CN106222286B - A method of research cotton bollworm larvae RNA jamming effectiveness - Google Patents
A method of research cotton bollworm larvae RNA jamming effectiveness Download PDFInfo
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- CN106222286B CN106222286B CN201610664530.5A CN201610664530A CN106222286B CN 106222286 B CN106222286 B CN 106222286B CN 201610664530 A CN201610664530 A CN 201610664530A CN 106222286 B CN106222286 B CN 106222286B
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Abstract
The present invention relates to field of biotechnology, specifically disclose a kind of method for studying cotton bollworm larvae RNA jamming effectiveness.The present invention is improved by the method to feeding cotton bollworm larvae siRNA, is established a kind of method for studying cotton bollworm larvae RNA jamming effectiveness, is improved the RNA jamming effectiveness to lepidopterous insects bollworm, stablizes experimental result.This method efficiently, simple, strong operability, the specific dosage of siRNA, can principal characteristic it is strong, provide technical support to study the function of lepidopterous insects bollworm gene.
Description
Technical field
The present invention relates to field of biotechnology, specifically, being related to a kind of side for studying cotton bollworm larvae RNA jamming effectiveness
Method.
Background technique
Bollworm Helicoverpa armigera (H ü bner) belongs to Lepidoptera noctuid, it is worldwide point a kind of
Cloth, polyphagous important agricultural insect, last century bollworm endangers cotton and causes huge economic loss.Transgenosis Bt
The extensive use of (Bacillus thuringiensist) cotton is effectively controlled causing harm for bollworm, but bollworm is to turning
The resistance problem of Bt cotton also increases significantly, and bollworm is to the harm of other non-transgenic crops also year by year at the same time
Aggravation.The functional gene of bollworm is studied using gene perturbation technique, can provide section for the prevention and treatment and improvement of bollworm
Foundation.
SiRNA (Small interfering RNA), is a kind of small RNA molecular, it is artificial during RNA interference
The small fragment RNA synthesized in vitro is made of about 21-25 nucleotide.The siRNA synthesized in vitro is imported cell by various means
Or in insect bodies, specific reduces the expression of some gene, to realize that the research of the gene function is called gene interference.
SiRNA plays central role in RNA silencing access, is to instruct element to what specific mRNA (mRNA) was degraded.At present it
It is a kind of powerful experimental tool in laboratory, is used for the research of gene function.Such as scientists with the technology for Bt
Acceptor gene interfered in insect, discovery receptor work in the mechanism of action of Bt.Some scientists also send out simultaneously
All dead after cotton bollworm larvae 7 days after the important gene such as HaNDUFV2 for now interfering bollworm, this is to turn base using new
Because technology prevention pest provides important clue.
People pass through injection method, feeding method, infusion method or Transgenic plant tissue training with having been able to comparative maturity at present
It is the methods of feeding that the related gene of bollworm is interfered.It is more commonly that the siRNA importing intracorporal method of bollworm is main
It is injection method and feeding method, wherein injection method requires have special equipment, higher cost, and mechanical damage when injection can draw
Play the higher death rate of bollworm；Feeding method is relatively simple, wherein also include many different feed modes, such as: drop is raised
Feeding method etc. is smeared on the method for feeding, mixed fodder feeding method, surface.Wherein the direct feeding siRNA of drop feeding method pipettor is to cotton
The oral cavity of earworm, it is complicated for operation, larva is easily injured, and it is difficult to ensure that feeding is complete；Traditional feed mixing method mixes siRNA
It closes in feed, is measured after a certain period of time, not quantitative, the amount of every group of processing or each test bay feeding siRNA can not
Control, and siRNA dosage is big；Surface smears feeding method and smears siRNA on man-made feeds surface, exist effect is relatively slow, efficiency compared with
In addition to this low disadvantage can not accurately import the intracorporal siRNA amount of bollworm, it is not significant that there are effects, experimental repeatability
Difference hardly results in stable and accurate experimental result.Therefore, quantitative feeding improves the jamming effectiveness of feeding method for further grinding
It is significant to study carefully related bollworm gene function.
Summary of the invention
In order to solve the problems in the existing technology, the object of the present invention is to provide a kind of research cotton bollworm larvae RNA
The method of jamming effectiveness.
In order to achieve the object of the present invention, technical scheme is as follows:
The present invention provides a kind of methods for studying cotton bollworm larvae RNA jamming effectiveness, carry out to cotton bollworm larvae hungry
After processing, quantitative feeding siRNA keeps bollworm feeding complete, the expression quantity of target gene in cotton bollworm larvae is detected, to study
The jamming effectiveness of cotton bollworm larvae RNA.
Experimental research find that being remarkably improved RNA when the siRNA food ingestion of cotton bollworm larvae is 0.4~0.8 μ g
Jamming effectiveness.In view of in the range of the siRNA food ingestion, RNA jamming effectiveness is without more apparent difference, therefore
The siRNA food ingestion of further preferred cotton bollworm larvae is 0.4 μ g, and the purpose for improving RNA jamming effectiveness can be realized.
Further, the cotton bollworm larvae selects 3 instar larvaes just completed after husking, and 3 instar larvaes just casted off a skin pass through
The survival rate screening of the first round, at this moment active 3 instar larvaes can be completed the normal history of life substantially, select just to complete husking
3 instar larvaes, can also verify and race against time for the subsequent interference experiment for larva.
Further, the Nature enemy is 11~13 hours, preferably 12 hours hungry.
More specifically, the method for the invention includes the following steps:
S1,8~10mg bollworm man-made feeds are put into the aperture of biometric fixed board, it is 0.08~0.16 that concentration, which is added dropwise,
The siRNA solution of μ g/ μ L obtains the man-made feeds containing 0.2~0.4 μ g siRNA；
S2, by after cotton bollworm larvae Nature enemy 12 hours, be placed in aperture described in S1, every Kong Yitou；
S3, after 6 hours, it is primary that the man-made feeds containing 0.2~0.4 μ g siRNA of S1 preparation are repeated into feeding；
S4, after repeating feeding 12 hours, the expression quantity of target gene in cotton bollworm larvae is detected.
The purpose that siRNA is added in two portions when feeding is to prevent siRNA (temperature: 27 ± 1 DEG C, wet under test conditions
Degree 60 ± 10%) under the conditions of degrade.
The present invention pass through a large number of experiments the study found that when bollworm man-made feeds be 8~10mg when, premenstruum (premenstrua) Nature enemy
Cotton bollworm larvae afterwards can be complete by the man-made feeds feeding containing siRNA in 6 hours.
According to the dosage of man-made feeds, the dripping quantity of the preferably described siRNA solution is 2.0~2.5 μ L, being capable of man-made feeds
To siRNA solution uniform absorption.And it is found by experiment that the siRNA solution of 2.5 μ L is that the man-made feeds of 8~10mg can carry
Maximum, siRNA can penetrate feed but not flow out at this time.
Further, the man-made feeds are the conventional feed that this field without containing NaOH feeds cotton bollworm larvae, are prevented
Controlling NaOH influences the possibility of siRNA rock-steady structure.
Further, in order to study RNA to the jamming effectiveness of the target gene of cotton bollworm larvae, the method for the invention may be used also
Be arranged control group, specifically, the control group using NControl siRNA solution replace siRNA solution, specific dosage with
Experimental group (bollworm of feeding siRNA in preceding method) is consistent.
Wherein, the solvent of the siRNA solution and NControl siRNA solution is DEPC water.
Preferably, the cotton bollworm larvae of feeding siRNA solution is as experimental group, it is real in order to guarantee the confidence level of experiment
It tests and is divided into 3 repetitions, every group repeats no less than 16, amounts to 48.
In the specific embodiment of the present invention, the present invention has studied RNA to cotton bollworm larvae using preceding method
The jamming effectiveness of ABCC2 gene (target gene), the siRNA are the nucleic acid that nucleotides sequence is classified as GGTGTTTACGGCGTTCCTT
Molecule.
The beneficial effects of the present invention are:
The present invention is improved by the method to feeding cotton bollworm larvae siRNA, establishes a kind of research cotton bollworm larvae
The method of RNA jamming effectiveness improves the RNA jamming effectiveness to lepidopterous insects bollworm, stablizes experimental result.This method is high
Effect, simple, strong operability, the specific dosage of siRNA, can principal characteristic it is strong, for the function for studying lepidopterous insects bollworm gene
Technical support can be provided.
Detailed description of the invention
Fig. 1 is the expression for interfering bollworm ABCC2 gene in the embodiment of the present invention 1 using fluorescence quantitative PCR detection RNAi
Result；Wherein, 0 the control of DEPC water process being represented, NC (0.5) represents feminine gender 0.5 μ g siRNA of feeding and compares, and 0.2,0.4,
0.5,0.6 and 0.8 represents the relative expression quantity of ABCC2 gene after bollworm difference feeding ABCC2siRNA.Between different letters
Indicate significant difference (18.0 software for of *: P < 0.05, Independent t-tests, SPSS version
Windows)。
Fig. 2 be in comparative example 1 of the present invention after 0.5 μ g siRNA of bollworm feeding in different time ABCC2 gene expression
Amount.
Fig. 3 is the expression quantity for detecting ABCC2 gene in comparative example 2 of the present invention after different feeding method processing.
Specific embodiment
The preferred embodiment of the present invention is described in detail below in conjunction with embodiment.It will be appreciated that following real
Providing merely to play the purpose of explanation for example is applied, is not used to limit the scope of the present invention.The skill of this field
Art personnel without departing from the spirit and purpose of the present invention, can carry out various modifications and replace to the present invention.
Experimental method used in following embodiments is conventional method unless otherwise specified.Such as qPCR
MasterMix Plus of SYBR Green I kit (Eurogentec, Freemont, CA) is RT-PCR used kit,
Or the condition according to manufacturer's specification suggestion.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Embodiment 1
The present embodiment is for illustrating influence of the different amounts of ABCC2siRNA of feeding to bollworm ABCC2 gene
Experimental subjects: 3 instar bollworm grubs, 48, point 3 repetitions.
With DEPC water respectively by ABCC2-siRNA and control Ncontrol siRNA dissolution, being made into concentration is 0.04-0.16
The solution of μ g/ μ l uses DEPC water as control；Prepare the artificial sample of bollworm, feed is cut into the fritter of 8~10mg.It will raise
Material is put into the aperture of biometric fixed board, and the siRNA solution (or control NControl and DEPC water) of 2.5 μ L is added dropwise in feed
On.Select that size is uniform, 3 instar larvaes after just casting off a skin are placed in the aperture after 12 hours hungry；After 6 hours, cotton
Earworm eats up all feeds completely, repeats above-mentioned feeding；Bollworm total serum IgE is extracted after 12 hours, reverse transcription at cDNA,
With the difference of fluorescence quantitative PCR detection gene expression amount.It is simultaneously that control is handled with traditional drop feeding method.Traditional
Drop feeding method is with the direct feeding siRNA of pipettor to the oral cavity of cotton bollworm larvae, and this method is it is difficult to ensure that bollworm meeting
The complete siRNA of feeding, therefore experimental repeatability is poor, it is more difficult to obtain stable experimental result.
It is double reference genes with EF-1 α and GAPDH, primer and probe implementation sequence is as follows:
ABCC2-Sense:5'-AAGTGTCGGTCTGGCTGTC-3'；
ABCC2-Antisense:5'-TGGTGGGTTAGTTGGTCCT-3'
EF-1 α-Sense:5'-GCCTGGTACCATTGTCGTCT-3'
EF-1α-Antisense:5'-GTAACCACGACGCAACTCCT-3'
GAPDH-Sense:5'-CGAACAGTCAAGTCAACG-3'
GAPDH-Antisense:5'-CAGAAGACAGTGGATGGA-3'
QPCR reaction system:
QPCR response procedures: 95 DEG C of initial denaturation 10min；95 DEG C of 15s, 60 DEG C of 1min, 40 circulations.
According to fluorescent quantitative PCR result (Fig. 1), after the amount of bollworm feeding ABCC2siRNA is greater than 0.4 μ g, the gene table
Up to amount compared with the control of feeding DEPC water and feeding the control of negative control (Ncontrol) siRNA, bollworm ABCC2 is raised
Gene expression quantity significantly reduce, and food ingestion be more than or equal to 0.4 μ g after, the gene without significant change.
Comparative example 1
The present embodiment the difference from embodiment 1 is that, repeat feed after, in the 3rd day extraction bollworm total serum IgE, reverse transcription
At cDNA, with the difference of fluorescence quantitative PCR detection bollworm ABCC2 gene expression amount, as a result as shown in Figure 2.
Comparative example 2
This comparative example is compared the method for the invention with traditional sessile drop method, as a result as shown in Figure 3；Wherein, Water
The control of DEPC water process is represented, Ncontrol represents feminine gender siRNA control, and ABCC2-1 represents ABCC2 gene in this experimental method
Relative expression quantity, ABCC2-2 represents the relative expression quantity of ABCC2 gene in drop feeding method.Indicate aobvious between different letters
It writes difference (18.0 software for Windows of *: P < 0.05, Independent t-tests, SPSS version).
According to fluorescent quantitative PCR result, compared with the control of feeding DEPC water, cotton boll is fed according to the method for the invention
The gene expression amount of the siRNA of worm ABCC2 gene significantly reduces 86.23%, with feeding negative control (Ncontrol) siRNA
Control compare, the gene expression amount for raising the siRNA of bollworm ABCC2 gene significantly reduces 86.41%, and feed it is negative right
It is not significantly different compared with DEPC water according to the gene expression amount of (Ncontrol) siRNA.And traditional drop feeding method is compared,
Drop feeding method due to the biology of experiment repeat between error it is very big (SE=0.31), so hardly resulting in stable effective
Experimental result.3 secondary pollutant of this method repeats test and stablizes, and error is small, and jamming effectiveness significantly improves.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.
Claims (4)
1. a kind of method for studying cotton bollworm larvae RNA jamming effectiveness, which comprises the steps of:
S1,8~10mg bollworm man-made feeds are put into the aperture of biometric fixed board, dropwise addition concentration is 0.08~0.16 μ g/ μ
The siRNA solution of L, obtains the man-made feeds containing 0.2~0.4 μ g siRNA, and the dripping quantity of the siRNA solution is 2.0~
2.5μL；
S2, by after cotton bollworm larvae Nature enemy 12 hours, be placed in aperture described in S1, every Kong Yitou；
S3, after 6 hours, it is primary that the man-made feeds containing 0.2~0.4 μ g siRNA of S1 preparation are repeated into feeding；
S4, after repeating feeding 12 hours, the expression quantity of target gene in cotton bollworm larvae is detected；
The cotton bollworm larvae is just to have completed 3 instar larvaes after husking；
The target gene is ABCC2 gene, the nucleotide sequence of the siRNA are as follows: GGTGTTTACGGCGTTCCTT.
2. the method according to claim 1, wherein setting control group, uses NControl siRNA solution generation
For siRNA solution.
3. according to the method described in claim 2, it is characterized in that, the siRNA solution and NControl siRNA solution
Solvent is DEPC water.
4. the method according to claim 1, wherein the cotton bollworm larvae of feeding siRNA solution is as experimental group,
Experimental group is no less than 48.
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| CN111387213B (en) * | 2020-03-24 | 2021-06-29 | 湖北大学 | Preparation method of virus-like particles for preventing and treating cotton bollworm, method for resisting cotton bollworm and application of virus-like particles |
| CN111500577B (en) * | 2020-04-22 | 2023-06-09 | 上海海洋大学 | Application of target gene in shellfish larva metamorphosis development process by RNA interference technology research |
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| CN101195821A (en) * | 2006-12-04 | 2008-06-11 | 中国科学院上海生命科学研究院 | Method for improving insect resistance of plant by using RNAi technique |
| CN103814950A (en) * | 2013-11-12 | 2014-05-28 | 河南大学 | Method for improving biological Helicoverpa armigera prevention and control effect of nuclear polyhedrosis viruses (NPVs) |
| CN105693839A (en) * | 2016-03-21 | 2016-06-22 | 中国农业大学 | Cotton bollworm P-glycoprotein gene and application thereof |
| CN105779502A (en) * | 2016-03-16 | 2016-07-20 | 中国农业科学院植物保护研究所 | Method for improving RNA interfering efficiency of mandibulate insects |
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| EP2438813A4 (en) * | 2009-06-05 | 2013-10-02 | Univ Nagoya Nat Univ Corp | Insect pest control method |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101195821A (en) * | 2006-12-04 | 2008-06-11 | 中国科学院上海生命科学研究院 | Method for improving insect resistance of plant by using RNAi technique |
| CN103814950A (en) * | 2013-11-12 | 2014-05-28 | 河南大学 | Method for improving biological Helicoverpa armigera prevention and control effect of nuclear polyhedrosis viruses (NPVs) |
| CN105779502A (en) * | 2016-03-16 | 2016-07-20 | 中国农业科学院植物保护研究所 | Method for improving RNA interfering efficiency of mandibulate insects |
| CN105693839A (en) * | 2016-03-21 | 2016-06-22 | 中国农业大学 | Cotton bollworm P-glycoprotein gene and application thereof |
Non-Patent Citations (1)
| Title |
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| 棉铃虫3种蜕皮相关基因的RNA干扰效应比较;杨静，韩召军;《南京农业大学学报》;20141231;第37卷(第1期);81-86 |
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