CN107988231A - Migratory locusts coria epidermal protein gene 6 and its application in locust control - Google Patents
Migratory locusts coria epidermal protein gene 6 and its application in locust control Download PDFInfo
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Abstract
The present invention provides a kind of migratory locusts coria epidermal protein gene 6 and its application in locust control.Specifically by bioinformatics method, a kind of migratory locusts coria epidermal protein gene is obtained from migratory locusts transcript profile, it is SEQ ID NO further to obtain sequence by migratory locusts genome sequence：1 full length gene.According to SEQ ID NO：1, design and synthesize the dsRNA of the gene, by dsRNA injections enter after migratory locusts body cavity can specific silence target gene so that husking occur difficult for polypide, coria can not close, and ultimately resulting in pest belly can not go deep into soil laying eggs and causing egg sac number and ovum grain number largely to reduce.Many experiments show that injecting dsRNA causes migratory locusts offspring's ovum grain number decrement to reach more than 70%.Due to the specificity and high efficiency of the present invention, there is remarkable result for reducing pest laying and offspring flocks quantity, new approach can be provided for control of insect.
Description
Technical field
The present invention relates to biological technical field, and in particular to a kind of migratory locusts coria epidermal protein gene (LmAbd6) and its
Applications of the dsRNA in control of insect.
Background technology
Migratory locusts are China's important agricultural insects, due to its with wing can between land long-distance migration fly so that
All it is greatly improved looking for food, seek a spouse, keeping away enemy and expand distribution efficiency etc., agricultural production is caused to seriously endanger.
The body length of migratory locusts adult, male worm are 32.4~48.1 millimeters, and female adult is 38.6~52.8 millimeters.Under normal circumstances, adult plumage
It can mate within average 7 days or so after change, female adult all one's life can carry out multiple mating, and post-coitum can lay eggs under soil for 4~7 days, deep
About 4~6 centimetres of degree.Mucus glue is secreted when ovum is given birth to and ovum particle shape into egg capsule.Every female adult can generally lay eggs 4~5 pieces of capsule, often
Block generally contains 50~80, ovum, grain sum average out to 200~400 of laying eggs throughout one's life.Migratory locusts can the deep enough lower oviposition of soil be by
There is extensibility in its female adult coria.Migratory locusts share 9 attached sections, 8 corias, contain a large amount of epidermal proteins in coria.
Epidermal protein is insect cuticle important composition component, is played an important roll in epidermal structure is formed, and the missing of its component or is contained
Amount change will cause epidermis dysfunction even function to be lost, so as to influence the growth and development of insect.In migratory locusts reproduction, when
Its internode membrane structure can be destroyed and cannot extend after epidermal protein content is reduced, and lead to not go deep into the lower oviposition of soil, and then lead
Its offspring's population quantity is caused to reduce.
Prevention to the plague of locusts at present relies primarily on chemical prevention, but chronic administration chemical insecticide causes a series of ask
Topic, such as environmental pollution, resistance to the action of a drug generation and the harm to non-target organism etc..Therefore, new, alternative chemical prevention is developed
Method becomes extremely urgent.RNA interference (RNAi) is gene silencing after a kind of specific transcriptional as caused by double stranded rna molecule
Phenomenon, since 2006 obtain the Nobel Prize, RNAi technology ranks among Environment Science always.RNAi is not only research gene work(
The powerful of energy, while also there is very high potential in terms of control of insect.Disturbed by RNA and carry out control of insect with as follows
Feature：1) desinsection has specificity, to non-target organism without lethal effect；2) RNA easily degrades in nature, noresidue；3)
It is nontoxic to environment, it is comparatively safe.Therefore scholar is referred to as forth generation insecticide.Disturbed based on RNA and carry out control of insect
Basic technology be Screening target sequence obtain with high lethal effect dsRNA.
The characteristics of for migratory locusts oviposition reproduction, the present invention is based on RNA perturbation techniques, chooses migratory locusts coria epidermal protein base
Because object, control of insect is carried out using its important biomolecule function, it is safe and efficient, be it is a kind of it is environmentally friendly, to the mankind
Nontoxic, specific novel molecular target calibration method.
The content of the invention
It is an object of the invention to provide a kind of migratory locusts coria epidermal protein gene (LmAbd6) full length sequence and its
Application in locust control.
The present invention provides a kind of migratory locusts coria epidermal protein full length gene sequence, its nucleotide sequence is respectively SEQ ID
NO：1.The full length gene sequence is to be based on migratory locusts transcript profile database, and the fragment for searching for acquisition is flown by the combination of GeneDoc softwares
Locust genome sequence splices it, is then obtained through the amplification of PCR total lengths, cloning and sequencing.The length of nucleotides is 624bp,
ORF sequence 303bp, encode 100 amino acid, molecular weight 9.8KD, and theoretical isoelectric point is 4.50, contains a signal
Peptide and a RR-1 type chitin binding domain.
A kind of migratory locusts coria epidermal protein gene provided by the invention, its nucleotide sequence are SEQ ID NO：2, it is root
According to SEQ ID NO：1 contains the sense primer SEQ ID NO of T7 promoters by primer premier5.0 Software for Design：3 Hes
Anti-sense primer SEQ ID NO：4, SEQ ID NO are obtained by PCR amplification：2, its product contains T7 promoters.Product is purified
Afterwards according to T7RiboMAXTMExpress RNAi System (Promega) kit illustrates that in-vitro transcription is respectively synthesized dsRNA
(dsLmAbd6)。
SEQ ID NO：Applications of the dsRNA of 2 synthesis in migratory locusts prevent：By microsyringe by SEQ ID NO：2
The dsRNA injections of synthesis enter in 2 days 5 ages migratory locusts nymph body cavity.The result shows that：Compared with control, SEQ ID NO：2 synthesis
DsRNA can specifically, a kind of mRNA of migratory locusts coria epidermal protein gene (LmAbd6) of extremely significant silence expression, and
Causing adult polypide to occur, husking is difficult, and coria can not close, and ultimately resulting in pest belly can not go deep into soil laying eggs and leading
Egg sac number and ovum grain number is caused to substantially reduce.
Beneficial effects of the present invention compared with prior art：The present invention shows that injecting dsRNA can cause to fly through many experiments
Locust offspring's ovum grain number decrement reaches more than 70%.The specificity and high efficiency of the present invention, for reducing pest laying with after
There is remarkable result for population quantity, new approach can be provided for control of insect.
Brief description of the drawings
Fig. 1：A kind of migratory locusts coria epidermal protein gene (LmAbd6) is in the female male imago diverse location coria of migratory locusts
Differential expression, β-actin are as reference gene.
Fig. 2：Migratory locusts injection SEQ ID NO：After when the dsRNA 24 of 2 synthesis is small, a kind of migratory locusts coria epidermal protein base
Because of the mRNA silence situations of (LmAbd6), β-actin are as reference gene, wherein * * P<0.01.
Fig. 3：5 age migratory locusts injection SEQ ID NO：The dsRNA of 2 synthesis, adult coria occurs dysplastic after husking
Phenotype.A left side is the control of injection dsGFP；The right side is the phenotypic map of injection dsLmAbd6 coria exceptions.
Fig. 4：Migratory locusts injection SEQ ID NO：The dsRNA of 2 synthesis, female adult egg sac number and ovum grain number are compared to control group
DsGFP is substantially reduced.A is female adult egg sac number and ovum grain number, is respectively from left to right the control for injecting dsGFP, injects SEQ ID
NO：The dsRNA of 2 synthesis；The control and injection SEQ ID NO that B is injection dsGFP：Average ovum grain number after the dsRNA of 2 synthesis.
Embodiment
Embodiment 1：
1st, migratory locusts coria epidermal protein full length gene sequence obtains
Transcript profile database based on migratory locusts, scans for migratory locusts epidermal protein gene using bioinformatics method,
After sequence analysis and combining the splicing of migratory locusts genome sequence and comparing, a kind of migratory locusts coria epidermal protein gene is obtained
(LmAbd6) sequence, using primer premier5.0 Software for Design upstream and downstream primer sequence and is sent to raw work bioengineering
(Shanghai) Co., Ltd. synthesizes.Healthy growth is chosen, the female male imago of migratory locusts of the same size, dissects internode membrane tissue, and freezes
In liquid nitrogen.6 first biology repeat, if 4 biology repeat, total serum IgE is extracted according to TaKaRa Trizol kits.
Using M-MLV reverse transcriptase by carried total serum IgE reverse transcription into the first chain cDNA, with this as template, with reference to the upstream and downstream of design
Primer, obtains LmAbd6 full length gene fragments by PCR amplification, obtained product is purified, Cloning Transformation to large intestine bar
In bacterium, and it is sent to Shanghai Invitrogen biology Co., Ltd and is sequenced, its sequence is SEQ ID NO：1.
2nd, migratory locusts coria epidermal protein gene is in the female male imago coria differential tissue expression of migratory locusts
Healthy growth is chosen, the female male imago of migratory locusts of the same size, dissects each internode membrane tissue, and be frozen in order
In liquid nitrogen.6 first biology repeat, if 4 biology repeat, using method for extracting total RNA in 1 and obtain different male and female into
Worm coria cDNA templates, a kind of migratory locusts coria epidermal protein gene is detected by real-time fluorescence quantitative PCR (RT-qPCR)
(LmAbd6) differential expression in female male imago difference coria, the results show LmAbd6 is in female adult the 4th, 5 and 6 coria groups
The expression quantity knitted is higher and pole is significantly higher than the expression quantity (Fig. 1) in correspondence position male worm coria, illustrates LmAbd6
May play an important roll in female adult the 4th, 5 and 6 corias.
3rd, migratory locusts coria epidermal protein gene specific dsRNA is synthesized
1) design of migratory locusts coria epidermal protein gene dsRNA primers
Based on a kind of migratory locusts coria epidermal protein gene order (SEQ ID NO：1), using primer premier5.0
Software for Design dsRNA primers, its primer sequence are respectively sense primer SEQ ID NO：3 and anti-sense primer SEQ ID NO：4.On
Anti-sense primer carries T7 promoter sequences.All primers are synthesized by Sangon Biotech (Shanghai) Co., Ltd..
2) synthesis of migratory locusts coria epidermal protein gene dsRNA
Using a kind of above-mentioned plasmid of migratory locusts coria epidermal protein gene extraction as template, sense primer SEQ ID are utilized
NO：3 and anti-sense primer SEQ ID NO：4 carry out PCR amplification.By the PCR product of amplification, through Gel Extraction Kit
(sigma) according to T7RiboMAX after kitsTMExpress RNAi System (Promega) kit explanation difference
In-vitro transcription synthesizes dsRNA.Quantified using NaNoDrop 2000 (Thermo scientific), reach its final concentration
2μg/μl.It is spare to be stored in -80 DEG C of super low temperature refrigerators.
Embodiment 2：The lethal migratory locusts experiments of migratory locusts coria epidermal protein gene dsRNA
1st, migratory locusts coria epidermal protein gene dsRNA is injected
Choose healthy growth, 30 first the 2nd day 5 age female nymph of the same size is tested.By 5 μ l (10 μ g) SEQ ID
NO：3 and SEQ ID NO：4 dsRNA is gently injected into nymph flank with 25 μ l specifications micro syringes along blood flow method
Between two, three uromeres in portion.30 female nymphs are chosen at the same time is established as control group.Inject the dsGFP of same volume and concentration
To control group body.Picking healthy growth, equivalent male worm of the same size mix with the female migratory locusts after injection respectively is placed in 30
(illumination is raised in DEG C constant temperature biochemical cultivation case：Interlunation=14h:10h, 30 ± 2 DEG C of temperature, humidity 60%), feeding daily
Fresh wheat seedling and wheat bran.
2nd, migratory locusts coria epidermal protein gene silencing detects
Each 15 of nymph after injection dsGFP and dsLmAbd6 24h is collected, dissection internode membrane tissue carries out total serum IgE and carries
Take, and reverse transcription is repeated into the first chain cDNA, 3 biology of every group of setting, each biology repeats 5 nymphs, using RT-
The relative expression quantity of qPCR methods difference testing goal gene (LmAbd6) and house-keeping gene (β-actin), calculates its silence effect
Rate.The result shows that compared with the control group, treatment group LmAbd6 expression quantity pole significantly reduces (Fig. 2).
3rd, adult Phenotypic Observation after injection dsRNA
After injecting dsGFP and dsLmAbd6, injection dsGFP control groups start to cast off a skin and all succeed 5 age nymphs after 6 days
Slough off to adult, polypide form vigor is normal after sloughing off to adult, and starts normal feed after half a day.Inject the processing of dsLmAbd6
The female nymph totally 15 of group, compared with control group nymph, wherein about 47% adult coria can not normally closed (Fig. 3), hand over
It can not normally go deep into soil laying eggs after matching somebody with somebody, wherein egg sac number and ovum grain number substantially reduces (Fig. 4).
Sequence table
<110>University Of Shanxi
<120>Migratory locusts coria epidermal protein gene 6 and its application in locust control
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 624
<212> DNA
<213>Migratory locusts (Locusta migratoria)
<400> 1
cgccagttcg cccgctatat aaacctcgct atacgggcta ccgacacact ccagctctca 60
gcaaccgact cgtctgctgc tcagcccagc ccagccaaca gccaacatga agctgctgct 120
cgttgtcgtc gccctcatcg ctgtcgccgc cgccagacca cagaagcctg gtgaggccgt 180
catcctggag cagagcatcg acaacgatgg actcggccag tacacgtttg gattcaagac 240
gagcgacggc ctgatccgcc aggagcaggg agtgctgaag aaccagggca cggagaacga 300
ggccatcgag gtccgcggca ccatcacgtg gaagggagcc gacggcaagg actacagcat 360
caacttcatc gccgacgaga acggcttcca gccccagtac aaccagtagg cgcgctccac 420
cctacaccgc ccgactgaca tgcccacgac gaccggcgcc gcgacgctgg aggcacgcgg 480
gggctgcacg ggcggactga gttcctccct cctcactcga cgaggggctg ccaccaacct 540
cacctaactc ctcacctcct ctctctctct ctctactcta aactgtacat aattgttcaa 600
agtgtcaata aaagttatta ccag 624
<210> 2
<211> 213
<212> DNA
<213>Migratory locusts (Locusta migratoria)
<400> 2
atgaagctgc tgctcgttgt cgtcgccctc atcgctgtcg ccgccgccag accacagaag 60
cctggtgagg ccgtcatcct ggagcagagc atcgacaacg atggactcgg ccagtacacg 120
tttggattca agacgagcga cggcctgatc cgccaggagc agggagtgct gaagaaccag 180
ggcacggaga acgaggccat cgaggtccgc ggc 213
<210> 3
<211> 41
<212> DNA
<213>Migratory locusts (Locusta migratoria)
<400> 3
taatacgact cactataggg atgaagctgc tgctcgttgt c 41
<210> 4
<211> 40
<212> DNA
<213>Migratory locusts (Locusta migratoria)
<400> 4
taatacgact cactataggg gccgcggacc tcgatggcct 40
Claims (5)
1. a kind of migratory locusts coria epidermal protein gene, its nucleotide sequence is SEQ ID NO：1.
2. a kind of migratory locusts coria epidermal protein gene, its nucleotide sequence is SEQ ID NO：2.
A kind of 3. dsRNA of migratory locusts coria epidermal protein gene chemical synthesis as claimed in claim 2.
4. the preparation method of dsRNA as claimed in claim 3, step are：According to SEQ ID NO：The sense primer SEQ of 1 design
ID NO：3 and anti-sense primer SEQ ID NO：4, SEQ ID NO are obtained by PCR amplification：2, its product contains T7 promoters；Production
According to T7 RiboMAX after thing is purifiedTMExpress RNAi System (Promega) kit illustrates that in-vitro transcription synthesizes
dsRNA。
A kind of 5. dsRNA of migratory locusts coria epidermal protein gene chemical synthesis as claimed in claim 3 answering in locust control
With.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109504684A (en) * | 2018-12-20 | 2019-03-22 | 山西大学 | The application of Ran gene and its dsRNA in control of insect |
| CN109734788A (en) * | 2019-02-25 | 2019-05-10 | 中国农业科学院植物保护研究所 | Migratory locusts mucoprotein Mucin17, its encoding gene and its application |
| CN113402596A (en) * | 2021-05-26 | 2021-09-17 | 中国农业科学院农业基因组研究所 | RR-2 family epidermal protein of scarab beetle, coding nucleotide sequence and application thereof |
| CN113981008A (en) * | 2021-10-14 | 2022-01-28 | 山西大学 | Method for preparing Lmzen homozygous mutant of migratory locust, RNP compound and application |
-
2017
- 2017-11-26 CN CN201711199240.9A patent/CN107988231A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109504684A (en) * | 2018-12-20 | 2019-03-22 | 山西大学 | The application of Ran gene and its dsRNA in control of insect |
| CN109504684B (en) * | 2018-12-20 | 2021-07-27 | 山西大学 | Application of Ran gene and dsRNA thereof in pest control |
| CN109734788A (en) * | 2019-02-25 | 2019-05-10 | 中国农业科学院植物保护研究所 | Migratory locusts mucoprotein Mucin17, its encoding gene and its application |
| CN113402596A (en) * | 2021-05-26 | 2021-09-17 | 中国农业科学院农业基因组研究所 | RR-2 family epidermal protein of scarab beetle, coding nucleotide sequence and application thereof |
| CN113402596B (en) * | 2021-05-26 | 2022-04-15 | 中国农业科学院农业基因组研究所 | RR-2 family epidermal protein of scarab beetle, coding nucleotide sequence and application thereof |
| CN113981008A (en) * | 2021-10-14 | 2022-01-28 | 山西大学 | Method for preparing Lmzen homozygous mutant of migratory locust, RNP compound and application |
| CN113981008B (en) * | 2021-10-14 | 2023-09-22 | 山西大学 | Method for preparing migratory locust Lmzen homozygous mutant, RNP complex and application |
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