Method for preventing and treating pests by using plant endogenous small RNA (Ribonucleic Acid) and application

Abstract

The invention relates to a method and application for positively regulating the defense response of plants to pests, and the method comprises the following steps: the plants are contacted with small RNA (Ribonucleic Acid) to enhance the resistance of the plants to the pests, and the small RNA comprises plant endogenous 22nt-siRNA. Researches find that the endogenous 22nt-siRNA of the plant can improve the resistance of the plant to phytophagous insects, and the 22nt-siRNA can specifically target a field phytophagous insect gene while enhancing the resistance of the plant, has the biological prevention and control advantage of no environmental pollution, and can be used for preventing and treating the phytophagous insects. Therefore, the method plays an important role in forward regulation of defense response of plants to pests and prevention and control of plant pests.

Description

Technical Field

[0001] The present application relates to the technical field of pest control, and in particular, to a method and application of controlling harmful organisms using plant endogenous small RNA. Background Art

[0002] RNA interference (RNAi) refers to the highly conserved phenomenon in the evolutionary process, which is induced by double-stranded RNA (dsRNA) and causes efficient and specific degradation of homologous mRNA.

[0003] The application of dsRNA in agriculture, especially in pest control, can be achieved in three ways: ① host-induced gene silencing (HIGS), ② virus-induced gene silencing (VIGS), and ③ spray-induced gene silencing (SIGS).

[0004] HIGS requires transgenic crops to express dsRNA targeting pests or pathogens. Plant-mediated insect RNAi technology overexpresses double-stranded RNA of insect gene fragments in plants. These double-stranded RNAs and the small RNAs produced enter insect cells during the insect feeding process and exert the function of RNAi.

[0005] VIGS is based on virus engineering to produce sufficient dsRNA in insects. This technology is applied to various microorganisms, such as bacteria, yeast or fungi, and these microorganisms are used as carriers of specific homologous sequences through scientific means to continuously produce si / dsRNA through the process of microbial infection of the host.

[0006] SIGS is more similar to the application method of traditional pesticides. It sprays or injects dsRNA directly into the environment through non-GMO means, and works through pest feeding or pathogen infection. Since dsRNA molecules can easily denature or degrade in the environment, the SIGS method requires special formulations to improve the stability of the active ingredients. If possible, the efficiency of RNAi can also be improved through appropriate formulations.

[0007] However, traditional host-mediated insect RNAi technology and exogenous dsRNA-mediated insect RNAi technology have obvious efficiency problems, which are manifested in the fact that dsRNA is easily degraded by dsRNase in insects and the dsRNA transport efficiency is low.

[0008] Therefore, how to improve the efficiency of RNAi of pests is a difficult point in plant pest control. Summary of the invention

[0009] In order to solve the above problems and improve the RNAi efficiency of plant pests, the first purpose of the present application is to provide a method for positively regulating the plant's defense response to pests, contacting the plant with small RNA to enhance the plant's resistance to pests, and the small RNA includes plant endogenous 22nt-siRNA.

[0010] In one embodiment, the plant is engineered to accumulate 22nt-siRNA in the plant;

[0011] Optionally, the transformation comprises constructing a plant mutant by at least one of gene mutation, gene editing, gene silencing and RNA degradation;

[0012] Optionally, the plant is selected from at least one of Arabidopsis thaliana, cabbage, corn, wheat, rice, millet, soybean, tomato, Brachypodium distachyon, Water lily, Cinnamomum camphora, Selaginella chinensis, Physcomitrella patens and Marchantia polyphylla.

[0013] In one embodiment, the plant mutant satisfies any one of the following conditions (1) to (3);

[0014] (1) Plant mutants lacking DCL4 protein or DRB4 protein;

[0015] (2) plant mutants lacking the bidirectional cytoplasmic RNA degradation pathway;

[0016] (3) plant mutants lacking the cytoplasmic RNA degradation pathway and at least one of the DCL4 protein and the DRB4 protein;

[0017] Optionally, the plant mutant includes at least one of dcl4-2 single mutant plants, drb4-1 single mutant plants, ein5-1 ski2-3 double mutant plants, ein5-1 dcl4-2 double mutant plants, ski2-2 dcl4-2 double mutant plants, ein5-1 drb4-1 double mutant plants and ski2-2 drb4-1 double mutant plants.

[0018] In one embodiment, the plant endogenous 22nt-siRNA includes a 22nt-siRNA derived from a plant encoding gene produced by a plant mutant;

[0019] Optionally, the plant encoding gene includes the NIA1 gene and the NIA2 gene.

[0020] The second objective of the present application is to provide the use of small RNA in the preparation of plant pest control agents, wherein the small RNA includes plant endogenous 22nt-siRNA.

[0021] In one embodiment, the plant pest control agent includes small RNA or a composition comprising small RNA;

[0022] Optionally, the composition further comprises at least one of a carrier, an excipient or a diluent for the small RNA;

[0023] Optionally, the plant pests include at least one of animals and microorganisms;

[0024] Optionally, the plant pests include phytophagous insects.

[0025] In one embodiment, the plant endogenous 22nt-siRNA includes a 22nt-siRNA derived from a plant encoding gene produced by a plant mutant;

[0026] Optionally, the plant mutant satisfies at least one of the following conditions (1) to (3);

[0027] (1) Plant mutants lacking DCL4 protein or DRB4 protein;

[0028] (2) plant mutants lacking the bidirectional cytoplasmic RNA degradation pathway;

[0029] (3) plant mutants lacking the cytoplasmic RNA degradation pathway and at least one of the DCL4 protein and the DRB4 protein;

[0030] Optionally, the plant encoding gene includes the NIA1 gene and the NIA2 gene;

[0031] Optionally, the plant mutant comprises at least one of a dcl4-2 single mutant plant, a drb4-1 single mutant plant, an ein5-1 ski2-3 double mutant plant, an ein5-1 dcl4-2 double mutant plant, a ski2-2 dcl4-2 double mutant plant, an ein5-1 drb4-1 double mutant plant, and a ski2-2 drb4-1 double mutant plant;

[0032] Optionally, the plant is selected from at least one of Arabidopsis thaliana, cabbage, corn, wheat, rice, millet, soybean, tomato, Brachypodium distachyon, Water lily, Cinnamomum camphora, Selaginella chinensis, Physcomitrella patens and Marchantia polyphylla.

[0033] The third object of the present application is to provide a plant pest control agent prepared according to the above application.

[0034] The fourth object of the present application is to provide a method for controlling plant pests, wherein the above-mentioned plant pest control agent is contacted with plant pests to inhibit the growth of the plant pests.

[0035] In one embodiment, the plant pests include at least one of animals and microorganisms;

[0036] Optionally, the plant pests include phytophagous insects. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the specific implementation methods of the present application or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0038] Figure 1 The experimental results of the 22nt-siRNA-enriched mutants provided in Example 1 of the present application for enhanced resistance to feeding by cotton bollworms are as follows: Figure 1 A in the middle shows the weight changes and size of cotton bollworms after feeding on rdr6-11 and sgs3-11 mutant plants for 4 days; Figure 1 Middle B shows the weight change of cotton bollworms after feeding on plants with different 22nt-siRNA-enriched mutants for 4 days and the size picture of cotton bollworms; Figure 1 Middle C represents GO cluster analysis of genes upregulated by ein5-1 dcl4-2; Figure 1 Middle D shows the statistics of defense-related gene expression in the ein5-1 dcl4-2 mutant;

[0039] Figure 2 The experimental results provided in Example 2 of the present application show that the resistance of siRNA-related mutants to cotton bollworm depends on RDR6 protein and DCL2 protein; Figure 2 A in the middle shows the weight change and size of cotton bollworms feeding on ein5-1 dcl4-2 phenotype recovery-related mutant plants 4 days later; Figure 2 Middle B shows the weight change and size of cotton bollworms feeding on ein5-1 ski2-3 phenotype recovery-related mutant plants 4 days later;

[0040] Figure 3 The insect test results of the mutants with DCL protein and DRB4 protein deficiency provided in Example 3 of the present application;

[0041] Figure 4The experimental results provided in Example 4 of the present application show that the mutant lacking anthocyanins and sinigrin cannot restore the insect-resistant phenotype of ein5-1dcl4-2;

[0042] Figure 5 The siRNA enrichment-related mutants provided in Example 5 of the present application have a broad spectrum of resistance to lepidopteran insects; Figure 5 A in the middle shows the insect test results and insect size of fall armyworm after feeding on the corresponding mutant enriched with 22nt-siRNA for 4 days; Figure 5 Middle B shows the insect test results and insect size of Plutella xylostella after feeding on the corresponding mutant enriched with 22nt-siRNA for 3 days. DETAILED DESCRIPTION

[0043] References to embodiments of the present application will now be provided in detail, one or more examples of which are described below. Each example is provided as an explanation rather than a limitation of the present application. In fact, it will be apparent to those skilled in the art that various modifications and variations may be made to the present application without departing from the scope or spirit of the present application. For example, a feature described or described as part of one embodiment may be used in another embodiment to produce a further embodiment.

[0044] Therefore, it is intended that the present application covers such modifications and variations that fall within the scope of the appended claims and their equivalents. Other objects, features and aspects of the present application are disclosed in or are apparent from the following detailed description. It will be appreciated by those of ordinary skill in the art that this discussion is merely a description of exemplary embodiments and is not intended to limit the broader aspects of the present application.

[0045] Small RNA refers to a class of highly conserved RNA molecules with a length of 18-32nt, mainly including miRNA, siRNA, snoRNA and piRNA. They are important regulatory factors of life activities and play an important role in physiological processes such as gene expression regulation, individual development, metabolism and disease occurrence.

[0046] RNA interference is an important mechanism for biological immunity against viral invasion. RNA interference regulates target gene RNA through small RNAs with a length of 20-24 nucleotides (nt).

[0047] However, the functions and mechanisms of some small RNAs, especially endogenous 22nt-siRNAs in plants, are still unclear.

[0048] In order to solve at least one of the above technical problems, the first aspect of the present application provides a method for positively regulating a plant's defense response to pests, wherein the plant is contacted with a small RNA to enhance the plant's resistance to pests, wherein the small RNA includes a plant endogenous 22nt-siRNA.

[0049] In the present application, plant endogenous 22nt-siRNA refers to plant RNA that is cleaved by Dicer-like 2 (DCL2) protein in vivo to form a small RNA with a length of 22nt.

[0050] Specifically, the source of plant endogenous 22nt-siRNA is well known, and its sequence can be obtained by known sequencing methods. In practical applications, plant endogenous 22nt-siRNA can be directly obtained in the plant by modifying the plant, or can be obtained by in vitro synthesis based on its sequence.

[0051] For the above-mentioned different pathways for producing small RNAs, the plant encoding genes from which the plant endogenous 22nt-siRNAs are derived are also different, but all can enhance the plant's resistance to insects.

[0052] Accordingly, in order to enhance the resistance of plants to harmful organisms, small RNA, i.e., endogenous 22nt-siRNA of plants, can be applied to the surface of plants, or plants can be modified to accumulate endogenous 22nt-siRNA in the plants. Specifically, the modification of plants can be to construct plant mutants by at least one of gene mutation, gene editing and gene silencing, or to inhibit the PTGS signaling pathway by targeted compounds.

[0053] In plants, due to the presence of DCL4 protein, DCL2 protein does not participate in the cleavage to produce 22nt-siRNA, and the activity of DCL2 protein will be significantly improved when the DCL4 protein function is lost. DRB4 protein helps DCL4 cleave double-stranded RNA to produce 21nt-siRNA by binding to DCL4 protein. The loss of DRB4 protein will also lead to a decrease in the activity of DCL4 protein, thereby increasing the activity of DCL2 protein.

[0054] In some specific embodiments, the plant mutant lacks at least one of the DCL4 protein and the DRB4 protein, which can enhance the activity of the DCL2 protein and thus produce accumulated 22nt-siRNA.

[0055] In some specific embodiments, the plant mutant lacks the cytoplasmic RNA degradation pathway and the DCL4 protein at the same time, which can enhance the activity of the DCL2 protein, thereby producing accumulated 22nt-siRNA, wherein the cytoplasmic RNA degradation pathway can be a unidirectional cytoplasmic RNA degradation pathway or a bidirectional cytoplasmic degradation pathway.

[0056] In some specific embodiments, the plant mutant lacks the cytoplasmic RNA degradation pathway and the DRB4 protein at the same time, which can enhance the activity of the DCL2 protein, thereby producing accumulated 22nt-siRNA, wherein the cytoplasmic RNA degradation pathway can be a unidirectional cytoplasmic RNA degradation pathway or a bidirectional cytoplasmic degradation pathway.

[0057] In some embodiments, plant mutants lacking the bidirectional cytoplasmic RNA degradation pathway can produce accumulated 22nt siRNA in plants via the classical RNAi pathway.

[0058] Among them, the "missing" of proteins or pathways mentioned above refers to the lack of protein function in individual plants and the obstruction of corresponding signal pathways.

[0059] In some specific embodiments, the plant mutants include at least one of dcl4-2 single mutant plants, drb4-1 single mutant plants, ein5-1ski2-3 double mutant plants, ein5-1 dcl4-2 double mutant plants, ski2-2 dcl4-2 double mutant plants, ein5-1drb4-1 double mutant plants and ski2-2 drb4-1 double mutant plants. The present application found that the above mutants can enhance resistance to pests, especially resistance to herbivorous insects.

[0060] According to existing studies, for ein5-1 dcl4-2 double mutant plants, the plant endogenous 22nt-siRNA includes 22nt-siRNA derived from 1182 protein-coding genes, and for ski2-2 dcl4-2 double mutant plants, the plant endogenous 22nt-siRNA includes 22nt-siRNA derived from 182 protein-coding genes, and ein5-1 dcl4-2 double mutant plants and ski2-2 dcl4-2 double mutant plants have 22nt-siRNA derived from the same 111 protein-coding genes. Most of the plant endogenous 22nt-siRNA comes from the plant coding genes NIA1 gene and NIA2 gene. The present application found that by constructing the above-mentioned plant mutants, it is possible to achieve the accumulation of 22nt-siRNA in the plant body and improve the resistance of the plant to harmful organisms.

[0061] In some embodiments, the plant is selected from at least one of Arabidopsis thaliana, Chinese cabbage, corn, wheat, rice, millet, soybean, tomato, Brachypodium distachyon, Water lily, Cinnamomum camphora, Selaginella chinensis, Physcomitrella patens and Marchantia polyphylla. All of the above plants can produce 22nt-siRNA that can enhance the insect resistance of plants.

[0062] Through research, the present application discovered that endogenous 22nt-siRNA in plants can improve the resistance of plants to herbivorous insects, and that 22nt-siRNA can not only enhance plant resistance, but also specifically target the genes of field herbivorous pests, and has the advantage of biological control without environmental pollution. Therefore, it plays an important role in positively regulating the defense response of plants to pests and controlling plant pests.

[0063] It should be noted that the plant endogenous 22nt-siRNA of the present application does not completely rely on the traditional anthocyanin and sinapinic acid glycoside signaling pathways for the prevention and control of pests. In the absence of anthocyanin and sinapinic acid glycoside signaling pathways, it still exhibits resistance to pests, especially resistance to herbivorous insects.

[0064] It is understandable that the endogenous 22nt-siRNA of the plant of the present application can specifically target the genes of plant pests, reduce the expression level of the targeted genes through RNA interference, thereby inhibiting the growth and development of plant pests, and further achieving the effect of biological control.

[0065] The second purpose of the present application is to provide the use of small RNA in the preparation of plant pest control agents, wherein the small RNA is RNA separated from the living plant and has the same sequence as the plant endogenous 22nt-siRNA, so as to reduce the expression level of the target gene through RNA interference, thereby inhibiting the growth and development of plant pests, thereby achieving the effect of biological control. Among them, the source of the plant endogenous 22nt-siRNA has been described in detail above and will not be repeated here.

[0066] In some embodiments, the plant pest control agent includes small RNA or a composition comprising small RNA to reduce the expression level of the targeted gene through RNA interference, thereby inhibiting the growth and development of the plant pests, thereby achieving a biological control effect.

[0067] In some specific embodiments, the composition also includes at least one of a carrier, excipient or diluent for small RNA, so that the small RNA can target specific genes of pests, reduce the expression level of the targeted genes through RNA interference, thereby inhibiting the growth and development of plant pests, and achieving the effect of biological control.

[0068] In some embodiments, the plant pest includes at least one of an animal and a microorganism.

[0069] In some embodiments, the plant pests are phytophagous insects.

[0070] In some embodiments, the plant is selected from at least one of Arabidopsis thaliana, Chinese cabbage, corn, wheat, rice, millet, soybean, tomato, Brachypodium distachyon, Nymphaea cyanea, Cinnamomum camphora, Selaginella chinensis, Physcomitrella patens and Marchantia polyphylla.

[0071] The third purpose of the present application is to provide a plant pest control agent prepared according to the above application, so as to reduce the expression level of targeted genes in pests through RNA interference, thereby inhibiting the growth and development of plant pests, and further achieving the effect of biological control.

[0072] The fourth object of the present application is to provide a method for controlling plant pests, by contacting the above-mentioned plant pest control agent with plant pests to inhibit the growth of plant pests, thereby inhibiting the growth and development of plant pests, and further achieving the effect of biological control.

[0073] In some embodiments, the plant pests include at least one of animals and microorganisms, and the plant pest control agent reduces the expression level of the targeted gene in the pest through RNA interference, thereby inhibiting the growth and development of the animals or microorganisms harmful to the plant, thereby achieving the effect of biological control.

[0074] In some embodiments, the plant pests are phytophagous insects.

[0075] The implementation scheme of the present application will be described in detail below with reference to examples.

[0076] This example uses existing 22nt-siRNA-enriched mutant plants and various small RNA-related genetic materials as well as herbivorous insects such as cotton bollworm, diamondback moth, and fall armyworm to conduct verification experiments. Among the plant mutant materials, single mutants were purchased from NASC (Nottingham Arabidopsis Stock Centre) or obtained by EMS (Ethylmethanesulfonate) mutagenesis in the laboratory, and double and multiple mutant materials were obtained by laboratory hybridization separation and identification.

[0077] The insect test was carried out using the following steps:

[0078] Select cotton bollworms or fall armyworms that have grown to the end of the 2nd instar and the beginning of the 3rd instar and have relatively consistent growth and group them. Each group has at least 24 cotton bollworms, which are raised in 12-well culture plates. Feed the cotton bollworms in each group with leaves of different mutant plants. Weigh and record the initial weight of the cotton bollworms before feeding. Change the food once a day during feeding. Start recording the weight changes after 3 days of feeding and continue to record until the 5th day. The experimental method of fall armyworm is basically the same as that of cotton bollworm.

[0079] The diamondback moth insect test experiment is basically the same. Diamondback moths with the same growth are selected and divided into groups, with at least 12 diamondback moths in each group. Every 6 diamondback moths are fed in a petri dish with leaves of various mutant Arabidopsis as food. The weight of the diamondback moths is weighed and recorded before feeding, and the weight gain of the diamondback moths is recorded after feeding for the corresponding number of days.

[0080] Example 1 22nt-siRNA Accumulating Mutants Enhance Plant Resistance to Herbivorous Insects

[0081] In order to clarify the role of siRNA in plant defense against herbivorous insects, especially chewing insects, this example firstly carried out insect test experiments using PTGS signal mutants rdr6-1 and sgs3-11 without siRNA, and found that the resistance of mutants rdr6-1 and sgs3-11 to cotton bollworm was significantly reduced. The specific experimental results are as follows: Figure 1 As shown in A.

[0082] Previous studies have shown that the double mutant plants ein5-1 ski2-3 exhibit growth and development defects and are accompanied by the accumulation of anthocyanins. At the same time, a large number of 21nt ct-siRNA and 22nt ct-siRNA from coding genes accumulate in the plant body. When the RNA degradation pathway and DCL4 protein are both missing, a large number of 22-nt siRNAs accumulate in the double mutant plants ein5-1 dcl4-2 and ski2-2 dcl4-2, resulting in severe growth and development defects and tolerance to nitrogen deficiency stress. DRB4 protein is a type of RNA binding protein that specifically binds to DCL4 protein and assists DCL4 protein in cleaving and producing 21nt-siRNA. The content of 22nt ct-siRNA in the double mutant plants ein5-1 drb4-1 and ski2-2 drb4-1 is also significantly increased.

[0083] In this example, the above 22nt-siRNA-enriched mutants were also fed to cotton bollworms for insect testing. The specific experimental results are as follows: Figure 1 As shown in B. The experimental results showed that the resistance of these 22nt-siRNA-enriched mutants to cotton bollworm was significantly enhanced.

[0084] like Figure 1As shown in C, enrichment analysis of differentially expressed genes between the wild type and ein5-1 dcl4-2 revealed that the up-regulated genes in ein5-1 dcl4-2 were enriched in items related to responses to adversity, including the items related to insect resistance, "response to mechanical damage" and "response to JA stimulation". This example further analyzed JA early response genes and defense-related genes and found that the expression levels of these genes were significantly increased in the ein5-1 dcl4-2 mutant, while the expression levels in ein5-1 dcl4-2 dcl2-1 were restored, as shown in the following figure. Figure 1 As shown in middle D. These results are also consistent with the insect-resistant phenotype of ein5-1 dcl4-2.

[0085] Example 2 Effect of 22nt-siRNA restoration on resistance of cotton bollworm

[0086] In order to verify that the insect resistance phenotype of siRNA-related mutants is mainly mediated by 22nt-siRNA, this example conducted an insect test on the three-mutant plants hybridized with 22nt-siRNA-related mutants and dcl2-1. The results showed that the resistance of ein5-1 dcl4-2dcl2-1 and ski2-2 drb4-1 dcl2-1 to cotton bollworm was not significantly different from that of the wild type. Figure 2 As shown in A. For the mutant plant ein5-1 ski2-3 that accumulates 21nt-siRNA and 22nt-siRNA, this example also hybridized the corresponding siRNA mutant plant with the rdr6-11 mutant plant to obtain the three-mutant ein5-1 ski2-3 rdr6-11 and the four-mutant plant ein5-1 ski2-3 dcl2-1dcl4-2 for cotton bollworm test experiments. The results showed that compared with the ein5-1ski2-3 double-mutant plant, the ein5-1ski2-3 rdr6-11 and ein5-1 ski2-3 dcl2-1 dcl4-2 plants had a certain degree of recovery in resistance to cotton bollworm, but still had differences with the wild type, as shown in Figure 2 As shown in B. These results indicate that 22nt-siRNA may play a more important role in plant defense against herbivorous insects.

[0087] Example 3 22nt-siRNA Accumulated PTGS Mutant for Plant Defense against Herbivorous Insects

[0088] 21nt-siRNA derived from endogenous plant genes is mainly produced by DCL4 protein-mediated cleavage, while 22nt-siRNA is mainly produced by DCL2 protein cleavage. Under normal circumstances, due to the presence of DCL4 protein, DCL2 protein does not participate in the cleavage to produce siRNA, and the activity of DCL2 protein will be significantly improved when the DCL4 protein function is lost. DRB4 protein helps DCL4 cut double-stranded RNA to produce 21nt-siRNA by binding to DCL4 protein. The loss of DRB4 protein will also lead to a decrease in the activity of DCL4 protein. In this example, mutants related to the PTGS signaling pathway, including mutants related to DCL protein and DRB4 protein, were subjected to insect tests. This example found that the mutants dcl4-2 and drb4-1 single mutant plants with only 22nt-siRNA accumulation had significantly enhanced resistance to cotton bollworm, while the dcl2-1 single mutant plants without 22nt-siRNA accumulation and the dcl2-1dcl4-2 double mutant plants had no obvious changes in resistance to cotton bollworm. The specific experimental results are as follows. Figure 3 shown.

[0089] Example 4 22nt-siRNA positively regulates plant defense responses to pests and is not completely dependent on anthocyanin and sinapinic acid signaling pathways

[0090] The mutants ein5-1 dcl4-2 and ski2-2 dcl4-2 plants, which accumulated a large amount of 22nt-siRNA, showed anthocyanin accumulation phenotypes. At the same time, the severe growth and development defects of the mutant plants may also be caused by the imbalance of secondary metabolites in the body. Anthocyanins and sinapinic acid glycosides are important secondary metabolites for Arabidopsis to resist herbivorous insects. In order to exclude the possibility that 22nt-siRNA only affects the accumulation of anthocyanins and sinigrin glycosides in plants to resist feeding by herbivorous insects, in this example, the anthocyanin synthesis mutant chs and the sinigrin glycoside mutant myb28 myb29 were hybridized with the ein5-1dcl4-2 plant to obtain the ein5-1 dcl4-2 chs triple-mutant plant and the ein5-1 dcl4-2 myb28 myb29 quadruple-mutant plant, and insect test experiments were carried out. The results showed that ein5-1 dcl4-2 chs and ein5-1 dcl4-2 myb28 myb29 still showed resistance to feeding by cotton bollworms compared with the control plants chs and myb28myb29. The specific experimental results are shown in Figure 2. Figure 4 These results indicate that 22nt-siRNA positively regulates plant defense responses to pests and is not entirely dependent on anthocyanin and sinapinic acid signaling pathways.

[0091] Example 5 22nt-siRNA enhances plant resistance to herbivorous insect feeding and is conservative

[0092] In order to verify whether 22nt-siRNA has a broad spectrum of resistance to herbivorous insects, this example also fed the relevant mutants enriched with 22nt-siRNA to the Lepidoptera insects Spodoptera frugiperda and Plutella xylostella. This example found that most of the mutants enriched with 22nt-siRNA still had strong resistance to these two herbivorous insects. The specific experimental results are as follows: Figure 5 As shown, it suggests that 22nt-siRNA is conserved in plants' defense against herbivorous insects.

[0093] The technical features of the above embodiments may be combined arbitrarily. To make the description concise, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0094] The above embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the attached claims.

Claims

1. A method for positively regulating a plant's defense response to harmful organisms, characterized in that: Plants are contacted with small RNAs, including 22nt-siRNAs endogenous to the plants, to enhance the resistance of the plants to pests.

2. The method according to claim 1, characterized in that Contacting the plant with the small RNA includes: modifying the plant to accumulate the plant's endogenous 22nt-siRNA in the plant; Optionally, the transformation comprises constructing a plant mutant by at least one of gene mutation, gene editing, gene silencing and RNA degradation; Optionally, the plant is selected from at least one of Arabidopsis thaliana, cabbage, corn, wheat, rice, millet, soybean, tomato, Brachypodium distachyon, water lily, Cinnamomum camphora, Selaginella chinensis, Physcomitrella patens and Marchantia polyphylla.

3. The method according to claim 2, characterized in that The plant mutant satisfies any one of the following conditions (1) to (3); (1) The plant mutant lacks DCL4 protein or DRB4 protein; (2) the plant mutant lacks a bidirectional cytoplasmic RNA degradation pathway; (3) the plant mutant lacks the cytoplasmic RNA degradation pathway and also lacks at least one of the DCL4 protein and the DRB4 protein; Optionally, the plant mutant includes at least one of dcl4-2 single mutant plants, drb4-1 single mutant plants, ein5-1 ski2-3 double mutant plants, ein5-1 dcl4-2 double mutant plants, ski2-2 dcl4-2 double mutant plants, ein5-1 drb4-1 double mutant plants and ski2-2 drb4-1 double mutant plants.

4. The method according to claim 2 or 3, characterized in that: The plant endogenous 22nt-siRNA includes the 22nt-siRNA derived from the plant coding gene produced by the plant mutant; Optionally, the plant encoding gene includes the NIA1 gene and the NIA2 gene.

5. Use of small RNA in the preparation of plant pest control agents, wherein the small RNA includes plant endogenous 22nt-siRNA.

6. The use according to claim 5, characterized in that: The plant pest control agent includes the small RNA or a composition comprising the small RNA; Optionally, the composition further comprises at least one of a carrier, an excipient or a diluent for the small RNA; Optionally, the plant pests include at least one of animals and microorganisms; Optionally, the plant pests include herbivorous insects.

7. The use according to claim 5 or 6, characterized in that: The plant endogenous 22nt-siRNA includes 22nt-siRNA derived from plant coding genes produced by plant mutants; Optionally, the plant mutant satisfies at least one of the following conditions (1) to (3); (1) The plant mutant lacks DCL4 protein or DRB4 protein; (2) the plant mutant lacks a bidirectional cytoplasmic RNA degradation pathway; (3) the plant mutant lacks the cytoplasmic RNA degradation pathway and also lacks at least one of the DCL4 protein and the DRB4 protein; Optionally, the plant encoding gene includes NIA1 gene and NIA2 gene; Optionally, the plant mutant comprises at least one of a dcl4-2 single mutant plant, a drb4-1 single mutant plant, an ein5-1 ski2-3 double mutant plant, an ein5-1 dcl4-2 double mutant plant, a ski2-2 dcl4-2 double mutant plant, an ein5-1 drb4-1 double mutant plant, and a ski2-2 drb4-1 double mutant plant; Optionally, the plant is selected from at least one of Arabidopsis thaliana, cabbage, corn, wheat, rice, millet, soybean, tomato, Brachypodium distachyon, water lily, Cinnamomum camphora, Selaginella chinensis, Physcomitrella patens and Marchantia polyphylla.

8. The plant harmful organism control agent prepared by the use according to any one of claims 5 to 7.

9. A method for controlling plant pests, characterized in that: The plant pest control agent according to claim 8 is contacted with the plant pest to inhibit the growth of the plant pest.

10. The method according to claim 9, characterized in that The plant pests include at least one of animals and microorganisms; Optionally, the plant pests include herbivorous insects.

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