CN107119052B - Artificially designed and synthesized miRNA molecules and their control effects on aphids - Google Patents

Abstract

The present invention relates to a miRNA molecule. The miRNA molecule is a double-stranded structure, including a sense strand and an antisense strand; wherein, the nucleotide sequence of the sense strand is as shown in SEQ ID NO: 1 in the sequence table, and the nucleotide sequence of the antisense strand is The nucleotide sequence is shown in SEQ ID NO: 2 in the sequence listing; or the miRNA molecule is a single-stranded structure, and its nucleotide sequence is shown in SEQ ID NO: 3 in the sequence listing. The miRNA avi-miR-263a agomir and miRNA avi-miR-263a antagomir designed and synthesized by the present invention are lethal to aphids, and provide new and effective products for the prevention and control of aphids; and their target genes can be used as ideal candidate genes Resources, used to create new RNAi-mediated insect-resistant crops, provide new research ideas for pest control, have high research value and broad application prospects.

Description

Artificially designed and synthesized miRNA molecules and their control effects on aphids

technical field

The invention relates to the field of biotechnology, in particular to an artificially designed and synthesized miRNA molecule and its effect on controlling aphids.

Background technique

Wheat aphids are a worldwide agricultural pest, mainly including Sitobion avenae (Fabricius), Schizaphis graminum (Rondani), Rhopalosiphum padi (Linnaeus) Acyrthosiphon dirhodum (Walker) and others. Wheat aphids have the characteristics of wide distribution, large numbers, strong fecundity, and serious damage. They can migrate over long distances and spread viruses during the migration process. The honeydew excreted by them can also breed a variety of molds and affect wheat plants. photosynthesis and respiration, leading to crop yield reduction, causing huge losses to agricultural production, which has become one of the hot spots in the research of pest control in the agricultural field. At present, the control methods for aphids in China mainly use chemical pesticides, and excessive spraying of chemical pesticides will bring serious consequences to the natural environment and human life. In recent years, many scholars at home and abroad have devoted themselves to the screening and utilization of wheat resistant varieties, hoping that the cultivation and utilization of wheat aphid-resistant varieties/lines can play a preventive role in the comprehensive control of wheat aphids. At present, due to the lack of effective aphid-resistant genes in the existing germplasm resources, it is urgent for researchers to develop new target genes for the control of wheat pests.

Contents of the invention

The object of the present invention is to provide a miRNA molecule: the miRNA molecule is a double-stranded structure or a single-stranded structure, and when the miRNA molecule is a double-stranded structure, it contains a sense strand and an antisense strand; wherein, the nucleotide sequence of the sense strand is as in the sequence listing shown in SEQ ID NO: 1, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO: 2 in the sequence listing; or when the miRNA molecule is a single-stranded structure, its nucleotide sequence is shown in SEQ ID NO in the sequence listing ID NO: 3, the details are as follows:

Sense strand (SEQ ID NO: 1): 5'-AAUGGCACUGAAAGAAUUCACGGG-3'

Antisense strand (SEQ ID NO: 2): 5'-CGUGAAUUCUUUCAGUGCCAUUUU-3'

Single strand (SEQ ID NO: 3): 5'-CCCGUGAAUUCUUUCAGUGCCAUU-3'

Preferably, in the double-strand structure, the chemical modification of the antisense strand includes: cholesterol modification at the 3' end, two thio-skeleton modifications at the 5'-end, and four thio-skeleton modifications at the 3'-end to obtain: 5'- CsGsUGAAUUCUUUCAGUGCCAUsUsUsUs-Chol 3', and the whole chain is methoxy modified; or the chemical modification of the single-chain structure includes: 3'-end cholesterol modification, 5'-end two thio-skeleton modifications, 3'-end four thiol Skeleton modification: 5'-CsCsCGUGAAUUCUUUCAGUGCCsAsUs Us-Chol 3', and the whole chain is modified with methoxy.

The miRNA molecule provided by the present invention is the corresponding miRNA avi-miR-263a agomir (miRNA agonist) and miRNA avi-miR-263a antagomir (miRNA antagonist) obtained by designing the mature sequence of aphid miRNAavi-miR-263a. The miRNA avi-miR-263a agomir has a double-stranded structure, the antisense strand (SEQ ID NO: 2) is modified, the 3' end is modified with cholesterol, the 5' end is modified with two sulfur skeletons, and the 3' end is modified with four sulfur groups. Skeleton modification and methoxy modification of the whole chain; the sense strand (SEQ ID NO: 1) is the active strand, and the antisense strand is the supplementary auxiliary strand. When designing, the first two bases are staggered for reverse complementary design, and in A UU overhang at the end facilitates unzipping. The miRNA avi-miR-263a antogimir has a single-chain structure (SEQ ID NO: 3), with cholesterol modification at the 3' end, two thio-skeleton modifications at the 5'-end, four thio-skeleton modifications at the 3'-end, and a full chain Methoxy modification. Both miRNA avi-miR-263a agomir and miRNA avi-miR-263a antagomir provided by the present invention were designed and synthesized by entrusting Suzhou Gemma Gene Co., Ltd.

The present invention protects a kit comprising miRNA molecules of double-stranded structure or miRNA molecules of single-stranded structure.

The present invention also protects a kit, which includes a miRNA molecule solution, and the miRNA molecule solution includes a sucrose solution with a mass fraction of 30% and the above-mentioned miRNA molecule with a double-stranded structure or a miRNA molecule with a single-stranded structure at a concentration of 300-500nM; Wherein, the solvent of the sucrose solution is water.

Wherein, the preparation process of miRNA molecule solution can be as follows: (1) use ultrapure water configuration mass fraction to be the sucrose solution of 30%, adopt the microporous membrane filtration sterilization of 0.2 μm then, the filtrate obtained will be packed in 1.5ml Store in a sterilized centrifuge tube at -20°C for later use; (2) Dilute the synthesized miRNA avi-miR-263a agomir or miRNA avi-miR-263a antagomir to a concentration of 300-500nM with a mass fraction of 30% for later use .

The present invention also protects the application of the above kit in the preparation of products for controlling aphids. Aphids include wheat aphids, and wheat aphids include Sitobion avenae (Fabricius), Schizaphis graminum (Rondani), Rhopalosiphum padi (Linnaeus) and Acyrthosiphon dirhodum (Walker) one or more of.

The present invention also provides a method for controlling aphids with the above-mentioned kit, comprising the steps of: taking a few nymphs and feeding them in an aphid feeder containing miRNA molecular solution for 72 hours; develop into aphids. It should be noted that it is preferred to grow on fresh potted wheat plants, and record the death and individual development of aphids during feeding until they develop into adult aphids about 6-7 days later, and count the survival rate of aphids; the preferred source of nymphs is Yu: Feed aphids with a single head continuously for N generations, and collect nymphs that reproduced within 24 hours of the first generation of aphidless adults of the N+1 generation, and the N generation is preferably 3 generations.

Preferably, the aphid feeding device containing the miRNA molecular solution is a semi-closed aphid feeding device containing the miRNA molecular solution, and the preparation method of the semi-closed aphid feeding device containing the miRNA molecular solution comprises the following steps: vertically sterilizing the cylindrical glass tube Place it on absorbent paper, seal the top with a paraffin film with a thickness of 0.8-2 μm, add the miRNA molecular solution on the upper surface of the paraffin film, and then seal with a paraffin film with a thickness of 8-15 μm to obtain a semi-closed sample containing the miRNA molecular solution. Aphid feeding device; wherein, the diameter of the cylindrical glass is 2cm, the height is 3cm, and the volume of the miRNA molecule solution is 200-300μL; the feeding conditions include: a semi-closed feeding system containing miRNA molecule solution that is connected to several nymphs. The bottom of the aphid is in airtight contact with absorbent paper, and then the entire rearing system is placed in the insect culture room, the temperature of the insect culture room is 20±1°C, the humidity is 60±10%, and the photoperiod L:D=14h:10h. It should be noted that the cylindrical glass tubes with openings at both ends were sterilized under high temperature and high pressure before use, placed vertically on absorbent paper, sealed with parafilm (Parafilm, USA) at the top, dripped the miRNA molecular solution on the parafilm, and then Seal a layer of paraffin film to make a sandwich structure of "paraffin film-miRNA molecular solution-paraffin film"; after inserting aphids, place the semi-closed aphid feeder on a tray covered with absorbent paper and raise them in the insect culture room , to ensure that the diameter of the bottom port of the aphid feeder is in airtight contact with the absorbent paper, in order to prevent the aphids from escaping.

Preferably, the number of heads is 10-20 heads.

Preferably, during the growth on the wheat plants, the wheat plants are covered with an insect breeding cover, the purpose of which is to avoid escape of aphids.

MiRNAs mainly participate in the life processes of organisms such as individual development, cell differentiation, cell apoptosis and proliferation at the post-transcriptional level through transcriptional regulation, transcriptional repression, and translational repression. Due to the important role of miRNAs in insect development, their expression inhibition and overexpression may both affect the normal development of organisms. Due to the limitation of miRNA size, direct knockout of miRNA cannot be well realized. Generally, the study of miRNA function is mainly identified by enhancing or weakening the expression of the target miRNA. Now the most widely used is the use of specific miRNAs sequences to chemically synthesize miRNA antagonists (miRNA antagomir) and miRNA agonists (miRNA agomir), compared with common miRNA inhibitors (miRNA inhibitor) and miRNA mimics (miRNA minics), they have Stronger cell affinity, stability and inhibition. miRNAantgomir is a single-stranded small RNA synthesized by a series of chemical modifications and special markers designed according to the mature sequence of miRNA. It can block the combination of miRNA and target gene by competing with the mature miRNA in vivo, so as to inhibit the function of miRNA. miRNA agomir is a double-stranded small RNA that mimics the miRNA in vivo and pairs with the target gene to enhance the expression of the target gene in vivo. By introducing miRNA agomir and miRNA antagomir into the organism, the expression of the target gene in the organism can be enhanced or weakened, thereby affecting the normal growth and development process of the organism. In particular, those pest control target genes are highly targeted and lethal to wheat pests after being silenced. These genes can be used as ideal candidate gene resources for the creation of new RNAi-mediated insect-resistant crops.

The miRNA avi-miR-263a agomir (miRNA agonist) and miRNA avi-miR-263a antagomir (miRNA antagonist) designed and synthesized by the present invention are lethal to aphids, and provide an effective product for the prevention and control of aphids; and , its target gene can be used as an ideal candidate gene resource for the creation of new RNAi-mediated insect-resistant crops, which provides a new research idea for the control of crops, especially wheat pests, and has high research value and broad application prospects.

Additional aspects and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.

Description of drawings

Fig. 1 is the feeding schematic diagram of wheat aphid in the semi-closed aphid feeding device in the embodiment of the present invention;

Fig. 2 is a graph showing the mortality rate of wheat aphids fed with miRNA avi-miR-263a agomir, miRNA avi-miR-263a antagomir, agomir NC, antagomir NC and NC in an embodiment of the present invention.

Detailed ways

The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the drawings in the embodiments of the present invention. The following examples are only used to illustrate the technical solution of the present invention more clearly, so they are only examples, and should not be used to limit the protection scope of the present invention.

The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores. In the quantitative experiments in the following examples, three repeated experiments were set up, and the data were the mean value or mean ± standard deviation of the three repeated experiments.

The aphid used in the embodiment of the present invention is Sitobion avenae (Fabricius).

Example 1: Design and synthesis of miRNA avi-miR-263a agomir and miRNA avi-miR-263a antagomir

The corresponding miRNA avi-miR-263aagomir (miRNA agonist) and miRNA avi-miR-263a antagomir (miRNA antagonist) were designed based on the mature sequence of aphid miRNAavi-miR-263a. The miRNAavi-miR-263a agomir has a double-stranded structure, and the nucleotide sequences of the two strands are shown in SEQ ID NO: 1 in the sequence listing and SEQ ID NO: 2 in the sequence listing; miRNA avi- miR-263a antogimir has a single-stranded structure, and its nucleotide sequence is shown in SEQ ID NO: 3 in the sequence listing.

In order to better characterize the effects of miRNA avi-miR-263a agomir and miRNA avi-miR-263a antagomir provided by the present invention, the present invention also designed negative controls agomir NC and antagomir NC, wherein the cores of the two chains of agomir NC The nucleotide sequences are respectively shown in SEQ ID NO: 4 in the sequence listing and SEQ ID NO: 5 in the sequence listing, and the nucleotide sequence of the antagomir NC single strand is shown in SEQ ID NO: 6 in the sequence listing.

The miRNA avi-miR-263a agomir, miRNA avi-miR-263a antagomir and the corresponding negative controls agomir NC and antagomir NC provided by the present invention were designed and synthesized by entrusting Suzhou Gemma Gene Co., Ltd. The specific sequence information is shown in Table 1 below.

Table 1 Sequence information of miRNA agomir and antagomir

The antisense strand (SEQ ID NO: 2) of miRNA avi-miR-263a agomir is modified to obtain: 5'-CsGsUGAAUUCUUUCAGUGCCAUsUsUsUs-Chol 3'. The modification process is as follows: modifying the 3' end of the antisense strand with cholesterol, Two thio-skeleton modifications are carried out at the 5' end, four thio-skeleton modifications are carried out at the 3' end, and finally the whole chain is modified with methoxy groups. The upper sense strand (SEQ ID NO: 1) is the active strand, and the lower antisense strand is the supplementary auxiliary strand. When designing, the first 2 bases are staggered for reverse complementary design, and UU is added to the end to facilitate unwinding.

The miRNA avi-miR-263a antogimir has a single-stranded structure (SEQ ID NO: 3), and it is modified to obtain: 5'-CsCsCGUGAAUUCUUUCAGUGCCsAsUsUs-Chol3'. The modification process is as follows: modifying the 3' end with cholesterol, Two thio-skeleton modifications are carried out at the 5' end, four thio-skeleton modifications are carried out at the 3' end, and methoxy modification is carried out on the whole chain.

Example 2: Using miRNA avi-miR-263a agomir and miRNAavi-miR-263a antagomir to feed aphids and the lethal effect

1. Preparation method of miRNA avi-miR-263a agomir, miRNA avi-miR-263a antagomi, agomir NC and antagomir NC:

(1) Use ultrapure water to prepare a sucrose solution with a mass fraction of 30%, then filter and sterilize it with a 0.2 μm microporous membrane, divide the obtained filtrate into 1.5ml sterilized centrifuge tubes, and store in a -20°C refrigerator spare;

(2) Dilute the synthesized and modified miRNA avi-miR-263aagomir, miRNA avi-miR-263a antagomir, agomir NC and antagomir NC to a concentration of 300nM with a 30% sucrose solution to obtain miRNA avi-miR -263a agomir (+ sucrose solution), miRNA avi-miR-263a antagomir (+ sucrose solution), agomir NC (+ sucrose solution) and antagomir NC (+ sucrose solution), set aside.

2. miRNA avi-miR-263a agomir, miRNA avi-miR-263a antagomi, agomir NC, antagomir NC and NC feeding aphids

(1) Feed the aphids continuously for 3 generations in a single head, collect the nymphs of the 4th generation of wingless adults that reproduced within 24 hours of the first generation, and put 10 of them into the miRNA avi-miR-263a agomir (+ sucrose solution) containing 200 μL , miRNA avi-miR-263aantagomir (+ sucrose solution), agomir NC (+ sucrose solution), antagomir NC (+ sucrose solution) and 30% sucrose solution were fed in a semi-closed aphid feeder. Among them, the preparation method of the semi-closed aphid feeder includes the following steps: vertically place the sterilized cylindrical glass tube on absorbent paper, seal the top with a paraffin film with a thickness of 1 μm, and add corresponding Solution (i.e. miRNA avi-miR-263a agomir (+ sucrose solution), miRNAavi-miR-263a antagomir (+ sucrose solution), agomir NC (+ sucrose solution), antagomir NC (+ sucrose solution), mass fraction of 30% sucrose solution), and then capped with a paraffin film with a thickness of 10 μm to make a sandwich structure of "paraffin film-miRNA molecule solution/sucrose solution-paraffin film" to obtain a semi-closed aphid feeding device containing different solutions; and, cylindrical The diameter of the glass is 2 cm, the height is 3 cm, and the volume of different solutions is 200 μL. After inserting the aphids, place the semi-closed aphid feeder on a tray covered with absorbent paper, as shown in Figure 1, and raise them in the insect culture room, ensuring that the lower end of the aphid feeder is in airtight contact with the absorbent paper to prevent aphids from escape. Feeding conditions include: place the semi-closed aphid feeder containing the corresponding solution connected to 10 nymphs respectively in the insect culture room, the temperature of the insect culture room is 20±1°C, the humidity is 60±10%, and the photoperiod L:D=14h:10h.

(2) After feeding for 72 hours as above, connect the nymphs in the aphid feeder to fresh potted wheat plants respectively, cover them with an insect cover to prevent the aphids from escaping, and record the death and individual development of the aphids during the feeding period until Develop into adult aphids after 6-7 days, count the survival rate of each treatment.

In order to ensure reproducibility, 10 operations were repeated for each treatment setting.

3. The lethal effect of feeding miRNA avi-miR-263a agomir, miRNA avi-miR-263a antagomir, agomirNC, antagomir NC and NC on aphids

The specific results are shown in Figure 2 and Table 2 below: After statistical analysis, it was found that the mortality rate of wheat aphids after feeding miRNA avi-miR-263aagomir was 90.00±3.74%, and that after feeding miRNA avi-miR-263a antagomir The mortality rate of wheat aphids was 69.00±5.38%, which was significantly different from the three controls; the mortality rate of wheat aphids after feeding the antagomir NC control group was 30.00±2.83%, and the mortality rate of wheat aphids after feeding the agomir NC control group was 16.00±2.90%, and the mortality rate of wheat aphids after feeding the NC control group was 9.00±2.63%. There were significant differences between the treatment group and the control group (P<0.05).

Table 2 The lethal effect of feeding different substances on aphids

The miRNA avi-miR-263a agomir (miRNA agonist) and miRNA avi-miR-263a antagomir (miRNA antagonist) designed and synthesized by the present invention are lethal to aphids, and provide an effective product for the prevention and control of aphids; and , its target gene can be used as an ideal candidate gene resource for the creation of new RNAi-mediated insect-resistant crops, which provides a new research idea for the control of crops, especially wheat pests, and has high research value and broad application prospects.

It should be noted that, unless otherwise specified, the technical terms or scientific terms used in this application shall have the usual meanings understood by those skilled in the art to which the present invention belongs. Relative steps, numerical expressions and numerical values of components and steps set forth in these embodiments do not limit the scope of the present invention unless specifically stated otherwise. In all examples shown and described herein, unless otherwise specified, any specific value should be construed as merely exemplary and not limiting, and thus other examples of the exemplary embodiments may have different values.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present invention, rather than limiting them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: It is still possible to modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the technical solutions of the various embodiments of the present invention. range, which should be included in the protection scope of the present invention.

SEQUENCE LISTING

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Claims (3)

1. a kind of method using kit pest controlling aphid, it is characterised in that:

The kit includes miRNA molecule solution, and the miRNA molecule is duplex structure, contains positive-sense strand and antisense strand；Its In, the nucleotide sequence of the positive-sense strand is as shown in the SEQ ID NO:1 in sequence table, and the nucleotide sequence of the antisense strand As shown in the SEQ ID NO:2 in sequence table；

Or the miRNA molecule is single-stranded structure, nucleotide sequence is as shown in the SEQ ID NO:3 in sequence table；

In the duplex structure, the chemical modification of the antisense strand includes: that 3 ' ends carry out cholesterol modification, and 5 ' ends carry out two sulphur For backbone modification, 3 ' ends carry out four thio backbone modifications, and full chain carries out methoxyl group modification；

Or the chemical modification of the single-stranded structure includes: that 3 ' ends carry out cholesterol modification, 5 ' ends carry out two thio backbone modifications, 3 ' ends carry out four thio backbone modifications, and full chain carries out methoxyl group modification；

The miRNA molecule solution includes the sucrose solution that mass fraction is 30% and concentration is the described of 300~500nM MiRNA molecule；Wherein, the solvent of the sucrose solution is water；

The method of the kit pest controlling aphid, includes the following steps:

Access head larvae aphid feeds 72h in the feeding aphid device containing miRNA molecule solution, is then linked into wheat strain and continues to raise；

The feeding aphid device containing miRNA molecule solution is the semiclosed feeding aphid device containing miRNA molecule solution, described to contain The preparation method of the semiclosed feeding aphid device of miRNA molecule solution includes the following steps: that the cylindrical glass tube after sterilizing is vertical It is placed on blotting paper, top utilizes seals with a thickness of 0.8~2 μm of olefin film, adds in the upper surface of the olefin film Enter miRNA molecule solution, then bound again with the olefin film with a thickness of 8~15 μm, obtains described containing miRNA molecule solution Semiclosed feeding aphid device；Wherein, the diameter of the cylindrical glass is 2cm, is highly 3cm, the body of the miRNA molecule solution Product is 200~300 μ L；

The condition of the feeding includes: by the bottom end for the semiclosed feeding aphid device containing miRNA molecule solution for accessing several larvae aphids With blotting paper close contact, then entire feeding system is placed in insectary, the temperature of the insectary is 20 ± 1 DEG C, Humidity 60 ± 10%, photoperiod L:D=14h:10h.

2. the method for kit pest controlling aphid according to claim 1, it is characterised in that:

Described several are 10~20.

3. the method for pest controlling aphid according to claim 2, it is characterised in that:

In the upper growth period of wheat strain, the wheat strain is covered with feeding worm cover.