CN105695476A - Migratory locust wing epidermis protein gene and application thereof in pest control - Google Patents

Migratory locust wing epidermis protein gene and application thereof in pest control Download PDF

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CN105695476A
CN105695476A CN201610186141.6A CN201610186141A CN105695476A CN 105695476 A CN105695476 A CN 105695476A CN 201610186141 A CN201610186141 A CN 201610186141A CN 105695476 A CN105695476 A CN 105695476A
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protein gene
migratory locust
wing
locustamigratoria
linnaeus
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赵小明
勾昕
秦忠玉
马恩波
张建珍
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Shanxi University
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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
    • 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
    • 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
    • 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

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
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  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Plant Pathology (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
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  • Biomedical Technology (AREA)
  • Toxicology (AREA)
  • Physics & Mathematics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Pest Control & Pesticides (AREA)
  • Environmental Sciences (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Insects & Arthropods (AREA)
  • Agronomy & Crop Science (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicinal Chemistry (AREA)
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Abstract

The invention provides a migratory locust wing epidermis protein gene and application thereof in pest control, particularly a migratory locust wing epidermis protein gene (LmACP19) full-length sequence and application thereof in migratory locust control. A biological informatical method is utilized to acquire an epidermis protein gene segment from a migratory locust transcriptome, and the gene full-length with the sequence of SEQ ID NO:1 is further acquired through the migratory locust genome sequence. The dsRNA (double-stranded ribonucleic acid) of the gene is designed and synthesized according to SEQ ID NO:1, and the dsRNA is injected into the migratory locust coelom to specifically silence the target gene, thereby causing difficulty in migratory locust ecdysis, tergum cracking, and developmental deformity and crimple of wings, and finally causing death. The repeated experiments indicate that the death rate reaches 69% or above. Due to the specificity and efficient lethality rate, the migratory locust wing epidermis protein gene has important practical meanings for pest control, and can provide a new way for pest control.

Description

Locustamigratoria (Linnaeus) wing epidermal protein gene and the application in Pest control thereof
Technical field
The present invention relates to biological technical field, be specifically related to Locustamigratoria (Linnaeus) wing epidermal protein gene (LmACP19) and the application in Pest control。
Background technology
Locustamigratoria (Linnaeus) is China Important Agricultural insect, due to its have wing can long-distance migration flight between land, thus looking for food, seek a spouse, keep away enemy and expand distribution efficiency etc. in be all greatly improved, agricultural production is caused serious harm。At present its preventing and treating is relied primarily on chemical prevention, but chronic administration chemical insecticide causes series of problems, such as environmental pollution, Drug resistance generation and the harm etc. to non-target organism。Therefore, development of new, alternative chemical prevention method become extremely urgent。
RNA interference (RNAi) is the phenomenon of gene silencing after a kind of specific transcriptional caused by double stranded rna molecule, and since within 2006, obtaining Nobel Prize, RNAi technology ranks among Environment Science always。RNAi is not only the powerful of research gene function, also has very high potential in Pest control simultaneously。Carrying out Pest control have a characteristic that 1 by RNA interference) parasite killing has specificity, to non-target organism without lethal effect；2) RNA very easily degrades at nature, noresidue；3) environment is nontoxic, comparatively safe。Therefore scholar is referred to as forth generation insecticide。The basic technology carrying out Pest control based on RNA interference is that Screening target sequence obtains the dsRNA with high lethal effect。
Insect cuticle is possible to prevent its internal water to scatter and disappear and from pathogen infection, and epidermal protein is insect cuticle important composition composition, and the disappearance of its composition or changes of contents all can cause epidermis dysfunction even afunction, thus affecting the growth promoter of insecticide。Feature for Locustamigratoria (Linnaeus) gregariousness He migrating property, the present invention is based on RNA perturbation technique, and choosing wing epidermal protein is object, utilizes its important biomolecule function to carry out Pest control, safe and efficient, it is a kind of novel molecular target calibration method harmless, specific environmentally friendly, non-toxic to humans。
Summary of the invention
It is an object of the invention to provide a kind of Locustamigratoria (Linnaeus) wing epidermal protein gene (LmACP19) full length sequence and the application in lethal Locustamigratoria (Linnaeus) thereof。
The present invention provides a kind of Locustamigratoria (Linnaeus) wing epidermal protein full length gene sequence, and its nucleotides sequence is classified as SEQIDNO:1。This full length gene sequence is based on Locustamigratoria (Linnaeus) transcript profile data base, and it is spliced in conjunction with Locustamigratoria (Linnaeus) genome sequence by the fragment that search obtains by GeneDoc software, then obtains through the amplification of PCR total length, cloning and sequencing。This length of nucleotides is 720bp, ORF sequence 531bp, encodes 176 aminoacid, and molecular weight is 18KD, and theoretical isoelectric point, IP is 7.88, containing a signal peptide and a RR-2 type chitin binding domain。
A kind of Locustamigratoria (Linnaeus) wing epidermal protein gene provided by the invention, its nucleotide sequence is SEQIDNO:2, it is the forward primer SEQIDNO:3 and downstream primer SEQIDNO:4 that contain T7 promoter by primerpremier5.0 software design according to SEQIDNO:1, obtaining SEQIDNO:2 by pcr amplification, its product contains T7 promoter。According to T7RiboMAX after product is purifiedTMExpressRNAiSystem (Promega) test kit illustrates in vitro transcription synthesis dsRNA。
The dsRNA of SEQIDNO:2 synthesis application in Locustamigratoria (Linnaeus) prevents and treats: the SEQIDNO:2 dsRNA synthesized injection is entered in Locustamigratoria (Linnaeus) body cavity by microsyringe。Result shows: compare with comparison, the dsRNA of SEQIDNO:2 synthesis can specifically, the mrna expression of extremely significant reticent Locustamigratoria (Linnaeus) wing epidermal protein gene LmACP19, and cause the backboard of adult to ftracture, wing development abnormal, show as wing development deformity, shrinkage can not launch, and ultimately results in death。
Accompanying drawing explanation
Fig. 1: Locustamigratoria (Linnaeus) injection SEQIDNO:2 synthesis dsRNA24 hour after, the mRNA silence situation of the special epidermal protein gene LmACP19 of Locustamigratoria (Linnaeus) wing, β-actin as reference gene, wherein * * * P < 0.001。
The dsRNA of Locustamigratoria (Linnaeus) injection SEQIDNO:2 synthesis in Fig. 2: 5 ages, after casting off a skin, there is dysplastic phenotype in adult wing。A: left side is the comparison of injection dsGFP, right side is the dsRNA, i.e. dsLmACP19 of injection SEQIDNO:2 synthesis；B: the phenotypic map of abnormal wing。
Detailed description of the invention
Embodiment 1: Locustamigratoria (Linnaeus) wing epidermal protein full length gene sequence obtains
1, Locustamigratoria (Linnaeus) wing epidermal protein full length gene sequence obtains
Transcript profile data base based on Locustamigratoria (Linnaeus), adopt bioinformatics method that Locustamigratoria (Linnaeus) epidermal protein gene is scanned for, splice and after comparison through sequence analysis and in conjunction with Locustamigratoria (Linnaeus) genome sequence, obtain Locustamigratoria (Linnaeus) wing epidermal protein gene (LmACP19) sequence, adopt primerpremier5.0 software design upstream and downstream primer sequence and be sent to Sangon Biotech's synthesis。Choose healthy growth, Locustamigratoria (Linnaeus) adult of the same size, dissect wing tissue, and be frozen in liquid nitrogen。3 first biologys repeated, if 3 biologys repeat, extracted total serum IgE according to TaKaRaTrizol test kit。Adopt M-MLV reverse transcription that carried total serum IgE reverse transcription is become the first chain cDNA, with this as template, upstream and downstream primer in conjunction with design, LmACP19 full length gene fragment is obtained by pcr amplification, be purified by the product obtained, Cloning Transformation is in escherichia coli, and be sent to Shanghai English Weihe River victory base biology company limited and check order, its sequence is SEQIDNO:1。
2, Locustamigratoria (Linnaeus) wing epidermal protein gene specific dsRNA synthesis
1) design of Locustamigratoria (Linnaeus) wing epidermal protein gene dsRNA primer
Based on Locustamigratoria (Linnaeus) wing epidermal protein gene order (SEQIDNO:1), adopt primerpremier5.0 software design dsRNA primer, its upstream and downstream primer sequence respectively SEQIDNO:3 and SEQIDNO:4。Upstream and downstream primer all carries T7 promoter sequence。All primers synthesize by Sangon Biotech (Shanghai) Co., Ltd.。
2) synthesis of Locustamigratoria (Linnaeus) wing epidermal protein gene dsRNA
With the plasmid of above-mentioned Locustamigratoria (Linnaeus) wing epidermal protein gene extraction for template, SEQIDNO:3 and SEQIDNO:4, as upstream and downstream primer, carries out pcr amplification。By the PCR primer of amplification, according to T7RiboMAX after GelExtractionKit (sigma) kitsTMExpressRNAiSystem (Promega) test kit illustrates in vitro transcription synthesis dsRNA。NaNoDrop2000 (Thermoscientific) is used to carry out quantitatively so that it is final concentration reaches 2 μ g/ μ l。It is stored in-80 DEG C of super cryogenic refrigerators standby。
Embodiment 2: the lethal Locustamigratoria (Linnaeus) experiment of Locustamigratoria (Linnaeus) wing epidermal protein gene dsRNA
1, Locustamigratoria (Linnaeus) wing epidermal protein gene dsRNA injection
Choose healthy growth, 2nd day 30 5 ages of the same size nymph test。The dsRNA of 5 μ l (10 μ g) SEQIDNO:2 is injected between two, three uromeres entering nymph flank portion along blood flow method gently with 25 μ l specification microsyringes。Choose 30 nymphs simultaneously and be established as matched group。The dsGFP of injection same volume and concentration is to matched group body。Be placed in 30 DEG C of constant temperature biochemical cultivation cases respectively to raise Locustamigratoria (Linnaeus) after injection (illumination: interlunation=14h:10h, temperature 30 ± 2 DEG C, humidity 60%), feed fresh wheat seedling and Testa Tritici every day。
2, Locustamigratoria (Linnaeus) wing epidermal protein gene silencing detection
Collect each 9 of nymph after injection dsGFP and dsRNA24h, dissect wing bud tissue and carry out Total RNAs extraction, and reverse transcription becomes the first chain cDNA, often group arranges and repeats 3 biologys, repeat 3 nymphs each biology, adopt the relative expression quantity of Real-timePCR method testing goal gene (LmACP19) and house-keeping gene (β-actin) respectively, calculate its silence efficiency。It is shown that compare with matched group, after injection dsRNA, process group Locustamigratoria (Linnaeus) wing epidermal protein gene expression amount pole significantly reduces (Fig. 1)。
3, adult Phenotypic Observation after injection dsRNA
5 age nymph after injection dsRNA, injection dsGFP matched group starts after 6 days to cast off a skin and all successfully slough off to adult, sloughs off to adult polypide form vigor normal, and starts normally to take food after half a day。The process group nymph totally 30 of injection dsLmACP19, compared with matched group nymph, wherein about 69% adult backboard cracking, front and rear wing shrinkage deformity, difficulty of casting off a skin, finally dead, it is in particular in forward and backward wing shrinkage, (Fig. 2) can not be launched。

Claims (5)

1. a Locustamigratoria (Linnaeus) wing epidermal protein gene, its nucleotide sequence is SEQIDNO:1。
2. a Locustamigratoria (Linnaeus) wing epidermal protein gene, its nucleotide sequence is SEQIDNO:2。
3. the dsRNA of Locustamigratoria (Linnaeus) wing epidermal protein gene chemical synthesis as claimed in claim 2。
4. the preparation method of dsRNA as claimed in claim 3, step is: the forward primer SEQIDNO:3 and downstream primer SEQIDNO:4 according to SEQIDNO:1 design, obtains SEQIDNO:2 by pcr amplification, and its product contains T7 promoter；According to T7RiboMAX after product is purifiedTMExpressRNAiSystem (Promega) test kit illustrates in vitro transcription synthesis dsRNA。
5. the dsRNA of Locustamigratoria (Linnaeus) wing epidermal protein gene as claimed in claim 3 application in Locustamigratoria (Linnaeus) prevents and treats。
CN201610186141.6A 2016-03-29 2016-03-29 Migratory locust wing epidermis protein gene and application thereof in pest control Pending CN105695476A (en)

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106636148A (en) * 2017-03-01 2017-05-10 山西大学 Locusta migratoria cytochrome P450 reductase gene dsRNA and application thereof
CN107699569A (en) * 2017-11-26 2018-02-16 山西大学 Migratory locusts coria epidermal protein gene 1 and its application in locust control
CN108949762A (en) * 2018-08-02 2018-12-07 中国农业大学 The dsRNA of wing development related gene optomotor-blind and its application in control of insect
CN109504684A (en) * 2018-12-20 2019-03-22 山西大学 The application of Ran gene and its dsRNA in control of insect
CN110129327A (en) * 2019-04-10 2019-08-16 山西大学 Migratory locusts duct forms application of the related gene Lmsnar and its dsRNA in migratory locusts prevention and treatment
CN112391390A (en) * 2020-11-19 2021-02-23 山西大学 Migratory locust clathrin heavy chain gene and synthesis and application of targeting dsRNA thereof
CN113321718A (en) * 2021-06-10 2021-08-31 中国农业科学院农业基因组研究所 Insect CPCFC family epidermal protein, coding nucleotide sequence and application thereof

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106636148A (en) * 2017-03-01 2017-05-10 山西大学 Locusta migratoria cytochrome P450 reductase gene dsRNA and application thereof
CN106636148B (en) * 2017-03-01 2019-07-09 山西大学 A kind of migratory locusts cytochrome P450 reductase gene dsRNA and its application
CN107699569A (en) * 2017-11-26 2018-02-16 山西大学 Migratory locusts coria epidermal protein gene 1 and its application in locust control
CN108949762A (en) * 2018-08-02 2018-12-07 中国农业大学 The dsRNA of wing development related gene optomotor-blind and its application in control of insect
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
CN110129327A (en) * 2019-04-10 2019-08-16 山西大学 Migratory locusts duct forms application of the related gene Lmsnar and its dsRNA in migratory locusts prevention and treatment
CN110129327B (en) * 2019-04-10 2022-05-31 山西大学 Migratory locust pore canal formation related geneLmsnarAnd application of dsRNA thereof in locusta migratoria control
CN112391390A (en) * 2020-11-19 2021-02-23 山西大学 Migratory locust clathrin heavy chain gene and synthesis and application of targeting dsRNA thereof
CN113321718A (en) * 2021-06-10 2021-08-31 中国农业科学院农业基因组研究所 Insect CPCFC family epidermal protein, coding nucleotide sequence and application thereof

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Application publication date: 20160622