CN110511936A - Ladybug of eggplant 28 stars growth and development related gene CHS1 and its application - Google Patents
Ladybug of eggplant 28 stars growth and development related gene CHS1 and its application Download PDFInfo
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Abstract
The invention discloses a kind of ladybug of eggplant 28 stars growth and development related gene chitin synthetase 1 (CHS1) and its applications.The research of the invention finds that the expression of cryptiogene CHS1 can significantly inhibit the growth and development of ladybug of eggplant 28 stars, ladybug of eggplant 28 stars can be efficiently prevented and treated.The dsCHS1 of silencing target gene CHS1 has also been devised simultaneously, can Direct-fed achieve the purpose that prevent and treat ladybug of eggplant 28 stars.This method is easy to operate, validity and sensitivity are good, insecticide efficiency is high, and has many advantages, such as environmental-friendly, there is good application prospect.
Description
Technical field
The invention belongs to insect pest Control Technology fields.More particularly, to a kind of ladybug of eggplant 28 stars growth and development phase
Correlation gene CHS1 and its application.
Background technique
Ladybug of eggplant 28 stars Henosepilachnavigintioctopunctata (Fabricius) belongs to coleoptera
Coccinellidae is a kind of important agricultural pests, and host plant is very extensive, the Solanaceaes such as eggplant of mainly causing harm, potato and tomato
Vegetables.Its larva and adult are food with blade, and happiness is clustered in vacuum side of blade, feeding lower epidermis, mesophyll, the aggrieved usual shape of blade
Cause the even whole strain of plant wilting dead at irregular speculum or perforation, when serious.Point of the ladybug of eggplant 28 stars in China
Cloth is in extensive range, and it is larger that density occurs especially on the south the Changjiang river.In recent years, due to climate warming, Trade Development and protecting field
The expansion of vegetable growing area, foodstuff is continuous throughout the year, the generation of ladybug of eggplant 28 stars and causes harm further serious.2015
Year, China starts potato staple food grainization strategy, will further expansion China potato cultivated area, 28 star wooden dipper of eggplant
The prevention and treatment of worm is very urgent.
Currently, the prevention and treatment of ladybug of eggplant 28 stars includes artificial capture method, attractant trapping, chemical pesticide.Wherein manually catch
Catch that not only effect is poor, there are also very heavy labour's problems；And the effect of attractant trapping is also not fully up to expectations, is not thorough；Cause
This is more also to rely on chemical pesticide, but chemical pesticide can cause environmental pollution and agricultural product quality and safety etc. are numerous to ask
Topic.
RNA interference (RNA interference, RNAi) is that one kind is guarded in evolution, the short-movie section dependent on generation
RNAs (siRNAs), thus the mechanism of action for promoting the degradation of homologous mRNA or it being inhibited to translate.RNAi provides one in elder brother
The important tool of functional genomics research is carried out in worm, and has established base to develop environment amenable pests control method
Plinth.Due to using RNAi technology can be with the expression of specific inhibition of gene, so the technology has been widely used for targeting interference
Pest gene to achieve the purpose that pest control, but at present both at home and abroad to ladybug of eggplant 28 stars gene functional research compared with
It is few, it is not found to have the target gene of insecticidal activity.
The present inventor team early-stage study shows (201710949193.9), utilizes the suitable external source dsRNA of Direct-fed
Mode can be realized the toxicity to ladybug, therefore be suitable for preventing and treating the external source of ladybug of eggplant 28 stars from the exploitation of gene level
DsRNA product, it is easy to use, at low cost, more due to the specificity of gene, it can be achieved that accurate, excellent control efficiency, to environment
Close friend has great application prospect in terms of the prevention and treatment of ladybug of eggplant 28 stars.However, the screening of associated target gene, with
And the design of good, special, the stable dsRNA of control efficiency is the biggest problem and critical issue.
Summary of the invention
The technical problem to be solved by the present invention is to overcome the defects and deficiency of existing ladybug of eggplant 28 stars Prevention Technique, mention
For a kind of high lethal gene of ladybug of eggplant 28 stars, i.e. CHS1 gene.And eggplant can efficiently be prevented and treated based on the Data mining
The technology of potato ladybug, i.e. Direct-fed have the target gene dsRNA of high lethal ability, benefit to ladybug of eggplant 28 stars
Reach prevention and treatment purpose with lethal effect of the dsCHS1 to ladybug of eggplant 28 stars.This method is easy to operate, validity and sensitivity
Good, insecticide efficiency height, and there is many advantages, such as environmental-friendly, there is good application prospect.
The object of the present invention is to provide a kind of ladybug of eggplant 28 stars CHS1 gene and its prevent and treat ladybug of eggplant 28 stars
Using.
Another object of the present invention be to provide a kind of CHS1 gene that can be used for preventing and treating ladybug of eggplant 28 stars dsRNA and its
Using.
Still a further object of the present invention is to provide a kind of method and kit for preventing and treating ladybug of eggplant 28 stars.
Above-mentioned purpose of the present invention is achieved through the following technical solutions:
The present invention is based on the transcript profile library of ladybug of eggplant 28 stars, screening obtains a high lethal gene --- CHS1 base
Cause, and develop the technology of dsRNA (dsCHS1) the prevention and treatment ladybug of eggplant 28 stars of feeding CHS1 gene.The present invention is eggplant leaf
Piece is separately immersed in the solution of the dsCHS1 and dsGFP of kit synthesis, is taken out and is fed the 1 of ladybug of eggplant 28 stars after drying
Instar larvae 2 days, then with unused dsRNA handle eggplant leaf feeding, observe and record ladybug of eggplant 28 stars the death rate and
Developmental condition；In addition, determining it to 1 age of ladybug of eggplant 28 stars, 3 ages and adult with the method for bacterium solution expression dsCHS1
Lethal ability, and then comprehensively external source dsCHS1 is evaluated to the insecticidal activity of ladybug of eggplant 28 stars.Finally, utilizing fluorescent quantitation
The method of PCR (qPCR) has tested and analyzed expression of the CHS1 gene in feeding dsCHS1 and dsGFP ladybug of eggplant 28 stars
Amount variation.The results show that Direct-fed external source dsCHS1 can significantly inhibit ladybug of eggplant 28 stars CHS1 gene expression, directly raise
Feeding external source dsCHS1 has significant lethal effect to ladybug of eggplant 28 stars.Therefore, following theme and application should all be in this hairs
Within bright protection scope:
A kind of ladybug of eggplant 28 stars CHS1 gene, sequence is as shown in SEQ ID NO.1.
The CHS1 gene answering in the product of prevention and treatment ladybug of eggplant 28 stars or preparation prevention and treatment ladybug of eggplant 28 stars
With.
The CHS1 gene is in the production for inhibiting ladybug of eggplant 28 stars growth or preparation to inhibit ladybug of eggplant 28 stars growth
Application in product.
The CHS1 gene is promoting ladybug of eggplant 28 stars dead or the production of preparation promotion ladybug of eggplant 28 stars death
Application in product.
Production of the inhibitor of the CHS1 gene in prevention and treatment ladybug of eggplant 28 stars or preparation prevention and treatment ladybug of eggplant 28 stars
Application in product.
A kind of dsRNA can be used for preventing and treating ladybug of eggplant 28 stars, the dsRNA can the above-mentioned CHS1 genes of targeted silent.
DsRNA sequence is as shown in SEQ ID NO.1.
A kind of kit for preventing and treating ladybug of eggplant 28 stars, contains CHS1 gene inhibitor.Preferably, the inhibitor is
Above-mentioned dsRNA.
Specifically, one of the approach of CHS1 gene prevention and treatment ladybug of eggplant 28 stars, i.e., a kind of 28 star of prevention and treatment eggplant are utilized
The method of ladybug, Direct-fed external source dsRNA so that dsCHS1 enters in ladybug of eggplant 28 stars body, the dsRNA can silencing/
Inhibit the CHS1 gene expression of ladybug of eggplant 28 stars, inhibits ladybug of eggplant 28 stars growth, promotes ladybug of eggplant 28 stars dead
It dies, to achieve the purpose that prevent and treat ladybug of eggplant 28 stars.
The invention has the following advantages:
The present invention screens to obtain a kind of high lethal gene CHS1 gene of ladybug of eggplant 28 stars, and it is efficiently heavy to develop it
Silent dsRNA, develops the technology that can efficiently prevent and treat ladybug of eggplant 28 stars, i.e. Direct-fed has ladybug of eggplant 28 stars
The target gene dsRNA of high lethal ability, reaches prevention and treatment purpose using lethal effect of the dsCHS1 to ladybug of eggplant 28 stars.It should
Method is easy to operate, validity and sensitivity are good, insecticide efficiency is high, and has many advantages, such as environmental-friendly, has and answers well
Use prospect.
Detailed description of the invention
Fig. 1 is dsGFP the and dsCHS1 electrophoretogram of bacterium solution expression.
Fig. 2 is influence of the dsCHS1 of various concentration to ladybug of eggplant 28 stars larval mortality.After being started using experiment
10 days larval mortality data establish survival curve with Cox regression routine.Different letters (such as a, b) indicate control curves with
Processing curve has significant difference.
Fig. 3 is that influence of the dsCHS1 of feeding bacterium solution expression to ladybug of eggplant 28 stars survival rate (schemes depositing for A:1 instar larvae
Motility rate；Scheme the survival rate of B:3 instar larvae；Scheme C: the survival rate of adult).It is utilized respectively 10 days, 10 days, 14 days 1 instar larvaes, 3
The Mortality data of instar larvae and adult establishes survival curve with Cox regression routine.Different letters (such as a, b) indicate that control is bent
Line and processing curve have significant difference.
Fig. 4 is the 3rd day that feeding dsRNA starts, the dsGFP control group (A) of normal development and the dsCHS1 processing of death
Group (B) ladybug of eggplant 28 stars phenotypic difference.
Fig. 5 is the 2nd day and the 4th day after feeding dsCHS1 and dsGFP, CHS1 gene expression amount in ladybug of eggplant 28 stars
Variation.
Specific embodiment
The present invention is further illustrated below in conjunction with Figure of description and specific embodiment, but embodiment is not to the present invention
It limits in any form.Unless stated otherwise, the present invention uses reagent, method and apparatus routinely try for the art
Agent, method and apparatus.
Unless stated otherwise, agents useful for same and material of the present invention are commercially available.
Ladybug of eggplant 28 stars used in following embodiments is raised in Department of Entomology, Agricultural University Of South China.Raise eggplant 20
The eggplant of eight star ladybugs is Wan Sheng marshal's circle eggplant seedling, ladybug is put with the culture dish containing filter paper, filter paper cotton balls moisturizing is put into
(25 ± 1 DEG C of temperature, humidity 70%-80%, photoperiod L:D=14:10) are bred in growth cabinet.
RNA extraction and application TRIzol extraction method (Invitrogen, USA), Reverse Transcription (PrimeScriptTMRT
Reagent Kit with gDNA Eraser) it is purchased from TAKARA Bioisystech Co., Ltd, dsRNA synthetic agent box
(MEGAscriptTMT7) it is purchased from Thermo Fisher Scientific, PCR reaction system used kit (EX TaqTM)
Purchased from TAKARA Bioisystech Co., Ltd, DNA purification and recovery kit (Universal DNA Purification Kit)
Purchased from TIANGEN Biotech (Beijing) Co., Ltd..
The data processing method of following embodiment: biology is carried out to ladybug of eggplant 28 stars for two kinds of dsRNA
The interpretation of result of measurement is counted using survival rate of the Excel 2010 to ladybug of eggplant 28 stars, soft using SPSS 19.0
Part is mapped using Cox regression analysis, and the difference analysis between various concentration uses single factor analysis.To target base after RNA interference
Because of the analysis of expression quantity variation, qPCR data use 2-△△CtMethod (Ct indicates recurring number) is calculated.It is soft using SPSS 19.0
Part carries out data analysis using one-way analysis of variance.
The acquisition of 1 growth and development related gene CHS1 kit of embodiment synthesis dsRNA
We construct its transcript profile library according to the genome of ladybug of eggplant 28 stars, then the text of the transcript profile based on building
Library, research screening and ladybug of eggplant 28 stars growth and development related gene, screening obtains CHS1 genetic fragment, such as SEQ ID
Shown in NO.1.Then dsRNA is synthesized.
1, the synthesis of ladybug of eggplant 28 stars Total RNAs extraction and the first chain cDNA.
It takes 2 instar larvaes 10 of ladybug of eggplant 28 stars only in 2ml centrifuge tube, TRIzol method is utilized to extract 28 star of eggplant
The total serum IgE of ladybug, the concentration and quality of RNA utilize instrument NanoDropOneCIt is measured, utilizes reverse transcription reagent box
(PrimeScriptTMRT reagent Kit with gDNA Eraser, TAKARA) it is inverted according to specification step
Record synthesizes the first chain of cDNA.
2, design of primers
The gene order of CHS1 is obtained from the transcript profile library of ladybug of eggplant 28 stars, designs the dsRNA of CHS1 gene
Primer P1 (table 1), green fluorescence protein gene (GFP) are expanded from the plasmid containing GFP that laboratory saves and are obtained, GFP base
The dsRNA primer P2 (table 1) of cause.Respectively plus related to restriction enzyme site on the primer P2 of the primer P1 and dsGFP of dsCHS1
Homology arm, design primer P3, to dsGFP construction of expression vector relevant primer P4 relevant to dsCHS1 construction of expression vector
(table 1).According to the sequence of CHS1 gene, the qPCR primer P6 of qPCR the primer P5, reference gene GAPDH of CHS1 gene are designed
(table 1).
Table 1:dsRNA synthesis and qPCR primer
3, the dsRNAs of kit synthesis CHS1 gene and GFP gene
PCR amplification is carried out using the primer P1 and P2 in table 1, the reaction system of PCR amplification is 10x EX Taq Buffer
5 μ L, 0.25 μ L of TaKaRa EX Taq, 4 μ L of dNTP Mixture, upstream primer (10 μm of oL/L) 1 μ L, downstream primer (10 μ
MoL/L) 1 μ L, cDNA/GFP plasmid 1 μ L, dd H2O polishing to 50 μ L.
The response procedures of PCR amplification are 94 DEG C of initial denaturation 3min；94 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C extend
1min, totally 30 recycle；72 DEG C of extension 5min.Amplified production is placed in 4 DEG C of preservations.Program after the reaction was completed, utilizes agarose
Gel electrophoresis detects amplification.With DNA purification and recovery kit (Universal DNA Purification
Kit, TIANGEN) recovery purifying two kinds of PCR products obtained above, as the template that dsRNA is transcribed in vitro, dsRNA's is external
Transcription system be 5 μ L of 10x Reaction Buffer, each 5 μ L of (ATP, GTP, CTP, UTP) solution, 5 μ L of Enzyme mix,
Template 20 μ L, ddH2O complements to 50 μ L.37 DEG C of placement 4h.The TURBO DNase removal residual of 2.5 μ L is added after reaction
Template DNA and single stranded RNA, dsRNA is then purified, finally with 50 μ L ddH2O dissolve dsRNA, respectively obtain dsCHS1 and
DsGFP, with the band of 1.5% agarose gel electrophoresis verifying dsRNA.
The CHS1dsRNA of ladybug of eggplant 28 stars is double-stranded RNA, by positive-sense strand and antisense strand at the core of positive-sense strand
Nucleotide sequence is the SEQ ID NO.1 in sequence table, and the nucleotides sequence of antisense strand is classified as the anti-of SEQ ID NO.1 in sequence table
To complementary series, the nucleotides sequence of CHS1dsRNA encoding gene is classified as the SEQ ID NO.1 in sequence table.GFP dsRNA is double
Chain RNA, by positive-sense strand and antisense strand at the nucleotides sequence of positive-sense strand is classified as the SEQ ID NO.2 in sequence table, antisense
The nucleotides sequence of chain is classified as the reverse complementary sequence of SEQ ID NO.2 in sequence table.
4, the acquisition of growth and development related gene CHS1 bacterium solution expression dsRNA
Two restriction enzyme sites, respectively BamHI (GGATCC) and SacI (GAGCTC) are selected in the sequence of L4440.Root
According to the sequence information (this sequence information has disclosed) of L4440, add respectively on the primer P2 of the primer P1 and dsGFP of dsCHS1
Upper homology arm relevant to two restriction enzyme sites, designs primer P3 relevant to dsCHS1 construction of expression vector, constructs with dsGFP
The relevant primer P4 (table 1) of expression vector.Using the cDNA of ladybug of eggplant 28 stars as template, the reaction system and expansion of PCR amplification
It is as indicated above to increase program, the target fragment of the dsCHS1 and dsGFP of carrier construction are obtained, with DNA purification and recovery kit
(Universal DNA Purification Kit, TIANGEN) recycles two kinds of PCR products obtained above.According to two enzymes
The sequence of enzyme site utilizes QuickCutTMSacI and QuickCutTMBamHI is by L4440 vector linearization, the reactant of digestion
It is to be detailed in specification, after the completion of endonuclease reaction, with DNA purification and recovery kit (Universal DNA Purification
Kit, TIANGEN) recycle the L4440 carrier linearized.
Utilize the Trelief of Guangzhou Qing Ke Biotechnology Co., LtdTMSoSoo Cloning Kit Ver.2 kit point
DsGFP and dsCHS1 is not reacted into 20min at 50 DEG C with the L4440 carrier of linearisation, is recombinated.DsCHS1 will then be contained
It is imported into HT115 competent cell with the recombinant expression carrier of dsGFP, is placed in and places 30min on ice, subsequent 37 DEG C of heat shocks
1min；3min is stood on ice, the 700 μ L of LB liquid medium for being free of ammonia benzyl is then added, at 37 DEG C, under conditions of 210rpm
1h is cultivated, is incubated overnight later with the LB plate containing ammonia benzyl and tetracycline.Picking single colonie is placed on 4ml and contains ammonia benzyl (100
μ g/mL) and the LB liquid medium of tetracycline (10 μ g/mL) in, 37 DEG C, 210rpm cultivate 12h, then take 50 μ L to be transferred to
5mL contains in the LB liquid medium of ammonia benzyl (100 μ g/mL) and tetracycline (10 μ g/mL), and 37 DEG C, 210rpm cultivates 3h, makes bacterium
The IPTG of 1mM is added between 0.5-0.8 in the OD value of liquid, and 37 DEG C, 120rpm cultivates 5h, induces dsRNA.
Two kinds of bacterium solutions containing dsGFP and dsCHS1 are collected into mycelia at 4 DEG C under conditions of 5000rpm, is utilized
TRIzol extraction method (Invitrogen, USA) extracts RNA and verifies whether successfully to induce with 1.5% agarose gel electrophoresis
dsRNA。
5, result
It is expanded with P1 primer, obtains the pcr amplification product that size is 458bp, be sequenced and leave out T7 promoter sequence
After column, obtaining the nucleotide sequence that size is 418bp is target gene CHS1, as shown in SEQ ID NO.1.To be loaded with GFP
Plasmid be template, expanded with P2 primer, obtain size be 507bp PCR product, the purpose item of dsGFP and dsCHS1
Band is consistent with sequencing result.
Using the dsRNA of bacterium solution expression target gene, the RNA of mycelia is extracted, is verified whether using agarose gel electrophoresis
The dsRNA for successfully inducing target gene can see according to electrophoresis result (Fig. 1) and successfully induce dsGFP's and dsCHS1
Purpose band.
Inhibiting effect of 2 dsRNA of embodiment to ladybug of eggplant 28 stars
1, the dsRNA of kit synthesis is inhibiting the application in ladybug of eggplant 28 stars growth and development
Ladybug of eggplant 28 stars dsCHS1 nursing group: 10 eggplants two are put into being placed with filter paper and the culture dish for humidifying cotton balls
1 instar larvae of 18 star ladybugs.Diameter is impregnated with the dsCHS1 solution of concentration 50ng/ μ L, 250ng/ μ L and 500ng/ μ L respectively
The round eggplant leaf dish 1min of 12mm, feeds larva after being air-dried at room temperature 1h, every replacing a leaf dish for 24 hours, continuously feeds
The leaf dish that dsCHS1 impregnates feeds larva two days later, with normal eggplant leaf.
Ladybug of eggplant 28 stars dsGFP nursing group: 10 eggplants two are put into being placed with filter paper and the culture dish for humidifying cotton balls
1 instar larvae of 18 star ladybugs.The dsGFP solution of the concentration 500ng/uL synthesized with kit impregnates the round eggplant of diameter 12mm
Blades 1min feeds larva after air-drying 1h, every replacing a leaf dish for 24 hours, continuously feeds leaf dish two days that dsGFP impregnates
Afterwards, larva is fed with untreated eggplant leaf.
5 repetitions of every group of setting, every the mortality for counting ladybug of eggplant 28 stars in each culture dish for 24 hours, and more
The blade renewed, culture dish are placed in growth cabinet (25 ± 1 DEG C of temperature, humidity 70%-80%, photoperiod L:D=14:10).
The dead number of ladybug of eggplant 28 stars in each culture dish of each group is counted, is calculated under control group and various concentration dsRNA processing
The survival rate of ladybug of eggplant 28 stars changes.
2, application of the dsCHS1 of bacterium solution expression to ladybug of eggplant 28 stars lethal effect
Ladybug of eggplant 28 stars dsCHS1 nursing group: 10 1 ages are put into being placed with filter paper and the culture dish for humidifying cotton balls
3 groups of experiments, 5 repetitions of every group of setting are arranged altogether in larva, 10 3 instar larvaes, 5 adults.With the bacterium solution leaching of expression dsCHS1
The round eggplant leaf 1min of bulb diameter 12mm, feeds larva after being air-dried at room temperature 1h.The each culture dish of 1 instar larvae in processing group
2 leaf dishes of middle placement；5 leaf dishes are placed in each culture dish of 3 instar larvaes；5 leaf dishes are placed in each culture dish of adult.
Every replacing a leaf dish for 24 hours, the continuous leaf dish for feeding the immersion of dsCHS1 bacterium solution two days later, is fed with normal eggplant leaf.
Ladybug of eggplant 28 stars dsGFP nursing group: 10 1 age childrens are put into being placed with filter paper and the culture dish for humidifying cotton balls
3 groups of controls, 5 repetitions of every group of setting are arranged altogether in worm, 10 3 instar larvaes, 5 adults.It is impregnated with the bacterium solution of expression dsGFP
The round eggplant leaf 1min of diameter 12mm, feeds larva after being air-dried at room temperature 1h.In each culture dish of 1 instar larvae in control group
Place 2 leaf dishes；5 leaf dishes are placed in each culture dish of 3 instar larvaes；5 leaf dishes are placed in each culture dish of adult.Often
Every replacing a leaf dish for 24 hours, the continuous leaf dish for feeding the immersion of dsGFP bacterium solution two days later, is fed with normal eggplant leaf.
Every the blade for counting the mortality of ladybug of eggplant 28 stars in each culture dish for 24 hours, and more renewing, culture dish
It is placed in growth cabinet (25 ± 1 DEG C of temperature, humidity 70%-80%, photoperiod L:D=14:10).Count each culture dish of each group
The dead number of middle ladybug of eggplant 28 stars calculates control and the variation of different disposal group ladybug of eggplant 28 stars survival rate.
3, according to the result of statistics it is found that continuously feeding 1 instar larvae dsCHS1 of ladybug of eggplant 28 stars two days later, eggplant 20
Downward trend is presented as time increases in the survival rate of eight star ladybugs, 1 instar larvae, and the feeding concentration of processing group is respectively
50ng/ μ L, 250ng/ μ L and 500ng/ μ L, the feeding concentration of control group are 500ng/ μ L.According to fig. 2 as a result, discovery is different
Exist between the processing group and control group of concentration significant difference (χ 2=98.373, df=3, P < 0.0001), the feeding of processing group
Concentration 50ng/ μ L (P < 0.0001, Exp (B)=4.185), 250ng/ μ L (P < 0.0001, Exp (B)=6.189) and 500ng/ μ
There is significant difference between the processing group of 3 various concentrations in L (P < 0.0001, Exp (B)=26.214).From the result of statistics
In it is available to draw a conclusion, it is dead compared with the control group when the concentration of processing group is respectively 50ng/ μ L, 10ng/ μ L, 5ng/ μ L
The rate of dying increases separately 4.185 times, 6.189 times and 26.214 times.
According to the result (such as Fig. 3) of statistics, continuous dsCHS1 two days for feeding the expression of ladybug of eggplant 28 stars bacterium solution
Afterwards, the survival rate of 1 instar larvae (P < 0.0001, Exp (B)=11.297) and 3 instar larvaes (P < 0.0001, Exp (B)=7.748)
The difference that there is conspicuousness is compared with control group, the survival rate of processing group adult compares no difference with control group.Processing group
1 instar larvae and 3 instar larvaes the death rate has increased separately 63.217 times and 65.528 times compared with the control group.
In addition, two days later with micro- sem observation feeding dsCHS1, the variation of ladybug of eggplant 28 stars phenotypic characteristic.It was found that from
It feeds the 3rd day that dsCHS1 starts, the ladybug of eggplant 28 stars of dsGFP control group normally enters 2 age grade sections, the children in processing group
Worm can not normally cast off a skin into 2 age grade sections and it is dead, phenotypic characteristic shows as the scolus of larva pronotary and spot can not shape
At as shown in figure 4, illustrate that feeding dsCHS1 can cause in vivo strong RNAi effect in ladybug of eggplant 28 stars, lead to wooden dipper
Worm is dead.
Embodiment 3dsCHS1 inhibits the expression of CHS1 gene in ladybug of eggplant 28 stars body
1, experimental method
The 2nd day after starting feeding dsRNA and the 4th day respectively are collected and are handled with 250ng/ μ L dsCHS1 and dsGFP
1 instar larvae of ladybug of eggplant 28 stars, each processing collects 3 biology and repeats.Extract the ladybug of eggplant 28 stars collected
RNA, then reverse transcription dilutes 10 times of templates as qPCR at cDNA.Relative quantification qPCR is carried out using P5 and P6 as primer
Analysis.QPCR system is the ddH that (15 μ L) includes 5.25 μ L22 × SYBR Green MasterMix (BIO-RAD of O, 7.5 μ L
Inc, Hercules, CA) 4 μM of primers and 1.0 μ L cDNA the first chain template.The reaction kit Bio-Rad of qPCR
C1000Real-Time PCR system(BIO-RAD,USA).Reaction condition is 95 DEG C of 5min；95 DEG C of 10s, 60 DEG C of 30s, 39
A circulation, 3 technologies of each sample repeat, and reaction carries out in 96 orifice plates (BIO-RAD, USA).
2, experimental result
To feed dsGFP as control, respectively after statistics feeding dsCHS1 on day 2 with 4 days, in ladybug of eggplant 28 stars body
The relative expression quantity of CHS1 gene changes (as shown in Figure 5).Show the 2nd day since feeding dsCHS1 in Fig. 5, CHS1's
Expression quantity and control group do not have marked difference (F1,4=0.901, P=0.396)；The 4th day since feeding dsCHS1, CHS1
Expression quantity have dropped 2.83 times of (F compared with the control group1,4=222.698, P < 0.0001), further explanation passes through feeding
DsCHS1 can cause strong RNAi effect in ladybug of eggplant 28 stars body, cause the expression quantity of internal CHS1 gene obvious
It reduces, and then ladybug of eggplant 28 stars development is caused to be suppressed or dead.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
SEQUENCE LISTING
<110>Agricultural University Of South China
<120>ladybug of eggplant 28 stars growth and development related gene CHS1 and its application
<130>
<160> 14
<170> PatentIn version 3.3
<210> 1
<211> 418
<212> DNA
<213>CHS1 genetic fragment
<400> 1
tttaccgacg gcagaaagag tagcatggat atggtgttta atttttgcgt tttgggtgcc 60
acaacttggt tcattctttc gatcaacaag aatgttattc ttcaaatcat ccagaaaaac 120
gccattatcc cactttttgg ttatttttac agctgaaacg gcacatacag ttggaattgc 180
ccttttaata ttcttcattt taccagaaat agatgtagta aaaggagcaa tgttaacaaa 240
ctgtctgtgt tttgtacctt cagtattagg attgctttct agaagcaaca aggagtctaa 300
acgcttttta aaagtaatcg ttgatctcat tgccatagca atccaagcaa caggatttgt 360
cttctggcct atagtagaac agaaagcaga tctatgggtc attccggttg ctacgatc 418
<210> 2
<211> 467
<212> DNA
<213>GFP genetic fragment
<400> 2
cttgaagttg accttgatgc cattcttttg cttgtcggcc atgatgtaca cattgtggga 60
gttatagttg tattccagct tgtggccgag aatgtttcca tcctccttaa agtcaatgcc 120
cttcagctcg attctattca ccagggtgtc accttcgaac ttgacttcag cgcgggtctt 180
gtagttcccg tcatctttga aaaagatggt tctctcctgc acatagccct cgggcatggc 240
gctcttgaaa aagtcatgct gcttcatatg gtctgggtat ctggaaaagc actgcacgcc 300
ataggtgaag gtagtgacca gtgttggcca tggcacaggg agctttccag tggtgcagat 360
gaatttcagg gtgagctttc cgtatgtggc atcaccttca ccctctccgc tgacagaaaa 420
tttgtgccca ttcacatcgc catccagttc cacgagaatt gggacca 467
<210> 3
<211> 40
<212> DNA
<213> P1-F
<400> 3
taatacgact cactataggg gatcgtagca accggaatga 40
<210> 4
<211> 40
<212> DNA
<213> P1-R
<400> 4
taatacgact cactataggg tttaccgacg gcagaaagag 40
<210> 5
<211> 40
<212> DNA
<213> P2-F
<400> 5
taatacgact cactatagga agttcagcgt gtccggcgag 40
<210> 6
<211> 40
<212> DNA
<213> P2-R
<400> 6
taatacgact cactataggt tcacgttgat gccgttcttc 40
<210> 7
<211> 40
<212> DNA
<213> P3-F
<400> 7
ctgatatcat cgatgaattc aaggggaacg tcacaacaac 40
<210> 8
<211> 39
<212> DNA
<213> P3-R
<400> 8
cgaattcctg cagcccgggt ggtctttaca ccaaagggc 39
<210> 9
<211> 41
<212> DNA
<213> P4-F
<400> 9
ctgatatcat cgatgaattc aagttcagcg tgtccggcga g 41
<210> 10
<211> 40
<212> DNA
<213> P4-R
<400> 10
cgaattcctg cagcccgggt tcacgttgat gccgttcttc 40
<210> 11
<211> 22
<212> DNA
<213> P5-F
<400> 11
gttgaatcgg agtagggatg ac 22
<210> 12
<211> 21
<212> DNA
<213> P5-R
<400> 12
tcggagtaga accgtcagta t 21
<210> 13
<211> 22
<212> DNA
<213> P6-F
<400> 13
agctcttctc atcatggctt ac 22
<210> 14
<211> 22
<212> DNA
<213> P6-R
<400> 14
gaaagaggtg cagaatgtgt tg 22
Claims (10)
1. a kind of ladybug of eggplant 28 stars CHS1 gene, which is characterized in that its sequence is as shown in SEQ ID NO.1.
2. a kind of dsRNA that can be used for preventing and treating ladybug of eggplant 28 stars, which is characterized in that the dsRNA silencing of target genes is power
Benefit requires the 1 CHS1 gene.
3. dsRNA described in CHS1 gene or claim 2 described in claim 1 is in prevention and treatment ladybug of eggplant 28 stars or preparation prevention and treatment
Application in the product of ladybug of eggplant 28 stars.
4. dsRNA described in CHS1 gene or claim 2 described in claim 1 is inhibiting ladybug of eggplant 28 stars growth or preparation
Inhibit the application in the product of ladybug of eggplant 28 stars growth.
5. dsRNA described in CHS1 gene or claim 2 described in claim 1 is promoting ladybug of eggplant 28 stars dead or preparation
Promote the application in the product of ladybug of eggplant 28 stars death.
6. the expression inhibiting agent of CHS1 gene described in claim 1 is in prevention and treatment ladybug of eggplant 28 stars or preparation prevention and treatment eggplant 28
Application in the product of star ladybug.
7. applying according to claim 6, which is characterized in that the expression inhibiting agent of the CHS1 gene is claim 2 institute
State dsRNA.
8. a kind of kit for preventing and treating ladybug of eggplant 28 stars, which is characterized in that the table containing CHS1 gene described in claim 1
Up to inhibitor.
9. kit according to claim 8, which is characterized in that the expression inhibiting agent is dsRNA described in claim 2.
10. a kind of method for preventing and treating ladybug of eggplant 28 stars, which is characterized in that feeding external source dsRNA, which can silencing/suppression
The expression of CHS1 gene described in claim 1 processed.
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Cited By (1)
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| CN112410359A (en) * | 2020-11-06 | 2021-02-26 | 华南农业大学 | Ladybug PP2A gene and application thereof in preventing and treating ladybug |
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