CN110358777A - The application of migratory locusts HMGR gene and its dsRNA in migratory locusts prevent and treat - Google Patents

The application of migratory locusts HMGR gene and its dsRNA in migratory locusts prevent and treat Download PDF

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CN110358777A
CN110358777A CN201910684482.XA CN201910684482A CN110358777A CN 110358777 A CN110358777 A CN 110358777A CN 201910684482 A CN201910684482 A CN 201910684482A CN 110358777 A CN110358777 A CN 110358777A
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migratory locusts
dsrna
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曾保娟
宋佳晟
周树堂
李湾湾
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Henan University
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Abstract

The invention belongs to field of biotechnology, a kind of application of migratory locusts HMGR gene and its dsRNA in migratory locusts prevent and treat is disclosed.After the dsRNA of the HMGR gene of synthesis is injected into migratory locusts body cavity, can specific silencing migratory locusts HMGR gene, cause migratory locusts to can not be successfully husking and enter next age, generate lethal effect, lethality is up to 99%.Due to high efficiency of the invention, specificity and safety, have broad application prospects in terms of migratory locusts control of insect, it is with good economic efficiency to protection China's agricultural production, ecological safety.

Description

The application of migratory locusts HMGR gene and its dsRNA in migratory locusts prevent and treat
Technical field
The invention belongs to field of biotechnology, are related to the application of migratory locusts HMGR gene and its dsRNA in migratory locusts prevention and treatment.
Background technique
Migratory locusts (Locusta migratoria) are fond of the cereal crops such as wheat, corn, for migrating property polyphagous pest-insect, are China or even worldwide important agricultural insect.Currently, the strategy of insect pest control based on chemical pesticide, leads to pest resistance to insecticide It improves, dosage increases, and control cost improves；Pesticide residue leads to environmental pollution, and agricultural product medicament residue endangers human health； Existing fungi microbe pesticide works slowly, and insecticide efficiency is low.Therefore, substitution chemical pesticide and fungi microbe pesticide are found Sustainability, high efficiency migratory locusts control strategy are extremely urgent.
RNA interference (RNA interference, RNAi) is one kind by small molecule double-stranded RNA (double stranded RNA, dsRNA) cause sequence specific post transcriptional gene silencing phenomenon, in the acquisition Nobel Prize in 2006.RNA interference is universal It is present in a variety of biologies such as nematode, fungi, insect and animals and plants, has the characteristics that efficient, special and gradually latent as one kind Control of insect technology.Control of insect based on RNA perturbation technique has the advantage that 1) specific: dsRNA only degrade and Its homologous mRNA reduces the toxic effect to non-target organism, especially to higher mammal and mankind's safety with higher Property；2) high efficiency: a small amount of dsRNA can effectively inhibit the expression of target gene, and dsRNA enter in pest body can cause it is complete The body even silencing of filial generation target gene.3) safety: RNA is easily degraded in nature, noresidue, to ecological environment and crops Product is pollution-free, comparatively safe.Currently, interfering the basic technology for carrying out control of insect based on RNA is that Screening target sequence obtains DsRNA with high lethal effect.
3- hydroxy-3-methyl glutaryl-CoA reductase (3-hydroxy-3-methylglutaryl CoA Reductase, HMGR) be mevalonic acid (Mevalonate, MVA) approach rate-limiting enzyme, under its effect, 3- hydroxyl -3- first Base glutaryl coenzyme A (HMG-CoA) is formed by the irreversible generation MVA of MVA approach, MVA through pyrophosphorylation and decarboxylation The isopentenyl pyrophosphate (IPP) of 5C, and IPP is the common precursor of terpenoid synthesis, therefore HMGR is cytoplasm terpene Important regulating and controlling point in compound metabolism.Terpenoid, also known as isoprenoid are one group of configurations and are related to cell hair Perhaps various compounds are educated, it is widely distributed in nature.In insect, by the juvenile hormone of corporal allata secretion (juvenile hormone, JH) is a kind of sesquiterpenoids, is one of hormone mostly important in insect bodies, it both existed Inhibit abnormal during insect larvae to pupa or adult, keep larval characters, and adult stage promotes adult raw after emergence It grows, has important regulating and controlling effect to insect growth, abnormal and reproduction.Therefore, HMGR is played in JH synthesis in regulation insect bodies Key effect, i.e. HMGR can control the source of insect JH biosynthesis raw material, and then influence by the synthesis of regulation terpene precursor The growth and development and reproduction of insect.Therefore, safe and efficient using HMGR important biomolecule function as control of insect target, it can New strategy is provided for the prevention and treatment of migratory locusts.
Summary of the invention
It is an object of that present invention to provide a kind of migratory locusts HMGR genes, can be used as drug target, carry out migratory locusts prevention and treatment.
Another object of the present invention is to provide the dsRNA based on above-mentioned migratory locusts HMGR gene chemical synthesis, injects the dsRNA to flying In locust body, migratory locusts can be made lethal, the outburst of prevention and control migratory locusts is caused disaster.
To achieve the above object, the invention adopts the following technical scheme:
The present invention provides a kind of migratory locusts HMGR gene, and ORF sequence is as shown in SEQ ID NO:1, ORF sequence 2742bp, Encode 913 amino acid.
The present invention provides the dsRNA based on above-mentioned migratory locusts HMGR gene chemical synthesis, is the justice as shown in SEQ ID NO:9 The double-stranded RNA of the antisense strand nucleotide sequence of chain nucleotide sequence and SEQ ID NO:9 composition.
The present invention also provides the primer pair for synthesizing the dsRNA, nucleotide sequence respectively such as SEQ ID NO:4 and Shown in SEQ ID NO:5.
The present invention also provides the DNA molecular for encoding the dsRNA, nucleotides sequence is classified as both ends with T7 promoter Sequence shown in SEQ ID NO:6.
The present invention also provides contain the kit for synthesizing the primer pair of the dsRNA.
The present invention also provides the synthetic methods of the dsRNA based on above-mentioned migratory locusts HMGR gene chemical synthesis, comprising the following steps: base The HMGR gene order shown in SEQ ID NO:1, using the whole worm total serum IgE reverse transcription of the migratory locusts of extraction at cDNA as template, benefit The upstream and downstream primer shown in SEQ ID NO:4 and SEQ ID NO:5 carries out PCR amplification, by PCR shown in SEQ ID NO:6 Amplified production connects acquisition recombinant plasmid pGEM-T-HMGR in pGEM-T carrier, then using recombinant plasmid pGEM-T-HMGR as mould Plate carries out PCR amplification with the primer pair for having T7 promoter sequence as shown in SEQ ID NO:7 and SEQ ID NO:8 and obtains For synthesizing the DNA profiling of dsRNA, then using the DNA with T7 promoter sequence as template, T7RiboMAX is usedTM Synthesis dsRNA is transcribed in vitro in Express RNAi System (Promega) kit.
The present invention also provides application of the above-mentioned dsRNA in migratory locusts prevention and treatment.
Compared with prior art, the beneficial effects of the present invention are:
Show that the dsRNA synthesized in migratory locusts internal injection can effectively inhibit the gene expression of HMGR by many experiments, causes Migratory locusts can not be successfully husking and enter next age, generate lethal effect, and dsRNA dosage is small (6 μ g), rapid-action (processing 3 days then start to cause migratory locusts largely lethal), lethality is up to 99%, significant effect.In addition, locust is prevented and treated with dsRNA, Species specificity is strong, gene will not be caused to interfere other species；DsRNA is degradable in the natural environment simultaneously, to ecological ring Border is friendly.Therefore, in view of high efficiency of the invention, specificity and safety, there is wide application in terms of migratory locusts control of insect Prospect, it is with good economic efficiency to protection China's agricultural production, ecological safety.
Detailed description of the invention
Fig. 1 is the table that 4 age migratory locusts inject female migratory locusts and HMGR gene in male migratory locusts behind dsRNA 48 hours synthesized Up to horizontal analysis, wherein Fig. 1-a is the HMGR relative expression quantity of female adult, and Fig. 1-b is the HMGR relative expression quantity of male worm, * * in figure P < 0.01, * * * P < 0.001.
Fig. 2 is survival rate statistical chart after the dsRNA that the injection of 4 age migratory locusts synthesizes.
Fig. 3 is the lethal phenotype after the dsRNA of 4 age migratory locusts injection synthesis, and wherein 3-a is the phenotypic map of control group dsGFP, Fig. 3-b is the processing group female adult phenotypic map of dsHMGR, and Fig. 3-c is the processing group male worm phenotypic map of dsHMGR.
Specific embodiment
The following examples are intended to illustrate the invention, but is not used to limit the scope of protection of the present invention.Unless otherwise specified, real Apply the conventional means that technological means used in example is well known to those skilled in the art.Test method in following embodiments, such as It is conventional method without special instruction.
Embodiment one: the sequence of migratory locusts HMGR gene and its acquisition of dsRNA
1. the acquisition of migratory locusts HMGR gene order
1.1 Total RNAs extraction
The female male imago of migratory locusts for choosing healthy growth, extracts whole worm total serum IgE, the specific steps are as follows:
(1) the whole worm of female male imago is milled in liquid nitrogen to powdered, 1ml TRIzol is added, and acutely concussion shakes up, and is stored at room temperature 20min cracks tissue sufficiently.
(2) 4 DEG C, 12000rpm is centrifuged 5 minutes, is transferred to the Tissue lysates of intermediate clear liquid newly with liquid-transfering gun after centrifugation In centrifuge tube；
(3) 500 μ l chloroforms are added, shake up manually 2 minutes, ice bath 5 minutes, 4 DEG C, 12000rpm is centrifuged 15 minutes；
(4) it takes supernatant into new centrifuge tube, 500 μ l acidic phenol chloroformic solutions (Sigma-Aldrich) is added, mix well 2 points Clock, ice bath 5 minutes, 4 DEG C, 12000rpm was centrifuged 10 minutes；
(5) take supernatant to new centrifuge tube, the isopropanol that 500 μ l are added turns upside down to mixing well, -20 DEG C of overnight precipitations；
(6) 4 DEG C, 12000rpm is centrifuged 10 minutes, is abandoned supernatant and is retained precipitating；
(7) the ice ethanol washing of 500 μ l 75% is added into precipitating, 4 DEG C, 12000rpm is centrifuged 5 minutes, abandons supernatant, and repeat It washed once；
(8) suitable ultrapure water (RNase free) dissolution is added after thoroughly drying up in superclean bench.Use 1% agar The integrality of sugared gel electrophoresis checking R NA, with the concentration of NanoDrop 2000 (Thermo Fisher) measurement RNA.
1.2 reverse transcription
According to FastKing cDNA the first chain synthetic agent box specification of Tiangeng, by above-mentioned mentioned total serum IgE reverse transcription at first CDNA is operated as follows:
(1) 2 μ g total serum IgEs, moisturizing to 8 μ l are taken, concussion mixes, and is incubated for 5 minutes for 65 DEG C, is placed on ice in PCR；
(2) it after cooling, is added after 2 μ 5 × gDNA of l Buffer are mixed and is centrifuged, be incubated for 3 minutes for 42 DEG C, be placed on ice in PCR；
(3) reaction system such as table 1 is configured, is mixed with the RNA handled well in (2).
1 total serum IgE reverse transcription of table at first cDNA reaction system
Constituent Volume (μ l)
10×Fast RT Buffer 2
RT Enzyme Mix 1
FQ-RT Primer Mix 1
Ultrapure water 5
Total volume 10
Reaction condition are as follows: 42 DEG C, PCR instrument is incubated for 15min, 95 DEG C of incubation 5min.
The acquisition of 1.3HMGR gene ORF sequence
It is scanned for, is obtained in migratory locusts transcript profile using homologous comparison according to the sequence information of the HMGR albumen of known insect The transcript of migratory locusts HMGR, further combined with migratory locusts genomic information, splicing obtains a complete migratory locusts HMGR gene.Using Primer premier5.0 software design upstream and downstream primer sequence (as shown in SEQ ID NO:2 and SEQ ID NO:3) is simultaneously sent It is synthesized toward Beijing six directions Hua Da Gene Tech. Company Limited.
The upstream and downstream primer sequence of PCR amplification HMGR gene is as follows:
HMGR-F (SEQ ID NO:2): 5 '-ATGGCGGCATCGCGAGTGTT-3 '；
HMGR-R (SEQ ID NO:3): 5 '-TCACGACTTTTCTTCACTTG-3 '.
The cDNA recorded using 1.2 procedure reverseds is as template, in conjunction with such as sequence SEQ ID NO:2 and SEQ ID NO:3 institute The upstream and downstream primer shown is obtained the ORF segment of HMGR gene by PCR amplification, gives this segment to Beijing six directions Hua Da gene section Skill Co., Ltd sequencing (subsequent all primer synthesis and sequencing are completed in Beijing six directions Hua Da Gene Tech. Company Limited), For ORF sequence as shown in SEQ ID NO:1, ORF sequence 2742bp encodes 913 amino acid.
2. synthesizing dsRNA
The DNA profiling of 2.1 amplification dsRNA
Based on HMGR gene order SEQ ID NO:1, the DNA molecular of synthesis dsHMGR is designed using primer premier5.0 The upstream and downstream primer of sequence (as shown in SEQ ID NO:9), primer sequence is respectively such as SEQ ID NO:4 and SEQID NO:5 institute Show.
Using step 1.2 reverse transcription at cDNA as template, using it is upper as shown in SEQ ID NO:4 and SEQ ID NO:5, Downstream primer carries out PCR amplification, and it is shown that reaction system such as table 2.
DsHMGR-F (SEQ ID NO:4): 5 '-TCCGCTACTTCTGTCTCTTCT-3 '；
DsHMGR-R (SEQ ID NO:5): 5 '-CACACACTGTTCTAAACTTCGA-3 '.
Table 2 carries out the reaction system of PCR amplification using cDNA as template
Constituent Volume (μ l)
10×Taq Buffer 2
dNTP(2.5mM) 1.6
DNA profiling 1.5
Upstream primer (10 μM) 0.8
Downstream primer (10 μM) 0.8
Taq Enzyme 0.1
Ultrapure water 13.2
Total volume 20
Reaction condition are as follows: (1) 95 DEG C, 5min；(2) 95 DEG C, 30s；58 DEG C, 30s；72 DEG C, 2.5min；35 circulations；(3)72℃ Extend 10min；4 DEG C of preservations.
The single product that PCR amplification obtains is connected in pGEM-T (Tiangen) carrier, recombinant plasmid pGEM-is obtained T–HMGR.Sequencing result shows that pcr amplification product size is 549bp, and nucleotide sequence is as shown in SEQ ID NO:6.
Using the recombinant plasmid pGEM-T-HMGR of above-mentioned acquisition as template, with the primer pair with T7 promoter sequence DsHMGR-T7-F/dsHMGR-T7-R (as shown in SEQ ID NO:7 and SEQ ID NO:8) carries out PCR amplification, obtains for closing At the DNA profiling (i.e. sequence shown in SEQ ID NO:6 of the both ends with T7 promoter) of dsRNA.PCR reaction system and condition are same Upper (table 2).It usesSV Gel and PCR Clean-Up System (Promega) kits purpose piece Section.NanoDrop 2000 surveys concentration, and determines its concentration in 125ng/ μ l-1mg/ μ l.
The upstream and downstream primer sequence for synthesizing the DNA profiling of dsRNA is sequence SEQ ID NO:4 and sequence SEQ ID NO:5 5 ' ends add T7 promoter sequence, it is (underscore mark T7 promoter sequence) specific as follows:
DsHMGR-T7-F (SEQ ID NO:7):
5’-TAATACGACTCACTATAGGTCCGCTACTTCTGTCTCTTCT-3'；
DsHMGR-T7-R (SEQ ID NO:8):
5’-TAATACGACTCACTATAGGCACACACTGTTCTAAACTTCGA-3’。
The synthesis of 2.2dsRNA
DNA of the both ends obtained using step 2.1 with T7 promoter sequence uses T7RiboMAX as templateTM Synthesis dsRNA is transcribed in vitro in ExpressRNAi System (Promega) kit.
It is as shown in table 3 that reaction system is transcribed in vitro.
Reaction system is transcribed in vitro in table 3
Reaction condition are as follows: 37 DEG C, 60min；70 DEG C, 10min；25 DEG C, 30min.
The purifying of 2.3dsRNA
(1) it in the reaction system of step 2.2 plus 160 μ l Nuclease-Free water and 200 μ l acidic phenol chloroforms, mixes well After place 5min on ice；
(2) 4 DEG C, 13000g is centrifuged 10min, and Aspirate supernatant body is put into new 1.5ml centrifuge tube；
(3) to the dehydrated alcohol of addition 0.1 times of volume sodium acetate (3.0M, pH 5.2) and 2.5 times of volumes in supernatant fluid, sufficiently After mixing, ice bath 5min on ice is placed；
(4) 4 DEG C, 13000g is centrifuged 10min, abandons supernatant；
(5) 75% ethyl alcohol of 500 μ L pre-cooling is added, 4 DEG C, 13000g is centrifuged 10min, abandons supernatant；
(6) step (5) are repeated, 75% ethyl alcohol washs one time again；
(7) it is put into the dry 20min of superclean bench, 40 μ L Nuclease-Free water are added, is placed on and dissolves 20min on ice, mix Even, NanoDrop 2000 surveys concentration, is put into -20 DEG C for use.
The dsRNA of acquisition is sequenced.Sequencing result shows: migratory locusts HMGR gene dsRNA is double-stranded RNA, by justice Chain and antisense strand are at as shown in SEQ ID NO:9, the nucleotides sequence of antisense strand is classified as the nucleotide sequence of positive-sense strand The reverse complementary sequence of SEQ ID NO:9.
Embodiment two: the lethal migratory locusts experiment of migratory locusts HMGR gene chemical synthesis dsRNA
1. migratory locusts dsRNA is injected
Healthy growth, 0 day 4 age of the same size migratory locusts are chosen as experimental material, half male and half female is randomly divided into dsHMGR processing group With dsGFP control group (because of the gene in migratory locusts body without green fluorescent protein GFP, therefore can be as negative control), often 30 larvas of group include female adult 15, male worm 15, are repeated 3 times, 90 larvas of every group of co-injection.With 10 μ l specifications it is micro into Sample device is by 6 μ g dsRNA out of migratory locusts abdomen third body segment injection migratory locusts body.Migratory locusts after injection are placed in draughty (25cm × 25cm × 25cm) is raised in metal cage, and temperature is 30 ± 2 DEG C, photoperiod 14L:10D, feed fresh wheat seeding and Wheat bran, twice daily.Per half a day is observed and counts larval mortality after injection.
The detection of 2.HMGR gene jamming effectiveness
After injecting dsRNA 48h, respectively in dsHMGR processing group and the random picking female adult of dsGFP control group 5, male worm 5, with Detect jamming effectiveness of the HMGR in female adult and male worm.
It is rounded the grinding of worm polypide, then extracts total serum IgE respectively, and be inverted to first cDNA, method is the same as embodiment one.It presses Reagent enhanced edition (SYBR Green) kit (PF205) specification method is premixed according to Tiangeng SuperReal fluorescent quantitation, is used The HMGR upstream and downstream primer as shown in SEQ ID NO:10, SEQ ID NO:11 and such as SEQ ID NO:12, SEQ ID NO:13 Shown in β-actin upstream and downstream primer, on LightCycler 96 (Roche) carry out qRT-PCR reaction, respectively testing goal The relative expression quantity of gene (HMGR) and reference gene (β-actin), then according to 2-ΔΔCtMethod is calculated, and analysis gene is heavy Silent efficiency.As a result as shown in Figure 1, compared with dsGFP control group, no matter in female migratory locusts or in male migratory locusts, dsHMGR The extremely significant reduction of the HMGR expression quantity of processing group, shows HMGR by effective reticence.
It is as follows that qRT-PCR reacts primer sequence:
HMGR-qRT-F (SEQ ID NO:10): 5 '-TAAAGGAGCGAATGAGGAATC-3 '；
HMGR-qRT-R (SEQ ID NO:11): 5 '-AGAGCAGACATGAGGGAGAGTT-3 '.
β-actin-qRT-F (SEQ ID NO:12): 5 '-AATTACCATTGGTAACGAGCGATT-3 '；
β-actin-qRT-R (SEQ ID NO:13): 5 '-TGCTTCCATACCCAGGAATGA-3 '.
QRT-PCR reaction system is as shown in table 4.
4 qRT-PCR reaction system of table
Reaction condition are as follows: (1) 95 DEG C of initial denaturation 10min；(2) 95 DEG C, 10s；58 DEG C, 30s；72 DEG C, 30s, totally 40 recycle； (3) solubility curve is analyzed, and 95 DEG C, 60s；65 DEG C, 30s；95 DEG C, 30s.
3. injecting migratory locusts mortality statistics and Phenotypic Observation after dsRNA
After 4 instar larvaes inject dsRNA, starts husking after injecting dsGFP control group 5 days of dsRNA and all successfully husking enters 5 Age, and posture vitality is normal, the 5 instar larvae no significant differences with normal development.Inject the dsHMGR processing group totally 60 of dsRNA Head, survival rate statistical result is as shown in table 5 and Fig. 2, it can be seen that after migratory locusts inject dsRNA, start mortality on day 3, The death rate is more than 90% up to 50%, the 4th day death rate, and almost all death in the 5th day, the death rate is up to 99%.Phenotypic map such as Fig. 3 institute Show.As seen from Figure 3, after dsHMGR processing group injection dsRNA, larva eventually leads to death because can not normally cast off a skin.
Table 5 injects migratory locusts survival rate statistical result after dsRNA
The embodiment of the above, only presently preferred embodiments of the present invention, is only used to explain the present invention, not limits this Invention practical range to those of ordinary skill in the art certainly can be according in technology disclosed in this specification Hold, makes other embodiments easily by way of replacing or changing, therefore all variations made in the principle of the present invention With improve etc., should be included in scope of the present invention patent.
SEQUENCE LISTING
<110>He'nan University
<120>application of migratory locusts HMGR gene and its dsRNA in migratory locusts prevent and treat
<130> 2019
<160> 13
<170> PatentIn version 3.3
<210> 1
<211> 2742
<212> DNA
<213>the ORF sequence of HMGR gene
<400> 1
atggcggcat cgcgagtgtt cggcgcgcac ggcgagctgt gcgccagcca cccctgggag 60
gtgatcgtgg ccacgctgac gctggccatc agcatgctga cggtggagca gcgagccggc 120
ggcgccacca ggctgcccgt cgccgccgca gcagcctccc aggccgagga gtacagcgct 180
gccgccgaca tgatcatgat gacggtcatc cgctgcgtcg ccgtgctgta ctgctattac 240
cagttctgca ggctccacaa gctgggctcc aaatacattc tcggtattgc aggactcttt 300
acagtcttct ccagttttgt attcagcact agtgtcatca atgttcttca cagtgacata 360
tctgatctca aagatgcttt atttttcttc ctattgctga ttgatctttc taaagcaagt 420
gctttagctc aattggccct aagtgccagc aacactgaag aagtgcgacg taatattgct 480
cgtgggatgg caactctggg acctgccctc acactggata cacttgttga ggcacttgtt 540
attggtgttg ggactctctc aggtgttcgc aggctggaga tactgtgctg ctttgcttgc 600
atgtctgtta ttgttaatta tatcgtcttc atgacattct atccagcatg tctttctctt 660
atcttagagt tatcaagagc tggtaagcat ggcaggcctg cttggcagga acgttcagct 720
atagtgcgtc atgtgctcca ggaggaaaag ccaaatccag tggtgcagag agtcaaagtg 780
ataatgtctg caggtcttgt gttggttcat gcacatagtc ggtggggact gggcggtgac 840
gatacaggca gtgtgccttc tgtggctgtc ccgggaacat ccctgccatt ggtggtttcc 900
tccgctactt ctgtctcttc tactgctgtc tacaaccaca ctgatgactt cacagcatcc 960
tccatccgcg agtacctcct caggtggctg actgtgagtg ctgaccacat cgtcattctt 1020
atcctcctgt tagcacttgc tgtaaagttt gttttctttg agagtgacga ggatatccga 1080
gaacaaatgc tggtgtctga ccgagctagt gacagggaac ggagacacag aagttacagc 1140
catggacacg cccttgatcg tatgcgacca atgcttgaac ctggttgggt cgaggtccca 1200
tctcgagaag aattcattgc aattaacaca gagcacaaat cagtccaaac tgatcatcag 1260
gatgagcagc agaagccaga ggaacaacag caagtacaac ctgaagaagt caaacatgtg 1320
actgaagcac ctccaacatg tcagcaacca cagcaagatg ttgcagcttc agaaataata 1380
atgaataaga tacctgttga tgacaataaa gaggagccac aagtttgtcg aagtttagaa 1440
cagtgtgtgg atatatatag gtccaaggag ggagcagcag ctttaacaga tgaagaaatg 1500
cttcagcttg ttcgagctag cgtggtacct tcttatcaac tggaagcagt tgttaaggat 1560
cctgagagag gtgttggcat tcgcagaaag ctttttgatg ctcacaaacc tggtgcactt 1620
tcagatctac cgtatactgg ctacgactat tcaaaggtgc ttggagcatg ctgtgaaaat 1680
gttgtgggct acttgccagt accagtaggt attgctggtc ctttgttatt ggatggtcaa 1740
caagtgcata ttcctatggc tacaacagag ggctgcttgg tggccagcac taatcgtggc 1800
tgccgtgcgt tgctttcgtg tggtgtcaca tcccgtgtcg ttgctgatgg tatgacacgt 1860
gggccagttg tccgcttccc atctgcagca agagccagtg aggctatgat gtggatgcaa 1920
acaccagaaa actttgaggg catcaaagct agctttgatg gtactagccg atttgcacgc 1980
ctaattaagt tgcatgtacg cattgcaggg aggcatctgt ttgtgagatt tgtagctacc 2040
acaggtgatg ccatgggcat gaacatgttg tcaaagggca ctgaagttgc tttgggattt 2100
gttcagaaaa catttccaga catggaacta ctcagtctta gtggaaattt ctgcactgac 2160
aagaagccag cagctgttaa ctggattgaa ggccgaggga agtctgttgt ctgtgaagcc 2220
gttgtgcctg gggaagtggt gtcaaaagtg cttaaaacga cagtgcaagc tcttgttgat 2280
gtaaatgtta gcaagaatat gataggctct gctgtggctg gaagtgtggg tggatttaat 2340
gcacatgctg caaatatagt aactgccatc ttcatagcaa caggccagga ccctgcacag 2400
aatgtgagta gcagcaactg tatgacacta atggaaccat ggggagaggg aggcaaggat 2460
cttcatgtgt cgtgtacaat gccatctctt gaaattggaa ctgttggtgg cggcacagtt 2520
ctgcctgcac agggtgcttg ccttgccatg ttgggtgtta aaggagcgaa tgaggaatca 2580
ccaggagaaa atgctaaaat gttagcaagg atagtatgtg gcactgtact agccggagaa 2640
ctctccctca tgtctgctct agctgcaggt cacctagtac gatcccacct gaggcataac 2700
agggcctctg ttacttcaag accaagtgaa gaaaagtcgt ga 2742
<210> 2
<211> 20
<212> DNA
<213> HMGR-F
<400> 2
atggcggcat cgcgagtgtt 20
<210> 3
<211> 20
<212> DNA
<213> HMGR-R
<400> 3
tcacgacttt tcttcacttg 20
<210> 4
<211> 21
<212> DNA
<213> dsHMGR-F
<400> 4
tccgctactt ctgtctcttc t 21
<210> 5
<211> 22
<212> DNA
<213> dsHMGR-R
<400> 5
cacacactgt tctaaacttc ga 22
<210> 6
<211> 549
<212> DNA
<213>cDNA template amplification product
<400> 6
tccgctactt ctgtctcttc tactgctgtc tacaaccaca ctgatgactt cacagcatcc 60
tccatccgcg agtacctcct caggtggctg actgtgagtg ctgaccacat cgtcattctt 120
atcctcctgt tagcacttgc tgtaaagttt gttttctttg agagtgacga ggatatccga 180
gaacaaatgc tggtgtctga ccgagctagt gacagggaac ggagacacag aagttacagc 240
catggacacg cccttgatcg tatgcgacca atgcttgaac ctggttgggt cgaggtccca 300
tctcgagaag aattcattgc aattaacaca gagcacaaat cagtccaaac tgatcatcag 360
gatgagcagc agaagccaga ggaacaacag caagtacaac ctgaagaagt caaacatgtg 420
actgaagcac ctccaacatg tcagcaacca cagcaagatg ttgcagcttc agaaataata 480
atgaataaga tacctgttga tgacaataaa gaggagccac aagtttgtcg aagtttagaa 540
cagtgtgtg 549
<210> 7
<211> 40
<212> DNA
<213> dsHMGR-T7-F
<400> 7
taatacgact cactataggt ccgctacttc tgtctcttct 40
<210> 8
<211> 41
<212> DNA
<213> dsHMGR-T7-R
<400> 8
taatacgact cactataggc acacactgtt ctaaacttcg a 41
<210> 9
<211> 549
<212> RNA
<213>dsRNA positive-sense strand
<400> 9
uccgcuacuu cugucucuuc uacugcuguc uacaaccaca cugaugacuu cacagcaucc 60
uccauccgcg aguaccuccu cagguggcug acugugagug cugaccacau cgucauucuu 120
auccuccugu uagcacuugc uguaaaguuu guuuucuuug agagugacga ggauauccga 180
gaacaaaugc uggugucuga ccgagcuagu gacagggaac ggagacacag aaguuacagc 240
cauggacacg cccuugaucg uaugcgacca augcuugaac cugguugggu cgagguccca 300
ucucgagaag aauucauugc aauuaacaca gagcacaaau caguccaaac ugaucaucag 360
gaugagcagc agaagccaga ggaacaacag caaguacaac cugaagaagu caaacaugug 420
acugaagcac cuccaacaug ucagcaacca cagcaagaug uugcagcuuc agaaauaaua 480
augaauaaga uaccuguuga ugacaauaaa gaggagccac aaguuugucg aaguuuagaa 540
cagugugug 549
<210> 10
<211> 21
<212> DNA
<213> HMGR-qTR-F
<400> 10
taaaggagcg aatgaggaat c 21
<210> 11
<211> 22
<212> DNA
<213> HMGR-qTR-R
<400> 11
agagcagaca tgagggagag tt 22
<210> 12
<211> 24
<212> DNA
<213> β-actin-qTR-F
<400> 12
aattaccatt ggtaacgagc gatt 24
<210> 13
<211> 21
<212> DNA
<213> β-actin-qTR-R
<400> 13
tgcttccata cccaggaatg a 21

Claims (7)

1. migratory locusts HMGR gene, which is characterized in that the ORF sequence of the HMGR gene is as shown in SEQ ID NO:1.
2. the dsRNA based on migratory locusts HMGR gene chemical synthesis described in claim 1, which is characterized in that as shown in SEQ ID NO:9 The double-stranded RNA of the antisense strand nucleotide sequence of positive-sense strand nucleotide sequence and SEQ ID NO:9 composition.
3. the primer pair for synthesizing dsRNA described in claim 2, which is characterized in that nucleotide sequence is respectively such as SEQ ID Shown in NO:4 and SEQ ID NO:5.
4. encoding the DNA molecular of dsRNA described in claim 2, which is characterized in that nucleotides sequence is classified as both ends with T7 promoter SEQ ID NO:6 shown in sequence.
5. the kit containing primer pair described in claim 3.
6. the synthetic method based on the dsRNA of migratory locusts HMGR gene chemical synthesis claim 2 described in claim 1, which is characterized in that The following steps are included: based on HMGR gene order shown in SEQ ID NO:1, with the whole worm total serum IgE reverse transcription of the migratory locusts of extraction at CDNA be template, using upstream and downstream primer shown in SEQ ID NO:4 and SEQ ID NO:5 carry out PCR amplification, by SEQ Pcr amplification product shown in ID NO:6 connects acquisition recombinant plasmid pGEM-T-HMGR in pGEM-T carrier, then to recombinate matter Grain pGEM-T-HMGR is template, with the primer for having T7 promoter sequence as shown in SEQ ID NO:7 and SEQ ID NO:8 The DNA with T7 promoter sequence is obtained to PCR amplification is carried out, then using the DNA with T7 promoter sequence as template, is used T7RiboMAXTMExpress RNAi System(Promega) kit in-vitro transcription synthesis dsRNA.
7. application of the dsRNA described in claim 2 in migratory locusts prevent and treat.
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