Application of arabidopsis thaliana source miR168a-5p in pest control

Abstract

The invention belongs to the technical field of pest control, discloses an application of Arabidopsis thaliana-derived miR168a-5p in pest control, and particularly discloses an application of Ath-miR168a-5p in pest control and / or preparation of a product for pest control. It is found for the first time that after injection of artificially synthesized Ath-miR168a-5p agomir into prodenia litura, compared with a control group injected with DEPC water, the survival rate of prodenia litura larvae can be remarkably reduced, and it is proved that Ath-miR168a-5p can be used for prevention and treatment of prodenia litura. Therefore, the Ath-miR168a-5p can be directly applied to field spraying and transgenic plants, and has a relatively good application value.

Description

Technical Field

[0001] The invention belongs to the technical field of pest control, and particularly relates to application of Arabidopsis thaliana-derived miR168a-5p in pest control. Background Art

[0002] miRNA is a type of small non-coding RNA with a length of about 21 to 24 nucleotides, which is widely present in animals and plants. A series of experiments have shown that miRNA may be a type of evolutionarily conserved molecule with important regulatory functions in organisms. miRNA is derived from the gene encoding miRNA in the cell nucleus and transcribed into pri-microRNA, which is then cut by the Drosha enzyme into a hairpin-shaped pre-miRNA with a length of about 70 bp, and then transported from the cell nucleus to the cytoplasm by the nucleoplasmic / cytoplasmic transporter, and then cut by the Dicer enzyme to grow into a miRNA double helix complex of 18 to 26 bp. One of the helices unwinds and binds to the AGO protein to form an asymmetric RISC complex. This complex binds to the 3'UTR of the target mRNA through the complementary pairing of the miRNA in it, thereby degrading the target mRNA or blocking its translation, thereby achieving negative regulation of the target gene. A large number of literature reports have shown that miRNA plays a vital role in gene expression, signal transduction, plant development, stress resistance, and disease prevention. In recent years, miRNA has been widely used in the field of pest control. RNAi is a conserved post-transcriptional gene silencing mechanism that exists in eukaryotes. Pesticides based on this mechanism can interfere with the normal growth, development, feeding, immunity, reproduction and other behaviors of pests by inhibiting gene expression, thereby achieving the purpose of pest control. Therefore, RNAi pesticides have also become a new type of pesticide recognized in the field of agriculture and forestry as being highly efficient, specific and environmentally friendly. Exploring sources of nucleic acid components that are better at insect control, safer for humans and animals, and more environmentally friendly will be beneficial to the application of RNAi pesticides. Screening miRNAs from plants that humans and animals eat daily for pest control has the advantage of being more environmentally friendly than traditional artificially synthesized dsRNA or insect-derived miRNAs. At present, miRNAs used for pest control are mainly derived from insect-derived miRNAs and artificially designed miRNAs. However, there are few studies on the use of plant-derived miRNAs for pest control, and no studies have reported that Ath-miR168a-5p can be used for pest control. Summary of the invention

[0003] The purpose of the first aspect of the present invention is to provide the use of Ath-miR168a-5p and related biological materials in pest control and / or the preparation of pest control products.

[0004] The second aspect of the present invention aims to provide a product.

[0005] The third aspect of the present invention aims to provide a method for controlling pests.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

[0007] The first aspect of the present invention provides the use of any one of the following substances (1) to (5) in pest control and / or in the preparation of pest control products;

[0008] (1) Ath-miR168a-5p;

[0009] (2) precursor of Ath-miR168a-5p;

[0010] (3) Ath-miR168a-5p agomir;

[0011] (4) a nucleic acid molecule encoding the Ath-miR168a-5p described in (1) or the precursor of the Ath-miR168a-5p described in (2);

[0012] (5) An expression cassette, recombinant vector or transgenic cell containing the nucleic acid molecule described in (4).

[0013] In some embodiments of the present invention, the nucleotide sequence of Ath-miR168a-5p is as shown in SEQ ID NO:1.

[0014] In some embodiments of the present invention, miRNA agomir is a specially chemically modified miRNA agonist, which can be directly injected into an animal body, simulates endogenous miRNA in an organism, and can enhance the function of endogenous miRNA. Compared with miRNA mimic for cells, miRNA agomir for animals has higher stability and miRNA activity, is easier to pass through the cell membrane and intercellular space and enrich in target cells, and can prevent the degradation of miRNA by nucleases in insects. Specifically, Ath-miR168a-5p agomir, i.e., Ath-miR168a-5p agonist, is a small fragment of double-stranded miRNA designed and synthesized for the mature body of Ath-miR168a-5p, and its effect is the same as that of mature Ath-miR168a-5p in vivo, which can upregulate the content of corresponding Ath-miR168a-5p in cells and enhance the function of endogenous Ath-miR168a-5p. Ath-miR168a-5p agomir contains the sequence shown in SEQ ID NO:1 and / or its complementary sequence.

[0015] In some embodiments of the present invention, the Ath-miR168a-5p of the present invention includes functional equivalents of the constituent nucleic acid molecules, i.e., variants, which show the same functions as the complete Ath-miR168a-5p nucleic acid molecules, and they may be mutated by deletion, substitution or insertion of nucleotide residues.

[0016] It is well known to those skilled in the art that in order to ensure the stability of miRNA, protective bases such as TT can be added to one or both ends of miRNA; miRNA bases can also be modified, but the function of miRNA is not affected. Therefore, it is well known to those skilled in the art that the sequence obtained by base modification of Ath-miR168a-5p or addition of bases at both ends without affecting the function of Ath-miR168a-5p is also included in the protection scope of the present invention, that is, the Ath-miR168a-5p can be any one or more ribonucleotides in the sequence shown in SEQ ID NO: 1 are modified.

[0017] In some embodiments of the present invention, the Ath-miR168a-5p is a mature miRNA, a precursor miRNA or a primary transcript.

[0018] In some embodiments of the present invention, the pests include insects of the order Lepidoptera.

[0019] In some embodiments of the present invention, the pest is one or more of Spodoptera litura, Manduca sexta, Cotton bollworm, Plutella xylostella, Spodoptera exigua and Spodoptera frugiperda.

[0020] In some embodiments of the present invention, the pest is Spodoptera litura.

[0021] In some embodiments of the present invention, the product is a pesticide or insecticide.

[0022] In some embodiments of the present invention, the product can be prepared into conventional dosage forms for use, such as dry powder, wettable powder, emulsifiable concentrate, microemulsion, paste, granules or suspensions.

[0023] In some embodiments of the present invention, the application is to kill pests.

[0024] The Arabidopsis-derived miRNA (Ath-miR168a-5p) has a control effect on the Arabidopsis pest Spodoptera litura. Since Ath-miR168a-5p is derived from Arabidopsis thaliana, a plant belonging to the cruciferous family like green leafy vegetables, it is safe for humans and can be used as a new type of insecticide for safe and effective control of Spodoptera litura. It can be used directly and in the construction of transgenic plants; therefore, Ath-miR168a-5p transgenic plants (i.e. plants overexpressing Ath-miR168a-5p) for the control of Spodoptera litura will have practical application value.

[0025] The second aspect of the present invention provides a product, comprising any one of the following substances (1) to (5);

[0026] (1) Ath-miR168a-5p;

[0027] (2) precursor of Ath-miR168a-5p;

[0028] (3) Ath-miR168a-5p agomir;

[0029] (4) a nucleic acid molecule encoding the Ath-miR168a-5p described in (1) or the precursor of the Ath-miR168a-5p described in (2);

[0030] (5) An expression cassette, recombinant vector or transgenic cell containing the nucleic acid molecule described in (4).

[0031] In some embodiments of the present invention, the nucleotide sequence of Ath-miR168a-5p is as shown in SEQ ID NO:1.

[0032] In some embodiments of the present invention, the product includes at least one of a reagent, a medicine, and a pesticide.

[0033] In some embodiments of the present invention, the product further comprises a pharmaceutically acceptable carrier, including but not limited to: a diluent, a buffer, a suspension, an emulsion, a granule, an encapsulation, an excipient, a filler, an adhesive, a spray, a transdermal absorbent, a wetting agent, a disintegrant, an absorption enhancer, a surfactant, a flavoring agent or an adsorption carrier.

[0034] The third aspect of the present invention provides a method for controlling pests, comprising applying the substance described in the first aspect of the present invention or the product described in the second aspect of the present invention to pests or pest habitats.

[0035] In some embodiments of the present invention, the method comprises treating pests, pest food (such as cruciferous plants), pest habitat (soil, area, material or environment where pests are growing or can grow, or materials to be protected from pest attack or infection, cultivated plants, plant propagation materials (such as seeds), soil, surface) with the substance or product. For example, transgenic plants expressing Ath-miR168a-5p or its agomir or precursor are constructed for pest control.

[0036] In some embodiments of the present invention, an effective dose of the substance described in the first aspect of the present invention or the product of the second aspect of the present invention is applied to pests or pest habitats.

[0037] In general, "effective amount" means the amount of active ingredient required to achieve an observable effect on growth, including necrosis, death, retardation, prevention and removal, destruction or reduction of the presence and activity of the target organism. For Ath-miR168a-5p or agomir used in the present invention, the effective amount can vary. The effective amount of Ath-miR168a-5p or agomir also varies according to the prevailing conditions, such as the desired insecticidal effect and duration, climate, target species, location, mode of application, etc.

[0038] In some embodiments of the present invention, the pests include insects of the order Lepidoptera.

[0039] In some embodiments of the present invention, the pest is one or more of Spodoptera litura, Manduca sexta, Cotton bollworm, Plutella xylostella, Spodoptera exigua and Spodoptera frugiperda.

[0040] In some embodiments of the present invention, the pest is Spodoptera litura.

[0041] The beneficial effects of the present invention are:

[0042] The present invention first discovered that after the artificially synthesized Ath-miR168a-5p agomir was injected into the Spodoptera litura, the survival rate of the nymphs of the Spodoptera litura could be significantly reduced compared with the control group injected with DEPC water, which proved that Ath-miR168a-5p can be used for the prevention and control of the Spodoptera litura. Therefore, Ath-miR168a-5p can be directly used for field spraying and transgenic plants, and has good application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 This is a conservation analysis of the Ath-miR168a-5p sequence in plants.

[0044] Figure 2Compare the Ath-miR168a-5p sequence with other plant-derived miR168a-5p sequences.

[0045] Figure 3 Target gene prediction analysis of Ath-miR168a-5p and Spodoptera litura full mRNA.

[0046] Figure 4 The survival rate of Spodoptera litura larvae injected with different concentrations of Ath-miR168a-5p agomir within 168 hours.

[0047] Figure 5 Injection of Ath-miR168a-5p agomir into Spodoptera litura larvae resulted in a lethal phenotype.

[0048] Figure 6 Statistics of 168h-old larvae of Spodoptera litura injected with different concentrations of Ath-miR168a-5p agomir. DETAILED DESCRIPTION

[0049] The present invention is further described in detail below through specific examples.

[0050] It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0051] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0052] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0053] Example 1

[0054] A miRNA Ath-miR168a-5p, whose nucleotide sequence is 5'-UCGCUUGGUGCAGGUCGGGAA-3' (SEQ ID NO: 1), is derived from Arabidopsis thaliana.

[0055] Example 2

[0056] Experimental methods

[0057] 1. Obtain the mature sequence of Ath-miR168a-5p from the miRBase database and use it as the query sequence. Perform a whole-genome Blast comparison with plants from 14 families through the plant genome database Ensembl Plants Home (https: / / plants.ensembl.org / index.html). Only plant sequences that are completely consistent with the query sequence (i.e., 100% match and 21nt in length) are obtained to evaluate the conservation of Ath-miR168a-5p in different plants. Subsequently, the NCBI Common Tree tool (https: / / www.ncbi.nlm.nih.gov / Taxonomy / CommonTree / wwwcmt.cgi) was used to construct an evolutionary tree of plant species with conserved miR168a.

[0058] 2. SnapGene software was used to perform homology analysis and comparison between the Ath-miR168a-5p sequence and the mature miR168a-5p sequences of other plant species identified in the miRBase database, confirming that Ath-miR168a-5p is a plant-derived miRNA.

[0059] Experimental Results

[0060] Homology analysis results showed that miR168a-5p was highly conserved in the plant kingdom, with homologous miR168a-5p ( Figure 1 ). In addition, during the comparison, it was found that miR168a-5p is widely distributed in dicotyledons, especially in Cruciferae and Leguminosae. In contrast, its distribution in monocotyledons is very rare. For example, in the largest group of monocotyledons, Poaceae, no homologous sequences that completely match (100%) were detected, and the highest match was 95%.

[0061] Furthermore, the mature miR168a-5p sequence from Arabidopsis was compared with the mature miR168a-5p sequences from 19 other plants identified in the miRBase database. The results showed that these sequences were highly conserved overall, with the mature miR168a-5p sequences of most plants being completely consistent with the sequence from Arabidopsis; only minor differences of 1 to 2 bases were observed in 7 species ( Figure 2 ). The analysis showed that Arabidopsis miR168a-5p is a highly conserved miRNA in the plant kingdom, and suggested that miR168a-5p from different plants may have similar biological functions.

[0062] Example 3

[0063] This example is used to predict the target gene of Ath-miR168a-5p in Spodoptera litura in Example 1, as follows:

[0064] Experimental methods

[0065] 1) The genome sequence and genome annotation information of Spodoptera litura (GenBank No. GCA_002706865.3) were obtained through the National Center for Bioinformation of the United States;

[0066] 2) Using TBtools (Chen et al., 2020) sequence processing tools, a 2000b sequence library of the CDS region and the CDS region downstream of the full-coding gene was formed;

[0067] 3) The three miRNA target gene prediction softwares miRanda, PITA and RNAhybrid were used to predict the target genes of Ath-miR168a-5p and the CDS region and the 2000b sequence downstream of the CDS region of Spodoptera litura. The screening thresholds of miRanda software were score less than -150, energy less than -20 and strict base pairing in the 5' end seed region of miRNA; the screening threshold of PITA software was 7 to 8 bases in the seed region of miRNA, and mismatches and stem-loop structures were not allowed in the seed region, and ddG was less than -10; the screening threshold of RNAhybrid software was energy less than -20 and p-value less than 0.05. Finally, the prediction results obtained by the three software were taken as the results of the intersection of any two softwares.

[0068] Experimental Results

[0069] Ath-miR168a-5p was predicted and analyzed with the CDS region and 3'UTR region of the full mRNA of Spodoptera litura (GenBank No. GCA_002706865.3). The results showed that Ath-miR168a-5p may target multiple reported lethal target genes in Spodoptera litura ( Figure 3 ), making Ath-miR168a-5p have potential anti-insect effect against Spodoptera litura.

[0070] Example 4

[0071] This example is used to verify the control effect of Ath-miR168a-5p in Spodoptera litura in Example 1, as follows:

[0072] Experimental methods

[0073] 1. Rearing of Spodoptera litura

[0074] The larvae were raised in an artificial climate box for a long time, and after stable reproduction for more than 30 generations, the feeding conditions were 26°C, 14 hours of light and 10 hours of darkness in every 24 hours, and a relative humidity of 60%. They were fed with artificial culture medium. Each 1L of artificial culture medium contained 150g wheat germ powder, 40g yeast powder, 13g agar powder, 4g vitamin C, 4g sorbic acid, 4g sucrose, 4g methylparaben and 200μL linoleic acid. The adults were fed with 10% honey water.

[0075] 2. Injection of artificially synthesized miRNA agomir into Spodoptera litura larvae

[0076] 1) Select the 3rd instar (pre-molting) larvae of Spodoptera litura in advance and place them in a plastic insect box to molt in a dark environment without artificial culture medium feed to ensure that the growth trend of the larvae is consistent during injection;

[0077] 2) Collect larvae that have molted normally and entered the first day of the fourth instar and place them on ice for 15 minutes;

[0078] 3) Use a microsyringe to inject DEPC water, 2.5 μg / μL Ath-miR168a-5p agomir, and 5 μg / μL Ath-miR168a-5p agomir, respectively, with an injection volume of 2 μL, and the injection site is the third to last pair of appendages of the larvae;

[0079] 4) The injected larvae were placed in new insect boxes and raised with artificial culture medium for Spodoptera litura;

[0080] 5) Count the survival rate of Spodoptera litura larvae within 0 to 168 hours after injection, and make statistics every 24 hours;

[0081] 6) The instars of Spodoptera litura larvae were counted 168 hours after injection.

[0082] Agomir is a double-stranded miRNA mimic that has been specially chemically modified and can be directly injected into animals to mimic endogenous miRNA. Compared with miRNA mimics for cells, animal miRNA agomir has higher stability and miRNA activity, and is more easily enriched in target cells through cell membranes and intercellular spaces. The use of miRNA agomir can achieve overexpression of target miRNA, thereby studying the function and mechanism of action of miRNA in vivo. Specifically, Ath-miR168a-5pagomir is a small fragment double-stranded miRNA designed and synthesized based on the mature sequence of Ath-miR168a-5p, and its function is the same as that of mature Ath-miR168a-5p in vivo, which can upregulate the content of corresponding Ath-miR168a-5p in cells and enhance the function of endogenous Ath-miR168a-5p. Ath-miR168a-5p agomir contains the sequence shown in SEQ ID NO:1 and / or its complementary sequence.

[0083] Experimental Results

[0084] 1. Effect of Ath-miR168a-5p agomir on the survival rate of Spodoptera litura larvae

[0085] After injecting Ath-miR168a-5p agomir into 4th instar 1-day-old larvae of Spodoptera litura, the mortality of Spodoptera litura was observed within 0 to 168 hours. Figure 4 As shown in the figure, the survival rate of Spodoptera litura larvae injected with Ath-miR168a-5p agomir was significantly lower than that of the control group injected with DEPC water during the 168h treatment period. Among them, when the dose of Ath-miR168a-5p agomir was higher, the survival rate of Spodoptera litura larvae was lower and the lethal effect was better. Figure 5 As shown, the lethal phenotype of Spodoptera litura caused by injection of Ath-miR168a-5p agomir was observed, and its growth was stagnant and its epidermis was black, dehydrated and shrunken. The above results suggest that heterologous expression of Ath-miR168a-5p in Spodoptera litura may act on important target genes related to growth and development or detoxification, achieving an insect-resistant effect on Spodoptera litura.

[0086] 2. Effects of Ath-miR168a-5p agomir on the developmental stages of Spodoptera litura larvae

[0087] By analyzing the larval age at 168 hours after treatment, it was found that most of the larvae in the control group were already at the 6th instar and 2 days old, while most of the larvae treated with Ath-miR168a-5p agomir were at the 6th instar and 1 day old, showing a significant delay in age and shortening of body length ( Figure 6 ). This suggests that Ath-miR168a-5p can delay the growth and development of Spodoptera litura larvae and can be used for pest control of Spodoptera litura larvae.

[0088] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. Use of any one of the following substances (1) to (5) in pest control and / or preparation of pest control products; (1) Ath-miR168a-5p; (2) precursor of Ath-miR168a-5p; (3) Ath-miR168a-5p agomir; (4) a nucleic acid molecule encoding the Ath-miR168a-5p described in (1) or the precursor of the Ath-miR168a-5p described in (2); (5) An expression cassette, recombinant vector or transgenic cell containing the nucleic acid molecule described in (4).

2. The use according to claim 1, characterized in that: The nucleotide sequence of Ath-miR168a-5p is shown in SEQ ID NO:

1.

3. The use according to claim 1, characterized in that: The pests include insects of the order Homoptera; The pests are preferably one or more of Spodoptera litura, Manduca sexta, Cotton bollworm, Plutella xylostella, Spodoptera exigua and Spodoptera frugiperda.

4. The use according to any one of claims 1 to 3, characterized in that: The application is to kill pests.

5. A product comprising any one of the following substances (1) to (5); (1) Ath-miR168a-5p; (2) precursor of Ath-miR168a-5p; (3) Ath-miR168a-5pagomir; (4) a nucleic acid molecule encoding the Ath-miR168a-5p described in (1) or the precursor of the Ath-miR168a-5p described in (2); (5) an expression cassette, a recombinant vector or a transgenic cell containing the nucleic acid molecule described in (4); Preferably, the nucleotide sequence of Ath-miR168a-5p is as shown in SEQ ID NO:

1.

6. The product according to claim 5, characterized in that The product includes at least one of a reagent, a medicine, and a pesticide.

7. A method for controlling pests, comprising applying the substance according to claim 1 or the product according to claim 5 or 6 to pests or pest habitats.

8. The method according to claim 7, characterized in that The method comprises treating the pests, the food of the pests, or the habitat of the pests with the substance or product.

9. The method according to claim 7 or 8, characterized in that: The pests include insects of the order Lepidoptera.

10. The method according to claim 9, characterized in that The pests are one or more of the group consisting of Spodoptera litura, Manduca sexta, Cotton bollworm, Plutella xylostella, Spodoptera exigua and Spodoptera frugiperda.

Images