CN114350661B

CN114350661B - Application of wheat source miRNA in insect control

Info

Publication number: CN114350661B
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: 2023-06-20
Anticipated expiration: 2041-12-08
Also published as: CN114350661A

Abstract

The invention discloses application of wheat source miRNA in insect control, and the gene sequence of the miRNA is shown as Seq ID No. 1. The miRNA is tae-miR9666a-3p derived from wheat, and can target a plurality of lethal target genes reported by brown planthoppers and other insects. The survival rate of the artificially synthesized tie-miR 9666a-3p miRNA mimic is obviously reduced compared with that of a control group within 7 days after the artificially synthesized tie-miR 9666a-3p miRNA mimic is injected into the insect body. Because tae-miR9666a-3p is miRNA of wheat source, wheat is daily life food, so the fragment is safe to human, can be used as a novel pesticide for safely and effectively controlling phytophagous pests, and can be directly used and transgenic plants.

Description

Application of wheat source miRNA in insect control

Technical Field

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

Background

microRNA (miRNAs) is a broad class of single-stranded non-coding RNA 19-24 nucleotides in length that negatively regulates gene expression at post-transcriptional levels. Starting from the discovery of lin-4 in caenorhabditis elegans (c.elegans), thousands of mirnas have been identified in plants, animals and other eukaryotes. miRNAs can effectively inhibit translation of target mRNA (messenger RNA) or mediate its degradation 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, whereas mirnas that bind to a single mRNA typically function in a synergistic manner. mirnas play a key role in a wide range of biological processes, including developmental time, 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 perform biological functions by regulating gene expression in various tissues. miR168a of rice origin was demonstrated to be able to modulate LDLRAP1 of mice by feeding into the blood of mammals to alter the physiological state of the mice; the miRNA of the honeysuckle source is helpful for resisting viruses of mice; the plant source miR167e-5p can target the beta-catenin of the mice to further inhibit the proliferation of intestinal epithelial cells. These studies indicate that uptake of plant-derived mirnas may contribute to physiological changes in animals. However, there are few studies on the interaction relationship between plant-derived miRNAs and insects, and no report has been made on the discovery of plant-derived miRNAs capable of controlling pests.

Brown planthoppers are monophagic hemiptera insects, have long-distance migratory habits, are primary pests on current rice in China and many Asian countries, mainly suck phloem juice of the rice through piercing-sucking mouthparts of the brown planthoppers, and can spread various rice diseases. In 9 months in 2020, brown planthoppers are listed in a list of crop diseases and insect pests by the rural department of China due to great harm to rice planting industry production. At present, a lot of target genes for preventing and controlling brown planthoppers are developed, but few miRNA small molecules for preventing and controlling brown planthoppers are available, and research reports for preventing and controlling brown planthoppers in a plant source miRNA mode are not available.

The statements made in the background section do not constitute an admission that they are prior art to the present disclosure.

Disclosure of Invention

The present invention aims to solve at least one of the technical problems in the prior art described above. Therefore, the invention provides application of wheat source miRNA in insect control, and the miRNA can be effectively applied to insect control.

According to one aspect of the invention, there is provided the use of a wheat-derived miRNA, the gene sequence of which is shown in Seq ID No.1, in insect control.

According to a preferred embodiment of the invention, there is at least the following advantageous effect: the miRNA is tae-miR9666a-3p from wheat: 5'-cgguagggcuguaugauggcga-3', which may target a number of lethal target genes that have been reported for insects such as brown planthoppers. The survival rate of the artificially synthesized tie-miR 9666a-3p miRNA mimic is obviously reduced compared with that of a control group within 7 days after the artificially synthesized tie-miR 9666a-3p miRNA mimic is injected into the insect body. Because tae-miR9666a-3p is miRNA of wheat source, wheat is daily life food, so the fragment is safe to human, can be used as a novel pesticide for safely and effectively controlling phytophagous pests, and can be directly used and 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-miR 9666a-3 p) of the wheat source has an inhibiting effect on the growth of the rice pest hemiptera monophagia insect brown planthopper. Because the tae-miR9666a-3p is derived from wheat and is a main crop, the novel pesticide is safe to human beings, can be used as a safe and effective novel pesticide for preventing brown planthoppers, and can be directly used and used for transgenic plants; therefore, the tae-miR9666a-3p transgenic plant (namely, the plant body overexpresses the tae-miR9666a-3 p) has practical application value in brown planthopper control.

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

An insecticide contains wheat source miRNA, wherein the sequence of the wheat source miRNA is shown as a Seq ID No. 1.

In still another aspect, the present invention provides a pest control method comprising the steps of: wheat-derived mirnas, the sequence of which is shown as Seq ID No.1, are injected, fed or sprayed on insects and transgenic plants.

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

Drawings

The invention is further described with reference to the accompanying drawings and examples, in which:

FIG. 1 is a diagram showing the sequence information of miRNA and the comparison result with wheat transcriptome used in the embodiment of the present invention;

FIG. 2 shows survival rates of brown planthoppers of the present invention after injection of tae-miR9666a-3p and PBS for 0h-168 h.

Detailed Description

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

In the description of the present invention, the descriptions of the terms "one embodiment," "some embodiments," "illustrative embodiments," "examples," "specific examples," or "some examples," etc., mean 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, schematic representations of the above terms do not necessarily refer to the same embodiments or examples. 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 the insect-resistant miRNA which can prevent and treat brown planthoppers and is safe and environment-friendly, a plant-source miRNA library is used as a small molecule library, and the brown planthopper full transcriptome is used as a target gene library to predict brown planthopper target genes of the plant-source miRNA. It was predicted that there were 61 possible lethal target genes for tae-miR9666a-3 p. Therefore, the survival rate of brown planthopper nymphs after the injection of the tae-miR9666a-3p is obviously reduced within 168 hours compared with a control group by taking the tae-miR9666a-3p as a follow-up study object. Therefore, the tie-miR 9666a-3p has potential insect resistance value.

Because the tae-miR9666a-3p is derived from wheat, the plant belongs to the class of human staple food plants and is safe to human beings. Therefore, constructing the transgenic plant based on the tee-miR 9666a-3p has practical application value for pest control.

Examples

Application of tae-miR9666a-3p in brown planthopper control. The following studies were performed to investigate the control mechanism:

screening the reported lethal genes of brown planthoppers, and extracting the 3' UTR sequence of the lethal genes. The installation package of PITA software in the Linux environment is downloaded on the PITA website (https:// genie.weizmann.ac.il/pubs/mir07/mir 07_exe.html) of target gene prediction software, and the installation is further decompressed. And calling a tee-miR 9666a-3p sequence and a 3' UTR database of the brown planthopper lethal target in a Linux system to predict target genes.

The experimental results are shown in table 1 below:

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

According to the 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 performed on the tae-miR9666a-3 p. The method comprises the following steps:

in this example, 5-year nymphs of hemiptera insects were injected with 0.5. Mu.g of artificially synthesized tae-miR9666a-3p (experimental group) and Phosphate Buffer (PBS) (control group), respectively, in a volume of 50nL. The injected insects are placed on rice at the initial stage of tillering for feeding, and are separated by a breathable plastic cover respectively, and the survival rate is counted every 24 hours. The observation results are shown in FIG. 2, and can be seen that the survival rate of the tae-miR9666a-3p is 25% at 168h after the injection of the tae-miR9666a-3p, and the survival rate of the control group is 88%, so that the survival rate of brown planthoppers can be obviously reduced by the tae-miR9666a-3 p. At the same time, it is also predicted that it can also 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 one of ordinary skill in the art without departing from the spirit of the present invention. Furthermore, embodiments of the invention and features of the embodiments may be combined with each other without conflict.

Sequence listing

<110> university of south China

<120> 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. An application of wheat source miRNA in insect control, which is characterized in that: the gene sequence of the miRNA is shown as a Seq ID No.1, and the insect is brown planthopper.

2. The use of a wheat-derived miRNA according to claim 1 for insect control, characterized in that: the application is to kill insect bodies.

3. The application of wheat source miRNA in the preparation of pesticides is characterized in that: the gene sequence of the miRNA is shown as the sequence of the Seq ID No.1, and the pesticide is used for killing brown planthoppers.

4. An insecticide, characterized in that: the pesticide contains wheat source miRNA, the sequence of the wheat source miRNA is shown as a Seq ID No.1, and the pesticide is used for killing brown planthoppers.

5. A pest control method characterized by: the method comprises the following steps: a wheat source miRNA, the sequence of which is shown as Seq ID No.1, is injected, fed or sprayed onto insects and transgenic plants, said pest being selected from brown planthoppers.