CN113106100A

CN113106100A - MiRNA, analogue and inhibitor for regulating and controlling lethality of lepidoptera pests and application thereof

Info

Publication number: CN113106100A
Application number: CN202110512128.6A
Authority: CN
Inventors: 朱斌; 李琳红; 梁沛; 高希武
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2021-05-11
Filing date: 2021-05-11
Publication date: 2021-07-13

Abstract

The invention belongs to the technical field of agricultural biology, in particular to miRNA (micro ribonucleic acid) capable of regulating and controlling the death of lepidoptera pests and delaying the growth and development of the lepidoptera pests, an analogue and an inhibitor thereof, and further discloses application of the miRNA in preparing a medicament for controlling the lepidoptera pests. The invention obtains lepidoptera insect plutella xylostella miRNA (pxy-miR-34-5 p) through sequence analysis of a lepidoptera insect miRNA gene family, and further synthesizes corresponding miRNA analogue agomiR-34 and inhibitor antagomiR-34 in vitro by marking and chemically modifying the mature body sequence of pxy-miR-34-5 p. Research by utilizing a microinjection method discovers that the pxy-miR-34-5p analogue agomimi R-34 and the inhibitor agomimi R-34 can delay the growth and development of lepidoptera pests or reduce the survival rate of the lepidoptera pests by regulating and controlling EcR of the lepidoptera pests, and theoretical guidance is provided for establishing a new strategy for controlling the lepidoptera pests based on a miRNA interference technology.

Description

MiRNA, analogue and inhibitor for regulating and controlling lethality of lepidoptera pests and application thereof

Technical Field

The invention belongs to the technical field of agricultural biology, in particular to miRNA (micro ribonucleic acid) capable of regulating and controlling the death of lepidoptera pests and delaying the growth and development of the lepidoptera pests, an analogue and an inhibitor thereof, and further discloses application of the miRNA in preparing a medicament for controlling the lepidoptera pests.

Background

Lepidoptera pests are important pests in vegetable crop production, and are often mixed with multiple pests, so that generation generations are more and generation overlapping is serious, and huge economic loss is caused every year. At present, the main means for preventing and controlling lepidoptera pests still depend on chemical prevention and control means of insecticides, but due to unreasonable use of a large amount of insecticides, the lepidoptera pests almost have resistance to all used insecticides, and only can continuously increase the use amount of pesticides to achieve better prevention and control effects, so that more serious environmental pollution is caused, and a vicious circle is formed. Therefore, there is an urgent need to find alternative means for controlling lepidopteran pests in agricultural production practices other than chemical pesticides.

MicroRNAs (miRNA) are non-coding single-stranded small RNA molecules with the length of about 22 nucleotides, which are coded by endogenous genes and are discovered in the 90 s of the 20 th century, and are processed from hairpin structure transcripts endogenously produced by cells. mirnas regulate expression levels by binding to specific sequences of 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 few by RNA polymerase iii, forming primary transcripts of hundreds or even thousands of bases in length (pri-mirnas) with a 5' end cap structure and a poly (a) tail, with a stem-loop structure inside. pri-miRNAs are cleaved in the nucleus by RNase III to generate miRNA precursors (pre-miRNAs). The pre-miRNA is transported from the nucleus to the cytoplasm via the transportation of the exputin-5 transporter. In the cytoplasm, pre-miRNA is cleaved into mature miRNA duplexes by Dicer enzyme. Finally, one functional single strand (5P or 3P) in the mature miRNA double strand is combined with other related proteins (including TRBP, Ago and the like) to form an RNA-induced silencing complex (RISC), and the RISC can identify and combine with a target gene to further regulate the expression of the gene. Functional studies have shown that miRNAs are involved in regulating a variety of important biological processes in insects, including insect growth and development, metamorphosis, pterotyl differentiation, reproduction, immunity, insecticide resistance, and the like. Sequence analysis also finds that the miR-34 of the lepidoptera insect has high conservation, and the 3' UTR region of the EcR of the lepidoptera insect has miR-34 conserved seed region binding sites. The invention obtains the miRNA of the lepidoptera insect through the sequence analysis of the miRNA gene family of the lepidoptera insect, and provides theoretical guidance for establishing a new strategy for controlling pests based on the miRNA interference technology.

Disclosure of Invention

Therefore, the technical problem to be solved by the invention is to provide a miRNA (pxy-miR-34-5 p) capable of regulating and controlling the killing of lepidoptera pests or delaying the growth and development of the insects, further synthesize an analogue agomiR-34 and an inhibitor antagomiR-34 of the miRNA, and further disclose the action of the analogue agomiR-34 and the inhibitor antagomiR-34 of pxy-miR-34-5p on the killing and development delaying of the lepidoptera pests through injection to the insects.

In order to solve the technical problems, the pxy-miR-34-5p for regulating and controlling delayed development or death of lepidoptera pests, disclosed by the invention, wherein pxy-miR-34-5p has a nucleotide sequence shown as follows:

5’-UGGCAGUGUGGUUAGCUGGUUGU-3’。

the invention also discloses an analogue agomiR-34 of pxy-miR-34-5p for regulating and controlling delayed development or death of lepidoptera pests, wherein the agomiR-34 has a nucleotide sequence shown as follows:

the invention also discloses an inhibitor antagomiR-34 of pxy-miR-34-5p for regulating and controlling delayed development or death of lepidoptera pests, wherein the antagomiR-34 has a nucleotide sequence shown as follows:

5’-ACAACCAGCUAACCACACUGCCA-3’。

the analogue agomoiR-34 and the inhibitor antagomoiR-34 are formed by labeling and chemically modifying a mature body sequence of pxy-miR-34-5 p.

The invention also discloses application of the pxy-miR-34-5p or the analog agomiR-34 or the inhibitor agomiR-34 in preparing a lepidoptera pest control medicament.

Specifically, the lepidopteran pests include insects of the noctuidae family or the plutella xylostella family.

Preferably, the lepidopteran pest comprises asparagus caterpillar, cotton bollworm or diamondback moth.

The invention also discloses a preparation or a kit for controlling lepidoptera pests, which comprises at least one of the pxy-miR-34-5p or the analogue agomiR-34 or the inhibitor agomiR-34.

Specifically, in the preparation or the kit for controlling lepidoptera pests:

the concentration of the analogue agomiR-34 is 1 mu g/mu L-5 mu g/mu L;

the concentration of the inhibitor antagomiR-34 is 1 mu g/mu L-5 mu g/mu L.

The invention also discloses a method for controlling lepidoptera pests, which comprises the step of injecting the pxy-miR-34-5p analogue agomiR-34 or the inhibitor agomiR-34 into the lepidoptera pests.

The invention also discloses application of the preparation or the kit for preventing and treating the lepidoptera pests in the field of preventing and treating the lepidoptera pests.

The invention obtains lepidoptera insect plutella xylostella miRNA (pxy-miR-34-5 p) through sequence analysis of a lepidoptera insect miRNA gene family, and further synthesizes corresponding miRNA analogue agomiR-34 and inhibitor antagomiR-34 in vitro by marking and chemically modifying the mature body sequence of pxy-miR-34-5 p. The research of a microinjection method finds that the pxy-miR-34-5p and the analogue agomimi R-34 and the inhibitor antagomiR R-34 can delay the growth and development of lepidoptera pests EcR or reduce the survival rate of the lepidoptera pests EcR through regulation. Particularly, the pxy-miR-34-5p analogue agomimi R-34 and the inhibitor agomimi R-34 are respectively injected into lepidoptera pests such as asparagus caterpillar, cotton bollworm and diamond back moth by a microinjection method, and the analogue and the inhibitor are found to have obvious lethal effects on the three lepidoptera pests, can obviously inhibit the growth and development of the lepidoptera pests, prolong the development period of the lepidoptera pests and provide theoretical guidance for establishing a new strategy for controlling the lepidoptera pests based on miRNA interference technology.

Drawings

In order that the present disclosure may be more readily and clearly understood, the following detailed description of the present disclosure is provided in connection with specific embodiments thereof and the accompanying drawings, in which,

FIG. 1 shows the expression level of EcR of Spodoptera exigua after injecting agomiR-34(A) and antagomiR-34(B) in example 1;

FIG. 2 is a graph showing the expression level of EcR of Helicoverpa armigera after injecting agomiR-34(A) and antagomiR-34(B) in example 2;

FIG. 3 shows the amount of EcR expression of Plutella xylostella after injecting the agomiR-34(A) and the antagomiR-34(B) in example 3.

Detailed Description

In the following embodiments of the invention, a lepidoptera insect miRNA gene family is analyzed by sequence analysis to obtain a lepidoptera insect miRNA pxy-miR-34-5p, wherein pxy-miR-34-5p has a nucleotide sequence shown as follows:

5’-UGGCAGUGUGGUUAGCUGGUUGU-3’。

further according to the pxy-miR-34-5p mature body sequence, corresponding miRNA analogue agomiR-34 and inhibitor antagomiR-34 are synthesized in vitro by Shanghai Jima pharmaceutical technology Limited company through marking and chemical modification, wherein,

the agomiR-34 has a nucleotide sequence shown as follows:

the antagomiR-34 has a nucleotide sequence shown as follows:

5’-ACAACCAGCUAACCACACUGCCA-3’。

example 1

In the present example, based on the pxy-miR-34-5p analog agomiR-34 and the inhibitor antagomiR-34 synthesized as described above, verification of the corresponding pest control effect was performed in beet armyworm while using the universal nonsense nucleic acid sequences agomiR-NC and antagomiR-NC as controls.

The above-mentioned agomiR-34, agomiR-NC, antagomiR-34 and antagomiR-NC were dissolved and diluted to 4. mu.g/. mu.L with nuclease-free water, respectively.

Selecting beet armyworm larvae with consistent age, and injecting the agomiR-34/antagomiR-34 into insect bodies by using a microinjector respectively, wherein the specific standards are as follows:

(1) selecting beet armyworm larvae of the late second instar, injecting 1.4 mu g of agomiR-34 solution into the abdomen (treatment group) of the beet armyworm, injecting the same amount of agomiR-NC into the beet armyworm of the control group, and feeding the beet armyworm with artificial feed in a dipping cup in a single head mode after injection;

(2) selecting beet noctuid larvae at the early stage of three instars, injecting 1.9 mu g of antagomiR R-34 solution into the abdomen of the beet noctuids (treatment group), injecting the same amount of antagomiR R-NC into the beet noctuids of a control group, and feeding the beet noctuids in a dipping cup by using an artificial feed in a single head mode after injection.

After 24h, 48h and 72h after injection, collecting samples of the beet armyworm treatment group and the control group, and respectively detecting the change of the expression level of the ecdysone receptor gene EcR of the target gene miR-34-5p in each group of beet armyworms, wherein the test result is shown in figure 1.

After injection, growth and development parameters of the beet armyworm such as body length, weight, death rate, pupation rate, eclosion rate and the like in each experimental group are observed and counted respectively, and the results are recorded in the following table 1.

TABLE 1 statistical chart of growth and development of beet armyworm after injection of agomiR-34 or antagomiR-34

As can be seen from the data in FIG. 1 (A), the expression level of EcR in beet armyworm is significantly reduced at 24h, 48h and 72h after injecting the agomiR-34; the results in table 1 show that, compared with the control group, after injecting the agomi r-34, the body length, the body weight and the pupa weight of the spodoptera exigua larvae are respectively and obviously reduced by 32.6%, 83.2% and 21.8%, the mortality rate is obviously increased by 44.7%, the pupation rate is obviously reduced by 52.8%, and the development duration is obviously prolonged by 3.5 days. The analogue agomiR-34 has obvious lethal effect on beet armyworm pests, can obviously inhibit the growth and development of the beet armyworm and prolong the development period of the beet armyworm.

As can be seen from the data in FIG. 1 (B), the expression level of EcR in beet armyworm is obviously increased after 24h and 48h of injecting antagomiR-34; the data in table 1 show that, compared with the control group, after the anti-demomir R-34 is injected, the body length, the body weight and the pupa weight of the beet armyworm larvae are respectively and obviously reduced by 39.4%, 62.1% and 38.9%, the mortality rate is obviously increased by 37.9%, the pupation rate and the eclosion rate are obviously reduced by 38.2% and 34.0%, and the development period is obviously prolonged by 4.7 days. The inhibitor antagomiR-34 has obvious lethal effect on the asparagus caterpillar pests, can obviously inhibit the growth and development of the asparagus caterpillar and prolong the development period of the asparagus caterpillar.

Example 2

In this example, based on the pxy-miR-34-5p analog agomiR-34 and the inhibitor antagomiR-34 synthesized as described above, verification of the corresponding pest control effect was performed in Helicoverpa armigera while using the universal nonsense nucleic acid sequences agomiR-NC and antagomiR-NC as controls.

Selecting cotton bollworm larvae with consistent age, and injecting the agomiR-34/antagomiR-34 into insect bodies by using a microinjection instrument, wherein the specific standards are as follows:

(1) selecting bollworm larvae at the late stage of two years old, injecting 2.2 mu g of agomi R-34 solution into the abdomen (treatment group) of the bollworm, injecting the same amount of agomi R-NC into the control group of the bollworm, and feeding the bollworm with artificial feed in a dipping cup in a single head mode after injection;

(2) the cotton bollworm larvae at the later stage of second instar are selected, 2.2 mu g of antagomiR R-34 solution is injected into the abdomen of the cotton bollworm (treatment group), the cotton bollworm of the control group is injected with the same amount of antagomiR R-NC, and the cotton bollworm is fed with single-head artificial feed in a dipping cup after injection.

After 24h, 48h and 72h after injection, collecting samples of the cotton bollworm treatment group and the control group, and respectively detecting the change of the EcR expression level of the target gene of miR-34-5p in the cotton bollworms of each group, wherein the test result is shown in figure 2.

After injection, the growth and development parameters of cotton bollworm, such as mortality, larval body length and weight, pupation rate, eclosion rate, and the like in each experimental group were observed and counted, and the results are recorded in the following table 2.

TABLE 2 statistical chart of the growth and development of Helicoverpa armigera after injection of agomiR-34 or antagomiR-34

As can be seen from the data in FIG. 2 (A), the expression level of EcR is significantly reduced 48h and 72h after injecting the agomiR-34; the results in table 2 show that, compared with the control group, after injecting agomi r-34, the body length, body weight and pupa weight of the cotton bollworm larvae are respectively and significantly reduced by 51.0%, 87.8% and 15.0%, the mortality rate is significantly increased by 60.6%, the pupation rate and the eclosion rate are respectively and significantly reduced by 33.8% and 58.8%, and the development period is significantly prolonged by 3 days. The agomiR-34 analogue has obvious lethal effect on cotton bollworm pests, can obviously inhibit the growth and development of the cotton bollworms and prolong the development period of the cotton bollworms.

As shown in (B) in the attached figure 2, the expression level of EcR is obviously increased 24h and 48h after the antagomiR-34 is injected; as can be seen from the data in Table 2, compared with the control group, the body length, the body weight and the pupa weight of the cotton bollworm larvae are respectively and obviously reduced by 33.6 percent, 74.9 percent and 23.1 percent after the anti-demomi R-34 is injected, the death rate is obviously increased by 31.7 percent, the emergence rate is obviously reduced by 16.9 percent, and the development duration is obviously prolonged by 3 days. The inhibitor antagomiR-34 has obvious lethal effect on cotton bollworm pests, can obviously inhibit the growth and development of the cotton bollworms and prolong the development period of the cotton bollworms.

Example 3

In the present example, based on the pxy-miR-34-5p analog agomiR-34 and the inhibitor antagomiR-34 synthesized as described above, verification of the corresponding pest control effect was performed in plutella xylostella while using the universal nonsense nucleic acid sequences agomiR-NC and antagomiR-NC as controls.

Selecting diamondback moth larvae with consistent age, and injecting the agomiR-34/antagomiR-34 into insect bodies by using a micro-injection instrument, wherein the specific standard is as follows:

(1) selecting diamondback moth larvae in the middle stage of three ages, injecting 207ng of the agomi R-34 solution into the abdomen (treatment group) of the diamondback moth, injecting the same amount of agomi R-NC into the diamondback moth of a control group, and feeding the diamondback moth in a culture dish by using cabbage leaves after injection;

(2) selecting diamondback moth larvae in the middle stage of three ages, injecting 690ng of antagomiR-34 solution into the abdomen of the diamondback moth (a treatment group), injecting the same amount of antagomiR-NC into the diamondback moth of a control group, and feeding the diamondback moth in a culture dish by using cabbage leaves after injection.

Samples of the diamondback moth treatment group and the control group are collected after 24h, 48h and 72h after injection, the change of the target gene EcR expression level of miR-34-5p in each group of diamondback moths is respectively detected, and the test result is shown in the attached figure 3.

After injection, growth and development parameters of diamondback moth such as death rate, larva body length and weight, pupation rate, eclosion rate and the like in each experimental group are observed and counted respectively, and the results are recorded in the following table 3.

TABLE 3 statistical chart of growth and development of Plutella xylostella after injection of agomiR-34 or antagomiR-34

As can be seen from the data in FIG. 3 (A), the expression level of the EcR of the diamondback moth in the treated group is significantly reduced 24h and 48h after injecting the agomiR-34; the results in table 3 show that, compared with the control group, the length, weight and pupa weight of the diamondback moth larvae are respectively and remarkably reduced by 7.3%, 16.8% and 53.8% after the injection of the agomiR-34, the death rate is remarkably increased by 47.1%, the pupation rate and the eclosion rate are respectively and remarkably reduced by 27.8% and 36.6%, and the development period is remarkably prolonged by 1 day. The analogue agomiR-34 has obvious lethal effect on diamondback moth pests, can obviously inhibit the growth and development of the diamondback moth, and prolongs the development period of the diamondback moth.

As can be seen from the data in FIG. 3 (B), the expression level of the EcR of the plutella xylostella in the treated group is remarkably increased 48h after the injection of the antagomiR-34; the results in table 3 show that, compared with the control group, after the anti-gomir-34 is injected, the body length, the body weight and the pupa weight of the diamondback moth larvae are respectively and obviously reduced by 26.6%, 52.5% and 42.0%, the death rate is obviously increased by 72.6%, the pupation rate and the eclosion rate are respectively and obviously reduced by 27.7% and 32.3%, and the development period is obviously prolonged by 1 day. The inhibitor antagomiR-34 has obvious lethal effect on the diamondback moth pests, can obviously inhibit the growth and development of the diamondback moth and prolong the development period of the diamondback moth.

In conclusion, in the embodiments of the invention, after injecting the analog agomis-34 of the plutella xylostella pxy-miR-34-5p, the expression level of EcR of beet armyworm, cotton bollworm and plutella xylostella in lepidoptera pests is remarkably reduced, and after injecting the inhibitor agomis-34, the expression level of EcR of the lepidoptera pests is remarkably increased. In addition, after the injection of the agomiR-34 and the antagomiR-34, the body length, the body weight and the pupal weight average of lepidoptera pests such as asparagus caterpillar, cotton bollworm, diamond back moth and the like are obviously reduced, the death rate is obviously increased, the pupal rate and the eclosion rate are also obviously reduced, and the development period is obviously prolonged. The abnormal expression of the miR-34-5p has high conservation on the lethal effect and the development delaying effect of lepidoptera pests, and the injection of the analogue agomiR-34 and/or the inhibitor antagomiR-34 can realize the effective control of the lepidoptera pests such as cabbage loopers, cotton bollworms, diamond back moths and the like.

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.

Claims

1. pxy-miR-34-5p for regulating delayed development or death of lepidoptera pests, wherein the pxy-miR-34-5p has a nucleotide sequence shown as follows:

5’-UGGCAGUGUGGUUAGCUGGUUGU-3’。

2. the pxy-miR-34-5p analogue agomiR-34 for regulating delayed development or death of lepidopteran pests according to claim 1, wherein the agomiR-34 has a nucleotide sequence shown as follows:

3. an inhibitor antagomiR-34 of pxy-miR-34-5p for regulating delayed development or lethality of lepidopteran pests according to claim 1, wherein the antagomiR-34 has a nucleotide sequence shown as follows:

5’-ACAACCAGCUAACCACACUGCCA-3’。

4. use of pxy-miR-34-5p of claim 1 or an analog agomiR-34 of claim 2 or an inhibitor antagomiR-34 of claim 3 for the preparation of a lepidopteran pest control agent.

5. Use according to claim 4, wherein the lepidopteran pest comprises a noctuidae or plutella insect.

6. Use according to claim 4 or 5, wherein the lepidopteran pest comprises beet armyworm, cotton bollworm or diamondback moth.

7. A formulation or kit for controlling lepidopteran pests, comprising at least one of pxy-miR-34-5p of claim 1 or an analog agomi r-34 of claim 2 or an inhibitor agomi r-34 of claim 3.

8. The formulation or kit for controlling a lepidopteran pest according to claim 7, wherein:

the concentration of the analogue agomiR-34 is 1 mu g/mu L-5 mu g/mu L;

the concentration of the inhibitor antagomiR-34 is 1 mu g/mu L-5 mu g/mu L.

9. A method for controlling lepidopteran pests, comprising the step of injecting pxy-miR-34-5p of claim 1 or agomiR-34 of the analog of claim 2 or the inhibitor of claim 3, agomiR-34, into a lepidopteran pest.

10. Use of the formulation or kit for controlling lepidopteran pests according to claim 7 or 8 in the field of lepidopteran pest control.