CN112369428A - miR-318 for regulating development of insect wings and application of miR-318 in pest control - Google Patents
miR-318 for regulating development of insect wings and application of miR-318 in pest control Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N57/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic phosphorus compounds
- A01N57/10—Biocides, pest repellants or attractants, or plant growth regulators containing organic phosphorus compounds having phosphorus-to-oxygen bonds or phosphorus-to-sulfur bonds
- A01N57/16—Biocides, pest repellants or attractants, or plant growth regulators containing organic phosphorus compounds having phosphorus-to-oxygen bonds or phosphorus-to-sulfur bonds containing heterocyclic radicals
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/033—Rearing or breeding invertebrates; New breeds of invertebrates
- A01K67/0333—Genetically modified invertebrates, e.g. transgenic, polyploid
- A01K67/0337—Genetically modified Arthropods
- A01K67/0339—Genetically modified insects, e.g. Drosophila melanogaster, medfly
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/70—Invertebrates
- A01K2227/706—Insects, e.g. Drosophila melanogaster, medfly
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Abstract
The invention discloses miRNA for regulating and controlling insect wing development and application thereof in pest control. Research shows that the wing area can be obviously reduced and the development of wings can be inhibited by over-expressing miR-318; further research shows that the action mechanism is as follows: miR-318 directly inhibits the expression of transcription factor cloned (sd) of a Hippo signal pathway, further inhibits the downstream of the pathway to promote cell proliferation and inhibit the expression of apoptosis target genes, and finally inhibits the development of wings. The miR-318 can obviously inhibit the development of insect wings, and provides an important target gene resource for the biological control of pests mediated by RNAi through artificially synthesizing the miR-318 or an analogue thereof or utilizing a transgenic plant expressing the miR-318. Provides important theoretical and practical significance for preventing and controlling the winged pests and has obvious economic and social benefits.
Description
Technical Field
The invention relates to the field of agricultural biotechnology and pest control, in particular to miR-318 for regulating development of insect wings and application thereof in pest control.
Background
The wings are used as important organs of the insects, endow the insects with the flying ability, and increase the space for feeding and mating of the insects. At present, the lepidoptera insect becomes a key point and a difficult point in agricultural pest control, a molecular mechanism for controlling the development of the insect wing is analyzed, a method for inhibiting the development of the insect wing is found, and important theoretical and practical significance is provided for the control of the lepidoptera pest.
MicroRNA (miRNA) is a small-molecule non-coding RNA with the length of about 22nt and is highly conserved in animal evolution. miRNA causes the transcription inhibition of target genes or the degradation of mRNA by recognizing the binding site on the 3' -UTR of the mRNA of the target genes, thereby regulating the expression of the target genes at the level of transcription and posttranscription. Studies have shown that mirnas are involved in a variety of biological processes, including cell differentiation, proliferation, senescence, apoptosis, migration, invasion, development, and signal transduction, among others.
Thousands of miRNAs have been isolated and found in nematode, drosophila, mouse and human cells, however, functional studies of miRNAs are relatively slow compared to frequent discovery of new miRNAs. Although bioinformatics approaches predict the effect of a series of miRNAs on target genes, few miRNAs have given direct evidence to demonstrate the target genes and their function of miRNAs. Therefore, how to determine the function of miRNA has become one of the most difficult and urgent tasks in this field.
In recent years, documents report that miRNA participates in the process of regulating and controlling insect wing development, wherein Drosophila research is the most extensive, and researchers find that miR-iab-4 and an antisense strand miR-iab-8 thereof can inhibit the activity of an ultra-bithorax gene (Ultrabithorax, Ubx) and further control the conversion between wings and balance bars; miR-7 influences the development of drosophila wing primordium by negatively regulating the expression of target gene yki; miR-9a causes wing tissue deletion and ectopic cell apoptosis by inhibiting expression of target gene LIM-only; miR-12 and miR-283 can cause wing development deformity by regulating a Hedgehog signal path. In other insects, miRNA is relatively less in research on wing development, and miR-263, miR-193 and miR-2788 are found to play a potential regulation role in the wing development process through the analysis of miRNA expression levels in different development stages of the sleeve disc.
miR-318 is a member of a plurality of miRNAs, but few researches on the function of miR-318 are carried out at present, and the regulation of insect wing development by miR-318 is not reported.
Disclosure of Invention
Aiming at the prior art, the invention aims to provide the application of miR-318 in the regulation of insect wing development. The research of the invention discovers that the overexpression of miR-318 can obviously reduce the wing area of the insect and inhibit the development of the wing; further research shows that the action mechanism is as follows: miR-318 directly inhibits the expression of transcription factor cloned (sd) of a Hippo signal pathway, further inhibits the downstream of the pathway to promote cell proliferation and inhibit the expression of apoptosis target genes, and finally inhibits the development of wings.
Based on the research, the invention provides the following technical scheme:
in a first aspect of the invention, the application of miR-318 in preparing an insect wing development inhibitor is provided.
Preferably, the insect is a lepidopteran insect.
More preferably, the lepidopteran insect is a cotton bollworm.
Preferably, the nucleotide sequence of the miR-318 is 5'-ggauacacac aguucaguuu g-3'; (SEQ ID NO. 1).
Preferably, the miR-318 inhibits insect wing development by inhibiting the expression of a Hippo signal pathway transcription factor Scalloped.
In a second aspect of the invention, the application of miR-318 in preparing pesticides is provided.
Preferably, the insecticide is a lepidopteran insect insecticide.
In the application, the pesticide can inhibit the development of insect wings, so that the feeding and mating spaces of insects are limited, the survival and the reproductive capacity of the insects are reduced, and the biological control of pests is realized.
In a third aspect of the present invention, there is provided a method for controlling pests, the method comprising the steps of:
over-expressing miR-318 in the body of the pest;
or the miR-318 is used as an effective component to feed or spray pests.
Preferably, the pests are lepidopteran insects; more preferably, the lepidopteran insect is a cotton bollworm.
In a fourth aspect of the present invention, there is provided an insect wing development inhibitor comprising an effective amount of miR-318 as an active ingredient.
In a fifth aspect of the invention, a pesticide is provided, wherein the pesticide comprises miR-318.
The invention has the beneficial effects that:
the invention discovers, for the first time, that the wing area can be obviously reduced and the development of wings can be inhibited by over-expressing miR-318. The action mechanism is that miR-318 inhibits the expression of transcription factor stressed (sd) of a Hippo signal channel, so that the downstream of the channel is inhibited to promote cell proliferation and inhibit the expression of apoptosis target genes, and finally the development of wings is inhibited. The miR-318 can obviously inhibit the development of insect wings, and provides an important target gene resource for the biological control of pests mediated by RNAi through artificially synthesizing the miR-318 or an analogue thereof or utilizing a transgenic plant expressing the miR-318. miR-318 inhibits development of insect wings, limits feeding and mating spaces of winged pests, can reduce survival rate and fertility of the winged pests, provides important theoretical and practical significance for prevention and treatment of the winged pests, and has remarkable economic and social benefits.
Description of the drawings:
FIG. 1: the effect of miR-318 overexpression on adult fin area; in the figure, "En-gal 4> UAS-GFP" indicates the filial generation of En-gal4 virgins and UAS-GFP male flies; "En-gal 4> UAS-miR-318" indicates the filial generation of En-gal4 virgins and UAS-miR-318 androgens.
FIG. 2: effect of miR-318 overexpression on Hippo signaling pathway target gene expression.
FIG. 3: bioinformatics predicts miR-318 target genes.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
As described in the background section, the functional studies of mirnas are relatively slow, and how to determine the function of mirnas has become one of the most difficult and urgent tasks in this field. Although the miRNA is reported to regulate the development of insect wings, the expression modes of different miRNAs are different, and the functions and the action mechanisms of the miRNAs are also obviously different. Therefore, the discovery of new miRNA for regulating insect wing development has important theoretical and practical significance for the control of winged pests.
Based on the method, the miRNA capable of regulating and controlling insect wing development is subjected to a large amount of screening and deep research. The result shows that the wing area can be obviously reduced and the development of wings can be inhibited by over-expressing miR-318.
Furthermore, the invention also researches the action mechanism of miR-318 for inhibiting insect wing development. The regulation of the size and volume of tissue and organs during the growth and development of organisms has been one of the most fundamental problems in biological research. The discovery of the Hippo signaling pathway begins to solve this biological puzzle. As a signaling pathway to control tissue growth and cell fate, the Hippo signaling pathway is evolutionarily highly conserved from arthropods to mammals. Therefore, the invention researches the action relationship between miR-318 and a Hippo signal pathway, and the result shows that miR-318 directly inhibits the expression of a transcription factor plated (sd) of the Hippo signal pathway, so that the downstream of the pathway is inhibited to promote cell proliferation and inhibit the expression of apoptosis target genes, and finally the development of wings is inhibited.
In order to make the technical solutions of the present application more clearly understood by those skilled in the art, the technical solutions of the present application will be described in detail below with reference to specific embodiments.
The test materials used in the examples of the present invention are all conventional in the art and commercially available.
Example 1:
one, miR-318 overexpression obviously reduces the area of drosophila wings
En-gal4 virgins specifically expressed in the Drosophila wing porterior (P) region were crossed with UAS-GFP and UAS-miR-318 androgens, respectively, and the offspring adult wing phenotype was observed. The wings were photographed using a stereomicroscope and the areas of the P and antigen (A) regions were measured by Image J software, the P/A ratio was calculated and compared with the control group and statistically analyzed. Because the individual sizes of the later-generation male drosophila melanogaster and the female drosophila melanogaster are different, the study respectively carries out statistics on the female and male individuals, and the study result only shows the wings of the male drosophila melanogaster imago and the statistical result.
The results are shown in FIG. 1, where the P/A value of the control group was 1.3, and the P/A value decreased to 0.8 after miR-318 was overexpressed. Further statistical analysis showed that overexpression of miR-318 significantly reduced the area of the P region of the wings compared to control drosophila (FIGS. 1A-C).
II, over-expression of miR-318 for inhibiting expression of target gene of Hippo pathway
In order to detect whether overexpression miR-318 regulates the activity of a Hippo signal channel, En-gal4 virgins are respectively hybridized with UAS-GFP and UAS-miR-318 androgens, offspring 3-instar larvae are dissected, pterogues are fixed by formaldehyde, PBST is used for rinsing for 3 times, and then the protein level of Cyclin E is detected by using a Cyclin E antibody.
Cyclin E is a target gene of the Hippo pathway. Overexpression of miR-318 significantly reduced the protein levels of Cyclin E (FIGS. 2B-B ") as compared to the control group (FIGS. 2A-A"). The GFP region in FIG. 2A is the region of En-gal4 expression, FIG. 2A 'is the protein level of Cyclin E in the wing primordia, and FIG. 2A "is a combined graph of FIGS. 2A and 2A'. The GFP region in FIG. 2B is the region where En-gal4 is expressed and also the region where miR-318 is overexpressed, FIG. 2B 'is the protein level of Cyclin E in the wing primordia, and FIG. 2B "is a combined graph of FIGS. 2B and 2B'.
Thirdly, miR-318 targets Hippo pathway transcription factor sd
The above results show that miR-318 is likely to be involved in Hippo signaling pathway homeostasis. To find a potential target of miR-318, we performed bioinformatics analysis, and found that the 3' -UTR region of the sd gene contains the binding site of miR-318 (FIG. 3).
Drosophila has its own advantages as a model insect, and very mature systems for overexpression and knock-down of genes have been developed in Drosophila today. The gal4-UAS system is a drosophila binary expression system commonly used at present, wherein gal4 is a transcription activator found in yeast, and can be combined with galactose Upstream Activating Sequence (UAS) at a special site in a DNA sequence to induce the transcription and translation of genes downstream of the UAS. Crossing with transgenic Drosophila lines carrying gal4 and UAS, respectively, produces progeny that conditionally express the gene of interest to which the UAS sequence is linked. This study was done with the aid of gal4-UAS system. Different gal4 can promote gene overexpression in different regions, and en-gal4 used in this study was expressed in Drosophila P region, and internal controls were performed in the A and P regions of the same wing, or wild-type comparison was performed. Compared with other non-model insects, the microinjection method is adopted to overexpress a certain gene, and the gal4-UAS overexpression gene is more durable and stable and has no mechanical damage. En-gal4 virgins, UAS-GFP males and UAS-miR-318 males adopted in the embodiment of the invention can be constructed by a conventional method.
Various elements of the Hippo signaling pathway in cotton bollworms have been identified and demonstrated to be conserved among fruit flies. Therefore, the miR-318 can be used as a potential target gene for preventing and controlling the cotton bollworm.
The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.
SEQUENCE LISTING
<110> Shandong university of agriculture
<120> miR-318 for regulating development of insect wings and application thereof in pest control
<130> 2020
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 21
<212> RNA
<213> Drosophila melanogaster
<400> 1
ggauacacac aguucaguuu g 21
Claims (10)
- Application of miR-318 in preparation of insect wing development inhibitor.
- 2. The use according to claim 1, wherein the insects are lepidopteran insects;preferably, the lepidopteran insect is a cotton bollworm.
- 3. The use of claim 1, wherein the nucleotide sequence of miR-318 is shown in SEQ ID No. 1.
- 4. The use of claim 1, wherein miR-318 inhibits insect wing development by inhibiting expression of Hippo signaling pathway transcription factor Scalloped.
- Application of miR-318 in preparation of pesticides.
- 6. The use according to claim 5, wherein the insecticide is a lepidopteran insect insecticide.
- 7. A method for controlling pests, said method comprising the steps of:over-expressing miR-318 in the body of the pest;or the miR-318 is used as an effective component to feed or spray pests.
- 8. The method according to claim 7, wherein the pest is a lepidopteran insect;preferably, the lepidopteran insect is a cotton bollworm.
- 9. An insect wing development inhibitor comprising an effective amount of miR-318 as an active ingredient.
- 10. An insecticide, wherein the insecticide comprises miR-318.
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