CN102586259A - Chilo venosatus walker ecdysis regulation transcription factor cDNA and cloning method and recombinant application thereof - Google Patents
Chilo venosatus walker ecdysis regulation transcription factor cDNA and cloning method and recombinant application thereof Download PDFInfo
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Abstract
The invention belongs to the field of biological gene engineering. A chilo venosatus walker ecdysis regulation transcription factor CsHR3 contains sequences shown by SEQ ID NO.1 and SEQ ID NO.2. The cloning method provided by the invention comprises the following steps of: extracting total RNA (ribonucleic acid) from chilo venosatus walker larvas in prepupal period, and carrying out reverse transcription to synthesize cDNA; designing a degenerate primer according to conserved sequences of different lepidopterous insect ecdysis regulation transcription factors, carrying out PCR (polymerase chain reaction) amplification on the intermediate segment of the chilo venosatus walker CsHR3 gene, designing a specific primer by adopting 3'-RACE (rapid-amplification of cDNA ends) technology, and then obtaining the 1266bp segment of the CsHR3 gene successfully. According to the invention, dsRNA is expressed in RNaseIII defective escherichia coli HT 115 by adopting a bacterium mediate RNAi technology to obtain dsRNA with biological activity, and the chilo venosatus walker ecdysis regulation transcription factor CsHR3 gene is silenced by feeding the chilo venosatus walker larvas, thus chilo venosatus walker can not complete ecdysis or normal growth and development process of the chilo venosatus walker is destroyed and biological prevention and control on the chilo venosatus walker is realized; or the chilo venosatus walker ecdysis regulation transcription factor is converted into a crop, and the crop with insect resistance is obtained.
Description
Technical field
The invention belongs to clone, expression technology in the technical field of biological genetic engineering, be specifically related to article sugarcane borer key gene---the molt-regulating transcription factor that grows
CsHR3Gene clone and bacteria mediated RNAi technological expression
CsHR3DsRNA comes reticent article sugarcane borer molt-regulating transcription factor
CsHR3Gene makes article sugarcane borer can not cast off a skin or destroy its normal growth growth course.
Background technology
Along with the sharp increase of world population, how improving crop yield is an extremely urgent problem.According to statistics, the grain loss that causes because of insect pest accounts for 20% of whole grain harvest.Therefore, how effectively controlling insect pest is an important factor that improves crop yield.Though a large amount of chemical pesticides that use can suppress insect pest, cause the resistance of insect easily, and very big to environmental disruption, are unfavorable for human Sustainable development.Along with the fast development of biotechnology, through the transgenic method modification of plants, the pest-resistant harmful ability that improves plant self has become the main flow of crop breeding.Last century the nineties, scientists is utilized the transgenic means to make farm crop express Bt albumen and to improve farm crop the biological controls of Agricultural pests is widely used in the whole world.Yet nearest discovers: lost one type of ability in the midgut epithelial cell of some insect and made insect produce resistance to Bt albumen with the protein bound acceptor of Bt.Recently, the RNAi technology has developed into a kind of novel method of controlling insect pest, improving crop quality on agricultural.
It is a kind of newfound gene regulating mechanism that RNA disturbs (RNAi).People suppress to have found that with the bacteriostatic action phenomenon of fungi RNA disturbs this ancient organism gene regulating mechanism from the genetic expression of plant altogether.As far back as 1998, people such as Fire found that double-stranded RNA (dsRNA) can cause target gene expression to suppress.Soon, RNA disturbs and has developed into a kind of mature technology, is widely used in the functional study of target gene.In insect, disturb (RNAi) to can be used to study the function of insect genes through dsRNA inductive RNA.Especially in recent years, the method for bacteria mediated or plant mediate rna i expression dsRNA has been induced the silence of target gene in various insects.Yet, also have a lot of problem in science need go to inquire into and research, can induce the appearance of the dead phenotype of insect as mediate the non-midgut gene of reticent insect through RNAi; Whether the RNAi mediation is expressed dsRNA amount and the dead phenotype of insect or is grown to hinder and be proportionate; Cycle and insect that RNAi mediates reticent target gene give birth to the dependency of surveying between the phenotype; Select which target gene cycle in biological control of insect pests short, give birth to survey effective, more efficient etc., these a series of problems all need test explore and research in checking.
The RNAi technology is studied functional gene in insect method mainly is an injection, generally imports embryo or the body cavity of insect through disturbing predry target gene dsRNA or siRNA with microsyringe, thereby realizes the inhibition to target gene mRNA expression.Though injection RNAi is obtaining huge achievement aspect the insect functional gene; But also there are following problems: at first; Injection RNAi very easily causes the inaccurate and insect death of data because of misoperation to the experiment operator technical requirements is very high, difficulty is big, intensity is big.Secondly, injection RNAi prepares dsRNA or siRNA all need accomplish by the external test kit of costliness, has increased experimental cost, and the amount of prepared dsRNA or siRNA but seldom.At last, injection RNAi often need carry out injection operation to single insect one by one, for its widespread use field biological control of insect pests has increased difficulty.To study insect functional gene needs easily in order satisfying,, to need better bacteria mediated or plant mediate rna i technology in insect functional gene Application Research in particular for exploring new biological control of insect pests method.
Molt-regulating transcription factor is one to start the early gene bunch expression of casting off a skin roughly the same the time, the regulatory factor of the late gene bunch expression that suppresses again to cast off a skin, performance important function of gene expression regulation effect in the insect molting process.Existing discovering can obtain from various insects
HR3The key gene that grows of family is like bollworm
HaHR3, maduca sexta
MHR3, fruit bat
DHR3Deng.Josefa etc. are cloned into molt-regulating transcription factor from Groton bug
BgHR3, and through the inhibition of RNAi technology
BgHR3Genetic expression, the result finds that larva can not take off old epidermis, and apolysis takes place, and shows
BgHR3Gene is participated in the Groton bug larval moult directly.Insect then can't become adult if can not normally cast off a skin, thereby forms the individuality that food ingestion reduces, do not have vitality even dead phenotype.Molt-regulating transcription factor is a key factor in the process of casting off a skin, and also receives the strictness regulation and control of body genes involved simultaneously, when expression level reduces or improves unusually, all can influence normally casting off a skin of insect.Therefore, break the balance of the gene of casting off a skin, interference insect is casted off a skin, and destroys its normal growth and development process, can reach the purpose of kill pests and Pest Control population.
Molt-regulating transcription factors such as fruit bat, maduca sexta, Groton bug gene clone and Study on mechanism have been carried out abroad.Fruit bat wherein
DHR3Study comparatively deeply, this gene is casted off a skin and metamorphosis plays a crucial role in growing fruit bat.Still have nothing to do at present in the GenBank DB in article sugarcane borer the cast off a skin record and the research report of regulatory factor gene.
Summary of the invention
The object of the invention is to provide a kind of article sugarcane borer molt-regulating transcription factor cDNA that from the article sugarcane borer larva, extracts; And provide the cloning process of article sugarcane borer molt-regulating transcription factor cDNA and reorganization to use, a kind of novel method of bacteria mediated RNAi technology prevention article sugarcane borer also is provided.
For realizing above-mentioned purpose, the present invention takes following technical scheme:
The article sugarcane borer molt-regulating transcription factor is cloned in the present invention from article sugarcane borer
CsHR3The cDNA of gene, it has the sequence shown in SEQ ID NO.1 and the SEQ ID NO.2:
(1) information of SEQ ID NO.1
(a) sequence signature:
* length: 1266 base pairs
* type: nucleic acid
* chain: two strands
* topological framework: linearity
(b) molecule type: cDNA
(c) suppose: not
(d) antisense: not
(e) initial source: article sugarcane borer (
Chilo sacchariphagus)
(f) sequence description: SEQ ID NO. 1
(2) information of SEQ ID NO.2
(a) sequence signature
* length: 421 amino acid
* type: amino acid
* chain: strand
* topological framework: linearity
(b) molecule type: protein
(c) sequence description: SEQ ID NO. 2.
The cloning process of article sugarcane borer molt-regulating transcription factor cDNA of the present invention, carry out through following steps:
(1) from article sugarcane borer larva prepupal period, extracts total RNA, utilize ThermoScript II to synthesize cDNA;
(2) conserved sequence according to different lepidopterous insects molt-regulating transcription factors designs degenerated primer:
CsHR3-middle?fragment-P1：5'-ACAGWGGTGAACTACCAGTG?-3'
CsHR3-middle?fragment-P2：5'-GACCATGRAATTGGTCGCT-3'
(3) with cDNA be template, the pcr amplification article sugarcane borer
CsHR3The intermediate segment of gene;
(4) purification step (3) gained PCR product, sepharose reclaim the purpose fragment, and get purified product and be connected on the pMD19-T simple carrier, transformed into escherichia coli competent cell Top10 then, 37 ℃ of dull and stereotyped cultivations, blue hickie screening contains
CsHR3Positive colony of intermediate segment;
(5) basis
CsHR3The sequencing result of intermediate segment, design 3 ' RACE Auele Specific Primer is a template with article sugarcane borer cDNA, pcr amplification
CsHR33 ' fragment sequence of gene;
CsHR3-3'?RACE-GSP1：5'?-?CGGGTCAACAGGAATAGATGTCA?-3'
CsHR3-3'?RACE-GSP2：5'-?CAGCGCCTGACTCAGTGTATGAC-3'
(6) purification step (5) gained PCR product, sepharose reclaim the purpose fragment, and get purified product and be connected on the pMD19-T carrier, transformed into escherichia coli competent cell Top10 then, 37 ℃ of dull and stereotyped cultivations, blue hickie screening obtains
CsHR3Positive colony of gene 3 ' end;
(7) positive colony with step (4) and (6) gained carries out sequencing, will measure institute's calling sequence then through information biology software DNAman splicing, obtains
CsHR3Gene 1266 bp sequences.
Concrete, said step (1) is: from the article sugarcane borer prepupal period larva that gather in the field, adopt single stage method to extract total RNA.Utilize total RNA reverse transcription to synthesize cDNA then, be specially: get total RNA 5 microlitres (50 nanogram), Random primer 1 microlitre (the every microlitre of 0.5 microgram), substrate dNTPs 2 microlitres (2.5 mmole), use RNase-free H
2O adds to 10 microlitres, mixing, and 65 ℃ of insulation 5min are put rapidly to cooled on ice 3min; In said mixture, add 5 * Primer Script Buffer, 4 microlitres, RNA enzyme inhibitors 0.5 microlitre (the every microlitre of 40 units), AMV ThermoScript II 0.5 microlitre (the every microlitre of 200 units) then, and use RNase-free H
2O adds to 20 microlitres, 42 ℃ of reaction 60min, and 70 ℃ of fire extinguishing 15min, 4 ℃ of coolings ,-20 ℃ of preservations are subsequent use.
In step (3) and (5) the used reaction reagent of pcr amplification form and reaction conditions following: at first together with following reagent mix,
Ex-Taq archaeal dna polymerase 0.5 μ l
10 * Taq DNA polymerase buffer, 5 μ l
Forward primer (10 μ M) 2 μ l
Reverse primer (10 μ M) 2 μ l
ddH
2O 35.5μl
TV 50 μ l
Reaction conditions is: at first 94 ℃ of preparatory sex change are 3 minutes; Circulate as follows then: 94 ℃ 30 seconds, 50 ℃ 30 seconds, 72 ℃ 2 minutes, carry out altogether 32 the circulation; Last 72 ℃ were extended 10 minutes, and 16 ℃ of preservations are subsequent use.
The reorganization of article sugarcane borer molt-regulating transcription factor cDNA according to the invention is used; Adopt bacteria mediated RNAi technology in RNase III defective escherichia coli HT115, to express dsRNA; Obtain the dsRNA of biologically active; Through the article sugarcane borer larva of feeding, come reticent article sugarcane borer molt-regulating transcription factor
CsHR3Gene makes article sugarcane borer can not cast off a skin or destroy its normal growth growth course, thereby is used for the biological control of article sugarcane borer insect.Perhaps change the article sugarcane borer molt-regulating transcription factor over to crop, obtain to have the crop of insect resistance capacity, be specially: will through gene engineering method
CsHR3Gene constructed one-tenth ihpRNAi genetic transformation carrier imports the sugarcane genome, studies the influence of molt-regulating transcription factor functional defect to the biological phenotype of article sugarcane borer, thereby is used for the control of borer pest article sugarcane borer.Because article sugarcane borer belongs to the lepidopteran Pyralidae, so the present invention is to other lepidoptera pest, and especially the biological control of Pyralidae insect has important value.
A kind of method of bacteria mediated RNAi control article sugarcane borer: select the article sugarcane borer molt-regulating transcription factor
CsHR3Gene is selected it as target
CsHR3Two dsRNA interference fragments that contain non-LBD functional domain and LBD functional domain on the gene, RNAi recombinant vectors L4440-CsHR3-I1, the L4440-CsHR3-I2 of structure bacteria mediated select green fluorescence protein gene simultaneously for use
EGFPAs negative control, clear water is as negative contrast.Through the heat shock method above-mentioned carrier is imported
Ecoli. among the HT115 (RNase III defective escherichia coli), add 0.8mM IPTG and carry out abduction delivering.Extract the feeding experiment that bacterial expression dsRNA carries out the article sugarcane borer larva, detected for 3 ages and after 5 ages, article sugarcane borer was fed 7 days phenotype change and body in
CsHR3Gene mRNA reduces level.
The article sugarcane borer cascade reaction that process is made up of the series of genes of hormone-mediated regulation and control of casting off a skin, molt-regulating transcription factor
CsHR3Be the direct inductive early gene of moulting hormone expression product, after this gene is expressed when being activated again with its target DNA on distinguished sequence combines, participate in the late gene that moulting hormone causes directly and regulate and control, be the important key factor in the cascade reaction of casting off a skin.If molt-regulating transcription factor is expressed and is suppressed, then insect can not be accomplished the process of casting off a skin normally.Therefore, the present invention is by the RNAi of bacteria mediated technology, and the dsRNA through the bacterial expression target gene comes the reticent article sugarcane borer key gene that grows
CsHR3MRNA expresses, and its normal growth is grown be suppressed, and interference insect is normally casted off a skin, and makes its larva can not take off old epidermis, breaks the balance of the gene of casting off a skin, thereby reaches the purpose of article sugarcane borer biological control of insect pests.This method is simple to operate, designs special RNAi target fragment to the key gene that grows, and the research basis is good, perspective height, and cost is low, and biological control of insect pests provides new method in the sugarcane agriculture prodn in order to explore.
Description of drawings
Fig. 1 is clone and the evaluation of three target gene CsHR3-I1, CsHR3-I2 and EGFP-I0; Wherein M is DL2000; No. 1 swimming lane does
CsHR3 Gene interference fragment 1 purpose band; No. 2 swimming lane does
CsHR3 Gene interference fragment 2 purpose bands; No. 3 negative contrasts of swimming lane
EGFPGene purpose band;
Fig. 2 is recombinant expression vector pL4440-CsHR3-I1, pL44440-CsHR3-I2, pL4440-EGFP-I0 structural representation;
Fig. 3 expresses in the bacterium
CsHR3With
EGFPThe evaluation figure of gene dsRNA; Wherein arrow is depicted as and expresses target gene dsRNA, and presents the different positions band and expect that size is consistent owing to target gene varies in size; (a) figure handles without the DNaseI enzyme, and (b) figure handles through the DNaseI enzyme; N representes not add IPTG and handles, and I representes to add IPTG and handles;
Fig. 4 surveys design sketch for article sugarcane borer 3 instar larvaes target gene dsRNA the giving birth to after the 7th day of feeding, and (a) and (b) are represented two repeating groups among the figure;
Fig. 5 surveys design sketch for article sugarcane borer 5 instar larvaes target gene dsRNA the giving birth to after the 7th day of feeding, and (a) and (b) are represented two repeating groups among the figure;
Fig. 6 is feeding the changes of weight situation of target gene dsRNA after the 7th day for article sugarcane borer 3 instar larvaes, and (a) and (b) are represented two repeating groups among the figure;
Fig. 7 article sugarcane borer behind the target gene dsRNA of feeding for qRT-PCR detects
CsHR3The gene mRNA changing conditions, (a) and (b) are represented two repeating groups among the figure.
Embodiment
Below through preferred embodiment the present invention is done further explain, but protection scope of the present invention is not limited thereto.
Article sugarcane borer (
Chilo sacchariphagus) key gene grows
CsHR3The clone.
1, extracts total RNA: adopt one step of Invitrogen company's T rizol method to extract total RNA of article sugarcane borer prepupal period larva.
2, synthetic cDNA: carry out with reference to the Dalian precious biotechnology cDNA of ltd test kit explanation, be specially: get total RNA 5 μ l, Random primer 1 μ l, substrate dNTPs 2 μ l, use RNase-free H
2O adds to 10 μ l, mixing, and 65 ℃ of insulation 5min are put rapidly to cooled on ice 3min; In said mixture, add 5 * Primer Script Buffer, 4 μ l, RNA enzyme inhibitors 0.5 μ l, AMV ThermoScript II 0.5 μ l then, and use RNase-free H
2O adds to 20 μ l, 42 ℃ of reaction 60min, and 70 ℃ of fire extinguishing 15min, 4 ℃ of coolings ,-20 ℃ of preservations are subsequent use.
3, pcr amplification article sugarcane borer
CsHR3The intermediate segment of gene: according to the conserved sequence design degenerated primer (primer sequence is following) of different lepidopterous insects molt-regulating transcription factors; With cDNA is template, the pcr amplification article sugarcane borer
CsHR3The intermediate segment of gene;
CsHR3-middle?fragment-P1：5'-ACAGWGGTGAACTACCAGTG?-3'
CsHR3-middle?fragment-P2：5'-GACCATGRAATTGGTCGCT-3'
The used reaction reagent of chain polymerization enzyme reaction (pcr amplification) form and reaction conditions following: at first together with following reagent mix,
Ex-Taq archaeal dna polymerase 0.5 μ l
10 * Taq DNA polymerase buffer, 5 μ l
Forward primer (10 μ M) 2 μ l
Reverse primer (10 μ M) 2 μ l
ddH
2O 35.5μl
TV 50 μ l
Reaction conditions is: at first 94 ℃ of preparatory sex change are 3 minutes, circulate as follows then: 94 ℃ 30 seconds, 50 ℃ 30 seconds, 72 ℃ 2 minutes, carry out 32 circulations altogether, last 72 ℃ were extended 10 minutes, 16 ℃ of preservations are subsequent use.
4, PCR product purification: utilize sepharose to reclaim test kit (available from sky, Beijing root biotechnology company) to the recovery of PCR product, purifying, the concrete operations step is undertaken by the test kit specification sheets.
5, the PCR product connects, transforms and identifies: get the above-mentioned PCR purified product of 5 μ l; Be connected in pMD19-T simple carrier (the precious biotechnology in Dalian ltd product); Reaction system is transformed into competent escherichia coli cell Top10 then shown in pMD19-T simple support agent box, 37 ℃ of dull and stereotyped cultivations; Indigo plant hickie screening (picking white reorganization bacterium colony is cultivated, and universal primer M13-47 and RV-M carry out bacterium liquid PCR evaluation on the use cloning vector pMD19-T simple) contains
CsHR3Positive colony of intermediate segment.
6, identify the sequencing result of positive colony according to the PCR of step 5, design 3 ' RACE Auele Specific Primer (primer sequence is following) is a template with article sugarcane borer cDNA, pcr amplification
CsHR33 ' fragment sequence of gene, the used reaction reagent of pcr amplification form and reaction conditions the same.
CsHR3-3'?RACE-GSP1：5'-?CGGGTCAACAGGAATAGATGTCA?-3'
CsHR3-3'?RACE-GSP2：5'-?CAGCGCCTGACTCAGTGTATGAC-3'
7, Purification step 6 gained PCR products, sepharose reclaim the purpose fragment, and get purified product and be connected in pMD19-T simple carrier, transformed into escherichia coli competent cell Top10 then, 37 ℃ of dull and stereotyped cultivations, blue hickie screening obtains
CsHR3Positive colony of gene 3 ' end.
8, positive colony with step 5 and 7 gained send the handsome Bioisystech Co., Ltd in Invitrogen-Shanghai to carry out sequencing, will measure institute's calling sequence then through information biology software DNAman splicing, obtains
CsHR3Gene 1266 bp sequences.Institute's calling sequence and GenBank database sequence are compared.
The gained gene has the sequence shown in SEQ ID NO.1 and the SEQ ID NO.2.
Make up RNAi recombinant vectors L4440-CsHR3-I1, the L4440-CsHR3-I2 of bacteria mediated, select green fluorescence protein gene simultaneously for use
EGFPAs negative control, clear water is as negative contrast.Bacterial expression dsRNA feeding experiment is divided into 4 treatment group, 3 ages that the growth of feeding respectively is consistent and 5 age the article sugarcane borer larva, repeats 3 times and tests.Concrete operations are following:
1,
CsHR3The target interference base is because of segmental selection
Consider molt-regulating transcription factor
HR3Be the gene expression regulation factor that plays a significant role in the insect molting process, so present embodiment is selected the article sugarcane borer molt-regulating transcription factor
CsHR3Gene is selected it as target
CsHR3Two dsRNA interference fragments that contain non-LBD functional domain and LBD functional domain on the gene (are interference fragment 1 and 2; Its nucleotide sequence is shown in SEQ ID NO.3 and SEQ ID NO.4); Two dsRNA fragment gene length are respectively 346 bp and 536bp, and both sequences all are derived from SEQ ID NO.1.
Get an article sugarcane borer prepupal period larva, press Invitrogen company's T rizol method (test kit explanation) step and extract total RNA.With reference to the synthetic cDNA of the Dalian precious biotechnology cDNA of ltd test kit explanation reverse transcription, be specially: get total RNA 5 μ l, Random primer 1 μ l, substrate dNTPs 2 μ l, use RNase-free H again
2O adds to 10 μ l, mixing, and 65 ℃ of insulation 5min are put rapidly to cooled on ice 3min; In said mixture, add 5 * PrimerScript Buffer, 4 μ l, RNA enzyme inhibitors 0.5 μ l, AMV ThermoScript II 0.5 μ l then, and use RNase-free H
2O adds to 20 μ l, 42 ℃ of reaction 60min, and 70 ℃ of fire extinguishing 15min, 4 ℃ of coolings ,-20 ℃ of preservations are subsequent use.
With the above-mentioned article sugarcane borer cDNA that obtains is template, to be directed against molt-regulating transcription factor
CsHR3Two pairs of special primers of gene mRNA design carry out pcr amplification, obtain respectively
CsHR3Two interference fragment genes of gene (be interference fragment 1 and 2, its nucleotide sequence is shown in SEQ ID NO.3 and SEQ ID NO.4); Simultaneously with green fluorescence protein gene
EGFPAs negative control, so that check dsRNA effects of jamming.More than three pairs of primer sequences as follows:
Amplification
CsHR3The primer of gene interference fragment 1 (wherein
CTCGAGBe restriction enzyme
XhoThe I site,
GGTACCBe restriction enzyme
KpnThe I site):
L4440-CsHR3-I1-P1:?5'-cc
CTCGAG?CAACACTGGACCTACATTGACA?-3'
L4440-CsHR3-I1-P2:?5'-gg
GGTACCTCGGCACAATCTAACCACAT-3'
The primer of amplification CsHR3 gene interference fragment 2:
L4440-CsHR3-I2-P1:?5'-?cc
CTCGAGGTCCTGGTGAAGAGTCTGGCTGAA-3'
L4440-CsHR3-I2-P2:?5'-?gg
GGTACCACAGGCACTGGATCTCGGGGTT-3'
The primer of amplification negative control EGFP gene interference fragment:
L4440-EGFP-I0-P1:?5'-?cc
CTCGAGCAGTGCTTCAGCCGCTACCC-3'
L4440-EGFP-I0-P2:?5'-?gg
GGTACCCTCCAGCAGGACCATGTGAT-3'
PCR reaction system: template cDNA 1 μ l, each 2 μ l of forward and reverse primer (concentration 10 μ M), dNTP 4 μ l, Ex-Taq 1 μ l, 10 * PCR Buffer, 5 μ l, ddH
2O 35 μ l, PCR reaction TV is 50 μ l, more than used dNTP, Ex-Taq, the 10 * PCR Buffer of reaction is Dalian precious biotechnology ltd Company products.
The PCR reaction conditions: 94 ℃ of 3min, 94 ℃ of 30s, 50 ℃ of 30s, 72 ℃ of 1min, 32 circulations, 72 ℃ are extended 10min.Obtain gene fragment 346 bp, 536bp and the 462bp of 3 expection sizes respectively, detect with 1.5% agarose gel electrophoresis, the result is as shown in Figure 1; Wherein M is DL2000 (2000bp, 1000bp, 750bp; 500bp, 250bp, 100bp) (the precious biotechnology in Dalian ltd product); No. 1 swimming lane does
CsHR3 Gene interference fragment 1 purpose band; No. 2 swimming lane does
CsHR3 Gene interference fragment 2 purpose bands; No. 3 negative contrasts of swimming lane
EGFPGene purpose band.
Respectively above-mentioned 3 purpose bands are cut glue and reclaim, reclaim test kit (sky, Beijing root bio tech ltd product) purifying with sepharose and obtain 3 purified product CsHR3-I1, CsHR3-I2 and EGFP-I0.3 purified products are connected respectively to the last 16 ℃ of reaction 12h of pMD19-T simple carrier (the precious biotechnology in Dalian ltd product), and reaction system is: purpose fragment 4.4 μ l, pMD19-T simple carrier 0.6 μ l, Solution I 5 μ l.Transformed into escherichia coli competent cell Top10 then, blue hickie screening, 3 positive colonies of each gene picking send the order-checking of the handsome Bioisystech Co., Ltd in Invitrogen-Shanghai.
2, prokaryotic expression dsRNA construction of recombinant vector
Plasmid L4440 contains the T7 bidirectional promoter; Because the T7 promotor is the strong promoter of RNA polymerase; Therefore each T7 promotor all can along 5 '-3 ' synthesizing single-stranded RNA (Single strand RNA, ssRNA), then two reverse T7 promotors just can form the ssRNA of two reverse complementals; These two complementary ssRNA combination just can formation double-stranded RNAs (double strand RNA, dsRNA).
Above-mentioned 3 gene fragment CsHR3-I1, CsHR3-I2 and EGFP-I0 and empty carrier L4440 (the precious biotechnology in Dalian ltd product) are used restriction enzyme respectively
XhoI with
KpnThe I enzyme is cut digestion; Reclaim test kit with sepharose then and carry out purifying; 3 fragments after enzyme is cut are connected respectively to the last 16 ℃ of reaction 12h of carrier L4440 with T4 dna ligase (the precious biotechnology in Dalian ltd product), and linked system is: purpose fragment 7 μ l, carrier 1 μ l; Connect buffer (the precious biotechnology in Dalian ltd product) 1 μ l, T4 dna ligase 1 μ l.To connect product and change competent escherichia coli cell Top10 over to through the heat shock method; Blue hickie screening; The picking positive transformant is incubated overnight in ammonia benzyl LB nutrient solution; Collect bacterium liquid, use plasmid extraction kit (Anxgen Company products) purification of Recombinant plasmid pL4440-CsHR3-I1, pL44440-CsHR3-I2 and pL4440-EGFP-I0.
Three recombinant plasmids are used respectively
XhoI with
KpnI is carried out double digestion, verifies whether it accurately is connected on the empty carrier L4440.Enzyme is cut time 2～3h, and enzyme is cut system: plasmid 5 μ l, XhoI 1.5 μ l, KpnI 1.5 μ l, 1 * M Buffer, 1 μ l, ddH
2O 1 μ l.Recombinant plasmid pL4440-CsHR3-I1, pL44440-CsHR3-I2 and pL4440-EGFP-I0 enzyme respectively are cut to 2790bp and 346 bp, 2790bp and 536bp, 2790bp and 462bp.Above-mentioned
XhoI with
KpnThe I restriction enzyme is all available from the precious biotechnology in Dalian ltd product.The structural representation of 3 recombinant expression vector pL4440-CsHR3-I1, pL44440-CsHR3-I2 and pL4440-EGFP-I0 is shown in accompanying drawing 2.
3, target gene dsRNA abduction delivering
With the recombinant plasmid pL4440-CsHR3-I1 that builds, pL44440-CsHR3-I2 and pL4440-EGFP-I0 adopt CaCl
2The heat shock method changes in recipient bacterium intestinal bacteria HT115 (DE3) competent cell, and this Pseudomonas is in the RNaseIII deficient strain.Because HT115 (DE3) has the Tetracycline resistant gene, and recombinant expression vector has the Ampicillin resistant gene,, cultivate 16h for 37 ℃ so select for use Tetracycline and Ampicillin twin antibiotic to screen.1 positive colony of each target gene picking shakes bacterium 12h at LB substratum (Tetracycline+Ampicillin), and 37 ℃, 200rpm.
3 bacterial strains after the activation are joined in the 1L LB substratum (containing 100 μ g/mL Ampicillin, 12.5 μ g/mL Tetracycline) in the 1:100 ratio be cultured to OD600=0.6 (about 2-3h) among 37 ℃ of 200rpm.Add sec.-propyl-β-D-sulfo-galactopyranoside (IPTG) and carry out abduction delivering dsRNA to final concentration 0.8mM.37 ℃ of 200rpm induce 4-8h, get 1mL bacterium liquid respectively, adopt bacterium total RNA extraction reagent box (giving birth to the worker available from Shanghai) to extract total RNA (process for extracting is undertaken by the test kit specification sheets) of bacterium, and the result sees shown in the accompanying drawing 3.Experimental result shows, 3 recombinant vectorss have all successfully induced the dsRNA of target gene to express, and when not adding IPTG and inducing, 3 recombinant vectorss all do not have dsRNA to express, and when DNaseI handled, bacteria total DNA was all digested.
4, the influence that bacterial expression dsRNA grows to article sugarcane borer of feeding
Get the dsRNA bacterium liquid that 1L induces respectively, at ambient temperature, the centrifugal 5min enrichment of 10000rpm thalline adds ddH according to 100:1 ratio (being 10mL)
2The bacterium liquid of O dissolving enrichment, fully mixing.Adopt the ultrasonic disruption appearance to destroy its bacteria cell wall again, its dsRNA is fully discharged.Reaction conditions: amplitude 25%, start 6s, stop 6s, total times 10 min, operation on ice.With this bacterium liquid as the life of the article sugarcane borer larva feeding dsRNA material of measuring and monitoring the growth of standing timber.
Article sugarcane borer larva feeding experiment is divided into four treatment group, is respectively L4440-CsHR3-I1 test group, L4440-CsHR3-I2 test group, L4440-EGFP-I0 negative control group and clear water control group.Article sugarcane borer is given birth to the test worm and is selected third-instar larvae and two growth period types of five-age larva.7 larvas are selected in each processing for use.More than the test repetition is 3 times.
Select 3 consistent ages of growth and 5 age the article sugarcane borer larva, respectively with fresh sweet corn kernel as giving birth to survey feed (adding 10 μ l dsRNA bacterium liquid in each sweet corn kernel approximately), regularly change the feed that contains dsRNA every day.Single head is raised, the raising condition be (25 ± 2 ℃, relative humidity 70%, every day light: secretly be 16h:8h).1-7 behind the feeding dsRNA days, accurate recording with the growth of observing the bar snout moth's larva, cast off a skin, body weight, pupate, emergence and dead phenotype change.
Test-results proves: feed expression colibacillary L4440-CsHR3-I1 of dsRNA and L4440-CsHR3-I2 test group article sugarcane borer 3 instar larvaes mainly show as: can not normally accomplish and cast off a skin, there is pigmentation in head, grows to be had a strong impact on; And the article sugarcane borer larval growth of L4440-EGFP-I0 negative control group and clear water control group is all right, and individual body weight gains is obvious, and normal the completion casted off a skin and transition in the length of time, and be as shown in Figure 4.Express simultaneously
CsHR3The L4440-CsHR3-I1 of dsRNA and L4440-CsHR3-I2 test group article sugarcane borer larva are individual; Developing body weight in time obviously reduces; And the article sugarcane borer larva of negative control group and clear water control group is individual, develops body weight in time and continues to increase, and is as shown in Figure 6.
Feed and express the colibacillary L4440-CsHR3-I1 of dsRNA and L4440-CsHR3-I2 test group article sugarcane borer 5 instar larvaes mainly show as: thus prepupa can not normally cast off a skin and forms lopsided pupa; Or weight and all less improper pupa of shape, have in addition can't form normal pupa and death.And the article sugarcane borer larva of L4440-EGFP-I0 negative control group and clear water control group turns to all right of pupa, forms full, individual big normal pupa, and is as shown in Figure 5.
5, article sugarcane borer behind the feeding dsRNA
CsHR3The gene mRNA changing conditions
Choose consistent article sugarcane borer 3 instar larvaes of growth; The 0th day and the 7th day two timing nodes behind feeding dsRNA; Get each 2 of L4440-CsHR3-I1 test group, L4440-CsHR3-I2 test group, L4440-EGFP-I0 negative control group and clear water control group larvas (amounting to 8 larvas) respectively, repeated experiments 3 times.Extract total RNA, through real-time quantitative qRT-PCR technology, with Auele Specific Primer (primer sequence is following)
CsHR3-qRT-PCR-P1：?5'-?AACCTCCGCCGCAGCAGCCTTAC?-3'
CsHR3-qRT-PCR-P2：?5'-?GATGTCGCCCTCCGCATGACTAA?-3'，
To four treatment group
CsHR3The changing conditions of gene mRNA detects.The RNA sample that extracts is so that article sugarcane borer β-the actin gene is as internal control gene, and primer sequence is following.Select
β-actinThe gene purpose is quality, homogeneity and the relative quantification particularity that checking R NA extracts.
The amplification article sugarcane borer
β-actinThe primer of gene:
Cs-β-actin-P1：5'-?ACC?AAC?TGG?GAC?GAT?ATG?GAG?AA?-3'
Cs-β-actin-P2：5'-?CCT?CAG?TCA?AGA?GGA?CTG?GGT?GC?-3'。
Through realtime RT-PCR technology for detection
CsHR3The gene changing conditions, as shown in Figure 7, article sugarcane borer 3 instar larvaes after getting food and expressing the intestinal bacteria of dsRNA, self
CsHR3Genetic expression receives great inhibition, the 7th day and comparison in the 0th day,
CsHR3The gene mRNA relative expression quantity has produced very big significant difference, and in negative control group and the clear water control group
CsHR3The mRNA expression level of gene is constant basically, thereby has verified foreign gene
EGFPGene dsRNA can not grow to article sugarcane borer and impact.This test-results shows that insect growth is grown key gene through feeding
CsHR3DsRNA can effectively suppress the transcriptional expression of insect self target gene mRNA.
To sum up, can draw the expression of feeding
CsHR3The intestinal bacteria of dsRNA not only grow the article sugarcane borer normal growth and have caused certain influence, and on target gene mRNA level, presented the significance reduction.According to above experimental result, express dsRNA through the RNAi method of bacteria mediated, can get into and effectively suppress target gene in the insect body
CsHR3Transcriptional expression, further make article sugarcane borer normally the process of casting off a skin had a strong impact on, larva can not be taken off epidermis of a specified duration; Apolysis takes place; Break the balance of the gene of casting off a skin, interference insect is casted off a skin, and causes dead phenotypes such as Dysecdysis; Destroy its normal growth growth course, the RNAi technological expression dsRNA that discloses bacteria mediated thus is probably as a kind of novel method, the New Policy of biological control of insect pests.
In addition, the insect that the inventive method comprised not only comprise article sugarcane borer (
Chilo sacchariphagus), also can for yellow top borer (
Chilo infuscatellus), the sugarcane wood noth (
Phragmataecia sp.), Pyrausta nubilalis (Hubern). (
Ostrinia furnacalis) waiting the important pests in the agriculture prodn, the gene of using this research not only comprises yellow top borer key gene---the molt-regulating transcription factor that grows
HR3, can also be
EcR,
Chitinase,
ActinGene etc.
Claims (7)
1. article sugarcane borer molt-regulating transcription factor
CsHR3CDNA, it is characterized in that its nucleotide sequence such as SEQ ID NO.1 are said.
2. the said article sugarcane borer molt-regulating transcription factor of claim 1
CsHR3, its aminoacid sequence such as SEQ ID NO.2 are said.
3. the cloning process of the said article sugarcane borer molt-regulating transcription factor cDNA of claim 1 may further comprise the steps:
(1) extracts total RNA from article sugarcane borer prepupal period larva, then the synthetic cDNA of reverse transcription;
(2) conserved sequence according to different lepidopterous insects molt-regulating transcription factors designs degenerated primer:
CsHR3-middle?fragment-P1：5'-ACAGWGGTGAACTACCAGTG?-3'
CsHR3-middle?fragment-P2：5'-GACCATGRAATTGGTCGCT-3'
(3) with cDNA be template, the pcr amplification article sugarcane borer
CsHR3The intermediate segment of gene;
(4) purification step (3) gained PCR product, sepharose reclaim the purpose fragment, and get purified product and be connected on the pMD19-T simple carrier, transformed into escherichia coli competent cell Top10 then, 37 ℃ of dull and stereotyped cultivations, blue hickie screening contains
CsHR3Positive colony of intermediate segment;
(5) basis
CsHR3The sequencing result of intermediate segment, design 3 ' RACE Auele Specific Primer is a template with article sugarcane borer cDNA, pcr amplification
CsHR33 ' fragment sequence of gene;
CsHR3-3'?RACE-GSP1：5'?-?CGGGTCAACAGGAATAGATGTCA?-3'
CsHR3-3'?RACE-GSP2：5'-?CAGCGCCTGACTCAGTGTATGAC-3'
(6) purification step (5) gained PCR product, sepharose reclaim the purpose fragment, and get purified product and be connected on the pMD19-T carrier, transformed into escherichia coli competent cell Top10 then, 37 ℃ of dull and stereotyped cultivations, blue hickie screening obtains
CsHR3Positive colony of gene 3 ' end;
(7) positive colony with step (4) and (6) gained carries out sequencing, will measure institute's calling sequence then through information biology software DNAman splicing, obtains
CsHR3Gene 1266 bp sequences.
4. like the cloning process of the said article sugarcane borer molt-regulating transcription factor cDNA of claim 3; It is characterized in that; The synthetic cDNA of reverse transcription is specially in the said step (1): get total RNA 5 microlitres, Random primer 1 microlitre, substrate dNTPs 2 microlitres, use RNase-free H
2O adds to 10 microlitres, mixing, and 65 ℃ of insulation 5min are put rapidly to cooled on ice 3min; In said mixture, add 5 * Primer Script Buffer, 4 microlitres, RNA enzyme inhibitors 0.5 microlitre, AMV ThermoScript II 0.5 microlitre then, and use RNase-free H
2O adds to 20 microlitres, 42 ℃ of reaction 60min, and 70 ℃ of fire extinguishing 15min, 4 ℃ of coolings ,-20 ℃ of preservations are subsequent use.
5. like the cloning process of the said article sugarcane borer molt-regulating transcription factor cDNA of claim 3, it is characterized in that, in step (3) and (5) the used reaction reagent of pcr amplification form and reaction conditions following: at first together with following reagent mix,
Template cDNA 1 μ l
Deoxynucleotide substrate dNTP 4 μ l
Ex-Taq archaeal dna polymerase 0.5 μ l
10 * Taq DNA polymerase buffer, 5 μ l
Forward primer (10 μ M) 2 μ l
Reverse primer (10 μ M) 2 μ l
ddH
2O 35.5μl
TV 50 μ l
Reaction conditions is: at first 94 ℃ of preparatory sex change are 3 minutes; Circulate as follows then: 94 ℃ 30 seconds, 50 ℃ 30 seconds, 72 ℃ 2 minutes, carry out altogether 32 the circulation; Last 72 ℃ were extended 10 minutes.
6. the reorganization of the said article sugarcane borer molt-regulating transcription factor cDNA of claim 1 is used; Adopt bacteria mediated RNAi technology in RNaseIII defective escherichia coli HT115, to express dsRNA; Obtain the dsRNA of biologically active; Through the article sugarcane borer larva of feeding, come reticent article sugarcane borer molt-regulating transcription factor
CsHR3Gene makes article sugarcane borer can not cast off a skin or destroy its normal growth growth course, thereby is used for the biological control of article sugarcane borer insect; Perhaps change the article sugarcane borer molt-regulating transcription factor over to crop, obtain to have the crop of insect resistance capacity through gene engineering method.
7. the method for a bacteria mediated RNAi control article sugarcane borer is characterized in that, selects the article sugarcane borer molt-regulating transcription factor
CsHR3Gene is selected it as target
CsHR3Two dsRNA interference fragments that contain non-LBD functional domain and LBD functional domain on the gene; Make up RNAi recombinant vectors L4440-CsHR3-I1, the L4440-CsHR3-I2 of bacteria mediated; Select for use green fluorescence protein gene EGFP as negative control simultaneously, clear water is as negative contrast; Through the heat shock method above-mentioned carrier is imported
Ecoli. among the HT115 (RNase III defective escherichia coli), add 0.8mM IPTG and carry out abduction delivering, extract the feeding experiment that bacterial expression dsRNA carries out the article sugarcane borer larva, write down for 3 ages and after 5 ages, article sugarcane borer was fed 7 days phenotype change and body in
CsHR3Gene mRNA reduces level.
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Cited By (3)
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| CN105063058A (en) * | 2015-07-31 | 2015-11-18 | 扬州大学 | Ostrinia furnacalis spinning gene segment as well as cloning method and application thereof |
| CN107760682A (en) * | 2017-10-18 | 2018-03-06 | 云南大学 | A kind of nucleotide sequence and its application available for molecule pest control |
| CN110042102A (en) * | 2019-03-20 | 2019-07-23 | 中国热带农业科学院热带生物技术研究所 | It is a kind of can it is mosquito-proof control mosquito double-stranded RNA, expression vector and its application |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105063058A (en) * | 2015-07-31 | 2015-11-18 | 扬州大学 | Ostrinia furnacalis spinning gene segment as well as cloning method and application thereof |
| CN107760682A (en) * | 2017-10-18 | 2018-03-06 | 云南大学 | A kind of nucleotide sequence and its application available for molecule pest control |
| CN110042102A (en) * | 2019-03-20 | 2019-07-23 | 中国热带农业科学院热带生物技术研究所 | It is a kind of can it is mosquito-proof control mosquito double-stranded RNA, expression vector and its application |
| CN110042102B (en) * | 2019-03-20 | 2022-12-06 | 中国热带农业科学院热带生物技术研究所 | Double-stranded RNA capable of preventing and controlling mosquitoes, expression vector and application thereof |
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