WO2015070765A1 - Rnai-based pest control method using irrigation, and application thereof - Google Patents

Abstract

An RNAi method for control of growth-related genes of plant-eating pests and applications thereof, a dsRNA preparation and method for the use thereof.

Description

RNAi technology for controlling pests by irrigation and its application Technical field

The present invention is in the field of biotechnology and agricultural applications, and in particular, the invention relates to RNAi technology for controlling pests by irrigation and uses thereof.

Background technique

A large number of studies have proved that the use of RNA interference (RNAi) technology can achieve the specific control of pest species. Therefore, the application of this technology will not affect other organisms, and it is an environmentally friendly new strategy for pest control. However, this technology still has some problems in practical applications. The most important problem is how to accurately deliver the double-stranded RNA (dsRNA) of the target gene to the target pest. There are two more feasible methods at present:

The first is to use the plant to express the dsRNA of the target pest target gene. When the pest feeds the transgenic plant, the dsRNA can be taken into the body to achieve the purpose of controlling the pest. However, under natural conditions, each crop has to face various diseases and insects. It is impossible for us to carry out transgenic operations on all crops to avoid the damage of many organisms.

The second is the mass production of double-stranded RNA preparations, which are sprayed like chemical pesticides to control pests. However, this method is used for some drill-bit pests (such as rice aphids), underground pests, insects with waxy protective layers on some body walls (such as brown planthopper), or insects hidden in the back, cracks, etc. of plant leaves. It is difficult to be treated in the process of spraying, and the effect of prevention and control is not achieved.

In addition, there are no good target gene screening and application techniques for sucking mouthparts pests, especially those with smaller individuals but faster reproduction speeds (such as aphids, whiteflies, etc.).

In order to solve the problems in the above technology, there is an urgent need in the art to develop an RNAi application technology that is easy to apply and widely effective, especially for drill-bit and needle-punched pests, thereby achieving the effects of controlling pests and protecting crops.

Summary of the invention

The object of the present invention is to provide a preparation and application new technology for controlling sucking mouthparts and drill collar harmful insects based on RNAi technology.

In a first aspect of the invention, there is provided a method of introducing a double-stranded RNA (dsRNA) into a plant, comprising the steps of: applying said double-stranded RNA to a root of a plant through which the root of the double-stranded RNA is absorbed The action is such that the double-stranded RNA is inhaled by the plant and transported to a portion of the plant other than the root.

In another preferred embodiment, the double-stranded DNA includes insect-resistant double-stranded DNA.

According to a second aspect of the present invention, a method for improving insect resistance of a plant, comprising the steps of: applying a pest-resistant double-stranded RNA (dsRNA) to a root of a plant such that the insect-resistant double-stranded RNA is the plant The roots are absorbed and transported to parts of the plant other than the roots, thereby increasing the insect resistance of the plants.

In another preferred embodiment, the plant portion other than the root portion includes stems, branches, leaves, flowers, fruits, or a combination thereof.

In another preferred embodiment, the delivery is carried out in the body of the plant.

In another preferred embodiment, the insect-resistant dsRNA is 50-2000 bp in length, preferably 100-1000 bp in length.

In another preferred embodiment, the insect-resistant dsRNA is administered at a concentration of 0.0001 to 5 mg/ml, preferably 0.001 to 1 mg/ml.

In another preferred embodiment, the portion of the plant other than the root is the upper 3/4 portion of the plant ground or surface portion. It is preferably the upper 2/3 part.

In another preferred embodiment, the insect-resistant dsRNA is a dsRNA directed against an insect growth-related gene.

In another preferred embodiment, the insect growth-related genes include the rice planthopper P450 gene (Cyp18A1), the rice locust carboxylasease (Ces), or the Asian corn borer K-serine protease inhibitor gene (KPIs). ).

In another preferred embodiment, the insect comprises Lepidoptera, Hemiptera, Diptera.

In another preferred embodiment, the rice planthopper P450 (Cyp18A1) gene fragment sequence is set forth in SEQ ID NO.

The sequence of the carboxylesterase (Ces) gene fragment is shown in SEQ ID NO.

The K-type serine protease inhibitor (KPIs) gene sequence of the Asian corn borer is shown in SEQ ID NO.

In another preferred embodiment, the dsRNA is represented by SEQ ID NOs.: 4-6.

In another preferred embodiment, the means of application includes irrigation, soaking, watering, drip irrigation.

In another preferred embodiment, the insect resistance comprises an anti-herbarian pest.

In another preferred embodiment, the herbivorous pest includes a sucking mouthparts pest, a chewing pest, a drill collar pest, and a ground pest.

In another preferred embodiment, the sucking mouthparts pest comprises rice brown planthopper, whitebacked planthopper or Laodelphax striatellus.

In another preferred embodiment, the drill collar pest comprises Asian corn borer, European corn borer or peach aphid.

In another preferred embodiment, the plant comprises a dicot, a monocot, or a gymnosperm.

In another preferred embodiment, the plant comprises a gramineous plant, a leguminous plant, a cruciferous plant, a solanaceous plant, and a cucurbitaceae plant.

In another preferred embodiment, the gramineous plant comprises corn, rice, barley, wheat, oats, sorghum, rye.

In another preferred embodiment, the legumes include soybeans, peanuts, broad beans, peas, red beans, mung beans, cowpeas, green beans, and lentils.

In another preferred embodiment, the cruciferous plant includes green vegetables, Chinese cabbage, cauliflower, radish, and kale.

A third aspect of the invention provides an agricultural composition comprising a pesticidally effective amount of an insect resistant double-stranded RNA and a pesticidally acceptable carrier.

In another preferred embodiment, the agricultural composition is applied to the root of the plant.

In another preferred embodiment, the dsRNA is administered at a concentration of 0.0001 to 5 mg/g or 0.0001-5 mg/ml, preferably 0.001 to 1 mg/ml or 0.001-1 mg/g.

In another preferred embodiment, the dsRNA is applied at a concentration of 0.01 to 5 mg/g or 0.01 to 5 mg/ml, preferably 0.1 to 1 mg/ml or 0.1 to 1 mg/g.

In another preferred embodiment, the agricultural composition is in a liquid or solid state.

In another preferred embodiment, the pharmaceutically or agrochemically acceptable carrier comprises water, a synergist, a surfactant or a stabilizer.

In another preferred embodiment, the agricultural composition further comprises an RNA stabilizer.

In another preferred embodiment, the RNA stabilizer comprises a solvent such as cyanogenic hydrochloride, an RNA protectant, deionized formamide or the like.

In a fourth aspect of the invention, there is provided the use of an insect-resistant double-stranded RNA sequence for the preparation of a composition for enhancing the insect resistance of a plant by root application.

In a fifth aspect of the invention, an isolated polynucleotide is provided, the polynucleotide being selected from the group consisting of:

(a) the sequence shown in any one of SEQ ID NO.: 1-6;

(b) a polynucleotide complementary to the sequence defined by (a); or

(c) having at least 70% (preferably at least 75%, 80%, 85%, 90%, more preferably at least 95%, 96%, 97%, 98%, 99%) or more of the sequence defined by (a) Any polynucleotide or complementary sequence of sequence identity, wherein the dsRNA of the strand complementary to any of the polynucleotide sequences has an activity of inhibiting the growth of the herbivorous pest after ingestion by the herbivorous pest.

In another preferred embodiment, the dsRNA of the strand complementary to any of the polynucleotide sequences can be taken up by the roots of the plant after irrigation, soaking, watering or drip irrigation, and is inhibited by the herbivorous pests. The activity of the growth of herbivorous pests.

In another preferred embodiment, the inhibiting the growth activity of the herbivorous pest comprises inducing or causing the death of the herbivorous pest.

In a sixth aspect of the invention, a dsRNA construct is provided, the construct of the dsRNA is double stranded, and the positive or negative strand thereof comprises the structure of formula I:

Seq _forward- X-Seq _reverse

Formula I

In the formula,

A nucleotide sequence Seq _forward growth-related genes or fragments of phytophagous insects;

Seq _reverses to a nucleotide sequence that is substantially complementary to the Seq _forward ;

X is a spacer sequence located between the _forward and Seq Seq the _reverse, and the spacer sequence Seq Seq _forward and _reverse are not complementary,

Wherein the plant-borne pest growth-related gene is selected from the group consisting of: a rice planthopper P450 gene (Cyp18A1), a rice locust carboxylasease (Ces), or an Asian corn borer K-serine protease inhibitor gene (KPIs). ).

In another preferred embodiment, the herbivorous pest comprises a sucking mouthparts pest or a drill collar pest.

In another preferred embodiment, the dsRNA construct can form a dsRNA of Formula II,

Formula II

In the formula,

Seq _{'Forward Forward} sequence corresponds to Seq RNA sequences or fragments of sequences;

Seq' _reverse is a sequence that is substantially complementary to the Seq'_forward;

X 'is absent; or located Seq' _forward and Seq 'spacer sequence between the _reverse, and the spacer sequence Seq' _forward and Seq 'no _reverse complementary,

|| represents a hydrogen bond formed between the Seq _forward and the Seq _reverse .

In another preferred embodiment, the Seq _forward and Seq _inverse lengths are at least 50 bp.

In another preferred embodiment, the spacer sequence X' has a length of 0-300 bp.

In a seventh aspect of the invention, an expression vector comprising the dsRNA construct of the sixth aspect of the invention is provided.

According to an eighth aspect of the invention, a genetically engineered host cell comprising the expression vector of the seventh aspect of the invention or the integration of the dsRNA corresponding to the sixth aspect of the invention in the host cell is provided The DNA sequence of the object.

In another preferred embodiment, the host cell is a plant cell, preferably a maize cell or a rice cell.

In a ninth aspect of the invention, there is provided a dsRNA sequence which is an insect resistant dsRNA and which is 100-800 bp in length, preferably 150-600 bp in length.

In another preferred embodiment, the sequence of the dsRNA is derived from a species selected from the group consisting of agricultural or forest pests or vegetable pests. Preferably, the species comprises the Asian corn borer of the Lepidoptera family and the rice brown planthopper of the Homoptera family.

In another preferred embodiment, the dsRNA sequence is synthetic or artificially produced.

In another preferred embodiment, the "derived from" means that the dsRNA has a homology (identity) to the nucleotide sequence of the target gene in the pest of 95-100%, preferably 99. -100%.

In another preferred embodiment, the target gene is specific to the pest.

In another preferred embodiment, the sequence of the dsRNA is selected from the group consisting of SEQ ID NOs.: 4-6; or a dsRNA of ≥ 50 bp in length.

According to a tenth aspect of the present invention, a method for controlling pests, which comprises applying the agricultural composition of the third aspect of the invention or the dsRNA of the ninth aspect of the invention to a plant in need of pest control, is provided.

It is to be understood that within the scope of the present invention, the various technical features of the present invention and the various technical features specifically described hereinafter (as in the embodiments) may be combined with each other to constitute a new or preferred technical solution. Due to space limitations, we will not repeat them here.

DRAWINGS

Figure 1 shows that rice roots absorb dsRNA and down-regulate the expression of related genes.

Figure 2 shows inhibition of brown planthopper related gene expression and lethality to brown planthopper.

Figure 3 shows the inhibition of Asian corn borer-related gene expression and the lethality rate of corn borer.

Figure 4 shows the stability and degradation rate of dsRNA.

Fig. 5 shows that the leaf surface at the distal end of the drip droplet after the dsRNA of the present invention was dripped on the surface of the rice leaf was observed 24 hours later, and the fluorescence phenomenon was also observed, and the gray portion in the figure was the displayed fluorescence.

Figure 6 shows the total RNA extracted by CK for the clear water treatment control; DS- is the total RNA extracted after 24 hours of immersion in different double-stranded RNA, and the arrow below it indicates that there is a specific band different from CK in the total RNA. It is the exogenous double-stranded dsRNA that the plant absorbs because there is no such specific band in all CK controls.

Figure 7 shows the extraction of total RNA from rice stems after immersion for 24 h in three different dsRNAs. After inversion into cDNA, the dsRNA fragment of the foreign gene was amplified, and the fragment with the same size as the target was obtained, but in the water control There is no strip in the middle.

detailed description

The present inventors conducted extensive and intensive research to screen important genes during the growth and development of herbivorous pests, and unexpectedly found that the dsRNA targeting the rice planthopper P450 (Cyp18A1) gene as shown in SEQ ID NO: 4, such as SEQ dsRNA targeting the carboxylesterase (Ces) gene represented by ID NO: 5 and dsRNA targeting the K-type serine protease inhibitor (KPIs) gene of Asian corn borer as shown in SEQ ID NO: 6. The dsRNA can be made into an irrigation liquid, which is absorbed by the plant roots and leaves, and then ingested by the herbivorous pests to interfere with the target genes of the pests, inhibit the expression of the target genes, and finally lead to the drilling and sucking pests. Death, to achieve the purpose of pest control. The dsRNA of the present invention can be simply formulated and applied: irrigation can achieve pest control effects. The invention can use the dsRNA to carry out pest control without the need of transgenic cultivation of the plant, especially for pests hidden in the root leaves and stems which cannot be controlled by spraying pesticides.

As used herein, "substantially complementary" means that the sequences of the nucleotides are sufficiently complementary to interact in a predictable manner, such as to form a secondary structure (eg, a stem-loop structure). Usually, two "basic At least 70% of the nucleotides of the complementary nucleotide sequences are complementary to each other; preferably, at least 80% of the nucleotides are complementary; more preferably, at least 90% of the nucleotides Compatible; further preferably, at least 95% of the nucleotides are complementary; such as 98%, 99% or 100%. In general, there may be up to 7 mismatched nuclei between two sufficiently complementary molecules. Glycosyl acid; preferably, having up to 6 unmatched nucleotides; more preferably, having up to 5 unmatched nucleotides; further preferably, having up to 4 unmatched nucleotides, such as having 0 1, 2, 3, 4 unmatched nucleotides.

As used herein, a "complementary" sequence generally refers to a sequence that converts a sequence in the 5'-3' direction to its 3'-5' direction (eg, 5'ATCG 3'->GCTA), and then takes its complementary sequence ( Such as GCTA → 5 'CGAT 3').

As used herein, a "stem loop" structure, also referred to as a "hairpin" structure, refers to a nucleic acid molecule that forms a secondary structure comprising a double-stranded region (stem), said double-stranded region Formed by two regions of the nucleic acid molecule (on the same molecule), the two regions are flanked by double-stranded portions; they also include at least one "loop" structure, including non-complementary nucleic acid molecules, ie, single-stranded regions. Even if the two regions of the nucleic acid molecule are not fully complementary, the double-stranded portion of the nucleic acid can remain in a double-stranded state. For example, insertions, deletions, substitutions, etc. may result in non-complementation of a small region or the formation of a stem-loop structure or other form of secondary structure by itself, however, the two regions may still be substantially complementary and are foreseeable Interaction occurs in the manner to form a double-stranded region of the stem-loop structure. Stem loop structures are well known to those skilled in the art, and typically after obtaining a nucleic acid having a nucleotide sequence of a primary structure, one skilled in the art will be able to determine whether the nucleic acid is capable of forming a stem-loop structure.

In the present invention, a preferred stem-loop structure can be represented by a dsRNA of Formula II, X' is a spacer sequence located between the Seq' _forward and Seq' _inversions , and the spacer sequence is Seq' _forward and Seq ' _Reverse non-complementary, when X' is absent, the dsRNA of Formula II is a complementary double-stranded RNA structure formed by Seq' _forward and Seq' _reverse .

As used herein, the term "nucleic acid inhibitor" refers to a class of target genes (fragments) useful in the control of herbivorous pests or fragments or truncated forms thereof, which are obtained by controlling the activity of herbivorous pests. The general name of the substance. The "nucleic acid inhibitor" is, for example, some interfering molecule, including dsRNA (also known as double-stranded RNA, double-stranded ribonucleic acid or double-stranded ribonucleotide sequence), antisense nucleic acid, small interfering RNA, microRNA, and the like. Or a construct that can express or form the dsRNA, antisense nucleic acid, small interfering RNA or microRNA.

In the present invention, the nucleic acid inhibitor is preferably a dsRNA.

As used herein, "operably linked" or "operably linked" refers to a spatial arrangement of the functionality of two or more nucleic acid regions or nucleic acid sequences. For example, the promoter region is placed at a specific position relative to the nucleic acid sequence of the gene of interest such that transcription of the nucleic acid sequence is directed by the promoter region such that the promoter region is "operably linked" to the nucleic acid sequence.

As used herein, "an RNA sequence corresponding to a DNA sequence" refers to an RNA sequence which is "AU" if the DNA sequence is "AT".

"Include", "has" or "includes" includes "including", "consisting essentially of", "consisting essentially of", and "consisting of"; "consisting primarily of", "Substantially composed of" and "consisting of" are subordinate concepts of "contains," "has," or "includes."

RNA interference (RNAi)

As used herein, the term "RNA interference (RNAi)" means that some small double-stranded RNA can efficiently and specifically block the expression of specific genes in the body, promote mRNA degradation, and induce cell expression. A phenotype of a specific gene deletion is also known, which is also referred to as RNA intervention or RNA interference. RNA interference is a highly specific mechanism of gene silencing at the mRNA level.

As used herein, the term "small interfering RNA (siRNA)" or "dsRNA" refers to a short-segment double-stranded RNA molecule that is capable of degrading specific mRNAs with mRNAs of homologous complementary sequences. This process is RNA. RNA interference pathway.

In the present invention, the basic principle of the RNA interference is to prepare the interfering RNA as an irrigation liquid, and after the plants are watered according to the conventional irrigation method, the insects are subjected to small interfering RNA (siRNA) or dsRNA which may interfere with the expression of the gene. Plants, thereby inducing the death of insects.

As a preferred manner, an intron sequence is ligated to the complementary gene sequence at both ends, and after introduction into the cell, a "stem-loop" structure can be produced, and the "stem"-like portion can be processed into an insect body. Small RNA of about 21-25 nt, this small RNA can effectively inhibit the expression of the target gene.

In the present invention, a preferred interfering RNA is a dsRNA. A dsRNA is a double-stranded RNA sequence formed by a positive strand and its complementary strand. Among them, the positive and negative chains can be completely matched or partially matched. A preferred dsRNA has dsDNA as shown in the formula II.

In the present invention, the length of the dsRNA sequence is not particularly limited, and is usually 100-800 bp in length, preferably 150-600 bp.

In another preferred embodiment, the sequence of the dsRNA is derived from a species selected from the group consisting of agricultural or forest pests or vegetable pests. Preferably, the species comprises the Asian corn borer of the Lepidoptera family and the rice brown planthopper of the Homoptera family.

In another preferred embodiment, the dsRNA sequence is synthetic or artificially produced.

In another preferred embodiment, the "derived from" means that the dsRNA has a homology (identity) to the nucleotide sequence of the target gene in the herbivorous pest of 95-100%, preferably It is 99-100%.

In another preferred embodiment, the target gene is unique to the herbivorous pest.

In another preferred embodiment, the sequence of the dsRNA is selected from the group consisting of SEQ ID NOs.: 4-6; or a dsRNA of ≥ 50 bp in length.

Insect growth related genes

As used herein, the terms "insect growth related gene", "phytophagous pest growth related gene" are used interchangeably and refer to a gene related to insect growth mainly in plants (especially crops). "Growth-related" means that low or no expression of the gene will cause abnormalities in the growth, development, metabolism, reproduction, etc. of the insect, and even lead to the death of the insect. In a preferred embodiment of the present invention, the herbivorous pest growth-related gene comprises a P450 gene (Cyp18A1), a carboxylesterase (Ces), or a K-serine protease inhibitor of Asian corn borer. Genes (KPIs).

"Insect growth related genes" are those genes closely related to insect growth. For example, in the P450 gene, many of them participate in the detoxification of foods. Once the expression of such genes is inhibited, insect poisoning is caused. Esterase is an important detoxifying enzyme system in insects. It can reduce the toxicity of toxic compounds by hydrolyzing ester bonds of ester toxic compounds or by combining with lipophilic toxic compounds to reduce their effective concentration. Many insecticides are ester compounds containing ester bonds, such as organophosphorus, carbamate and pyrethroid pesticides. Esterases also play important roles in insect resistance, such as carboxylesterases. Therefore, interfering with the expression of esterase in insects can not only reduce its detoxification ability, but also prevent its resistance. Protease inhibitors are a class of substances that inhibit protease activity. Serine protease inhibitors can regulate a variety of physiological reactions in the body, inhibit the catalytic action of the enzyme, prevent the conversion of the zymogen into an active enzyme, and therefore affect the normal insects. Growth and development.

Preferably, in the brown planthopper, the dsRNA designed for the P450 gene (Cyp18A1) fragment and the carboxylesterase (Ces) fragment is used to soak the rice roots and then the plant organs other than the soaking part are fed to the brown planthopper, corresponding genes. Expression was down-regulated (Fig. 2a, 2b); the mortality of brown planthopper after 5 days of feeding on the corresponding dsRNA-treated rice was 26.67% and 48.33%, respectively (Fig. 2c). In Asian corn borer, the expression of this gene was significantly down-regulated by feeding dsRNAs designed for K-serine protease inhibitor gene (KPIs) fragments to Asian corn borer after watering the maize (Fig. 3a). The mortality rate of Asian corn borer after 5 days of feeding on the corresponding dsRNA-treated corn was 43.33% (Fig. 3b).

As a preferred aspect of the present invention, the preferred fragment of the herbivorous pest growth-related gene of the present invention has a length of at least 50 bp, and may be, for example, 60 bp, 80 bp, 100 bp, 200 bp, 500 bp, or 1000 bp. The gene may be a full-length gene or a gene fragment when used in the present invention, preferably, the dsRNA of the P450 gene (Cyp18A1) against rice planthopper is as shown in SEQ ID NO: 4; the carboxylic acid against rice planthopper The dsRNA of the esterase gene (Ces) is shown in SEQ ID NO: 5; the dsRNA of the K-type serine protease inhibitor gene (KPIs) against Asian corn borer is shown in SEQ ID NO: 6.

The present invention also provides a dsRNA directed against a non-insect-derived EYFP gene, the sequence of which is set forth in SEQ ID NO: 7. Compared with the rice planthopper P450 gene, the carboxylesterase gene and the Asian corn borer K-serine protease inhibitor gene, the EYFP gene is not effective.

The present invention provides a dsRNA for a herbivorous pest growth-related gene, which can be irrigated by the dsRNA to irrigate the root of the crop and absorbed through the root of the plant, and the insect is inhibited from growth and a large number of deaths are observed after the crop having absorbed the dsRNA.

The dsRNA construct of the present invention is represented by Formula I, and the dsRNA is represented by Formula II, and the length of the spacer sequence X or X' employed is not particularly limited as long as it forms a construct with a forward sequence and a reverse sequence. After being introduced into the body, the dsRNA represented by Formula II can be formed. As a preferred mode of the present invention, the spacer sequence of the present invention has a length of 80 to 300 bp; more preferably 100 to 250 bp.

In a preferred embodiment of the present invention, the construct expressing the herbivorous pest growth-related gene dsRNA is introduced into a host, and the host may be a plant cell, a tissue or an organ, and the construct may be expressed in a plant. The insect gene dsRNA, dsRNA, is processed into siRNA. Generally, the length of the siRNA is about 21-25 nt.

Typically, the construct is located on an expression vector. Accordingly, the present invention also encompasses a vector comprising the construct. The expression vector typically also contains a promoter, an origin of replication, and/or a marker gene, etc., operably linked to the construct. Methods well known to those skilled in the art can be used to construct the expression vectors required for the present invention. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The expression vector preferably comprises one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as kalatinmycin, gentamicin, hygromycin, ampicillin resistance.

Vectors comprising the appropriate gene sequences described above, as well as appropriate promoters or control sequences, can be used to transform a suitable host. In the methods of the invention, the host may be any host suitable for carrying the expression vector and capable of delivering the expression vector to a plant cell. Preferably, the host is Agrobacterium.

Although the herbivorous pests exemplified in the examples of the present invention are Asian corn borer and rice planthopper. However, it should be understood that the present invention is not particularly limited to insects suitable for use in the present invention, and the insect may be any herbivorous pest that can feed on plants, such as lepidopteran, hemipteran, Homoptera or Diptera insects.

The present invention is not particularly limited to plants suitable for use in the present invention, and may be various common economic crops. Or ornamental plants, such as Gramineae, Cruciferae, Leguminosae, etc., preferably corn or rice.

Nucleotide sequence of the target gene

The P450 (Cyp18A1) gene fragment of rice planthopper is shown in SEQ ID NO. 1; the carboxylesterase (Ces) gene fragment of rice planthopper is shown in SEQ ID NO. 2: the type K of Asian corn borer The full-length cDNA sequence of the serine protease inhibitor (KPIs) gene is shown in SEQ ID NO.

Target sequence of dsRNA

In one embodiment of the present invention, based on the RNAi technology, an interfering RNA fragment targeting the P450 gene is screened using the P450 gene of rice planthopper (Cyp18A1) as a target, and preferably, the dsRNA sequence of the P450 gene fragment is SEQ ID. NO: 4 (dsRNA1: dsCyp18A1).

In one embodiment of the present invention, an interfering RNA fragment directed against a carboxylesterase gene, preferably the carboxylic acid, is screened based on the RNAi technology using a carboxylesterase (Ces) gene as a target. The dsRNA sequence of the esterase gene fragment is set forth in SEQ ID NO: 5 (dsRNA2: dsCes).

In one embodiment of the present invention, interfering RNA fragments targeting K-serine protease inhibitors (KPIs) are screened based on RNAi technology using K-type serine protease inhibitors (KPIs) genes of Asian corn borer as targets. The dsRNA sequence of the K-type serine protease inhibitor (KPIs) gene fragment is set forth in SEQ ID NO: 6.

In one embodiment of the invention, the enhanced yellow fluorescent protein gene EYFP of jellyfish is used as a foreign gene control (SEQ ID NO.: 7) based on RNAi technology. The dsRNA sequence of the EYFP gene fragment is set forth in SEQ ID NO: 41 (dsRNA: dsEYFP).

dsRNA construct and its application

Any substance obtained based on the target gene (fragment) provided by the present invention or a fragment thereof or a truncated form, which has activity for controlling herbivorous pests, can be used as a nucleic acid inhibitor for controlling herbivorous pests. Preferably, the nucleic acid inhibitor is a construct of a plurality of interfering molecules, eg, dsRNA, antisense nucleic acid, small interfering RNA or microRNA, or may express or form the dsRNA, antisense nucleic acid, small interfering RNA or micro RNA construct. More preferably, it is a dsRNA or a construct that can express the dsRNA.

According to the genes and sequences thereof provided by the present invention, constructs for expressing dsRNA, antisense nucleic acids, small interfering RNAs or microRNAs can be designed. Accordingly, the present invention provides an artificially constructed construct. Designing the constructs according to the genes provided by the present invention and their sequences is well known to those skilled in the art, and generally allows the construct to comprise an intron sequence (not complementary to the flanking sequences), ligated at both ends A complementary gene sequence that, when introduced into a cell, produces a "stem loop" structure, and the "stem" portion can form dsRNA, antisense nucleic acid, small interfering RNA or microRNA, such dsRNA, antisense nucleic acid, small interfering RNA or MicroRNAs are particularly effective in inhibiting the expression of genes of interest.

A preferred method for controlling the herbivorous pest of the present invention comprises directly irrigating or soaking the nucleic acid inhibitor for a subject (such as a plant) to be controlled to achieve the purpose of controlling a herbivorous pest.

The invention provides a dsRNA construct, the construct of the dsRNA is double stranded, and the positive or negative strand thereof comprises the structure of formula I:

Seq _forward- X-Seq _inverse I

In the formula,

A nucleotide sequence Seq _forward growth-related genes or fragments of phytophagous insects;

Seq _reverses to a nucleotide sequence that is substantially complementary to the Seq _forward ;

X is none, or is a sequence of intervals between the Seq _forward and Seq _reverse , and the interval sequence is not complementary to the Seq _forward and Seq _reverse ,

The plant-related pest growth-related genes are the P450 gene (Cyp18A1), the carboxylesterase (Ces), and the K-serine protease inhibitor gene (KPIs) of the Asian corn borer.

In a preferred embodiment of the invention, the Seq _forward and Seq _inverse lengths are at least 50 bp.

In a preferred embodiment of the present invention, the dsRNA construct is taken up by the root of the crop and ingested by the herbivorous pest to form a dsRNA of the formula II.

Formula II

In the formula,

Seq _{'Forward Forward} sequence corresponds to Seq RNA sequences or fragments of sequences;

Seq' _reverse is a sequence that is substantially complementary to the Seq'_forward;

X 'is absent; or located Seq' _forward and Seq 'spacer sequence between the _reverse, and the spacer sequence Seq' _forward and Seq 'no _reverse complementary,

|| represents a hydrogen bond formed between the Seq' _forward and Seq' _reverse .

Of course, the construct can also be located on an expression vector. Accordingly, the present invention also encompasses a vector comprising the construct. The expression vector typically also contains a promoter, an origin of replication, and/or a marker gene, etc., operably linked to the construct. Methods well known to those skilled in the art can be used to construct the expression vectors required for the present invention. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The expression vector preferably comprises one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as kanamycin, gentamicin, hygromycin, ampicillin resistance .

Vectors comprising the appropriate gene sequences described above, as well as appropriate promoters or control sequences, can be used to transform a suitable host. In the methods of the invention, the host can be any host suitable for carrying the expression vector and capable of expressing a nucleic acid inhibitor. For example, the host is Escherichia coli, fungi, yeast, plant cells, animal cells, and the like.

Transformation of the host with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art, depending on the type of plant. When the host is a prokaryote such as E. coli, competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated by the CaCl ₂ method, and the procedures used are well known in the art. Another method is to use MgCl _2. Conversion can also be carried out by electroporation if desired. Transformation of fungal and yeast cells, plant cells, animal cells is also well known to those skilled in the art.

A host carrying the construct or expression vector and capable of expressing a nucleic acid inhibitor can be directly administered to a subject (such as a plant) in need of control for the purpose of controlling a herbivorous pest.

The "stem"-like portion of the stem-loop structure described above is formed by the reverse interaction of Seq forward and Seq and can be processed to form a nucleic acid inhibitor. The formed nucleic acid inhibitor has the following structure:

Wherein, Seq' is _positively selected from the RNA sequence or sequence fragment corresponding to the sequence shown in any one of SEQ ID NO: 1-3; and Seq' is _reversed to a sequence substantially complementary to the Seq' _forward . X 'is positioned Seq' _forward and Seq 'spacer sequence between the _reverse, and the spacer sequence Seq' _forward and Seq 'is not complementary to _{the reverse.} The X' sequence can be excised in vitro or not, and the dsRNA is processed and excised by an enzyme (such as nuclease Dicer) in the insect after it enters the insect body.

Composition and its application

The invention also provides a composition comprising a dsRNA construct and/or dsRNA, and a pharmaceutically or agrochemically acceptable carrier. In another preferred embodiment, the composition is a composition for interfering with the growth of a herbivorous pest, inducing or causing death of a herbivorous pest.

In another preferred embodiment, the dsRNA has the following sequence:

dsRNA1: as shown in SEQ ID NO.: 4;

dsRNA2: as shown in SEQ ID NO.: 5;

dsRNA3: as shown in SEQ ID NO.: 6;

In a preferred embodiment of the invention, the composition is an aqueous solution, typically having a pH of from about 5 to about 8, preferably a pH of from about 6 to about 8.

The term "carrier," as used herein, includes various excipients and diluents. Such carriers include, but are not limited to, water, saline, buffer, dextrose, glycerol, ethanol, and combinations thereof.

The composition of the present invention can be directly irrigated or watered to a crop organ, wherein the organ includes roots, stems, leaves, flowers, and fruits of the plant; in addition, organs of the plant can be immersed in the composition of the present invention to achieve The composition of the invention is absorbed by plants. Of course, it is preferred that the agricultural composition of the present invention is formulated or added to an irrigation liquid and irrigated at the root of the plant, so that the dsRNA of the present invention is absorbed through the roots and brought to the non-irrigated portion, thereby allowing the pest to feed the non-irrigated portion of the plant. After that, growth inhibition or death occurs to achieve the insect resistance of the plant.

Water and an aqueous solution containing other adjuvants in the composition of the present invention are prepared by a conventional method. Preferably, the composition is made under sterile or RNase free conditions.

The plant-feeding pest growth-related gene is derived from the P450 gene (Cyp18A1), the carboxylesterase (Ces), and the K-serine protease inhibitor gene (KPIs) of Asian corn borer;

In another preferred embodiment, the insect is a sucking mouthparts pest or a borer pest, preferably from a herbivorous pest, preferably from rice brown planthopper and Asian corn borer.

Advantages of the invention

(1) The present invention applies a dsRNA preparation of an effective target gene to a field pest control simply by irrigation or soaking, so that the plant can effectively absorb the dsRNA of the present invention and a composition thereof, thereby avoiding complicated transgenic operation of all crops. ;

(2) The present invention performs high-throughput screening of herbivorous pest target genes for sucking, drilling, and underground pests;

(3) The present invention solves the problem of resistance that may occur during pest control through the rotation and mixing application of the target;

(4) The dsRNA preparation of the present invention has relative stability and ensures the function of playing an insecticide during the growth period of the crop;

(5) Under field light conditions, the dsRNA preparation of the present invention can be rapidly reduced after the crop growth period Solution, with good bio-safety and ecological safety.

The invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are not intended to limit the scope of the invention. The experimental methods in the following examples which do not specify the specific conditions are usually carried out according to the conditions described in conventional conditions such as Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer. The suggested conditions.

General method

Planting and cultivation of plant materials

The plant materials used in this study were rice variety Zhonghua 11 (ZH11, purchased from China Rice Research Institute) and corn variety Zhengdan 958 (ZD958, purchased from Henan Agricultural Science Research Institute). Rice seeds were sown in a cylindrical plastic cup 10 cm high and 5 cm in diameter; corn seeds were sown in a 15 cm cylindrical plastic cup with a diameter of 10 cm. All plants are grown in a greenhouse after planting. The day/night temperature in the greenhouse is 28/22 ° C, the day/night cycle is 14/10 hours, and the relative humidity is 50-60%.

Insect breeding

Rice brown planthopper and Asian corn borer were collected from Shanghai Songjiang Wuyi Farm. The brown planthopper population was raised on the ZH11 rice variety planted above, and 2-3 instar nymphs were used for this experiment.

Asian corn borer is bred in a light incubator with artificial feed. The incubator temperature was 25 ° C, the day/night cycle was 14/10 hours, and the relative humidity was 75%.

The corn carp feeding formula is: corn poulte 120.0 g, corn flour 32.0 g, soybean powder 120.0 g, vitamin C 4.0 g, agar 12.0 g, yeast powder 72 g, sorbic acid 4.0 g, glucose 60.0 g, formaldehyde 1.6 ml, water 1 liter .

Example 1

Screening target gene

1. Extraction of total RNA from rice, corn, brown planthopper and Asian corn borer

The leaves of rice and maize were taken, the nymphs of 2-3 years old were taken from brown planthopper, and the total RNA was extracted from 3rd instar larvae of Asian corn borer. RNA was extracted by conventional Trizol method, purified by conventional methods and treated with DNase to obtain concentration ≥300ng/ul. Total RNA samples with a total of ≥6ug and OD260/280 of 1.8-2.2.

2.Isolation of mRNA and synthesis of cDNA

The mRNA with polyA was isolated using magnetic beads with oligo-dT, and then the first strand of cDNA was synthesized using a random hexamer and Invitrogen's Superscript II reverse transcriptase kit.

3. Gene amplification and sequencing

Using the target gene-specific primers shown in Table 1, the above-prepared cDNA was used as a template to amplify the genes listed in the table, and the obtained gene fragment was purified, ligated into pMD18-T vector (Takara), and transformed into E. coli Top10 strain, blue-white spot screening, positive strain sequencing, used as a template for dsRNA synthesis after correct testing.

Table 1. Primers used for gene synthesis

Example 2

Synthesis of dsRNA

The target gene sequence of the synthetic dsRNA was amplified and sequenced using a primer with a T7 adaptor (Table 2), and the dsRNA was synthesized and purified using a MEGAscript RNAi kit (Ambion, Huntingdon, UK).

Specific steps and methods

1. Extract the total RNA of the target species by Trizol method and purify it.

(1) 100 mg of tissue was ground to a powder with liquid nitrogen (using a DEPC-treated mortar);

(2) Pour the dry powder into the loading

In a centrifuge tube;

(3) After standing at room temperature for 5 min, add 200 μl of chloroform, and mix by inversion for 15 s;

(4) After standing at room temperature for 5 min, centrifuge at 12000 rpm for 15 min at 4 °C;

(5) Take the supernatant liquid into a new centrifuge tube, add an equal volume of isopropyl alcohol (~500μl), and mix and invert several times;

(6) After standing at room temperature for 10 min, centrifuge at 10 ° C for 10 min at 4 ° C;

(7) gently pour off the supernatant, retain the precipitate, add 500 ~ 1000μl 75% ethanol and slightly rinse;

(8) After centrifugation at 7600 rpm for 5 min at 4 ° C, the ethanol was decanted, and the ethanol was inverted on the absorbent paper to maintain the ethanol for 10 min;

(9) Add appropriate amount of DEPC water and dissolve for 10 minutes.

(10) Expand the RNA sample plus DEPC water to 100 μl, add 100 μl (equal amount) of phenol/chloroform/isoamyl alcohol (25:24:1), and mix well;

(11) centrifugation at 13,000 rpm for 5 min at room temperature, the upper layer of liquid was transferred to another centrifuge tube;

(12) Add 100 μl (equal amount) of chloroform/isoamyl alcohol (24:1) and mix well;

(13) centrifugation at 13,000 rpm for 5 min at room temperature, the upper layer liquid is transferred to another centrifuge tube;

(14) Add 10 μl (1/10 amount) of 3M NaOAc (pH 5.2);

(15) Add 250 μl (2.5 times) cold absolute ethanol, precipitate at -70 ° C overnight (or precipitate at -20 ° C for 2 h);

(16) Centrifuge at 13000 rpm for 5 min at 4 ° C, remove the supernatant to leave a precipitate, rinse with 1 ml of 75% cold ethanol, and dry for 5 min;

(17) Add appropriate amount of DEPC water and dissolve for 10 minutes;

(18) Dilute 100 times the measured concentration and measure the quality by electrophoresis.

2. Reverse the cDNA.

(1) Mix the following reagents:

(2) plus Oligo dT Primer (25pmol / μl) 1μl;

(3) plus template RNA (1 μg or less) x μl;

(4) plus reverse transcriptase 1 μl;

(5) 30 ° C 10 min, 42 ° C 20 min, 99 ° C 5 min;

(6) Immediately placed on ice for 5 min;

(7) After transient centrifugation, the cDNA was stored at -20 °C.

3. The dsRNA template was amplified using the dsRNA primers in Table 2, and the product was cut into gels.

(1) Electrophoresis and tapping;

(2) Add an equal volume of Binding Buffer (XP2), incubate at 55-60 ° C for 7 min or until the gel is completely dissolved, shaking once every 2 to 3 min;

(3) will

Put the DNA column into a 2ml collection tube;

(4) Add 700 μl of gelatin to the column and centrifuge at 10,000 × g for 1 min at room temperature;

(5) Dispose of the filtered liquid, and the column is placed in the original collection tube;

(6) Add 300 μl Binding Buffer (XP2) to the column, centrifuge at 10,000 × g for 1 min at room temperature, discard the filtered solution, and place the column into the original tube;

(7) Add 700 μl SPW Wash Buffer (diluted with absolute ethanol) to the column, centrifuge at 10,000 × g for 1 min at room temperature, discard the filtered solution, and place the column into the original tube;

(8) OPTIONAL: repeat step (7);

(9) discard the filtered liquid, and the empty column is centrifuged at the highest speed (≥13,000×g) for 2 min;

(10) Put the column into a clean 1.5ml centrifuge tube, add 30-50μl Elution Buffer to the center of the column, leave it at room temperature for 1min, and centrifuge at the highest speed (≥13,000×g) for 1min to elute the DNA.

4, dsRNA synthesis

(1) 8 μL ATP/CTP/UTP/GTP solution

(2) 1 μg of purified DNA

(3) 2μL Enzyme Mix

(4) The mixture was incubated in a 37 ° C incubator for 4 h, and the product was dsRNA.

(5) Add 1 μL of TURBO DNase, mix and incubate at 37 ° C for 15 min, the product is dsRNA

Table 2 Primers used to synthesize dsRNA

Among them, EYFP is an enhanced yellow fluorescent protein gene as a foreign gene control.

Example 3 Absorption of exogenous dsRNA by plant organs

This experiment uses blank (CK), dsRNA of the present invention (SEQ ID NOs.: 4-6), dsRNA of rice endogenous gene Actin (SEQ ID NO.: 40), and control exogenous dsRNA, dsEYFP (SEQ ID). NO.: 41) After the dsRNA was absorbed by the root system, the expression of dsRNA in the stem (the stem not in contact with the dsRNA soaking solution) was observed and measured, and the final concentration of all dsRNAs was 0.5 mg/ml, which were grouped as follows:

a. blank control group: recorded as CK, this control is planted (rice or corn), the same method of planting, the growth conditions are the same, but not treated with dsRNA solution, as in the present invention, the rice is soaked with water, Corn is irrigated with clear water;

b. The dsRNA of the present invention: a dsRNA (dsCyp18A1), a carboxylesterase (Ces) dsRNA (dsCES), or a K-serine endogenously expressed in Asian corn borer, which is endogenously expressed by the brown planthopper, P450 gene (Cyp18A1) The dsRNA (dsKPI) of the protease inhibitor gene (KPIs) is used to irrigate or soak the roots of the plants, and the dsRNA expression of the stems of the dsCES, dsCyp18A1, and dsKPI of the present invention after root absorption is measured;

c. Soaking rice roots with dsRNA of rice endogenous gene Actin, and measuring the expression of Actin in the stem after dsRNA absorption through the root;

d. Exogenous genes not found in insects and plants: The enhanced yellow fluorescent protein (EYFP) dsRNA (dsEYFP) of Victoria multi-tube jellyfish is used to irrigate or soak the roots of plants to determine the expression of dsRNA in stems.

result:

After soaking the rice roots with FAM fluorescently labeled dsEYFP at a final concentration of 0.5 mg/ml for 24 h, obvious green fluorescence was observed in the leaf sheath of rice under the fluorescence microscope (Fig. 1a). Apparent green fluorescence was observed in the stem of rice (Fig. 1b), and the rice stem RNA after dsRNA was extracted to amplify the dsRNA as a template, and a band of the same size as the exogenous gene dsEYFP was obtained (Fig. 1c). Moreover, after soaking rice roots with the dsRNA of the rice endogenous gene Actin for 24 h, the growth of rice roots was significantly inhibited (Fig. 1d, e), and the expression of this gene was also significantly inhibited (Fig. 1f). These results indicate that exogenous dsRNA can enter the plant through the root system and inhibit the expression of related genes.

As shown in Figure 6, CK is the total RNA extracted from the water treatment control; DS- is the total RNA extracted from the stem after irrigation or soaking the roots of the plants for 24 hours with different double-stranded RNA, and the arrow below it indicates that there is A specific band different from CK is the exogenous double-stranded dsRNA that the plant absorbs because there is no such specific band in all CK controls. Figure 7 shows that after soaking rice with three different dsRNAs such as dsCYP18A1; dsCes; dsEYFP for 24h, the total RNA was extracted from the stem of rice, and then inverted into cDNA, the dsRNA fragment of the foreign gene was amplified, and the result was obtained. Fragments of consistent size, but no bands in the clear water control.

In addition, the dsRNA of the present invention was spotted on the surface of rice leaves, and it was found that after 24 hours, the leaf surface at the distal end of the drip was observed, and fluorescence phenomenon was also observed, indicating that the dsRNA of the present invention can be absorbed by the foliage of the plant. (Figure 5)

Conclusion: Fluorescently labeled exogenous dsRNA can be absorbed into the stem of plants by plant organs, especially roots. The dsRNA of the plant gene itself can be absorbed by roots and affect the growth of plants, while the dsRNAs for insect growth in the present invention are also Both can be absorbed by plant organs, especially the roots, and transported to the stems of plants. And leaves can also transport dsRNA. Thus, exogenous dsRNA can be absorbed into the interior of the plant through organs of the plant, such as roots, leaves, and the like.

Example 4 Test for lethal effects of insects by exogenous dsRNA absorbed by the roots.

In this experiment, synthetic dsRNA was used to simulate rice and maize, and its effects on insects were observed. The gene expression of the samples was detected by qPCR. The primers are shown in Table 3. The sequence and gene source information of dsRNA are shown in Table 4. Shown.

Table 3. Primers for quantitative PCR detection

Table 4 dsRNA name and source information

4.1 Lethal effect of rice roots on the absorption of dsRNA against brown planthopper

After germination, the seeds of rice ZH11 were grown in the nutrient solution to the two-leaf stage. The rice was transplanted into a 0.5 mg/ml dsRNA solution, and the rice roots were all immersed in the solution. After soaking for 24 hours, the soaked rice was obtained.

Then, 1-2 instar brown planthoppers were connected to the above-mentioned soaked rice, and each plant had 10 heads. The planthoppers were mainly concentrated in the stems of rice, and were fed from the roots of 1-4 cm (non-soaked parts). After 24 hours of inoculation, the planthoppers were taken to detect gene expression. During the entire test, protective measures were taken to prevent the planthopper from directly contacting the dsRNA solution.

Among them, the dsRNA solution is a synthetic P450 gene (Cyp18A1) and or a carboxylesterase (Ces) dsRNA or dsEYFP (concentration of 0.5 mg/mL, respectively) expressed by the brown planthopper.

The results showed that the expression of the two genes of brown planthopper was significantly down-regulated (Fig. 2a, b). After 24 hours of treatment with double-stranded RNA, the mortality rates of brown planthoppers on the corresponding dsRNA-treated rice for 5 days after continuous feeding were 26.67% and 48.33%, respectively (Fig. 2c and Table 5).

Table 5. Lethality rate of rice planthopper after watering rice with brown planthopper gene dsRNA

4.2 Lethality of Asian corn borer after absorption of dsRNA by maize roots

The Zhengdan 985 corn seed was planted in the nutrient mash. When the corn grows to the 3-leaf stage, the roots are irrigated with 0.5 mg/ml dsRNA solution. After 24 hours, the corn mash is connected, and each corn seedling is connected to the 2nd-instar corn mash. There are 10 larvae, and the corn borer mainly feeds on the corn leaf parts and the upper part of the stem, and does not penetrate into the soil to feed. After 24 h, corn mash was taken to detect gene expression. Among them, the dsRNA solution is a dsRNA or dsEYFP (concentration of 0.5 mg/mL) of K-type serine protease inhibitor gene (KPIs) endogenously expressed in synthetic Asian corn borer.

The results showed that the expression of this gene in corn borer was significantly down-regulated (Fig. 3a). After 24 hours of treatment with double-stranded RNA, the mortality of Asian corn borer on the corresponding dsRNA-treated corn after continuous feeding 5 was 43.33% (Fig. 3b and Table 6).

Table 6. Lethality rate of corn borer after watering corn with Asian corn borer gene dsRNA

Example 5 dsRNA stability and degradability test

The synthesized dsRNA was placed under 4 conditions of 4 ° C, natural light or shading, and the results showed that the dsRNA was stored at 4 ° C for 90 days, and no significant degradation was observed ( FIG. 4 a ). Partial degradation was observed at room temperature in the dark, and 50% of dsRNA was also detected at 90 days (Shading in Figures 4b and 4c). At room temperature, significant degradation began at 45 days, and dsRNA was not detected at 90 days (sunlight in Figures 4b and 4c). On the one hand, these results indicate that double-stranded RNA is a relatively stable preparation, which can ensure the stability of the storage of the preparation and play a role in a certain period of time after application. On the other hand, under light conditions, it can be degraded faster, thus not It will cause environmental pollution.

All documents mentioned in the present application are hereby incorporated by reference in their entirety in their entireties in the the the the the the the the In addition, it should be understood that various modifications and changes may be made by those skilled in the art in the form of the appended claims.

Claims (17)

1. A method for introducing a double-stranded RNA (dsRNA) into a plant, comprising the steps of: applying said double-stranded RNA to a root of a plant, said root-absorbing effect of said plant on double-stranded RNA, said Double-stranded RNA is inhaled by the plant and delivered to a portion of the plant other than the root.
2. A method for improving plant resistance to insects, comprising the steps of: applying insect-resistant double-stranded RNA (dsRNA) to a root of a plant such that the insect-resistant double-stranded RNA is absorbed by the root of the plant, and It is transported to the parts of the plant other than the roots to increase the insect resistance of the plants.
3. The method according to claim 1 or 2, wherein the dsRNA is a dsRNA directed against an insect growth related gene.
4. The method of claim 3, wherein said dsRNA is as set forth in SEQ ID NOs.: 4-6.
5. A method according to claim 1 or 2, wherein said means of application comprises irrigation, soaking, watering, or drip irrigation.
6. The method of claim 2 wherein said insect resistance comprises an herbivorous pest.
7. The method according to claim 1 or 2, wherein the plant comprises a dicot, a monocot, or a gymnosperm.
8. An agricultural composition characterized in that the agricultural composition comprises a pesticidally effective amount of an insect resistant double-stranded RNA and a pesticidally acceptable carrier.
9. The agricultural composition according to claim 8, wherein the dsRNA is applied at a concentration of 0.0001 to 5 mg/g or 0.0001-5 mg/ml, preferably 0.001 to 1 mg/ml or 0.001-1 mg/g.
10. Use of an insect-resistant double-stranded RNA sequence for preparing a composition for enhancing insect resistance of a plant by root application.
11. An isolated polynucleotide, characterized in that said polynucleotide is selected from the group consisting of:

    (a) the sequence shown in any one of SEQ ID NO.: 1-6;

    (b) a polynucleotide complementary to the sequence defined by (a); or

    (c) having at least 70% (preferably at least 75%, 80%, 85%, 90%, more preferably at least 95%, 96%, 97%, 98%, 99%) or more of the sequence defined by (a) Any polynucleotide or complementary sequence of sequence identity, wherein the dsRNA of the strand complementary to any of the polynucleotide sequences has an activity of inhibiting the growth of the herbivorous pest after ingestion by the herbivorous pest.
12. A dsRNA construct characterized in that the construct of the dsRNA is double-stranded and the positive or negative strand thereof comprises the structure of formula I:

    Seq _forward- X-Seq _reverse

    Formula I

    In the formula,

    A nucleotide sequence Seq _forward growth-related genes or fragments of phytophagous insects;

    Seq _reverses to a nucleotide sequence that is substantially complementary to the Seq _forward ;

    X is a spacer sequence located between the _forward and Seq Seq the _reverse, and the spacer sequence Seq Seq _forward and _reverse are not complementary,

    Wherein the plant-borne pest growth-related gene is selected from the group consisting of: a rice planthopper P450 gene (Cyp18A1), a rice locust carboxylasease (Ces), or an Asian corn borer K-serine protease inhibitor gene (KPIs). ).
13. The dsRNA construct of claim 12, wherein the dsRNA construct forms a dsRNA of formula II,

    

    In the formula,

    Seq _{'Forward Forward} sequence corresponds to Seq RNA sequences or fragments of sequences;

    Seq' _reverse is a sequence that is substantially complementary to the Seq'_forward;

    X 'is absent; or located Seq' _forward and Seq 'spacer sequence between the _reverse, and the spacer sequence Seq' _forward and Seq 'no _reverse complementary,

    || represents a hydrogen bond formed between the Seq _forward and the Seq _reverse .
14. An expression vector comprising the dsRNA construct of claim 12.
15. A genetically engineered host cell comprising the expression vector of claim 14 or a DNA sequence incorporating a dsRNA construct according to claim 12 in a chromosome.
16. A dsRNA sequence characterized in that the dsRNA sequence is an insect resistant dsRNA and has a length of from 100 to 800 bp, preferably from 150 to 600 bp.
17. A method for controlling pests, which comprises applying the agricultural composition of claim 8 or the dsRNA of claim 16 to a plant in need of pest control.

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