larvae of gypsy moth after hungry for 12 h. The injection dose was 24 μ g.
The invention has the beneficial effects that:
the tanning hormone is a heterodimer consisting of BURS (alpha subunit) and PBURS (beta subunit), is combined with a receptor thereof under specific conditions, plays a role in regulating tanning of a new epidermis after exuviation of an insect, extension and maturation of a wing, muscle contraction, migration of ovum marginal cells and the like, and is mainly synthesized in a thoracoabdominal ganglion. BURS and PBURS have independent biological activities under certain conditions, and the biological functions of homodimers of the BURS and the PBURS are unknown.
The invention inhibits the transcriptional level of tannase alpha subunit BURS gene by RNA interference technology, leads to the developmental deformity of gypsy moth imago wings to prevent and control the gypsy moth which is an important pest in agriculture and forestry.
The invention discloses a gypsy moth BURS gene full-length nucleic acid sequence and a sequence applied to synthesizing dsRNA, the gene full-length and the sequence used for synthesizing dsRNA provided by the invention can obviously inhibit the expression of BURS gene, so that the phenomenon that the wing phenotype of female and male adults of gypsy moth can not be normally extended is caused, and the wing development deformity indirectly influences the flight ability, mating and egg laying amount of the gypsy moth adults, thereby providing a new method for controlling gypsy moth pests without the problems of drug resistance and environmental pollution.
Drawings
FIG. 1 shows the BURS gene expression level of 6 th instar larvae of gypsy moth after dsRNA injection;
FIG. 2 is a graph of the effect of RNAi on the phenotype of gypsy moth male adult wings; wherein the left panel is a control drone and the right panel is a treated drone;
FIG. 3 is a graph of the effect of RNAi on the phenotype of gypsy moth female adult wings; wherein the left panel is a control female and the right panel is a treated female;
FIG. 4 is photographs of the front and back sides of dsRNA blocking the development of gypsy moth female adult wings; the left panel is control females and the right panel is treated females.
Detailed Description
The technical solution of the present invention is not limited to the following specific embodiments, but includes any combination of the specific embodiments.
The first embodiment is as follows: the nucleotide sequence of the BURS gene of the gypsy moth is shown as SEQ ID NO: 1, the nucleotide sequence of the coding region CDS is shown as SEQ ID NO: 2, respectively.
The second embodiment is as follows: the amino acid sequence of CDS coding protein of the coding region of the BURS gene of the gypsy moth is shown as SEQ ID NO: 3, respectively.
The third concrete implementation mode: the dsRNA sequence of the BURS gene of the gypsy moth of the embodiment is shown as SEQ ID NO: 4, respectively.
The invention takes gypsy moth BURS gene fragment as a template, designs a specific dsRNA primer pair, and synthesizes BURS gene dsRNA through a MEGAscript RNAi kit (Ambion).
The fourth concrete implementation mode: the dsRNA of the buds gene of the gypsy moth is applied to pest control.
The fifth concrete implementation mode: the fourth difference between this embodiment and the specific embodiment is that: the specific method for using dsRNA of the gypsy moth BURS gene for pest control comprises the following steps: and injecting dsRNA of the gypsy moth BURS gene into 6-instar larvae of gypsy moth after hungry for 12 h. The rest is the same as the fourth embodiment.
The sixth specific implementation mode: the fifth embodiment is different from the fifth embodiment in that: the injection dose was 24 μ g. The rest is the same as the fifth embodiment.
The following examples are given to illustrate the present invention, and the following examples are carried out on the premise of the technical solution of the present invention, and give detailed embodiments and specific procedures, but the scope of the present invention is not limited to the following examples.
Example 1: gypsy moth BURS gene full-length clone
Extracting gypsy moth larva total RNA according to the operational instruction of a TRIzol RNA animal and plant tissue total RNA extraction reagent of Invitrogen company, performing DNA digestion on the extracted RNA, detecting the RNA by using an ultraviolet spectrophotometer and electrophoresis, selecting qualified RNA, and performing PrimeScript according to TaKaRa companyTMThe RT-PCR Kit (model DRR014A) reverse transcription Kit operation stepsFirst strand cDNA Synthesis was performed. Designing a primer (a forward primer: 5'-TCGGAGTAAACAAACATAGCAAAG-3'; a reverse primer: 5'-TAGGGGTCCTTCTTCAGCAT-3') according to the nucleotide sequence of the annotated gene BURS of the gypsy moth larva transcriptome library, cloning the BURS sequence by using a reverse transcription-polymerase chain reaction (RT-PCR) method by taking a first strand of the cDNA as a template, and carrying out agarose gel electrophoresis detection on a PCR product. Recovering BURS gene segments according to the operation method of the E.Z.N.A. glue recovery kit, connecting the recovered PCR product with T-18(pMD18-T plasmid connecting cloning kit) overnight, transforming the connecting product into escherichia coli DH5 alpha competent cells, sending positive cloning bacterial liquid to Shanghai workers for sequencing through bacterial liquid PCR detection, and verifying a gypsy moth BURS gene coding frame.
The nucleic acid sequence of the gypsy moth BURS gene is 646bp, and is shown as SEQ ID NO: 1 is shown. The length of a coding region CDS is 480bp, and the coding region CDS is shown as SEQ ID NO: 2, respectively. Encodes 159 amino acids as shown in SEQ ID NO: 3, respectively. The encoded protein has a molecular weight of 17.79479kDa and a pI (theoretical isoelectric point) of 7.97, and is a basic protein.
Example 2: synthesis of gypsy moth BURS gene dsRNA
Designing and synthesizing dsRNA primers of the BURS gene according to the full length of the BURS gene cloned in the example 1, and adding a T7 promoter sequence with the size of 20bp, a forward primer (5'-TAATACGACTCACTATAGGGTGTTTCGTTTTGACGACTTTGT-3') and a reverse primer (5'-TAATACGACTCACTATAGGGAGGGGTCCTTCTTCAGCATA-3') to the 5 ' end of each specific primer; using the first chain of cDNA as a template, obtaining a fragment sequence with the length of 451bp by PCR amplification, carrying out agarose gel electrophoresis detection on a PCR product, and recovering the target fragment of 451bp by adopting an operation method of an E.Z.N.A. gel recovery kit. The recovered 451bp target fragment was used as a template to obtain dsRNA of BURS gene by using an in vitro dsRNA synthesis Kit (MEGAscript T7 Kit). An ultraviolet spectrophotometer and 2% agarose gel electrophoresis are used for detecting the concentration and the quality of the dsRNA. And (4) storing the mixture in a refrigerator at the temperature of minus 80 ℃ for later use.
Example 3: detection of gypsy moth BURS gene silencing effect
The dsRNA (24. mu.g) of BURS gene synthesized in example 2 was microinjected into dance virusThe 6 th larva of the moth takes an RNase-free water treatment group as a control, active larva is selected for 12h, 24 h, 36 h, 48 h and 72h respectively to extract total RNA, DNA is digested by DNase I (Promega), a PrimeScriptTM RT kit (TaKaRa) is adopted to synthesize a first strand of cDNA, the synthesized cDNA is diluted into 100 mu L and used as a real-time fluorescent quantitative RT-PCR template, and a fluorescent quantitative detection primer (a forward primer: 5'-GTAAGGGCGCAATCAGTGGA-3'; a reverse primer: 5'-CATGTGCGTTCCATTTGCCA-3') is designed to detect the expression quantity of the BURS gene after dsRNA injection. The expression level of the BURS gene of 6 th instar larvae of gypsy moth after dsRNA injection is shown in figure 1 (in figure 1)
Which is represented by CK and is represented by CK,
indicating injection of dsRNA), the results indicate: after the RNase-free water treatment group is used as a control, after dsRNA injection, the BURS gene mRNA expression levels are respectively 26.22%, 7.18%, 10.17%, 45.55% and 39.65% of those of the control at 12h, 24 h, 36 h, 48 h and 72h, and are remarkably silenced, RNAi interference efficiency is high, wherein the BURS expression level reaches the lowest at 12h, and the silencing effect is the best.
Real-time fluorescent quantitative PCR was performed using the kit SYBR Green Real-time PCR Master mix Plus (Toyobo). The internal reference genes are Actin, EF1 alpha and TUB, and the primer sequences are shown in Table 1. The real-time fluorescent quantitative PCR reaction system is as follows: 10 uL 2 × SYBR premix ExTaq enzyme, 1 uL each of forward and reverse primers (10 umol/L), 2 uL each of cDNA template, and deionized water to make up 20 uL; the reaction conditions are as follows: 30s at 94 ℃ and then 44 cycles: plates were read at 94 ℃ for 12s, 59 ℃ for 30s, 72 ℃ for 40s, and finally 81 ℃ for 1 s. Repeat 3 times per treatment with 2-△△CtThe method is used for analyzing the expression level of the gene.
TABLE 1 RNAi-related primer sequences
Example 4: dsRNA of gypsy moth BURS gene hinders development of gypsy moth adult wings
In vitro synthesized dsRNA (24. mu.g) from example 2 was microinjected into 6 th instar gypsy moth larvae after 12h starvation, 30 larvae per group were treated, repeated 3 times, and RNase-free water treated larvae were used as controls to replace fresh leaves daily while recording their effect on the gypsy moth adult fin phenotype. FIG. 2 is a graph of the effect of RNAi on the phenotype of gypsy moth male adult wings; the left panel is normal male moth, the right panel is male moth after dsRNA injection. FIG. 3 is a graph of the effect of RNAi on the phenotype of gypsy moth female adult wings; the left panel shows normal female moths, and the right panel shows female moths after dsRNA injection. FIG. 4 is photographs of the front and back sides of dsRNA blocking development of gypsy moth female adult wings, the left side is the front side, and the right side is the back side.
The results show that: the expression of BURS gene is reduced by micro-injection of dsRNA, which causes the development of wings of male and female gypsy moths to have deformity, thus preventing the wings of the adults from extending and influencing the flight capability of the adults. Therefore, the dsRNA of the gypsy moth BURS gene can be applied to the control of gypsy moth pests.
Sequence listing
<110> Neze college
<120> gypsy moth BURS gene, encoding protein thereof and application of dsRNA thereof in pest control
<160>16
<210> 1
<211> 646
<212> DNA
<213> Lymantria dispar Linnaeus)
<220>
<223> gypsy moth BURS gene full length
<400> 1
cttcggagta aacaaacata gcaaagttta agttcgcttc tttatactaa gatgtttcgt 60
tttgacgact ttgttatttt aagttttgta tttgcatttg cttgtcatat acatgatagg 120
ccagtaaggg cgcaatcagt ggaagtgccc ttatcaattg gtcaagaatg tcagatgaca 180
cctgttattc atgtcctaaa acatccagga tgtataccta aagctatacc ttcatttgcc 240
tgtatcggaa agtgtaccag ttacgtgcag gtttccggta gtaaaatttg gcaaatggaa 300
cgcacatgta actgttgcca agaatctggt gagcgggaag cttctgttgt actattgtgc 360
cctaaagcta agagcgaaga taagaagtta agaaggatta caaccaaagc tccccttgaa 420
tgtatgtgca gaccatgtgg tagtattgaa gaaagcgcta ttattcctca agaagtagct 480
ggatatgctg aagaaggacc cctatataat catttcagaa aaacattgta aaaaggttat 540
attcaaattt gtcaatgtgt tagtgattat taaattatta tccgatattg aagttattgt 600
agtaccatgt gaatttgata catactaaat aaataccttt gaccaa 646
<210> 2
<211> 480
<212> DNA
<213> Lymantria dispar Linnaeus)
<220>
<223> Lymantria dispar BURS gene CDS sequence
<400> 2
atgtttcgtt ttgacgactt tgttatttta agttttgtat ttgcatttgc ttgtcatata 60
catgataggc cagtaagggc gcaatcagtg gaagtgccct tatcaattgg tcaagaatgt 120
cagatgacac ctgttattca tgtcctaaaa catccaggat gtatacctaa agctatacct 180
tcatttgcct gtatcggaaa gtgtaccagt tacgtgcagg tttccggtag taaaatttgg 240
caaatggaac gcacatgtaa ctgttgccaa gaatctggtg agcgggaagc ttctgttgta 300
ctattgtgcc ctaaagctaa gagcgaagat aagaagttaa gaaggattac aaccaaagct 360
ccccttgaat gtatgtgcag accatgtggt agtattgaag aaagcgctat tattcctcaa 420
gaagtagctg gatatgctga agaaggaccc ctatataatc atttcagaaa aacattgtaa 480
<210> 3
<211> 159
<212> PRT
<213> Lymantria dispar Linnaeus)
<220>
<223> BURS gene-encoded protein
<400> 3
Met Phe Arg Phe Asp Asp Phe Val Ile Leu Ser Phe Val Phe Ala
5 10 15
Phe Ala Cys His Ile His Asp Arg Pro Val Arg Ala Gln Ser Val
20 25 30
Glu Val Pro Leu Ser Ile Gly Gln Glu Cys Gln Met Thr Pro Val
35 40 45
Ile His Val Leu Lys His Pro Gly Cys Ile Pro Lys Ala Ile Pro
50 55 60
Ser Phe Ala Cys Ile Gly Lys Cys Thr Ser Tyr Val Gln Val Ser
65 70 75
Gly Ser Lys Ile Trp Gln Met Glu Arg Thr Cys Asn Cys Cys Gln
80 85 90
Glu Ser Gly Glu Arg Glu Ala Ser Val Val Leu Leu Cys Pro Lys
95 100 105
Ala Lys Ser Glu Asp Lys Lys Leu Arg Arg Ile Thr Thr Lys Ala
110 115 120
Pro Leu Glu Cys Met Cys Arg Pro Cys Gly Ser Ile Glu Glu Ser
125 130 135
Ala Ile Ile Pro Gln Glu Val Ala Gly Tyr Ala Glu Glu Gly Pro
140 145 150
Leu Tyr Asn His Phe Arg Lys Tyr Leu
155 159
<210>4
<211> 451
<212> DNA
<213> Lymantria dispar Linnaeus)
<220>
<223> Lymantria dispar BURS gene dsRNA sequence
<400> 4
tgtttcgttt tgacgacttt gttattttaa gttttgtatt tgcatttgct tgtcatatac 60
atgataggcc agtaagggcg caatcagtgg aagtgccctt atcaattggt caagaatgtc 120
agatgacacc tgttattcat gtcctaaaac atccaggatg tatacctaaa gctatacctt 180
catttgcctg tatcggaaag tgtaccagtt acgtgcaggt ttccggtagt aaaatttggc 240
aaatggaacg cacatgtaac tgttgccaag aatctggtga gcgggaagct tctgttgtac 300
tattgtgccc taaagctaag agcgaagata agaagttaag aaggattaca accaaagctc 360
cccttgaatg tatgtgcaga ccatgtggta gtattgaaga aagcgctatt attcctcaag 420
aagtagctgg atatgctgaa gaaggacccc t 451
<210> 5
<211> 24
<212> DNA
<213> Artificial sequence
<220>
<223> BURS gene cloning forward primer
<400> 5
tcggagtaaacaaacatagcaaag 24
<210> 6
<211> 20
<212> DNA
<213> Artificial sequence
<220>
<223> BURS gene cloning reverse primer
<400> 6
taggggtccttcttcagcat 20
<210> 7
<211> 42
<212> DNA
<213> Artificial sequence
<220>
<223> dsRNA forward primer of BURS gene
<400> 7
taatacgactcactatagggtgtttcgttttgacgactttgt 42
<210> 8
<211> 40
<212> DNA
<213> Artificial sequence
<220>
<223> dsRNA reverse primer of BURS gene
<400> 8
taatacgactcactatagggaggggtccttcttcagcata 40
<210>9
<211> 24
<212> DNA
<213> Artificial sequence
<220>
<223> Actin Forward primer
<400> 9
atgttagtatgatcgagcgtatcg 24
<210>10
<211> 21
<212> DNA
<213> Artificial sequence
<220>
<223> Actin reverse primer
<400> 10
gcatgatctgaggagcatctt 21
<210>11
<211> 22
<212> DNA
<213> Artificial sequence
<220>
<223> EF1 alpha forward primer
<400> 11
tttgccttccttgcgctcaaca 22
<210>12
<211> 22
<212> DNA
<213> Artificial sequence
<220>
<223> EF1 alpha reverse primer
<400> 12
tgtaaagcagctgatcgtgggt 22
<210>13
<211> 22
<212> DNA
<213> Artificial sequence
<220>
<223> TUB Forward primer
<400> 13
aatgcaagaaagccttgcgcct 22
<210>14
<211> 22
<212> DNA
<213> Artificial sequence
<220>
<223> TUB reverse primer
<400> 14
atgaaggaggtcgacgagcaaa 22
<210>15
<211> 20
<212> DNA
<213> Artificial sequence
<220>
<223> q BURS forward primer
<400> 15
gtaagggcgcaatcagtgga 20
<210>16
<211> 20
<212> DNA
<213> Artificial sequence
<220>
<223> q BURS reverse primer
<400> 16
catgtgcgttccatttgcca 20
Claims (5)
1. The gypsy moth BURS gene is characterized in that the nucleotide sequence of the gene is shown as SEQ ID NO: 1, the nucleotide sequence of the coding region CDS is shown as SEQ ID NO: 2, respectively.
2. The codifying protein of the buds gene of gypsy moth according to claim 1, wherein the amino acid sequence of the codifying protein of the coding region CDS of the BURS gene of gypsy moth is as shown in SEQ ID NO: 3, respectively.
3. Use of the gypsy moth BURS gene of claim 1 to affect gypsy moth adult wing development; the influence on the development of the wings of the gypsy moth adults refers to the inhibition of the expression of BURS genes, which causes the development deformity of the wings of the gypsy moth adults, and the wings cannot be normally stretched.
4. Use according to claim 3, characterized in that the specific method of inhibiting the expression of the BURS gene is: injecting dsRNA of the gypsy moth BURS gene into 6-instar larvae of gypsy moth after hungry for 12 h; the dsRNA sequence of the gypsy moth BURS gene is shown as SEQ ID NO: 4, respectively.
5. Use according to claim 4, characterized in that the injected dose is 24 μ g/bar.
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| CN104561007A (en) * | 2015-02-03 | 2015-04-29 | 东北林业大学 | Lymantria dispar linnaeus CYP6B53 gene dsRNA and application thereof in nuisanceless control |
| CN104561018A (en) * | 2015-02-03 | 2015-04-29 | 东北林业大学 | Lymantria dispar linnaeus heatshock protein Hsp23 gene and application of dsRNA thereof in nuisanceless control |
| CN107287244A (en) * | 2017-07-17 | 2017-10-24 | 福建农林大学 | A kind of method using short single stranded DNA restraint insect gene expression |
| CN107446953A (en) * | 2017-07-17 | 2017-12-08 | 福建农林大学 | A kind of method using PCR primer restraint insect gene expression |
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| CN104561007A (en) * | 2015-02-03 | 2015-04-29 | 东北林业大学 | Lymantria dispar linnaeus CYP6B53 gene dsRNA and application thereof in nuisanceless control |
| CN104561018A (en) * | 2015-02-03 | 2015-04-29 | 东北林业大学 | Lymantria dispar linnaeus heatshock protein Hsp23 gene and application of dsRNA thereof in nuisanceless control |
| CN107287244A (en) * | 2017-07-17 | 2017-10-24 | 福建农林大学 | A kind of method using short single stranded DNA restraint insect gene expression |
| CN107446953A (en) * | 2017-07-17 | 2017-12-08 | 福建农林大学 | A kind of method using PCR primer restraint insect gene expression |
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