Primer pair, DNA, dsRNA, kit for interfering with the feeding of Odontothrips loti, and application and prevention and control method thereof

Abstract

A primer pair, DNA, dsRNA, kit, application and control method for interfering with feeding of thrips. The primer pair for interfering with feeding of thrips has nucleotide sequences as shown in SEQ ID NO: 2 and SEQ ID NO: 3. The DNA for interfering with feeding of thrips is obtained by amplification of gDNA of thrips and primer pair dsO1PLF and dsO1PLR. The dsRNA for interfering with feeding of thrips is synthesized using the DNA for interfering with feeding of thrips as a template. The kit for interfering with feeding of thrips, the active ingredient of which includes dsRNA for interfering with feeding of thrips. The application of the dsRNA in preventing and controlling thrips reduces the degree of damage by interfering with feeding of thrips, and causes the death of thrips. The dsRNA is used in combination with pyrethroid insecticides to significantly improve the control effect of the insecticide.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a primer pair, DNA, dsRNA, a kit, and an application and prevention and control method for interfering with the feeding of horn flower thrips. Background Art

[0002] Thrips tataricus Odontothrips loti ) is one of the main pests of alfalfa in China. It is seriously occurring in the main alfalfa producing areas in my country, with a damage rate of 70%-100%, seriously affecting the yield and quality of alfalfa. At the same time, alfalfa virus disease is one of the important diseases of alfalfa fields. It is increasingly occurring in the field, with an incidence rate of 20%-100%, causing alfalfa yield reduction or even complete failure, and is an important factor restricting alfalfa production. Insects are the main carriers of plant viruses, among which the ox-horned flower-toothed thrips is an important vector insect that carries and spreads alfalfa virus disease in my country. Plant virus diseases are called the "cancer" of crops, and there is still a lack of effective prevention and control methods. Therefore, the effective prevention and control of thrips is a key link in cutting off the transmission pathways of viral diseases and achieving effective prevention of alfalfa virus diseases.

[0003] At present, the control of thrips mainly relies on chemical pesticides, including pyrethroids, neonicotinoids and other pesticides. Due to the strong reproductive capacity and short life cycle of thrips, their field resistance develops very rapidly. The resistance of thrips to pesticides has seriously affected the control of viral diseases and pests in alfalfa fields. RNAi is a new pest management method with strong targeting and environmental friendliness, and has been widely used in the agricultural field. However, there are no reports on products and methods for controlling thrips by interfering with thrips feeding using RNAi technology.

[0004] Chinese patent application CN116606851A discloses a primer set dsFoTOF / Rs, DNA, dsRNA, kit and application and method for controlling thrips. The invention controls thrips by reducing the egg-laying amount of western flower thrips. Although it can reduce the number of thrips offspring, it cannot directly kill them, and the period of control effect is long. Chinese patent application CN115136959A discloses a feeding inhibitor of bean thrips and its screening method and application. The feeding inhibitors described in the invention are all plant-derived extracts, the extraction process is complicated, and the control cost is high. There is no report on the technology of affecting the feeding of horn flower tooth thrips by gene interference to achieve control effect.

[0005] Therefore, there is an urgent need in the prior art for a primer set, DNA, dsRNA, kit, application and method for interfering with the feeding of Cynoglossum sphaerocephalus. Summary of the invention

[0006] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a primer pair, DNA, dsRNA, a kit and an application and prevention and control method for interfering with the feeding of Cynoglossum sphaerocephalus.

[0007] The objective of the present invention is achieved through the following technical solutions:

[0008] By performing selection and elimination analysis and differential expression analysis on populations of thrips with different insecticide resistance levels, a pectinase gene containing multiple non-synonymous mutation sites and with significantly different RNA expression levels was screened out in resistant thrips, and the nucleotide sequence is shown in SEQ ID NO: 1. A primer pair that interferes with feeding by thrips was designed based on the target sequence fragment SEQ ID NO: 1.

[0009] The nucleotide sequence of the primer pair that interferes with feeding of Thrips nigromaculata is as follows:

[0010] dsOlPLF: 5'-taatacgactcactatagggGAAACGGAGGGCTACGAGAC-3', SEQ ID NO: 2;

[0011] dsOlPLR: 5'-taatacgactcactatagggTGCTGGCGACCTATGTTATGG-3', SEQ ID NO: 3.

[0012] The DNA that interferes with feeding by the thrips is obtained by amplifying the gDNA of the thrips and the primer pair dsO1PLF and dsO1PLR. The nucleotide sequence of the DNA that interferes with feeding by the thrips is shown in SEQ ID NO: 1.

[0013] The dsRNA that interferes with feeding of the thrips is synthesized using the DNA that interferes with feeding of the thrips as a template. The dsRNA can prevent and control the thrips by affecting feeding, reduce the degree of damage caused by thrips in the field, reduce the losses caused by feeding of the thrips, and cause the death of the thrips.

[0014] A kit for interfering with feeding of B. cerana, wherein the active ingredient comprises dsRNA interfering with feeding of B. cerana.

[0015] Application of dsRNA that interferes with feeding of Bulrushia spp. in the prevention and control of Bulrushia spp.

[0016] Furthermore, the dsRNA that interferes with feeding of Cynoglossum sphaerocephalus is used in combination with a pyrethroid insecticide.

[0017] A method for preventing and controlling thrips of Botrytis cinerea, which uses the dsRNA that interferes with the feeding of thrips of Botrytis cinerea to prevent and control the thrips of Botrytis cinerea.

[0018] Furthermore, the dsRNA that interferes with feeding of Cynoglossum sphaerocephalus is used in combination with a pyrethroid insecticide.

[0019] The invention has the following beneficial effects: the dsRNA provided by the invention has strong targeting property, can specifically control the field thrips, and has no effect on the beneficial natural enemies in the field. The dsRNA provided by the invention reduces the losses caused by the thrips feeding, reduces the spread of alfalfa virus disease, reduces the degree of damage, and causes the death of the thrips by interfering with the feeding of the thrips. The dsRNA provided by the invention is used in combination with a pyrethroid insecticide to significantly improve the control effect of the insecticide. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is the mortality of Cyprinus sphaerocephalus 48 hours after feeding with dsRNA;

[0021] Figure 2 is the mortality of Cyperus sphaerocephalus after treatment with lambda-cyhalothrin;

[0022] Figure 3 This is the agarose gel electrophoresis image of the PCR amplification product of gDNA of Thrips tatarinowii. DETAILED DESCRIPTION

[0023] The present invention is described in detail below in conjunction with the accompanying drawings.

[0024] Example 1 Synthesis of dsRNA that interferes with feeding by Cynocephalus serratus

[0025] Step 1: Extract gDNA from 20 female Thrips nigrohornense:

[0026] 1) Add 200ul CTAB mixed solution to 20 female adults of Thrips nigromaculata, grind them thoroughly and add 800ul CTAB mixed solution;

[0027] 2) Place in a water bath at 65°C for 30 minutes, inverting every 10 minutes; centrifuge at 25°C, 12,000 rpm for 10 minutes;

[0028] 3) Take the supernatant to a new centrifuge tube, add a chloroform-isoamyl alcohol mixture (volume ratio 24:1) with an equal volume (1 ml) of CTAB, shake horizontally for 5 minutes, and centrifuge at room temperature (25°C) and 12,000 rpm for 10 minutes;

[0029] 4) Take the supernatant, add 1 ml of chloroform-isoamyl alcohol mixture (volume ratio 24:1), centrifuge at 25°C, 12000 rpm for 10 min, and take the supernatant;

[0030] 5) Add 1 ml of isopropanol to the supernatant of step 4), gently invert to mix, and let stand at -80°C for 10 min;

[0031] 6) Centrifuge at room temperature (25°C), 12,000 rpm for 10 min and pour out the supernatant;

[0032] 7) Add 1 ml of 70% ethanol (precooled at -20°C) to the precipitate to precipitate DNA, centrifuge at 25°C, 12,000 rpm for 10 min, repeat once;

[0033] 8) Invert on filter paper to dry, add 20ul ddH 2 O was dissolved and stored at -20℃ to obtain gDNA of Thrips taurifolia.

[0034] Step 2: PCR amplification:

[0035] According to the target sequence fragment SEQ ID NO: 1, a specific primer was designed using Primer-BLAST, and the specific primer was synthesized by Sangon Biotech (Shanghai) Co., Ltd. The nucleotide sequence of the specific primer is as follows:

[0036] dsOlPLF: 5'-taatacgactcactatagggGAAACGGAGGGCTACGAGAC-3' (SEQ ID NO: 2);

[0037] dsOlPLR:5'-taatacgactcactataggggTGCTGGCGACCTATGTTATGG-3' (SEQ ID NO:3).

[0038] Establish the PCR amplification system of the target sequence fragment SEQ ID NO: 1, see Table 1;

[0039]

[0040] The PCR reaction conditions are shown in Table 2 ;

[0041]

[0042] Step 3: Use 1% agarose gel and run electrophoresis at 120V for 20 minutes. Observe the electrophoresis bands on a gel imaging system. Figure 3As shown, if a 368 bp band appears, the PCR product and primers are sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing, and the sequencing result is compared with the target fragment SEQ ID NO: 1. After the comparison result is confirmed as the target fragment, the PCR product is purified.

[0043] Step 4: Using the purified PCR product as a template, synthesize dsRNA in a metal bath at 37° C. for 4 hours. The synthesis system is shown in Table 3;

[0044]

[0045] Step 5: Purify dsRNA product:

[0046] 1) The material to be purified is treated with DEPC H 2 O to 200 μL;

[0047] 2) Add half equal volume (100 μL) of water-saturated phenol reagent and half equal volume (100 μL) of chloroform;

[0048] 3) Mix gently and centrifuge (12000 rpm, 4°C) for 15 min;

[0049] 4) Take the upper phase, add an equal volume of chloroform (200 μL), mix gently, and centrifuge (12000 rpm, 4°C) for 15 min;

[0050] 5) Take the upper phase, add 1 / 10 volume (20 μL) of 3M sodium acetate (pH 5.2) and 2.5 volumes (500 μL) of -20℃ precooled 100% ethanol, mix gently and place at -20℃ for half an hour;

[0051] 6) Centrifuge (12000 rpm, 4°C) for 30 min;

[0052] 7) At this time, there is a white precipitate at the bottom of the centrifuge tube. Discard the supernatant, add -20℃ pre-cooled 80% ethanol, mix gently, and wash the precipitate;

[0053] 8) Centrifuge (7500 rpm, 4°C) for 5 min;

[0054] 9) Slowly aspirate the ethanol, and slowly aspirate the supernatant close to the precipitate with a 10μL pipette. To prevent the precipitate from being aspirated together, open the centrifuge tube with a small amount of residual ethanol and place it in a 37℃ constant temperature incubator. After about 10 minutes, all the ethanol will evaporate;

[0055] 10) Add 20 μL DEPC HO 2 Tap the bottom of the tube gently to allow the precipitate to fully and evenly dissolve in DEPC H 2O in;

[0056] 11) Dissolve 0.5 μL in 9.5 μL DEPC H 2 O, run gel test, NANOdrop test concentration and OD value. The detected band is single and bright, and its A260 / 280 OD value is between 1.8-2.3, and its A260 / 230 OD value is between 1.8-2.5, which proves that the dsRNA is of good quality. Calculate the concentration, dilute the dsRNA to the required concentration, and store it at -80℃. Dilute to 500ng / μL for prevention and treatment.

[0057] Example 2 Effect of dsRNA on the mortality of Thrips tatarinus

[0058] The dsRNA synthesized in Example 1 with a volume of 50 ul and a concentration of 500 ng / μL was used to feed female adults of Thrips tatarica. Each treatment had 15 thrips and 4 replicates were set. After 48 hours, the mortality rate was calculated. Figure 1 As shown, the mortality rate of S. cerana was 42% 48 hours after feeding with dsRNA, while the mortality rate of the control group (dsGFP) was 20%. Compared with the control group, the mortality rate of S. cerana doubled after feeding with dsRNA 48 hours.

[0059] Example 3 Application of dsRNA in the control of Cynoglossum sphaerocephala

[0060] Pyrethroids are the most commonly used insecticides in the control of thrips. In order to explore the application of the dsRNA synthesized in Example 1 in the control of thrips, the thrips that survived 48 hours after being fed with dsRNA in Example 2 and the thrips that survived in the control group of Example 2 were treated with a highly effective cyhalothrin agent at a concentration of 11.56 mg / L, and the mortality rate was calculated after 48 hours. Figure 2 As shown, the mortality rate of the thrips that survived 48 hours after feeding dsRNA reached 96% after being treated with the highly effective cyhalothrin agent for 48 hours, while the mortality rate of the control group (dsGFP) was only 32%, indicating that the sensitivity of the thrips to insecticides after dsRNA treatment was significantly increased. Therefore, when pyrethroid insecticides are applied in the field, the use of the dsRNA of the present invention can significantly improve the control effect.

[0061] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A dsRNA that interferes with feeding by Botrytis cinerea, which is synthesized using a DNA that interferes with feeding by Botrytis cinerea as a template, wherein the nucleotide sequence of the DNA that interferes with feeding by Botrytis cinerea is shown in SEQ ID NO: 1, and the DNA that interferes with feeding by Botrytis cinerea is amplified by gDNA of Botrytis cinerea and a primer pair dsO1PLF and dsO1PLR; The nucleotide sequence of the dsO1PLF is: 5'-taatacgactcactatagggGAAACGGAGGGCTACGAGAC-3', SEQ ID NO: 2; The dsO1PLR nucleotide sequence is: 5'-taatacgactcactatagggTGCTGGCGACCTATGTTATGG-3', SEQ ID NO:

3.

2. A kit for interfering with feeding of Cyprinus serratus, characterized in that: The active ingredient comprises the dsRNA for interfering with feeding of the horned flower thrips according to claim 1.

3. Use of the dsRNA that interferes with feeding of B. sphaerocephala according to claim 1 in preventing and controlling B. sphaerocephala.

4. The use according to claim 3, characterized in that: The dsRNA that interferes with feeding of thrips is used in combination with a pyrethroid insecticide.

5. A method for preventing and controlling Botrytis cinerea, characterized in that: The dsRNA that interferes with feeding of B. sphaerocephala as claimed in claim 2 is used to prevent and control B. sphaerocephala.

6. The control method according to claim 5, characterized in that: The dsRNA that interferes with feeding of thrips is used in combination with a pyrethroid insecticide.

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