CN114350661B - Application of a wheat-derived miRNA in insect control - Google Patents

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 a wheat-derived miRNA in insect control

technical field

The invention belongs to the technical field of insect control, and in particular relates to the application of wheat-derived miRNA in insect control.

Background technique

microRNAs (miRNAs) are a broad class of single-stranded non-coding RNAs, 19-24 nucleotides in length, that negatively regulate gene expression at the post-transcriptional level. Starting with the discovery of lin-4 in C. elegans, thousands of miRNAs have been identified in plants, animals and other eukaryotes. Through the principle of complementary base pairing, miRNAs can effectively inhibit the translation of target mRNA (messenger RNA) or mediate its degradation. A single miRNA is often capable of recognizing hundreds of different mRNA transcripts. Likewise, an mRNA molecule often has multiple miRNA binding sites, and miRNAs bound to a single mRNA often function in a cooperative 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 were reported to pass through 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 has been shown to regulate LDLRAP1 in mice to change the physiological state of mice by eating into the blood of mammals; honeysuckle-derived miRNA helps mice resist viruses; plant-derived miR167e-5p can target mice β-catenin further inhibits intestinal epithelial cell proliferation. These studies suggest that intake of plant-derived miRNAs may contribute to physiological changes in animals. However, there are very few studies on the interaction between plant-derived miRNAs and insects, let alone the discovery of plant-derived miRNAs that can control pests.

The brown planthopper is a monophagous hemiptera insect with the habit of long-distance migration. It is currently the primary pest of rice in my country and many Asian countries. It mainly sucks the phloem juice of rice through its piercing-sucking mouthparts. Spread many rice diseases. In September 2020, the brown planthopper was included in the list of first-class crop diseases and insect pests by the Ministry of Agriculture and Rural Affairs of China because of its great harm to the rice planting industry. At present, many target genes have been developed for the control of BPH, but there are very few small miRNA molecules that can be used to control BPH, and there is no research report on the control of BPH in the form of plant-derived miRNA.

The content stated in this background technology does not represent an admission that it belongs to the disclosed prior art.

Contents of the invention

The present invention aims to solve at least one of the technical problems in the above-mentioned prior art. For this reason, the present invention proposes the application of a wheat-derived miRNA in insect control, and the miRNA can be effectively used in insect control.

According to one aspect of the present invention, an application of wheat-derived miRNA in insect control is proposed, and the gene sequence of the miRNA is shown in Seq ID No.1.

According to a preferred embodiment of the present invention, it has at least the following beneficial effects: the miRNA is tae-miR9666a-3p: 5'-cgguagggcuguaaugauggcga-3' derived from wheat, which may target multiple insects such as brown planthoppers, which have been reported lethal target genes. Injecting artificially synthesized tae-miR9666a-3p miRNA mimics into worms, the survival rate within 7 days was significantly lower than that of the control group. Since tae-miR9666a-3p is a wheat-derived miRNA, and wheat is a daily food for humans, this fragment is safe for humans and can be used as a safe and effective new type of insecticide for the control of phytophagous pests, and can be directly used and transgenic plants .

In some embodiments of the present invention, the application is to kill insect parasites and/or inhibit the growth of insect parasites.

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

In some embodiments of the present invention, the rice pest is brown planthopper. Wheat-derived miRNA (tae-miR9666a-3p) has inhibitory effect on the growth of brown planthopper, a monophagous insect of Hemiptera, which is a rice pest. Since tae-miR9666a-3p is derived from wheat, a major crop, it is safe for humans and can be used as a safe and effective new insecticide for controlling brown planthopper, which can be directly used and transgenic plants; therefore tae-miR9666a-3p transgenic plants (that is, overexpression of tae-miR9666a-3p in plants) will have practical application value in the control of BPH.

Another aspect of the present invention also provides an application of wheat-derived miRNA in insect control, the gene sequence of the miRNA is shown in Seq ID No.1.

An insecticide, the insecticide contains wheat-derived miRNA, and the sequence of the wheat-derived miRNA is shown in Seq ID No.1.

Another aspect of the present invention provides a pest control method, comprising the following steps: injecting, feeding or spraying wheat-derived miRNA to insects and transgenic plants, the sequence of the wheat-derived miRNA is shown in Seq ID No.1.

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

Description of drawings

The present invention will be further described below in conjunction with accompanying drawing and embodiment, wherein:

Fig. 1 is the miRNA sequence information used in the embodiment of the present invention and the comparison result figure with wheat transcriptome;

Fig. 2 is the survival rate of brown planthoppers injected with tae-miR9666a-3p and PBS according to the embodiment of the present invention from 0h to 168h.

Detailed ways

The conception and technical effects of the present invention will be clearly and completely described below in conjunction with the embodiments, so as to fully understand the purpose, features and effects of the present invention. Apparently, the described embodiments are only some of the embodiments of the present invention, rather than all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative efforts belong to The protection scope of the present invention. The test methods used in the examples are conventional methods unless otherwise specified; the used materials, reagents, etc., are commercially available reagents and materials unless otherwise specified.

In the description of the present invention, reference to the terms "one embodiment," "some embodiments," "exemplary embodiments," "examples," "specific examples," or "some examples" is intended to mean that the embodiments are A specific feature, structure, material, or characteristic described by 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 embodiment or example. Furthermore, the specific 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 insect-resistant miRNAs that can control BPH and are safe and environmentally friendly, the plant-derived miRNA library was used as a small molecule library, and the whole transcriptome of BPH was used as a target gene library to predict the BPH target genes of plant-derived miRNAs. After prediction, it was found that there were 61 possible lethal target genes of tae-miR9666a-3p. Therefore, taking tae-miR9666a-3p as the follow-up research object, the survival rate of N. lugens nymphs after injection of tae-miR9666a-3p was significantly lower than that of the control group within 168h. Therefore, tae-miR9666a-3p has potential insect resistance value.

Since tae-miR9666a-3p is derived from wheat, it belongs to a class of human staple food plants and is safe for humans. Therefore, constructing transgenic plants based on tae-miR9666a-3p for pest control has practical application value.

Example

Application of tae-miR9666a-3p in the control of brown planthopper. In order to study its prevention and control mechanism, the following studies have been carried out:

The reported lethal genes of N. lugens were screened, and the 3'UTR sequences of the lethal genes were extracted. Download the installation package of the PITA software in the Linux environment from the target gene prediction software PITA website (https://genie.weizmann.ac.il/pubs/mir07/mir07_exe.html), and further decompress and install it. In the Linux system, the tae-miR9666a-3p sequence and the 3'UTR database of the lethal target of brown planthopper were called to predict the target gene.

The experimental results are shown in Table 1 below:

Table 1 Prediction of target gene binding sites between tae-miR9666a-3p and 3’UTR of reported lethal genes in N. lugens

According to the results in Table 1, tae-miR9666a-3p may target the 3'UTR sites of multiple lethal target genes of N. lugens. Therefore, tae-miR9666a-3p will be followed up to verify the insecticidal effect. details as follows:

In this example, 0.5 μg of artificially synthesized tae-miR9666a-3p (experimental group) and phosphate buffered saline (PBS) (control group) were injected into 5th instar nymphs of Hemiptera insects respectively, with an injection volume of 50 nL. The injected worms were reared on paddy rice at the early stage of tillering, separated by air-permeable plastic covers, and the survival rate was counted every 24 hours. The observation results are shown in Figure 2, from which it can be seen that at 168h after injection of tae-miR9666a-3p, the survival rate of tae-miR9666a-3p was 25%, while the survival rate of the control group was 88%, thus indicating that tae- miR9666a-3p can significantly reduce the survival rate of N. lugens. 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 above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned embodiments, and within the scope of knowledge of those of ordinary skill in the art, various modifications can be made without departing from the spirit of the present invention. Variety. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other if there is no conflict.

sequence listing

<110> South China Normal University

<120> Application of a wheat-derived 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 (5)

1. An application of wheat-derived miRNA in insect control, characterized in that: the gene sequence of the miRNA is as shown in SeqID No.1, and the insect is brown planthopper. 2. The application of wheat-derived miRNA in insect control according to claim 1, characterized in that: the application is to kill insects. 3. An application of wheat-derived miRNA in the preparation of insecticide, characterized in that: the gene sequence of the miRNA is shown in SeqID No.1, and the insecticide is used to kill brown planthopper. 4. An insecticide, characterized in that: the insecticide contains wheat-derived miRNA, the sequence of which is shown in Seq ID No.1, and the insecticide is used to kill brown planthopper. 5. A pest control method, characterized in that: comprising the steps of: injecting, feeding or spraying wheat source miRNA on insects and transgenic plants, the sequence of said wheat source miRNA is as shown in Seq ID No.1, said The pests are selected from brown planthoppers.

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