CN117965545A - MiR-8492-3p for preventing lepidoptera pests and application thereof - Google Patents
MiR-8492-3p for preventing lepidoptera pests and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of agricultural biology, and provides miR-8492-3p for preventing lepidoptera pests and application thereof. According to the invention, miRNA of a key gene for the growth and development of plutella xylostella is analyzed, plutella xylostella lethal miRNA (pxy-miR-8492-3 p) is screened, and the fact that the plutella xylostella chitinase (Chi 10) and the plutella xylostella ecdysone receptor (EcR) are inhibited by agomir-8492 after analogues agomir-8492 are injected is found, so that plutella xylostella dies. In addition, prokaryotic expressed amiRNA-8492 or pxy-miR-8492-3p transgenic rape have high toxicity to various lepidopteran pests. The invention provides a new target and technical support for establishing a new strategy for preventing and controlling lepidoptera pests such as plutella xylostella and the like based on miRNA-RNAi.
Description
Technical Field
The invention belongs to the technical field of agricultural biology, and particularly relates to miRNA (miR-8492-3 p) for preventing lepidoptera pests and application thereof.
Background
The plutella xylostella is an important pest on cruciferous vegetables, algebra occurs in many years, generation overlap is serious, and huge economic loss is caused each year. At present, the main means for preventing and controlling the plutella xylostella still depend on chemical prevention and control means of pesticides, but due to unreasonable use of the chemical pesticides, the plutella xylostella almost has resistance to all used pesticides, and the usage amount of pesticides can only be increased continuously to achieve better prevention and control effects, so that more serious environmental pollution is caused, and vicious circle is formed. Therefore, in agricultural production practice, there is an urgent need to find alternative means for controlling plutella xylostella other than chemical pesticides.
MicroRNAs (miRNA) is a class of non-coding single-stranded small RNA molecules of about 22 nucleotides in length, which were found to be encoded by endogenous genes, processed from hairpin transcripts produced endogenously by cells, found in the 90 th century. mirnas regulate expression levels by binding to specific sequences in the 5 'non-coding region (5' utr), open Reading Frame (ORF) or 3 'non-coding region (3' utr) of the target gene mRNA, cleaving the mRNA, or blocking translation. Transcription of most mirnas is mediated by RNA polymerase ii, and a minority is mediated by RNA polymerase iii, forming a primary transcript (pri-miRNA) of several hundred or even thousands of bases in length, where pri-miRNA has a 5' end cap structure and a poly (a) tail with a stem-loop structure inside. pri-miRNA is sheared into miRNA precursors (pre-miRNA) under the action of RNase III in cell nuclei. The pre-miRNA is transported from the nucleus into the cytoplasm via the Expotin-5 transporter transport. In the cytoplasm, the pre-miRNA is cleaved by Dicer enzyme into mature miRNA duplex. Finally, one functional single strand (5 p or 3 p) of the mature miRNA duplex forms a "RNA-induced silencing complex" (RISC) by binding to other related proteins (including TRBP, ago, etc.), which recognizes and binds to the target gene, thereby regulating gene expression. Functional studies have shown that miRNAs are involved in regulating a variety of important biological processes in insects, including insect growth, metamorphosis, wing differentiation, reproduction, immunity, pesticide resistance, and the like.
Disclosure of Invention
The invention aims to provide miR-8492-3p for preventing lepidoptera pests and application thereof.
Another object of the invention is to provide analogs agomir-8492 of the miR-8492-3p and application thereof.
To achieve the object of the present invention, in a first aspect, the present invention provides a miRNA for controlling lepidopteran pests, which is miR-8492-3p (i.e. pxy-miR-8492-3 p), the nucleotide sequence is as follows: 5'-AACACACGCGGGGGUGUCCAGU-3' (SEQ ID NO: 1).
In a second aspect, the invention provides analogs of the miRNA, which are agomir-8492, are double-stranded, and have the following nucleotide sequences (SEQ ID NOS: 2-3):
the invention also provides a precursor sequence of the miRNA, which is pxy-mir-8492, and is amiRNA-8492 (amiR-8492) after being expressed by a prokaryotic system, and the nucleotide sequence is as follows （SEQ ID NO:4）：5'-AAGGUGGUGGAAAAACUGGAUACCCUUUCGUAUGAUUCUCAGAGGAAACUGACAAACACACGCGGGGGUGUCCAGUAAUUUCAGCACAGA-3'.
In a third aspect, the invention provides a method for the preparation of said miRNA or said analogue or said precursor sequence by chemical synthesis or by use of a prokaryotic expression system.
In a fourth aspect, the invention provides a nucleic acid molecule encoding said miRNA or said analogue or said precursor sequence.
In a fifth aspect, the invention provides biological materials comprising the nucleic acid molecules, including but not limited to recombinant DNA, expression cassettes, transposons, plasmid vectors, viral vectors, engineering bacteria, or transgenic cell lines.
In a sixth aspect, the invention provides the use of said miRNA or said analogue or said precursor sequence or said nucleic acid molecule or said biological material for the preparation of a lepidopteran pest control agent.
In a seventh aspect, the invention provides a formulation or kit for controlling lepidopteran pests, the formulation or kit comprising any one of the miRNA or the analogue or the precursor sequence.
Further, the concentration of the analog in the injection method is 200-800 ng/head.
Further, the concentration of the precursor sequence in the spraying method is 1-6 ng/. Mu.L.
In an eighth aspect, the present invention provides a method for controlling lepidopteran pests, using the miRNA, the analogue and/or the precursor sequence, by injection or spraying.
In a ninth aspect, the present invention provides a method for controlling lepidopteran pests by constructing a transgenic plant expressing the miRNA, the analogue and/or the precursor sequence for controlling lepidopteran pests.
Lepidopteran pests according to the present invention include, but are not limited to, plutella xylostella, spodoptera frugiperda, spodoptera exigua, spodoptera litura, cotton bollworm, and the like.
By means of the technical scheme, the invention has at least the following advantages and beneficial effects:
According to the invention, through the predictive screening analysis of the plutella xylostella miRNA and target genes thereof, the plutella xylostella lethal miRNA of the vegetable insect, namely pxy-miR-8492-3p, is synthesized into corresponding miRNA analogues agomir-8492 in vitro through marking and chemical modification of mature body sequences of the pxy-miR-8492-3 p. The microinjection method is used for research, and the pxy-miR-8492-3p and analogues agomir-8492 thereof can reduce the survival rate of the plutella xylostella Chi10 and the EcR by inhibiting the expression of the plutella xylostella Chi10 and the EcR. In particular, the analogues agomir-8492 of pxy-miR-8492-3p are injected into plutella xylostella by using a microinjection method, the analogues are found to have obvious lethal effect on the plutella xylostella, and the precursor sequence of pxy-miR-8492-3p is further expressed through an HT115-L4440 prokaryotic expression system, so that amiR-8492 is prepared. The biological test shows that amiR-8492 has high toxicity to larvae of lepidoptera pests such as plutella xylostella, spodoptera frugiperda, asparagus caterpillar and the like. The transgenic rape expressing pxy-miR-8492-3p is constructed, and has good control effect on larvae of plutella xylostella, spodoptera frugiperda and spodoptera exigua. The invention provides a new target and technical support for establishing a new strategy for controlling lepidopteran pests based on miRNA interference technology.
Drawings
FIG. 1 is a schematic representation of the predicted target binding sites of pxy-miR-8492-3p in the Chi10 and EcR sequences of plutella xylostella in a preferred embodiment of the invention.
FIG. 2 shows the expression level of plutella xylostella pxy-miR-8492-3p and target genes Chi10 and EcR thereof after injection agomir-8492 in the preferred embodiment of the invention. Wherein,Represents P <0.05.
FIG. 3 shows mortality of plutella xylostella after injection agomir-8492 according to the preferred embodiment of the present invention.
FIG. 4 shows mortality of 3 lepidopteran pests feeding amiR-8492 spray-treated potted rape and corn leaves in a preferred embodiment of the invention. Wherein,Represents P <0.05.
FIG. 5 shows mortality after feeding of transgenic rape expressing pxy-miR-8492-3p by 3 lepidopteran pests in a preferred embodiment of the invention.
Detailed Description
The invention aims to provide miRNA for preventing and controlling plutella xylostella and even lepidoptera pests, namely pxy-miR-8492-3p, and further synthesizes analogues agomir-8492 thereof, wherein the inhibition effect of pxy-miR-8492-3p on plutella xylostella growth and development key genes chitinase (Chi 10, NCBI number XM_ 048623717.1) and ecdysone receptor (EcR, NCBI number XM_ 011566004.1) and the lethal effect of analogues agomir-8492 thereof on plutella xylostella are disclosed through an injection method.
The invention adopts the following technical scheme:
The invention provides pxy-miR-8492-3p for inhibiting growth and development key genes Chi10 and EcR of plutella xylostella so as to be lethal, wherein the pxy-miR-8492-3p has a nucleotide sequence shown as follows:
5’- AACACACGCGGGGGUGUCCAGU-3’（SEQ ID NO:1）
the invention also provides an analogue agomir-8492 of the plutella xylostella lethal miRNA, pxy-miR-8492-3p, wherein the agomir-8492 has a nucleotide sequence (SEQ ID NO: 2-3) shown as follows:
the analogues agomir-8492 are formed by marking and chemically modifying mature body sequences of pxy-miR-8492-3 p.
The invention also provides application of pxy-miR-8492-3p or the analogue agomir-8492 in preparing a vegetable pest plutella xylostella control medicament.
Specifically, the pests include, but are not limited to, plutella xylostella, spodoptera frugiperda, spodoptera exigua, cotton bollworms, and the like.
The invention also provides a preparation for controlling lepidopteran pests, which comprises at least one of pxy-miR-8492-3p or analogues agomir-8492.
Specifically, in the preparation for preventing and controlling lepidopteran pests, the concentration of agomir-8492 in the analogue injection method is 200-800 ng/head; the concentration of amiR-8492 in the spraying method is 1-6 ng/. Mu.L.
The invention also provides a method for controlling lepidopteran pests, which comprises the step of controlling lepidopteran pests by spraying the mature body or analogue agomir-8492 or amiRNA of pxy-miR-8492-3 p.
The invention also provides a method for controlling lepidopteran pests, comprising the step of controlling lepidopteran pests by constructing transgenic plants expressing the miRNA.
The invention discloses the inhibiting effect of pxy-miR-8492-3p on key genes chitinase (Chi 10) and ecdysone receptor (EcR) for the growth and development of plutella xylostella and the lethal effect of analogues agomir-8492 thereof on plutella xylostella through an injection method.
By the spray method, the virulence of amiR-8492 on plutella xylostella, spodoptera frugiperda and spodoptera exigua, which are prepared in large quantities by using a prokaryotic expression system, was revealed.
Construction of transgenic rape expressing pxy-miR-8492-3p by transgenic technology reveals the control effect on plutella xylostella, spodoptera frugiperda and spodoptera exigua.
The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Unless otherwise indicated, the technical means used in the examples are conventional means well known to those skilled in the art, and all raw materials used are commercially available.
In the following examples pxy-miR-8492-3p has the nucleotide sequence shown below: 5'-AACACACGCGGGGGUGUCCAGU-3'.
Further according to the pxy-miR-8492-3p mature body sequence, a corresponding miRNA analogue agomir-8492 is synthesized in vitro by the Shanghai Ji Ma pharmaceutical technology Co., ltd through marking and chemical modification, wherein the agomir-8492 has a nucleotide sequence shown as follows:
Examples 1 agomir to 8492 high virulence against Plutella xylostella
In this embodiment, miRNA target prediction software RNAhybrid is used to predict the binding sites of plutella xylostella pxy-miR-8492-3p on potential target genes Chi10 and EcR, and the result is shown in FIG. 1.
Based on the analogues agomir-8492 of pxy-miR-8492-3p synthesized by the method, corresponding pest control effects are verified in plutella xylostella, and meanwhile, a general nonsensical nucleic acid sequence agomir-NC is used as a control. agomir-NC sequence is as follows:
The above agomir-8492 and agomir-NC were dissolved and diluted to 4. Mu.g/. Mu.L with nuclease-free water, respectively. Selecting plutella xylostella larvae in the middle of three ages, injecting 552ng agomir-8492 solution into the abdomen (treatment group) of plutella xylostella, injecting the plutella xylostella in the control group with the same amount of agomir-NC, and feeding the plutella xylostella larvae in a culture dish after injection; samples of the plutella xylostella treated group and the control group were collected 24 h and 48 h after injection, and the change of the expression levels of the target genes Chi10 and ec R of pxy-miR-8492-3p in the plutella xylostella treated group and the control group were detected respectively, and the results are shown in FIG. 2. Parallel experiments are carried out simultaneously, and the mortality of the plutella xylostella in each experimental group is observed and counted after injection. The results are shown in FIG. 3.
From the data in FIG. 1, it can be seen that the binding sites of plutella xylostella pxy-miR-8492-3p on its potential target genes Chi10 and EcR are at 4671 and 4181 bp, respectively, of the target gene sequences.
As can be seen from the data in fig. 2, 24-h and 48-h after agomir-8492 are injected, the expression level of the treated group plutella xylostella pxy-miR-8492-3p is obviously increased by 2736.6 and 110.3 times; the expression level of the Chi10 and the EcR genes is obviously reduced by 68.1-85.8%.
As can be seen from the data in fig. 3, the mortality rate of the plutella xylostella larvae after injection agomir-8492 was initially significantly higher than that of the control group, and the cumulative mortality rate of the plutella xylostella larvae after 6 d was increased by 58.9% compared to the control group.
In conclusion, after the analogues agomir-8492 of the plutella xylostella pxy-miR-8492-3p are injected, the expression level of the plutella xylostella growth and development key genes Chi10 and EcR is obviously reduced, and the plutella xylostella mortality is obviously increased, so that agomir-8492 has obvious lethal effect on plutella xylostella larvae.
According to the invention, through predictive analysis of the plutella xylostella miRNA and the target thereof, the plutella xylostella lethal miRNA is obtained, and pxy-miR-8492-3p is obtained. The invention provides a new target for establishing a new strategy for controlling lepidoptera pests such as plutella xylostella based on miRNA interference technology.
EXAMPLE 2 prokaryotic expression of amiRNA-8492 for control of lepidopteran pests
This example shows the sequence of amiR-8492 (amiRNA-8492) obtained by extracting total RNA by HT115-L4440 prokaryotic expression system (HT 115 competent cells and L4440 vector were supplied by the institute of plant protection Liang Pei, university of agricultural China, see Artificial miRNA-mediated silencing of ecdysone receptor (EcR) affects larval development and oogenesis inHelicoverpa armigera,Sneha Yogindran, Manchikatla Venkat Rajam,Insect Biochemistry and Molecular Biology） expression pxy-miR-8492) as follows: 5'-AAGGUGGUGGAAAAACUGGAUACCCUUUCGUAUGAUUCUCAGAGGAAACUGACAAACACACGCGGGGGUGUCCAGUAAUUUCAGCACAGA-3'.
The Chinese cabbage and the corn are respectively planted in 10 cm small flowerpots, and 9 plants are planted in each pot. Spraying 20mL of amiR-8492 with a concentration of 2.4 ng/. Mu.L onto potted cabbage after the cabbage grows 40 d, and transferring 2-year-old plutella xylostella onto the potted cabbage after the leaves are dried; after corn grows 20 d, 20mL of 5.6 ng/. Mu.L amiR-8492 is sprayed on potted corn, after leaves are dried, spodoptera exigua or spodoptera exigua at 1 age are transferred to the potted corn, 40 larvae are respectively connected on each pot, and 3 times of repetition are carried out. Potted cabbage and corn were treated with HT115-L4440 empty vector bacterial liquid total RNA spray as controls. Cumulative mortality of 3 pests was observed and recorded daily, and the results are shown in fig. 4.
As can be seen from the data in fig. 4, the cumulative mortality of plutella xylostella, spodoptera frugiperda and spodoptera exigua larvae fed amiR-8492 spray-treated potted cabbage or potted corn 8 d was significantly increased by 65.0%, 80.0% and 64.2%, respectively, compared to the control group. As can be seen, amiR-8492 are highly virulent against a variety of lepidopteran pests. The result also shows that miRNA pesticide prepared by prokaryotic expression and other systems can effectively control various lepidoptera pests.
EXAMPLE 3 construction of transgenic plants expressing pxy-miR-8492-3p for control of lepidopteran pests
In the embodiment, firstly, transgenic rape expressing pxy-miR-8492-3p is constructed, then rape plants B#13 and B#14 with high miR-8492-3p expression level are selected, after rape grows 60 d, leaves are taken and placed in a culture dish of 9 cm, 20 head 2-year-old plutella xylostella, 1-year-old spodoptera frugiperda or 1-year-old beet nocturnal larvae are respectively placed in the culture dish, 3 repeats are arranged for each transgenic rape line, and Wild (WT) rape is used as a control. Every 2d old leaves were replaced with fresh canola leaves until all surviving larvae pupate, and the mortality of the larvae was recorded daily, as shown in figure 5.
From the data in FIG. 5, the cumulative mortality of plutella xylostella, spodoptera frugiperda and spodoptera exigua larvae following continuous feeding of miR-8492-3p transgenic rape B#13 and B#14 for 6 d was increased by 60.0% -61.7%, 77.7% -88.9% and 71.7% -83.3%, respectively, compared to feeding of WT rape. Thus, miR-8492-3p transgenic rape has high toxicity to various lepidopteran pests. The results also show that the construction of transgenic plants by miRNA which is critical to the growth and development of insects can also well control various lepidoptera pests.
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Claims (10)
1. A miRNA for preventing and controlling lepidopteran pests, which is characterized by being miR-8492-3p and having the following nucleotide sequence: 5'-AACACACGCGGGGGUGUCCAGU-3'.
2. The analogue of miRNA of claim 1, which is agomir-8492, is a double-stranded structure and has the nucleotide sequence:
。
3. The precursor sequence of the miRNA according to claim 1, which is pxy-mir-8492, and which is amiRNA-8492 after expression by a prokaryotic system, the nucleotide sequence is as follows: 5'-AAGGUGGUGGAAAAACUGGAUACCCUUUCGUAUGAUUCUCAGAGGAAACUGACAAACACACGCGGGGGUGUCCAGUAAUUUCAGCACAGA-3'.
4. A nucleic acid molecule encoding the miRNA of claim 1 or the analog of claim 2 or the precursor sequence of claim 3.
5. A biological material comprising the nucleic acid molecule of claim 4, wherein the biological material is a recombinant DNA, an expression cassette, a transposon, a plasmid vector, a viral vector, an engineering bacterium or a transgenic cell line.
6. Use of a miRNA according to claim 1 or an analogue according to claim 2 or a precursor sequence according to claim 3 or a nucleic acid molecule according to claim 4 or a biological material according to claim 5 for the preparation of a lepidopteran pest control agent;
The lepidopteran pests include plutella xylostella, spodoptera frugiperda, spodoptera exigua, prodenia litura and cotton bollworm.
7. A formulation or kit for controlling lepidopteran pests, characterized in that it comprises any one of the miRNA of claim 1, the analogue of claim 2, the precursor sequence of claim 3.
8. A method for controlling lepidopteran pests, characterized in that the miRNA of claim 1, the analogue of claim 2 and/or the precursor sequence of claim 3 are used for lepidopteran pest control by injection or spraying;
The lepidopteran pests include plutella xylostella, spodoptera frugiperda, spodoptera exigua, prodenia litura and cotton bollworm.
9. The method of claim 8, wherein the concentration of the analog in the injection process is 200-800 ng/head;
the concentration of the precursor sequence in the spraying method is 1-6 ng/mu L.
10. A method for controlling lepidopteran pests, characterized in that the method is used for controlling lepidopteran pests by constructing transgenic plants expressing the miRNA of claim 1, the analogue of claim 2 and/or the precursor sequence of claim 3;
The lepidopteran pests include plutella xylostella, spodoptera frugiperda, spodoptera exigua, prodenia litura and cotton bollworm.
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