CN104450755A

CN104450755A - Oriental migratory locust ATP synthase alpha subunit gene and application of dsRNA of oriental migratory locust ATP synthase alpha subunit gene in pest control

Info

Publication number: CN104450755A
Application number: CN201410665503.0A
Authority: CN
Inventors: 夏玉先; 胡军
Original assignee: Chongqing University
Current assignee: Chongqing University
Priority date: 2014-11-14
Filing date: 2014-11-14
Publication date: 2015-03-25
Anticipated expiration: 2034-11-14
Also published as: CN104450755B

Abstract

The invention relates to an oriental migratory locust ATP synthase alpha subunit gene and application of dsRNA of the oriental migratory locust ATP synthase alpha subunit gene in pest control. The oriental migratory locust ATP synthase alpha subunit gene disclosed by the invention provides the conserved sequence of ATP synthetase alpha subunit cDNA of a locust, can be used for constructing the constructs, namely RNA, DNA and the like, and obviously inhibits the expression of the ATP synthetase alpha subunit inside a locust body by silencing the ATP synthetase alpha subunit inside the locust body by utilizing an RNAi technology to cause the lethal effect on the locust, thus being applied to the RNAi technology to prevent and control oriental migratory locust pests.

Description

A kind of Asiatic migrotory locust atp synthase α subunit gene and the application of dsRNA in pest control thereof

Technical field

The present invention relates to biological technical field, particularly relate to a kind of atp synthase α subunit gene, dsRNA and the application in Asiatic migrotory locust control thereof.

Background technology

Insect serious harm agriculture production, due to chemical insecticides such as long-term application organochlorine, organophosphoruss, causes series of problems: pesticide residue cause environmental pollution serious; Toxic pesticides harm humans is healthy; There is resistance in insect, cost accounting raising etc.Have at present and biotic pesticide are applied to pest control, but the desinsection time is longer, insecticidal effect is unstable, therefore in the urgent need to novel insect pest control method.

RNA interference (RNAi) is gene silencing phenomenon after a kind of specific transcriptional usually caused by double stranded rna molecule, obtains the Nobel prize in 2006.The functional study that this discovery is not only gene provides the breakthrough in method, simultaneously also for the disease treatment of the mankind and pest control open new approach.2008, after Price and Gatehouse summarizes numerous experimental result, put forward the strategy of insect pest control based on RNAi.Pest control based on RNA perturbation technique has following advantage: 1) selecting the gene single-minded to insect to disturb, is safe to higher animal and the mankind; 2) pest-resistant have specificity, to target organisms without lethal effect; Nontoxic to environment.

Summary of the invention

The object of this invention is to provide a kind of nucleic acid molecule, can the gene of code East Asia migratory locusts ATP synthetase alpha subunit, and as drug target, make locust lethal.

A kind of nucleic acid molecule, is characterized in that: sequence is SEQ ID NO.1 and complementary sequence thereof.

Preferably, in above-mentioned nucleic acid molecule, SEQ ID NO.2 sequence and complementary sequence thereof can as effective drug targets, interference base because of transcriptional expression.

Another object of the present invention is to for above-mentioned nucleic acid molecule, the dsRNA (double stranded RNA) of effectively its transcriptional expression of interference is provided.

RNA construct, it comprises at least one double-stranded RNA district, and its at least one chain comprises and above-mentioned arbitrary nucleic acid molecule complementary nucleotide sequence.

Above-mentioned RNA construct, at least one double-stranded RNA district wherein said at least has 17bp.

Preferably, RNA construct, its sequence is SEQ ID NO.3 or its complementary sequence.

DNA construct, contains above-mentioned nucleic acid molecule, or comprises the region of above-mentioned RNA construct of encoding.

Expression construct, contains above-mentioned DNA construct.

Host cell, comprises above-mentioned RNA construct, DNA construct or expression construct.

Insect-killing composition, contains above-mentioned RNA construct and/or above-mentioned DNA construct and/or above-mentioned expression construct and/or above-mentioned host cell and applicable carrier.

Above-mentioned any one RNA construct and/or DNA construct and/or expression construct and/or host cell and applicable carrier and/or the application of insect-killing composition in control locust.

Beneficial effect

1. present invention obtains the conserved sequence of the ATP synthetic enzyme enzyme α subunit eDNA of locust, and build atp synthase α subunit in the construct silences such as its RNA, DNA locust body, in empirical tests body, the expression of atp synthase α subunit is obviously suppressed, and causing locust to produce lethal effect, the present invention can be applicable to RNAi technology to Asiatic migrotory locust pest control.

2. the present invention is for preventing and treating locust especially Asiatic migrotory locust, and species specificity is strong, can not cause interference, as honeybee, people etc. to other species; Gene specific of the present invention is strong, can not produce disturbing influence to the gene except atp synthase or α subunit.

3. the present invention prevents and treats locust and has high efficiency, especially Asiatic migrotory locust, dosage little (dsRNA consumption is 100ng), rapid-action (process cause for about 2 days locust lethal).

Accompanying drawing explanation

Lethal cases after the dsRNA of migratory locusts injection in Fig. 1: five ages SEQ ID NO:2 synthesis.

Fig. 2: five age migratory locusts infusion sequence be the different time points mrna expression of atp synthase α subunit gene (LmATPSA) after the dsRNA that synthesizes of SEQ ID NO:3, RP49 is as reference gene.12,24,36h is the hours after injection dsRNA.

Embodiment

Below in conjunction with drawings and Examples, the present invention is described in further details.Following examples are only limitted to illustrate that the present invention and being not used in limits the scope of the invention.

Embodiment 1: the acquisition of migratory locusts atp synthase α subunit gene and dsRNA thereof

The acquisition of i, migratory locusts atp synthase α subunit gene

1) migratory locusts atp synthase α subunit gene is in the search of migratory locusts est database

Based on the est database of migratory locusts, adopt bioinformatics method to search for migratory locusts atp synthase α subunit gene, after sequence assembly and comparison, obtain 1 migratory locusts atp synthase α subunit gene altogether.

2) design of migratory locusts atp synthase α subunit gene primer

Based on the base sequence obtaining LmATPSB, adopt primer premier5.0 software design primer.All primers are by the synthesis of Nanjing Jin Sirui biological company limited.

3) migratory locusts total serum IgE obtains

Choose male and female half and half migratory locusts 5 nymph in age in the same size, 3 first groups, be frozen in liquid nitrogen, RNA to be extracted, concrete operation step is with reference to TRIzol (Invitrogen) specification sheets.

4) synthesis of the first chain cDNA

With reference to PromegaM-MLV ThermoScript II reagent specification sheets, carry out the synthesis of the first chain cDNA.

5) acquisition of migratory locusts atp synthase α subunit gene

Carry out PCR according to Thermo Fusion specification sheets, clone obtains migratory locusts atp synthase α subunit gene cDNA sequence further.

6) analysis of migratory locusts atp synthase α subunit gene base sequence

Utilize relevant online software and the software analysis gained sequence such as GENEDOC, gene seeker in Expasy website, in NCBI website, use BLAST function to carry out sequence homology comparison afterwards.After sequence verification, atp synthase α subunit (LmATP5A) the sequence 2100bp of acquisition, open reading frame 1656bp.

II, migratory locusts atp synthase α subunit gene fragment and dsRNA thereof synthesize

1) acquisition of migratory locusts atp synthase α subunit gene fragment

The sequence SEQ ID NO:1 of migratory locusts atp synthase α subunit gene is obtained based on this institute;

1, design dsRNA primer, primer sequence is respectively SEQ ID NO:4 and SEQ ID NO:5.All primers are by the synthesis of Nanjing Jin Sirui biological company limited.

Choose male and female half and half Asiatic migrotory locust 5 nymph in age in the same size, three first groups, frozen in liquid nitrogen, RNA to be extracted, concrete operation step is with reference to TRIzol (Invitrogen) specification sheets.Carried RNA reverse transcription is become the first chain cDNA by M-MLV ThermoScript II, as template, with SEQ ID NO:4 and SEQ ID NO:5 for primer carries out pcr amplification, obtains atp synthase α subunit gene fragment.

2) synthesis of migratory locusts atp synthase α subunit dsRNA

Glue reclaims kits gene fragment, according to MEGAscript highyield transcription kit (Ambion) specification sheets synthesis dsRNA after kits, dsRNA concentration adopts ultraviolet spectrophotometer to measure, and to be diluted to final concentration be 20ng/ μ l.Described dsRNA fragment is as shown in SEQ ID NO:3.

Embodiment 2: migratory locusts atp synthase α subunit dsRNA lethal five age migratory locusts

1, migratory locusts atp synthase α subunit dsRNA injects

Choose the 3rd day five ages, size is homogeneous, healthy state is consistent nymph for injecting the dsRNA (SEQ ID NO:3) of above-mentioned synthesis, 25 μ l specification microsyringes are used for injection, can not be firmly excessive during injection, along the direction of blood flow, valve, as injection point, is avoided in the junction of flank portion the 2 to 3 uromere.The amount of injection dsRNA is 100ng, and arranges the control group of dsGFP (100ng), often organizes 20 cephalonts, and 3 biology repeat, and amount to 60.After injection, examination worm the first day of the lunar month material cage is entered to raise (illumination: interlunation is 14h: 10h, temperature 30 ± 2 DEG C), gives fresh wheat seedling.

2, to inject after dsRNA five age migratory locusts phenotype observation

Five-age larva is after injection dsRNA, and injection dsGFP control group starts to cast off a skin after 5 days and all successfully casts off a skin to adult, and form vigor to adult of casting off a skin is normal.The treatment group nymph totally 60 of injection dsATP5A, within the 2nd day, start dead, within the 4th day, mortality ratio is more than 90%, and raw mapping as shown in Figure 1.

3, migratory locusts atp synthase α subunit gene mRNA level in-site detects

The silence efficiency of the rear different time points of migratory locusts nymph dsRNA injection in 5 ages is detected, selects the polypide of injection rear 12h, 24h and 36h to extract object as RNA, often organize each time point and 3 biology repetitions are set.Extract the total serum IgE of each sample and reverse transcription becomes the first chain cDNA, with the expression amount (Fig. 2) of RT-PCR testing goal gene (LmATP5A) and house-keeping gene (RP49).Find through data analysis, in migratory locusts body after injection atp synthase α subunit double-stranded RNA, LT ₅₀it is 2.4 ± 0.4 days; Mrna expression amount in turn reduces 91.7%, 93.6% and 95.7% in 12 hours after injection, 24 hours and 36 hours; .And the mRNA injecting the atp synthase α subunit in the Asiatic migrotory locust body of GFP dsRNA does not have the change of significance, do not cause lethal yet.

According to method described in embodiment 2, dsRNA (SEQ ID NO:3) is injected in honeybee, spider body respectively, does not find lethality.

α subunit relative expression quantity in body is injected after dsRNA in table 1 embodiment 2

Survival rate data (unit is %) after injecting dsRNA in table 2 embodiment 2

The <110> University Of Chongqing summer is first beautiful

<120> Asiatic migrotory locust atp synthase α subunit gene fragment and the application of dsRNA in pest control thereof

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<211>2100

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<220>

<221>

<222>（229）..（1881）

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tcttgtcgtc ataaacagac tgttcttaac ccaggatggc cttgccgtag gcgggggcgg 180

cgtaggcaac gggggcggcc atctttctgc aagggcagaa cagtcgaaat ggctctcctc 240

tccttccgtt tagcctctgc tgtggcgaaa catttgcctg cggctacgcc tcagataagt 300

ggtttaacat ggcctgcagc acagataaca aaccgcaata tccatgtttc gtgcagtcag 360

agagctgcag aaatatcttc aattttggag gagaggatac ttggtgctgc cccaaaggct 420

gatttagagg aaactggaag agtcttgagt attggagatg gtattgctcg agtgtatggt 480

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ctgaacttgg aaccagacaa tgttggtgtt gtagtatttg gtaatgacag gctcataaag 600

gaaggagaca ttgtaaaacg aacgggagcc attgtagatg tacctgttgg agaggaactt 660

ttggggcgtg ttgttgatgc tttgggaaat cctattgatg gtaaagggcc attgaaggct 720

tcaaagaggt ttagggttgg tatcaaggct ccaggaatca ttcctcgtat ctctgtgagg 780

gaacctatgc agacaggaat taaagctgta gattctctgg tgcctattgg acgaggacaa 840

cgagaattga taattggaga caggcagact ggcaaaacag ccttggctat tgacacaatt 900

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gtggctattg gtcagaagcg atcaactgtt gcccagattg tgaagaggtt gacagattca 1020

ggtgctattg gttattcaat cattgtatct gctactgcat ctgatgctgc acctctacag 1080

tatttggcac cttattcagg atgcgctatg ggagaattct tccgtgataa tggaaagcat 1140

gccttgatca tctatgatga tctgtcgaag caggctgtgg cctacagaca gatgtctctg 1200

ttgctgcgac gaccaccagg tcgtgaagcc tatcctggtg atgtcttcta cctgcattct 1260

agattattgg agagagctgc taaaatgaat gatgctcatg gaggtggctc acttactgct 1320

cttcctgtca ttgagacaca ggcaggtgat gtgtctgcat acattccaac aaatgtaatt 1380

tccattactg atggtcagat cttcttggag acagagttgt tttacaaggg tatccggcct 1440

gcgatcaacg taggtttgtc tgtctctaga gtaggctcag ctgctcagac aagagctatg 1500

aagcaggttg ctggttccat gaagctggaa ttggcacagt accgagaggt tgcagcattt 1560

gcccagtttg gttcagatct ggatgctgca acccagcaac tgctgaacag aggtgtgagg 1620

ttaacagagc ttctgaagca gggacagtat gttccaatgg caattgagga acaagttgca 1680

gttatttact gtggtgtgag ggggcatctt gacaaattgg atccctccaa aatcactgca 1740

tttgaaaagg agttcctcca acatataaag acatctgaag ctgcattgtt ggcaaatatt 1800

gcaaaggagg gaaagatcac agatgagatc gatgtgaaac tgaagaaaat tgtgacagat 1860

tttgttgcaa acttccaggg ttgaaactct gcaagattgt aggcaaaaac attatttaaa 1920

atattttgtc cagcatttag gtgtgacgac tgagaaagtg ccagctttgt gttgaaactg 1980

attttttcag ttagagtatt gtgtatatgt ggaattagat tactgccact gtaaagtaat 2040

tgttaaattg ttaataaaat tacttcccca taggaaaaaa aaaaaaaaaa aaaaaaaaaa 2100

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ggtttgaaaa atatacaggc tgatgagatg gtggaattct cttcaggttt aaagggtatg 240

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aaggaaggag acattgtaaa acgaacggga gccattgtag atg 343

<210>3

<211>343

<212>DNA

<213> artificial sequence

<400>3

agugguuuaa cauggccugc agcacagaua acaaaccgca auauccaugu uucgugcagu 60

cagagagcug cagaaauauc uucaauuuug gaggagagga uacuuggugc ugccccaaag 120

gcugauuuag aggaaacugg aagagucuug aguauuggag augguauugc ucgaguguau 180

gguuugaaaa auauacaggc ugaugagaug guggaauucu cuucagguuu aaaggguaug 240

gcucugaacu uggaaccaga caauguuggu guuguaguau uugguaauga caggcucaua 300

aaggaaggag acauuguaaa acgaacggga gccauuguag aug 343

<210>4

<211>40

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<213> artificial sequence

<400>4

taatacgact cactataggg agtggtttaa catggcctgc 40

<210>5

<211>40

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taatacgact cactataggg catctacaat ggctcccgtt 40

Claims

1. a nucleic acid molecule, is characterized in that: sequence is SEQ ID NO.1 or SEQ ID NO.2, or the complementary sequence of SEQ ID NO.1 or SEQ ID NO.2.

2. a RNA construct, it comprises at least one double-stranded RNA district, and its at least one chain comprises and nucleic acid molecule complementary nucleotide sequence arbitrary described in claim 1 or 2.

3. the RNA construct of claim 2, at least one double-stranded RNA district wherein said is at least 17bp.

4. the RNA construct of claim 2, sequence is SEQ ID NO.3 or its complementary sequence.

5. a DNA construct, contains claim 1 or 2 nucleic acid molecule, or comprises the region of RNA construct described in coding claim 3 or 4.

6. expression construct, contains DNA construct as claimed in claim 5.

7. a host cell, comprises expression construct described in DNA construct described in RNA construct described in claim 3 or 4 or claim 5 or claim 6.

8. an insect-killing composition, contains host cell and applicable carrier described in expression construct described in DNA construct described in RNA construct described in claim 3 or 4 and/or claim 5 and/or claim 6 and/or claim 7.

9. the application of insect-killing composition in control locust described in host cell described in expression construct described in DNA construct described in RNA construct described in claim 3 or 4 and/or claim 5 and/or claim 6 and/or claim 7 and applicable carrier and/or claim 8.

10. locust as claimed in claim 9 is Asiatic migrotory locust.