CN104630247B - Insect chitin deacetylate enzyme gene 1 and its application in control of insect - Google Patents
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Abstract
The present invention provides a kind of 1 (CDA1) full length sequence of insect chitin deacetylate enzyme gene and its application in agricultural insect pests control.Specifically by bioinformatics method, 1 segment of chitin deacetylate enzyme gene is obtained from migratory locusts transcript profile, it is SEQ ID NO further to transfer and obtain sequence：1 full length gene.According to SEQ ID NO：1, design and synthesize the dsRNA of the gene, injected into after migratory locusts body cavity can specific silence target gene, lead to death to make migratory locusts be difficult to slough old epidermis.Many experiments show that its lethality reaches 94.7% or more.Due to the specific and efficient lethality of the present invention, has important practical significance for control of insect, new approach can be provided for control of insect.
Description
Technical field
The present invention relates to biotechnologies, and in particular to migratory locusts chitin deacetylate enzyme gene 1 (CDA1) and in evil
Application in worm prevention.
Background technology
Migratory locusts are China's important agricultural insects, migrate cause to seriously endanger to agricultural production on a large scale.It is anti-to it at present
It controls and relies primarily on chemical prevention.Chronic administration chemical insecticide leads to a series of problems, such as environmental pollution, and drug resistance generates and right
The harm of non-target organism.Therefore the method for developing novel, alternative chemical prevention becomes particularly urgent.
The phenomenon that RNA interference (RNAi) is gene silencing after a kind of specific transcriptional caused by double stranded rna molecule, from
Since 2006 obtain the Nobel Prize, RNAi technology is always the research hotspot of life science.RNAi is not only research gene work(
The powerful of energy, while also there is very high potential in terms of control of insect.It is interfered by RNA and carries out control of insect with as follows
Advantage：1) desinsection specificity, to non-target organism without lethal effect；2) RNA is easily degraded in nature, noresidue；3) to ring
Border is nontoxic, comparatively safe.Therefore scholar is referred to as forth generation insecticide.It is interfered based on RNA before carrying out control of insect
Carry is that Screening target sequence obtains the dsRNA for having high lethal effect to insect.
Its internal water can be prevented to scatter and disappear for insect cuticle and from pathogen infection, chitin is the important of insect cuticle
Constituent, it is essential that chitin dense arrangement develops insect growth.Chitin deacetylate enzyme gene 1 (CDA1) is negative
Duty hydrolyzes the acetamido of chitin N- acetyl glucosamines, formation chitan (or Chitosan, shell are poly-
Sugar), to change its physical property, other components such as epidermal protein is made to be easier to be combined with chitin, to form densification
Epidermal structure.Insect occurs casting off a skin and be obstructed and lethal phenomenon after the silence target gene.Simultaneously due to people and other higher mammals
There is no chitin, therefore, the present invention uses RNA perturbation techniques, and chitin deacetylate enzyme is screened for chitin metabolic system
The dsRNA molecular targets of gene 1 are comparatively safe, play an important roll in migratory locusts prevent.
Invention content
The purpose of the present invention is to provide a kind of 1 full length sequence of insect chitin deacetylate enzyme gene and its dsRNA, with
And they synthetic method and the application in control of insect.
The present invention provides a kind of 1 full length sequence of migratory locusts chitin deacetylate enzyme gene, and nucleotides sequence is classified as SEQ ID
NO：1.The full length gene sequence is to be based on migratory locusts transcript profile database, search for the segment of acquisition by GeneDoc softwares to its into
Row splicing, design sense primer CAACGTCACAACCAGTGAGTGTC (SEQ ID NO：And downstream primer 3)
GCGGTACACGATGAAAGATGG(SEQ ID NO：4) it, is obtained by PCR amplification.The length of nucleotides is 1753bp, core
Nucleotide sequence is SEQ ID NO：1.
The present invention provides a kind of amino acid sequence that migratory locusts chitin deacetylate enzyme gene 1 encodes, it is characterized in that nucleosides
Acid sequence is SEQ ID NO：2 sequence.The amino acid sequence is to SEQ ID NO：It is predicted after 1 progress bioinformatic analysis
It obtains.Bioinformatic analysis shows that 535 amino acid of coding of CDA1 genes, molecular weight 61KD, theoretical isoelectric point are
5.11。
The dsRNA and its application in control of insect that the present invention provides 1 synthesis of migratory locusts chitin deacetylate enzyme gene：
Based on 1 sequence of migratory locusts chitin deacetylate enzyme gene, T7 promoters are contained by primer premier5.0 Software for Design
Sense primer taatacgactcactatagggTCTGTAACGGCGAGAAGGAC (SEQ ID NO：And downstream primer 5)
taatacgactcactatagggCCATCATGGTGAACTGGTTG(SEQ ID NO：6) segment length, is obtained by PCR amplification
Both ends for 586bp are DNA fragmentation (the SEQ ID NO of T7 promoters：7).According to T7RiboMAX after kitsTM
Express RNAi System (Promega) kit illustrates the dsRNA of in-vitro transcription synthesis.It will be closed by microsyringe
At dsRNA injection enter migratory locusts body cavity in.The result shows that：Migratory locusts chitin deacetylate enzyme gene 1 after injection dsRNA
MRNA expression significantly reduces, and migratory locusts husking difficulty occur and lead to death.
Beneficial effects of the present invention：After migratory locusts five ages nymph injects dsRNA, nymph occurred phenomenon of casting off a skin at the 7th day, but only
There are wing bud opening, back to arch, old epidermis is difficult to slough until death, and the death rate reaches 94.7% or more.Migratory locusts of the present invention are several
The dsRNA that fourth matter deacetylate enzyme gene 1 synthesizes has high lethality to migratory locusts, has important reality for control of insect
Meaning can provide new approach for control of insect.
Description of the drawings
Fig. 1：Ago-Gel detects 1 full length cDNA sequence length (M DL5000DNA of chitin deacetylate gene
Marker, band is followed successively by 100,250,500,750,1000,1500,2000 from small to large, 3000,5000bp, 1 is chitin
1 stripe size of deacetylate gene)
Fig. 2：On the transcription of chitin deacetylate enzyme gene 1 influence, (1 is pair for injecting dsGFP afterwards for 24 hours for dsRNA injections
According to group, 2 be the experimental group for injecting dsRNA).β-actin are reference gene.Wherein * P<0.05, * * P<0.01.
Fig. 3：(1 is the control group for injecting dsGFP, and 2 and 3 be note for the influence of dsRNA pairs of 5 ages migratory locusts nymph growth and development
Penetrate the experimental group of dsRNA).There is the difficult dead phenotype of husking in experimental group migratory locusts.
Specific implementation mode
Embodiment 1：Migratory locusts chitin deacetylate enzyme gene 1cDNA full length sequences obtain and amino acid sequence analysis
1. migratory locusts chitin deacetylate enzyme gene 1cDNA segments obtain
Transcript profile database based on migratory locusts, scans for its Unigene, and after NCBI Blastx analyses, determination obtains
Obtain the segment of 1 migratory locusts chitin deacetylate enzyme gene 1.
2. migratory locusts chitin deacetylate enzyme gene 1cDNA full length sequences obtain
Said gene segment is spliced by GeneDoc softwares, and uses primer premier5.0 Software for Design
Sense primer CAACGTCACAACCAGTGAGTGTC (SEQ ID NO：And downstream primer GCGGTACACGATGAAAGATGG 3)
(SEQ ID NO：4) it, is synthesized by Shanghai Invitrogen biology Co., Ltd.
Healthy growth, 5 age migratory locusts nymph in the same size, half male and half female are chosen, it is under Stereo microscope that its epidermis is fast
Speed is dissected, and is frozen in liquid nitrogen.4 first biology repeat, and RNA is extracted according to TaKaRa Trizol kits.It adopts
With M-MLV reverse transcriptase by carried RNA reverse transcriptions at the first chain cDNA.Led in conjunction with design upstream and downstream primer as template with this
It crosses PCR amplification and obtains chitin deacetylate enzyme gene full length fragment (Fig. 1), pass through Gel Extroaction Kit (Omega)
PCR product is purified, by product cloning after purification to pEASY-T3Cloning vector (Quan Shi King Companies), is turned
Enter competent cell, expand bacterium solution culture, send bacterium solution using after Plasmid Mini Kit1 (Omega) extraction plasmids detections
It is sequenced toward invitrogen companies sequencing company.Sequencing obtains nucleotides sequence and is classified as SEQ ID NO：1 sequence.
3. 1 amino acid sequence analysis of migratory locusts chitin deacetylate enzyme gene
It is translated by ExPaSy online softwares to having obtained chitin deacetylate enzyme gene 1, prediction chitin goes second
The open reading frame of acyl group enzyme gene 1 encodes 535 amino acid, molecular weight 61KD, isoelectric point 5.11.Functional domain prediction hair
Existing chitin deacetylate enzyme gene 1 has signal peptide, chitin binding domain (CBD), LDL receptor domain (LDLa)
With catalytic activity domain (CDA).1 amino acid of migratory locusts chitin deacetylate enzyme gene is same with red flour beetle CDA1 amino acid sequences
Source degree reaches 90%.
Embodiment 2：The dsRNA of migratory locusts chitin deacetylate enzyme gene 1 is synthesized
1. the design of migratory locusts chitin deacetylate enzyme gene 1dsRNA primers
Based on migratory locusts chitin deacetylate enzyme gene sequence, using primer premier5.0 Software for Design.Design
DsRNA primers, sequence are respectively taatacgactcactatagggTCTGTAACGGCGAGAAGGAC (SEQ ID NO：5) and
taatacgactcactatagggCCATCATGGTGAACTGGTTG(SEQ ID NO：6) (italicized item is T7 promoters).Institute
There is primer to be synthesized by Shanghai Invitrogen biology Co., Ltd.
2,1 specificity dsRNA synthesis of migratory locusts chitin deacetylate enzyme gene
Plasmid is extracted as template using above-mentioned chitin deacetylate enzyme gene 1, is drawn with containing T7 promoter sequence upstream and downstream
Object carries out PCR amplification.It is 586bp genetic fragments (SEQ ID NO to obtain a segment length：7), using Gel Extraction Kit
(Omega) kit by PCR product after purification according to T7RiboMAXTMExpress RNAi System (Promega) kit
Illustrate that in-vitro transcription synthesizes dsRNA.It is quantified using NaNoDrop 2000 (Thermo scientific), makes its final concentration
Reach 2.5 μ g/ μ l.It is spare to be stored in -80 DEG C of super low temperature refrigerators.
Embodiment 3：The lethal migratory locusts experiments of dsRNA of migratory locusts chitin deacetylate enzyme gene 1
1, specificity dsRNA is injected
Healthy, in the same size, half male and half female the 2 days 5 ages nymph of 28 head growths is chosen to be tested.It is micro- using 25 μ l specifications
Amount syringe gently injects the dsRNA that 2.5 μ l (6.25 μ g) are synthesized between two, three uromeres in nymph flank portion.Simultaneously
It chooses 28 nymphs and is established as control group, in the dsGFP to control group body for injecting same volume and concentration.Migratory locusts after injection are set
(illumination is raised in 30 DEG C of constant temperature biochemical cultivation cases：Interlunation=14h:10h, 30 ± 2 DEG C of temperature, humidity 60%), daily
Feed fresh wheat seedling and wheat bran.
2,1 silence of migratory locusts chitin deacetylate enzyme gene detects
It is each to collect 9 injection dsGFP and dsLmCDA124h teleonymph polypides progress Total RNAs extractions, and reverse transcription is at first
Chain cDNA, using Real-time PCR methods difference testing goal gene (LmCDA1) and house-keeping gene (β-actin) it is opposite
Expression quantity, to calculate its silence efficiency.The result shows that compared with the control group, after injecting dsRNA, processing group chitin
Matter deacetylate enzyme gene expression significantly reduces (Fig. 2).3 biology of every group of setting repeat, if each biology repeats 3
Worm.
3, five age nymph Phenotypic Observations after injection dsRNA
After five age nymphs inject dsRNA, control group polypide all successfully casted off a skin to adult the 7th day 5 ages, and after casting off a skin at
Worm developmental condition is good.After injecting ds LmCDA1, equally there is husking phenomenon at the 7th day in nymph, but only wing bud is opened, carried on the back
Portion arches, and old epidermis is difficult to be sloughed from polypide until dead (Fig. 3), and the death rate reaches 94.7% or more.
Claims (5)
1. a kind of migratory locusts chitin deacetylate enzyme gene 1, it is characterised in that nucleotides sequence is classified as SEQ ID NO：1.
2. the amino acid sequence that migratory locusts chitin deacetylate enzyme gene 1 as described in claim 1 encodes, it is characterised in that ammonia
Base acid sequence is SEQ ID NO：2.
3. the dsRNA that migratory locusts chitin deacetylate enzyme gene 1 as described in claim 1 synthesizes.
4. the synthetic method of dsRNA as claimed in claim 3, it is characterised in that include the following steps：It is gone according to migratory locusts chitin
The nucleotide sequence of acetyl group enzyme gene 1 designs the sense primer containing T7 promoters
TaatacgactcactatagggTCTGTAACGGCGAGAAGGAC and downstream primer
TaatacgactcactatagggCCATCATG GTGAACTGGTTG obtain the mould that both ends are T7 promoters by PCR amplification
Plate, the T7RiboMAX produced according to Promega after kitsTMExpress RNAi System kits explanation is external
Transcription synthesis obtains.
5. applications of the dsRNA of method synthesis as claimed in claim 4 in migratory locusts prevent.
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| CN105462996A (en) * | 2015-12-23 | 2016-04-06 | 太原理工大学 | Gene silencing technology-based gypsymoth chitin deacetylase gene |
| CN105755006A (en) * | 2016-03-29 | 2016-07-13 | 山西大学 | Migratory locust wing specific cuticle protein gene and application of dsRNA thereof |
| CN105695476A (en) * | 2016-03-29 | 2016-06-22 | 山西大学 | Migratory locust wing epidermis protein gene and application thereof in pest control |
| CN108795911A (en) * | 2018-07-03 | 2018-11-13 | 南京林业大学 | Chitin deacetylase, chitin nanofiber dispersion liquid and its preparation method and application |
| CN109504684B (en) * | 2018-12-20 | 2021-07-27 | 山西大学 | Application of Ran gene and dsRNA thereof in pest control |
| CN113122540B (en) * | 2021-05-14 | 2022-10-11 | 河北农业大学 | RNAi target gene capable of killing grubs efficiently and application of RNAi target gene |
| CN114317568B (en) * | 2021-12-31 | 2023-12-26 | 山西大学 | Method for synthesizing dsRNA of cricket CDA1 and CDA2 genes and application thereof |
| CN117384894B (en) * | 2023-12-04 | 2024-02-09 | 中国水产科学研究院黄海水产研究所 | Deacetylase and encoding gene, recombinant plasmid, recombinant bacterium and application thereof |
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