CN114350661A

CN114350661A - Application of wheat source miRNA in insect prevention and treatment

Info

Publication number: CN114350661A
Application number: CN202111492457.5A
Authority: CN
Inventors: 郑思春; 林羿光; 华丽君
Original assignee: South China Normal University
Current assignee: South China Normal University
Priority date: 2021-12-08
Filing date: 2021-12-08
Publication date: 2022-04-15
Anticipated expiration: 2041-12-08
Also published as: CN114350661B

Abstract

The invention discloses an application of a wheat source miRNA in insect prevention and treatment, wherein the gene sequence of the miRNA is shown as Seq ID No. 1. The miRNA is a tae-miR9666a-3p derived from wheat, and can target a plurality of lethal target genes reported by insects such as brown planthopper and the like. Injecting artificially synthesized tae-miR9666a-3p miRNA mimetibody into the polypide, wherein the survival rate is obviously reduced in 7 days compared with that of a control group. Because the tae-miR9666a-3p is miRNA of wheat source, and the wheat is food for daily life of people, the fragment is safe to people, can be used as a safe and effective novel pesticide for preventing and controlling phytophagous pests, and can be directly used for transgenic plants.

Description

Application of wheat source miRNA in insect prevention and treatment

Technical Field

The invention belongs to the technical field of insect prevention and treatment, and particularly relates to application of wheat source miRNA in insect prevention and treatment.

Background

microRNAs (miRNAs) are a wide range of single-stranded non-coding RNAs with a length of 19-24 nucleotides, and can negatively regulate gene expression at the post-transcriptional level. Thousands of mirnas have been identified in plants, animals and other eukaryotes starting with the discovery of lin-4 in caenorhabditis elegans (c. miRNAs can effectively inhibit translation or mediate degradation of target mRNA (messenger RNA) by the principle of complementary base pairing. A single miRNA is typically capable of recognizing hundreds of different mRNA transcripts. Likewise, one mRNA molecule typically has multiple miRNA binding sites, while mirnas that bind to a single mRNA typically act in a synergistic manner. mirnas play key roles in a wide range of biological processes, including developmental timing, cell differentiation, proliferation, apoptosis, and metabolism.

In past studies, plant-derived mirnas have been reported to cross the strict gastrointestinal barrier of animals, enter the blood circulation, and further exert biological functions by regulating gene expression in various tissues. Rice-derived miR168a was shown to be able to modulate LDLRAP1 in mice by feeding into the blood of mammals to alter the physiological state of the mice; the miRNA of the honeysuckle source is beneficial to the mice to resist viruses; the plant-derived miR167e-5p can target the beta-catenin of the mouse to further inhibit the proliferation of intestinal epithelial cells. These studies indicate that the uptake of plant-derived mirnas may contribute to physiological changes in animals. However, the research on the interaction relationship between plant-derived mirnas and insects is very few, and the discovery of plant-derived mirnas with pest control functions is not reported.

The brown planthopper is a monophagous hemipteran insect, has the habit of long-distance migration, is the leading pest on the current rice in China and many Asian countries, mainly sucks phloem juice of the rice through a piercing-sucking mouthpart, and can transmit various rice diseases. In 9 months of 2020, brown planthopper is listed as a category of crop pest and disease records in rural parts of agriculture in China due to great harm to the production of rice planting industry. At present, a lot of target genes for controlling the brown planthopper are developed, but few miRNA micromolecules which can be used for controlling the brown planthopper exist, and no research report for controlling the brown planthopper in a plant source miRNA form exists.

Statements in this background are not admitted to be prior art to the present disclosure.

Disclosure of Invention

The present invention is directed to solving at least one of the problems of the prior art described above. Therefore, the invention provides an application of a wheat source miRNA in insect control, and the miRNA can be effectively applied to insect control.

According to one aspect of the invention, the application of the wheat source miRNA in insect control is provided, wherein the gene sequence of the miRNA is shown as Seq ID No. 1.

According to a preferred embodiment of the present invention, at least the following advantages are provided: the miRNA is a gene Tae-miR9666a-3p derived from wheat: 5'-cgguagggcuguaugauggcga-3', which can target multiple lethal target genes reported by insects such as Nilaparvata lugens. Injecting artificially synthesized tae-miR9666a-3p miRNA mimetibody into the polypide, wherein the survival rate is obviously reduced in 7 days compared with that of a control group. Because the tae-miR9666a-3p is miRNA of wheat source, and the wheat is food for daily life of people, the fragment is safe to people, can be used as a safe and effective novel pesticide for preventing and controlling phytophagous pests, and can be directly used for transgenic plants.

In some embodiments of the invention, the application is killing and/or inhibiting the growth of insect bodies.

In some embodiments of the invention, the insect is a rice pest.

In some embodiments of the invention, the rice pest is brown planthopper. The miRNA (tae-miR9666a-3p) derived from wheat has an effect of inhibiting the growth of the rice pest hemiptera monotrophic insect brown planthopper. Because the tae-miR9666a-3p is derived from wheat and is a main crop, the pesticide is safe to human beings, can be used as a safe and effective novel pesticide for preventing and treating brown planthopper, and can be directly used and used as a transgenic plant; therefore, the tae-miR9666a-3p transgenic plant (namely the plant over-expresses tae-miR9666a-3p) has practical application value in controlling brown planthopper.

The invention also provides application of the wheat source miRNA in insect control, wherein the gene sequence of the miRNA is shown as Seq ID No. 1.

The pesticide contains a wheat source miRNA, and the sequence of the wheat source miRNA is shown in Seq ID No. 1.

In still another aspect of the present invention, there is provided a pest control method including the steps of: wheat source miRNA is injected, fed or sprayed to insects and transgenic plants, and the sequence of the wheat source miRNA is shown as Seq ID No. 1.

In some embodiments of the invention, the pest is brown planthopper.

Drawings

The invention is further described with reference to the following figures and examples, in which:

FIG. 1 is a diagram of miRNA sequence information and alignment results with wheat transcriptome used in an embodiment of the invention;

FIG. 2 shows the survival rate of brown planthopper at 0-168 h after injection of tae-miR9666a-3p and PBS.

Detailed Description

The concept and technical effects of the present invention will be clearly and completely described below in conjunction with the embodiments to fully understand the objects, features and effects of the present invention. It is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments, and those skilled in the art can obtain other embodiments without inventive effort based on the embodiments of the present invention, and all embodiments are within the protection scope of the present invention. The test methods used in the examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are commercially available reagents and materials unless otherwise specified.

In the description of the present invention, reference to the description of the terms "one embodiment," "some embodiments," "an illustrative embodiment," "an example," "a specific example," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

In order to screen safe and environment-friendly insect-resistant miRNA capable of preventing and treating Nilaparvata lugens, a plant-derived miRNA library is used as a small molecule library, a Nilaparvata lugens complete transcriptome is used as a target gene library, and Nilaparvata lugens target gene prediction of plant-derived miRNA is carried out. 61 possible lethal target genes of the tae-miR9666a-3p are predicted. Therefore, the survival rate of the brown planthopper nymphs is remarkably reduced within 168h compared with that of the control group after the injection of the tae-miR9666a-3p by taking the tae-miR9666a-3p as a follow-up research object. Therefore, the tae-miR9666a-3p has potential insect-resistant value.

The tae-miR9666a-3p is derived from wheat, belongs to one of staple food plants of human, and is safe to human. Therefore, the construction of transgenic plants for pest control based on the tae-miR9666a-3p has practical application value.

Examples

Application of tae-miR9666a-3p in controlling brown planthopper. The following studies were carried out to study the control mechanism:

screening lethal genes reported by brown planthopper, and extracting a 3' UTR sequence of the lethal genes. The installation package of the PITA software in the Linux environment is downloaded at the target gene prediction software PITA website (https:// genie. weizmann. ac. il/pubs/mir07/mir07_ exe. html) and installation is further decompressed. Calling a sequence of tae-miR9666a-3p and a 3' UTR database of a brown planthopper lethal target in a Linux system to perform target gene prediction.

The results of the experiment are shown in table 1 below:

TABLE 1 Tae-miR9666a-3p prediction of target Gene binding site with Brown planthopper reported lethal Gene 3' UTR

According to results shown in Table 1, the tae-miR9666a-3p can target 3' UTR sites of a plurality of brown planthopper lethal target genes, so that subsequent insecticidal effect verification is carried out on the tae-miR9666a-3 p. The method comprises the following specific steps:

in this example, 0.5. mu.g of artificially synthesized tae-miR9666a-3p (experimental group) and Phosphate Buffered Saline (PBS) (control group) were injected into 5-year-old nymphs of hemiptera insects, respectively, and the injection volume was 50 nL. The injected polypide is put on rice at the early tillering stage for feeding, and is separated by a breathable plastic cover, and the survival rate is counted every 24 hours. The observation results are shown in FIG. 2, and it can be seen that the survival rate of the tae-miR9666a-3p is 25% at 168h after the injection of the tae-miR9666a-3p, while the survival rate of the control group is 88%, thereby indicating that the survival rate of the brown planthopper can be obviously reduced by the tae-miR9666a-3 p. At the same time, it is also expected that it can be used for controlling similar insects.

The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the gist of the present invention. Furthermore, the embodiments of the present invention and the features of the embodiments may be combined with each other without conflict.

Sequence listing

<110> university of south China

Application of wheat source miRNA in insect control

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 22

<212> RNA

<213> Triticum aestivuml

<400> 1

cgguagggcu guaugauggc ga 22

Claims

1. The application of the wheat source miRNA in insect control is characterized in that: the gene sequence of the miRNA is shown as Seq ID No. 1.

2. The use of a wheat-derived miRNA according to claim 1 for insect control, wherein: the application is to kill and/or inhibit the growth of insect bodies.

3. The use of a wheat-derived miRNA according to claim 2 for insect control, wherein: the insects are rice pests.

4. The use of a wheat-derived miRNA according to claim 3 for insect control, wherein: the rice pest is brown planthopper.

5. An application of wheat source miRNA in pesticide preparation is characterized in that: the gene sequence of the miRNA is shown as Seq ID No. 1.

6. An insecticide, characterized by: the pesticide contains wheat source miRNA, and the sequence of the wheat source miRNA is shown in Seq ID No. 1.

7. A method for controlling pests, characterized by: the method comprises the following steps: wheat source miRNA is injected, fed or sprayed to insects and transgenic plants, and the sequence of the wheat source miRNA is shown as Seq ID No. 1.

8. The method of controlling insects of claim 7, wherein: the pests are selected from brown planthopper.