CN104293810A

CN104293810A - Gene silencing technology based gypsy moth chitinase gene and dsRNA

Info

Publication number: CN104293810A
Application number: CN201410439310.3A
Authority: CN
Inventors: 范晓军; 张建栋; 赵秋勇; 张常; 刘建红; 糜艳霞
Original assignee: Taiyuan University of Technology
Current assignee: Taiyuan University of Technology
Priority date: 2014-09-01
Filing date: 2014-09-01
Publication date: 2015-01-21
Anticipated expiration: 2034-09-01
Also published as: CN104293810B

Abstract

The invention provides a gene segment of an insect chitinase 5 gene and application of dsRNA thereof. Through cloning and sequencing of the gypsy moth chitinase 5 gene, a full-length chitinase 5 gene with a nucleotide sequence as SEQIDNO:1 can be obtained, and a chitinase 5 gene segment with a sequence as SEQIDNO:2 is selected therefrom to be applied in dsRNA synthesis. After injection of the dsRNA into the body cavity of gypsy moth, gypsy moth dies due to ecdysis difficulty. Thus, the invention provides a new approach for development of safe and nuisanceless pest control methods.

Description

Based on gypsymoth chitinase gene and the dsRNA of gene silent technology

Technical field

The present invention relates to biological technical field, be specifically related to the gypsymoth lethal gene fragment Chitinase5 based on gene silent technology and the application of dsRNA and dsRNA in lethal insect thereof.

Background technology

In China, the continuous single life-time service of chemical agent has caused insect to create resistance in various degree to Multiple Pesticides, such as, needs to continue to increase Pesticide use amount just can reach satisfied prevention effect for insect gypsymoth, cause even more serious environmental pollution, form vicious cycle.In addition, gypsymoth also can affect human health, and when the direct dirty dancing poison moth of people, redness often can occur and to itch phenomenon, severe patient even produces fash.Therefore, in agricultural production practice, be badly in need of the alternative means of prevention outside chemical pesticide.

RNA disturbs (RNA interference, RNAi) be a kind of gene silencing phenomenon by double chain RNA mediate, double-stranded RNA is finally processed to the tiny RNA (siRNA) that size is about 22nt, the mode of being matched by sequence is combined with protein coding gene, according to the degree degraded target gene mRNA of sequence pairing or the protein translation process of suppressor gene.RNAi is present in fungi, plant and animal widely.Andre Fire in 2006 and Craig Mello are because finding that RNAi phenomenon is awarded Nobel's medical science and physiology is encouraged.

In organism, it is necessary that some important gene pairss sustain life.Therefore, theoretically, if utilize RNA perturbation technique the expression of important gene in Agricultural pests to be disturbed, then can cause the teratogenesis of insect or lethal, thus reach the object of Control pests.

Chitin degrading and metabolism are distinctive biological phenomenas in insect constant pitch main drive object, and owing to not containing chitin in the mankind and other higher animal bodies, therefore insect chitin degeneration system has been acknowledged as the target of novel pesticide effect.Chitinase plays keying action in the degradation process of insect chitin, adopts RNA perturbation technique, carries out the application of chitinase dsRNA in pest control significant.

Summary of the invention

Object of the present invention provides chitinase 5 gene and its lethal gene fragment of a kind of insect gypsymoth, the application of dsRNA and dsRNA synthesized by this gene fragment in lethal insect gypsymoth.

The present invention adopts following technical scheme to realize:

Chitinase 5 gene for gypsymoth, its nucleotide sequence is as shown in SEQ ID NO:1; Being that after the degenerated primer by designing insect chitinase, pcr amplification obtains its conserved regions, obtaining total length, called after LdCHT5 by RACE-PCR amplification.Its total length is 1895bp, wherein open reading-frame (ORF) 1737bp, and 578 amino acid of encoding, the length of 5 ＇-UTR and 3 ＇-UTR is respectively 42bp and 116bp.

The lethal fragment of chitinase 5 gene of above-mentioned gypsymoth, its nucleotide sequence, as shown in SEQ ID NO:2, is design upstream primer SEQ ID NO:3 and downstream primer SEQ ID NO:4 according to SEQ ID NO:1, obtains through pcr amplification.

Utilize related kit further, according to the chitinase 5 gene lethal fragment synthesis dsRNA of gypsymoth.

The application of dsRNA in lethal insect: injection dsRNA is to the prepupal period larva body cavity of gypsymoth, and result shows: in insect body, dsRNA can the mrna expression of specific reticent chitinase 5 gene, causes gypsymoth because of difficult death of casting off a skin.

The present invention is reasonable in design; obtain the lethal gene fragment of chitinase 5 gene of gypsymoth; and the dsRNA to be synthesized by this gene fragment; provide a kind of biological control method for insect gypsymoth; be expected to a large amount of uses reducing agricultural chemicals, protection of the environment, reduce production cost; improve economic return, there is market application foreground.

Accompanying drawing explanation

The impact that Fig. 1 grows on it after representing prepupal period Lymantria dispar larvae injection dsRNA.The experimental group (in figure A, B) of the injection dsRNA difficulty that occurs casting off a skin cannot turn to the phenotype of adult and death, and the control group (in figure C, D) of injection DEPC process water is grown normally.

Embodiment

embodiment 1

The preparation method of gypsymoth chitinase 5 full length gene is as follows:

(1), the design of PCR primer

According to the amino acid consensus sequence of known insect chitinase 5, devise following degenerated primers as follows:

Upstream primer: CHT5F 5'-GAYTGGGAGTACCCHGG-3',

Downstream primer: CHT5R 5'-TCCATRTCRATRGCCCA-3';

(2), the design of gypsymoth chitinase 5 gene RACE primer

As follows based on fragment design primer obtained above:

5'RACE upstream primer: 5'-CGCTACGTAACGGCATGACAGTG (C) ₁₆-3'

5'RACE downstream primer: 5'-GGTGTACGGAGCAGGATCGCCACC-3'

3'RACE upstream primer: 5'-GCTGGATGCTATCCACGTGATGTCG-3'

3'RACE downstream primer: 5'-CGCTACGTAACGGCATGACAGTG (T) ₁₈-3'

All primers are by the synthesis of the English Weihe River, Shanghai prompt base biological company limited.

(3) acquisition of gypsymoth total serum IgE

Choose gypsymoth 4 instar larvae of the same size, 3 first groups, be frozen in liquid nitrogen, total serum IgE to be extracted, extract the Trizol test kit of concrete operation step according to TaKaRa of total serum IgE.

(4), the synthesis of gypsymoth cDNA first chain

The operation steps of the synthesis of cDNA first chain is with reference to PrimeScriptTM RT reagent Kit with gDNA Eraser test kit.

(5), pcr amplification

With above-mentioned cDNA first chain for template, carry out pcr amplification according to Taq enzyme (TIANGEN) specification sheets, increased fragment is cloned further, checked order.

(6) sequential analysis

Utilize ExPASy to analyze the sequence obtained, and in NCBI website, use BLAST function to carry out sequence homology comparison, determine that obtained gene fragment is gypsymoth chitinase 5 gene.

Chitinase 5 gene of above-mentioned gypsymoth, its nucleotide sequence is as shown in SEQ ID NO:1.

embodiment 2

The preparation method of gypsymoth chitinase 5 gene lethal fragment is as follows:

According to the nucleotide sequence of gypsymoth chitinase 5 gene that embodiment 1 obtains, adopt the specific primer of primerpremier5.0 software design, upstream primer sequence is SEQ ID NO:3, downstream primer sequence is SEQ ID NO:4, and all primers are by the synthesis of the English Weihe River, Shanghai prompt base biological company limited; Wherein, upstream primer and downstream primer need the nucleotide sequence of gypsymoth chitinase 5 gene obtained for embodiment 1 to design, applicant, after the conscientiously screening to multiple upstream and downstream design of primers and multiple gene fragment, just finally determines the lethal gene fragment with practical application effect.

To successfully connect the restructuring pEASY-T1 plasmid of gypsymoth chitinase 5 gene that embodiment 1 obtains as template, pcr amplification obtains gypsymoth chitinase 5 gene lethal fragment, uses MicroElute Cycle Pure Kit(OMEGA) test kit carries out purifying.

The chitinase 5 gene lethal fragment of above-mentioned gypsymoth, its nucleotide sequence is as shown in SEQ ID NO:2.

embodiment 3

The acquisition of the dsRNA of gypsymoth chitinase 5 gene lethal fragment

With the chitinase 5 gene lethal fragment that embodiment 2 obtains, according to T7RiboMAX Express RNAi System(Promega) test kit specification sheets, in-vitro transcription synthesis dsRNA.The dsRNA the obtained agarose gel electrophoresis of 1.5% detects its unicity, with microplate reader (Thermo Scientific Multiskan GO, Thermo Fisher Scientific, USA) detect its concentration, and be concentrated into final concentration 1 μ g/ μ l be saved to-80 DEG C for subsequent use.

embodiment 4

The application of dsRNA in lethal insect gypsymoth is as follows:

The lethal gypsymoth experiment of the dsRNA of gypsymoth chitinase 5 gene lethal fragment

(1) the dsRNA injection of gypsymoth chitinase 5 gene fragment

Choose prepupal period larva for injecting the dsRNA of above-mentioned synthesis.The microsyringe of 5ul specification is used for injection, can not be firmly excessive during injection, and flank portion, as injection point, notes avoiding valve.The amount of injection dsRNA is 5 μ g, and arranges control group, experimental group and each 20 of the control group of the water of the DEPC process of injection equivalent.After injection, larva to be positioned in biochemical cultivation case with carrying out purchased from Chinese forest-science academy forest ecological environment and Protective strategy raising (illumination: interlunation=16h:8h, temperature 26 ± 1 DEG C, humidity 70%).

(2) observation of gypsymoth phenotype after dsRNA, is injected

Continue to raise after larva injection, and observe in time.The experimental group of injection dsRNA gypsymoth has part larva to be adult and dead because sloughing off nymphosis.

(3) observation of gypsymoth death condition after dsRNA, is injected

The gypsymoth experimental group mortality ratio of injection dsRNA is 25%, and this is compared with the control group (mortality ratio is 1.67%) of injection DEPC process, and lethal effect is obvious.

specification sheets Nucleotide and aminoacid sequence table

SEQUENCE LISTING

<110> Institutes Of Technology Of Taiyuan

<120> is based on gypsymoth chitinase 5 gene of gene silent technology and dsRNA

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 1895

<212> DNA

<213> gypsymoth (Lymantria dispar)

<400> 1

ctcggatcca ctagtaacgg ccgccagtgt gctggaattg cccttagagt gatactagcg 60

acgttggccg tcctggcggt cgcaacgact gcaattgaag cggacagcaa agcgcgcata 120

gtatgctact tcagcaactg ggcggtgtac agacccggcg tgggtcgcta cggcatcgag 180

gacatccccg tggacctctg cacgcacatc atctactcct tcatcggcgt cactgagaag 240

agcaatgagg tcctcatcat tgatcctgag ctggacgtag acaagaacgg ttttcggaac 300

ttcacagctc ttcgaaagtc gcaccctaac gtgaagttca cggtggctgt gggtggctgg 360

gccgaggggg gctctaagta ctcgcacatg gttgcgcaga aacaaaccag aatggctttc 420

gttaggagcg ttgttgattt cttgaagaaa tacgactttg atggtttgga cttggattgg 480

gagtaccctg gtgcggctga ccgtggtggc tccttctcag acaaggatcg gttcctcttc 540

ctcgtccagg agttgaggag agccttcatc agggagaaga ggggatggga actgactgct 600

gctgtgcctc tcgctaactt cagactgatg gagggatacc acgtacctga tctttgccaa 660

gagctggatg ctatccacgt gatgtcgtac gatttgcgcg gcaactgggc tggctttgca 720

gatgtgcact cacctttata taagcgtcct catgatcagt gggcctatga gaagttgaat 780

gtgaatgatg gcctagcgct ctgggaagag aagggttgtc ccagcaacaa gctcgtcgtc 840

ggcattccgt tctacggtcg ctccttcaca ctctccgctg gtaacaacaa ttacggactt 900

ggtacctaca tcaacaagga ggctggaggt ggcgatcctg ctccgtacac caatgctact 960

gggttctggg cttattatga gatctgtacc gaagtcgaca aagaaggctc aggctggacc 1020

aagaaatggg atgacgccgg caaatgtccc tacgcctaca agggcaccca gtgggtcggc 1080

tacgaggacc ctcgcagtgt cgagatcaag atgaactgga tcaaggagaa gggctacctg 1140

ggcgccatga cctgggccat cgatatggac gacttcaagg gactttgtgg tgatgagaac 1200

cctctgatca agctcctgca taagcatatg agcacttaca ctgtcccacc acctcgctct 1260

ggaaatacta ctcctacgcc tgaatgggcg cggcccccgt ccacaacgtc cgacccggct 1320

gagggggaga tcgtcactac tgtcaagtcc acgactgcga agccagctac gacgaaacca 1380

agcacagcca agccaacgac ggccaagcct acgacggcca agccaacaac ggccaagcct 1440

acgacgacca aagcacccca agccgtaaca atcccagatg atgagaatga catcgctgtg 1500

agacctgaac ctccgaaaaa acctgtaact ccagaaaccc ctgtggtacc tgaagttcct 1560

gaatctgctg aaacaccaac tgaaaatgaa atagataacc acgacgtttg caattctgaa 1620

gaggattacg tgcctgacaa gaaaaagtgc gataagtact ggcgatgcgt caacggacga 1680

ggaatgctgt tcacatgcca accaggaact gtgttcaacg tgaagctgaa tgtctgcgac 1740

tggccagaca acgccgaccg tagtgactgc gagccctaaa ctgaagggca attctgcaga 1800

tatccatcac actggcggcc gctcgagcat gcatctagag ggcccaattc gccctatagt 1860

gagtcgtatt acaattcact ggccgtcgtt ttaca 1895

<210> 2

<211> 472

<212> DNA

<213> gypsymoth (Lymantria dispar)

<400> 2

taagtactcg cacatggttg cgcagaaaca aaccagaatg gctttcgtta ggagcgttgt 60

tgatttcttg aagaaatacg actttgatgg tttggacttg gattgggagt accctggtgc 120

ggctgaccgt ggtggctcct tctcagacaa ggatcggttc ctcttcctcg tccaggagtt 180

gaggagagcc ttcatcaggg agaagagggg atgggaactg actgctgctg tgcctctcgc 240

taacttcaga ctgatggagg gataccacgt acctgatctt tgccaagagc tggatgctat 300

ccacgtgatg tcgtacgatt tgcgcggcaa ctgggctggc tttgcagatg tgcactcacc 360

tttatataag cgtcctcatg atcagtgggc ctatgagaag ttgaatgtga atgatggcct 420

agcgctctgg gaagagaagg gttgtcccag caacaagctc gtcgtcggca tt 472

<210> 3

<211> 41

<212> DNA

<213> gypsymoth (Lymantria dispar)

<400> 3

taatacgact cactataggg taagtactcg cacatggttg c 41

<210> 4

<211> 44

<212> DNA

<213> gypsymoth (Lymantria dispar)

<400> 4

taatacgact cactataggg gaatgccgac gacgagcttg ttgc 44

Claims

1. chitinase 5 gene for gypsymoth, its nucleotide sequence is as shown in SEQ ID NO:1.

2. a chitinase 5 gene lethal fragment for gypsymoth, its nucleotide sequence is as shown in SEQ ID NO:2.

3. a preparation method for chitinase 5 gene of gypsymoth according to claim 1, is characterized in that: comprise the steps:

(1), the design of PCR primer

Upstream primer: CHT5F 5'-GAYTGGGAGTACCCHGG-3',

Downstream primer: CHT5R 5'-TCCATRTCRATRGCCCA-3';

(2), the design of gypsymoth chitinase 5 gene RACE primer

As follows based on fragment design primer obtained above:

5'RACE upstream primer: 5'-CGCTACGTAACGGCATGACAGTG (C) ₁₆-3'

5'RACE downstream primer: 5'-GGTGTACGGAGCAGGATCGCCACC-3'

3'RACE upstream primer: 5'-GCTGGATGCTATCCACGTGATGTCG-3'

3'RACE downstream primer: 5'-CGCTACGTAACGGCATGACAGTG (T) ₁₈-3'

(3), the acquisition of gypsymoth total serum IgE

Extract the Trizol test kit of concrete operation step according to TaKaRa of total serum IgE;

(4), the synthesis of gypsymoth cDNA first chain

The operation steps of the synthesis of cDNA first chain is with reference to PrimeScriptTM RT reagent Kit with gDNA Eraser test kit;

(5), pcr amplification

With above-mentioned cDNA first chain for template, carry out pcr amplification according to Taq enzyme specification sheets, increased fragment is cloned further, checked order;

(6), sequential analysis

Utilize ExPASy to analyze the sequence obtained, and in NCBI website, use BLAST function to carry out sequence homology comparison, determine that obtained gene fragment is gypsymoth chitinase 5 gene, its nucleotide sequence is as shown in SEQ ID NO:1.

4. a preparation method for the chitinase 5 gene lethal fragment of gypsymoth according to claim 2, is characterized in that: comprise the steps:

According to the nucleotide sequence of gypsymoth chitinase 5 gene that claim 3 obtains, design upstream primer sequence is SEQ ID NO:3, and downstream primer sequence is SEQ ID NO:4; To successfully connect the restructuring pEASY-T1 plasmid of gypsymoth chitinase 5 gene that claim 3 obtains as template, pcr amplification obtains chitinase 5 gene lethal fragment, its nucleotide sequence, as shown in SEQ ID NO:2, uses MicroElute Cycle Pure Kit test kit to carry out purifying.

5. the dsRNA of an insect chitinase 5 gene lethal fragment synthesis according to claim 2.

6. the application of dsRNA in lethal insect gypsymoth utilizing gypsymoth chitinase 5 gene lethal fragment to synthesize according to claim 5.