Laodelphax striatellus ecdysis gene LsMH and application thereof in biological control of Laodelphax striatellus

Abstract

According to the laodelphax striatellus ecdysis gene LsMH and the application thereof in biological prevention and control of laodelphax striatellus, the laodelphax striatellus ecdysis gene LsMH serves as one of key genes for ecdysis of laodelphax striatellus, the laodelphax striatellus ecdysis gene LsMH can be subjected to targeted interference, laodelphax striatellus can be remarkably inhibited from forming adults through eclosion, and laodelphax striatellus death is avoided, so that laodelphax striatellus population is the harm of the laodelphax striatellus to food crops is reduced. The control of laodelphax striatellus is carried out through a biological means, the method is safe and harmless to environment ecology and food, and a new strategy is provided for green control of laodelphax striatellus. The dsRNA or the interference reagent is prepared through transcription of the laodelphax striatellus ecdysis gene LsMH, the LsMH gene can be effectively interfered after the dsRNA or the interference reagent is injected into the laodelphax striatellus, the phenomena that wings are bent or incomplete, the abdomen is wizened and ecdysis is incomplete occur in the ecdysis process of laodelphax striatellus nymphs, laodelphax striatellus eclosion into adults is inhibited, laodelphax striatellus death is avoided, and biological control over laodelphax striatellus is achieved.

Description

Technical Field

[0001] The present application relates to the field of agricultural science and technology, and in particular to a molting gene LsMH of the gray leafhopper and its application in biological control of the gray leafhopper. Background Art

[0002] As a major pest in my country and other crop-growing areas in Asia, the gray leafhopper not only directly harms rice, wheat, corn and other food crops by sucking plant sap and laying eggs, but also spreads a variety of crop viruses to indirectly harm crops, seriously affecting the quality, stable and high yield of food. Since the 1960s, gray leafhoppers have frequently broken out in many areas of my country, causing a 30-35% reduction in food crop yields each year, posing a serious threat to my country's food production safety. At present, my country mainly relies on chemical pesticides to control gray leafhoppers. However, the unreasonable use of chemical pesticides in large quantities has not only caused the rapid development of gray leafhoppers' resistance to pesticides, but also aggravated environmental pollution and ecosystem damage, seriously hindering the process of my country's agricultural modernization and posing a serious threat to food, ecological and economic security.

[0003] Ecdysone is the main metabolic enzyme of insect juvenile hormone, which is synthesized by fat body cells and epithelial cells and distributed in insect hemolymph, midgut and fat body. Insect ecdysone has the function of highly recognizing and decomposing bound or free juvenile hormone, balancing the level of juvenile hormone in insects, and participating in physiological and biochemical behaviors such as insect molting, pupation and reproduction. Therefore, promoting or inhibiting the activity of ecdysone can disrupt the level of juvenile hormone in insects, causing disorder in insect development and reproduction, and achieving the purpose of pest control. Ecdysone has great potential in green pest control. Summary of the invention

[0004] The present application embodiment provides a molting gene LsMH of Laodelphax striatellus and its application in biological control of Laodelphax striatellus to solve the problems existing in the related technologies. The technical solution is as follows:

[0005] In a first aspect, an embodiment of the present application provides a molting gene LsMH of the gray leafhopper, and the nucleotide sequence of the molting gene LsMH of the gray leafhopper is shown in SEQ ID NO.1.

[0006] In one embodiment, the amino acid sequence encoded by the Laodelphax striatellus molting gene LsMH is shown as SEQ ID NO.2.

[0007] In a second aspect, an embodiment of the present application provides a primer set for amplifying the above-mentioned planthopper molting gene LsMH, including an upstream primer SEQ ID NO.3 and a downstream primer SEQ ID NO.4.

[0008] In a third aspect, an embodiment of the present application provides a dsRNA, which targets and silences the molting gene LsMH of the small brown planthopper.

[0009] In one embodiment, the nucleotide sequence of the dsRNA is shown as SEQ ID NO.5.

[0010] In a fourth aspect, the embodiments of the present application provide a method for preparing the above-mentioned dsRNA, which is prepared by using the nucleotide sequence SEQ ID NO.5 of the molting gene LsMH of the small brown planthopper as a template and transcribing using a T7 High Yield RNA transcription kit.

[0011] In one embodiment, the reaction system is: 10x Reaction Buffer 2μl, Enzyme mix 2μl, ATP solution 2μl, UTP solution 2μl, CTP solution 2μl, GTP solution 2μl, 1μg DNA template, add water to a total volume of 20μl; mix well, incubate at 37°C overnight, add DNase and mix well, then treat at 37°C for 15min and 65°C for 5min to obtain dsRNA of the molting gene LsMH of the gray leafhopper.

[0012] In a fifth aspect, the embodiments of the present application provide the use of the above-mentioned gray leafhopper molting gene LsMH or the above-mentioned dsRNA in the control of gray leafhopper.

[0013] In a sixth aspect, an embodiment of the present application provides an interference reagent for the molting gene LsMH of the gray leafhopper, wherein the interference reagent includes the above-mentioned dsRNA; or includes a gene fragment transcribed from the above-mentioned molting gene LsMH of the gray leafhopper.

[0014] In one embodiment, the interference agent for the molting gene LsMH of the Laodelphax striatellus acts on the Laodelphax striatellus by injection.

[0015] The advantages or beneficial effects of the above technical solution include at least:

[0016] The present application discloses a new molting gene LsMH of gray leafhopper, which is one of the key genes of gray leafhopper molting. By targeted interference with the molting gene LsMH of gray leafhopper, it can significantly inhibit the emergence of gray leafhopper into adults, causing the death of gray leafhopper, thereby controlling the gray leafhopper population and reducing the harm of gray leafhopper to food crops. The prevention and control of gray leafhopper by biological means is safe and harmless to the environment, ecology and food, providing a new strategy for green prevention and control of gray leafhopper.

[0017] The dsRNA or interference agent of the present application is prepared by transcribing the molting gene LsMH of the gray planthopper. After being injected into the gray planthopper, it can effectively interfere with the LsMH gene. During the molting process of the gray planthopper nymphs, the wings become bent or incomplete, the abdomen becomes shriveled, and the molting is incomplete. This can significantly inhibit the gray planthopper from emerging into an adult, causing the death of the gray planthopper, thereby achieving biological control of the gray planthopper.

[0018] The above summary is for illustrative purposes only and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments and features described above, further aspects, embodiments and features of the present application will be readily apparent by reference to the accompanying drawings and the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In the accompanying drawings, unless otherwise specified, the same reference numerals throughout the multiple drawings represent the same or similar parts or elements. These drawings are not necessarily drawn to scale. It should be understood that these drawings only depict some embodiments disclosed in the present application and should not be regarded as limiting the scope of the present application.

[0020] Figure 1 This is the result of PCR amplification of the LsMH fragment of the molting gene of Laodelphax striatellus;

[0021] Figure 2 Synthesis of dsRNA of the molting gene LsMH of Laodelphax striatellus;

[0022] Figure 3 The comparison of the eclosion morphology of the Laodelphax striatellus after the dsRNA of the molting gene LsMH was introduced into the Laodelphax striatellus and that of the control group;

[0023] Figure 4 This is the emergence rate of the small brown planthopper after the dsRNA of the molting gene LsMH was introduced into the small brown planthopper. DETAILED DESCRIPTION

[0024] In the following, only some exemplary embodiments are briefly described. As those skilled in the art will appreciate, the described embodiments may be modified in various ways without departing from the spirit or scope of the present application. Therefore, the drawings and descriptions are considered to be exemplary and non-restrictive in nature.

[0025] RNA interference (RNAi) technology, by introducing small double-stranded RNA into the target pest, specifically binds to the target gene mRNA of the pest, degrades or inhibits the protein translation of the target gene, disrupts the normal physiological and biochemical behavior of the pest, and achieves the purpose of controlling the pest population or reducing the pest's ability to spread viruses. RNAi technology has the advantages of high specificity, good effect, and green environmental protection in pest control, and has been widely used in the control of various agricultural pests.

[0026] Promoting or inhibiting the activity of molting hormone can disrupt the level of juvenile hormone in insects, causing insect development and reproduction disorders, thereby achieving the purpose of pest control. The molting gene LsMH of Laodelphax striatellus and its application in biological control of Laodelphax striatellus are described in detail below with specific examples.

[0027] Example 1 Cloning of the Molting Gene LsMH of Laodelphax striatellus

[0028] 1. Amplification of the LsMH fragment of the molting gene of the Laodelphax striatellus:

[0029] (1) Take 4-5 adults or nymphs of Laodelphax striatellus, wash them three times with 1x PBS (2.68 mM KCl, 37 mM NaCl, 8.1 mM Na2HPO4, 1.47 mM KH2PO4, pH 7.4), and extract total RNA using the TrizoL method: add 1 mL TrizoL to fully grind the insect body; add 500 μL chloroform and mix vigorously; centrifuge at 12000 rpm and 4°C for 15 min; carefully transfer the supernatant to a new centrifuge tube; add an equal volume of isopropanol, mix well and let stand for 10 min; centrifuge at 12000 rpm and 4°C for 10 min and remove the supernatant; add 1 mL 75% ethanol, centrifuge at 9600 rpm and remove the supernatant; dissolve the precipitate in RNase-free water and measure the RNA solubility.

[0030] (2) Using total RNA as template, One-Step gDNA Removal and cDNA Synthesis SuperMix Reverse Transcription Kit was used to reverse transcribe RNA: 5 μg total RNA, 1 μL Oligo(dT), 10 μL 2xTS Reaction Mix, 1 μL Enzyme Mix, 1 μL gDNA Remover, and RNase-free water was added to make up the total volume to 20 μL; after mixing, incubate at 42°C for 30 min; denature at 85°C for 5 s to inactivate the reverse transcriptase and obtain the cDNA of the Laodelphax striatellus.

[0031] (3) Primers were designed based on SEQ ID NO.1 of the LsMH gene of Laodelphax striatellus and SEQ ID NO.2 of the amino acid encoded by the LsMH gene.

[0032] SEQ ID NO.1:

[0033]

[0034] SEQ ID NO.2:

[0035] MTESLSTTQLMLKTHILPVIRAGNTSTYTLVQSYHITRLVTAVKTLPPMEVYQFVPSKTLNEFNTRLDEAGSELHLELEFGDNSNNQEGDDLPSAKAFPPDLDLANVGSDFDLSEMDKTKFPDMHLRLKKAHQNSPQQNQFQPVPTTPQEQATPSLSPEQLQQL ALYRFLNPNAPLPPQLLTTSRPVLKVETIYESHVIPLFNGQSTLFSTISRPIATVSKTEYEVVTNSLALPPVQPPVNTINPFLQQPQPQFAITSTPIVTQTMVTATDSKVLKLTFGAKTAYTTLYSTKVVPTVLTTYLTANVPVQPTVAAFPGYFPAPFNPYLG.

[0036] The primers are shown in SEQ ID NO.3 and SEQ ID NO.4,

[0037] SEQ ID NO.3(LsMH-F):AAGTAACTGGCAGATTCAACC;

[0038] SEQ ID NO.4 (LsMH-R): CTCGAATAGATGTGCTGCAGG;

[0039] (4) Using the cDNA of gray leafhopper as a template, SEQ ID NO.3 and SEQ ID NO.4 primers were used to perform PCR amplification of the LsMH gene. The system was as follows: 5 μL 2x ApexHF Mix, 1 μL upstream primer LsMH-F (SEQ ID NO.3), 1 μL downstream primer LsMH-R (SEQ ID NO.4), 2 μL gray leafhopper cDNA, 1 μL ddH2O, a total of 10 μL; PCR amplification reaction conditions were as follows: 95°C, 2 min; 95°C 20 s, 55°C 20 s, 72°C 1 min, 35 cycles; 72°C 10 min extension.

[0040] 2. Monocloning of the LsMH gene of Laodelphax striatellus:

[0041] (1) Separate the PCR products by 1% agarose gel electrophoresis. Figure 1As shown; cut the band with a blade and recover the PCR product with a DNA agarose gel kit; obtain the purified DNA fragment of the L. striatellus LsMH gene; connect the L. striatellus LsMH gene DNA fragment to the pClone007 vector using a blunt-end cloning kit, and the reaction system is as follows: 2 μL 10xBuffer, 2 μL pClone007 Blunt simple vector, 2 μL L. striatellus LsMH gene DNA fragment, 14 μL ddH2O, and a total volume of 20 μL. After the reagents are mixed, the ligation reaction is carried out at 25°C for 5 minutes.

[0042] (2) Transform the ligation product into competent cells. The transformation procedure is as follows: add the ligation product to the competent cells, flick the tube wall with your finger to mix, and place on ice for 30 minutes; place at 42°C for 90 seconds and then quickly place on ice for 2 minutes; transfer the competent cells into LB medium without antibiotics, and culture at 180 rpm for 1 hour; take 150 μL of the resuscitated bacterial solution and spread it on an LB medium plate containing ampicillin overnight; pick a single colony and place it in LB liquid medium containing ampicillin, and culture the colony at 180 rpm for 6 hours.

[0043] (3) Colony PCR was used to verify the DNA fragment of the LsMH gene. The reaction system was as follows: 5 μL 2×ApexHF Mix, 1 μL upstream primer LsMH-F (SEQ ID NO.3), 1 μL downstream primer LsMH-R (SEQ ID NO.4), 2 μL gray leafhopper cDNA, 1 μL ddH2O, and the total volume was 10 μL; the PCR amplification reaction conditions were as follows: 95°C, 2 min; 95°C 20 s, 55°C 20 s, 72°C 1 min, 35 cycles; 72°C 10 min extension. The PCR amplification products were separated by 1% agarose gel electrophoresis; the bands were cut with a blade, and the PCR products were recovered with a DNA agarose gel kit; the product was sent to the company for sequencing, and the sequence fragment of the gray leafhopper LsMH gene was obtained as shown in SEQID NO.5;

[0044] SEQ ID NO.5:

[0045] AAGTAACTGGCAGATTCAACCGGTTCCAACCACTCCTCAGGAACAGGCCACTCCCTCACTAAGCCCTGAACAACTCCAACAGTTGGCGTTGTACAGGTTTTTTGAATCCCAACGCACCCCTACCACCACAATTGCTAACCACGTCCAGGCCAGTACTGAAAGTGGAGACCATCTACG AGAGTCATGTGATACCTCTGTTCAACGGACAAAGTACTCTGTTTAGTACCATCTCACGACCGATTGCAACTGTCAGCAAAACGGAATACGAAGTCGTGACCAATTCTTTGGCGCTACCTCCTGTACAACCTCCTGTAAATACTATCAACCCCTTCCTGCAGCACATCTATTCGAG.

[0046] Example 2 Synthesis of dsRNA of the Molting Gene LsMH of Laodelphax striatellus

[0047] (1) PCR amplification of the LsMH gene fragment as shown in SEQ ID NO.5 using primers with T7 promoter. The bacterial solution containing the LsMH fragment of the molting gene of the gray leafhopper as shown in SEQ ID NO.5 was used as a template, and the target gene was amplified using primers SEQ ID NO.6 and SEQ ID NO.7 with T7 promoter sequence. The reaction system was as follows: 50 μL 2×ApexHF Mix, 10 μL upstream primer LsMH-F (SEQ ID NO.6), 10 μL downstream primer LsMH-R (SEQ ID NO.7), 20 μL bacterial solution containing the gray leafhopper gene LsMH, 10 μL ddH2O, and the total volume was 100 μL; the PCR amplification reaction conditions were as follows: 95℃, 2 min; 95℃20 s, 55℃20 s, 72℃1 min, 35 cycles; 72℃10 min extension.

[0048] SEQ ID NO.6 (dsLsMH-F):

[0049] TAATACGACTCACTATAGGGGAAGTAACTGGCAGATTCAACC;

[0050] SEQ ID NO.7 (dsLsMH-R):

[0051] TAATACGACTCACTATAGGGGCTCGAATAGATGTGCTGCAGG.

[0052] (2) PCR product recovery and purification: The PCR product was separated by 1% agarose gel electrophoresis, the target band was cut with a blade, and the PCR product was recovered by a DNA agarose gel kit to obtain a large number of single L. striatellus molting gene LsMH fragments containing the T7 promoter.

[0053] (3) Synthesis and purification of dsRNA of the gray leafhopper gene LsMH. The synthesis of dsRNA of the gray leafhopper gene LsMH was performed using the T7 High Yield RNA Transcription kit as follows: the gray leafhopper molting gene LsMH fragment containing the T7 promoter was used as the DNA template, 10x Reaction Buffer 2μl, Enzyme mix 2μl, ATP solution 2μl, UTP solution 2μl, CTP solution 2μl, GTP solution 2μl, 1μg DNA template, and water was added to a total volume of 20μl. The reaction system was prepared and mixed, and incubated at 37°C overnight. DNase was added and mixed, and then incubated at 37°C for 15 min and 65°C for 5 min to obtain the dsRNA of the gray leafhopper molting gene LsMH. The solubility of the dsRNA of the gray leafhopper molting gene LsMH was determined, and the quality of the dsRNA was determined by 1% agarose gel electrophoresis. Figure 2 shown.

[0054] Example 3 Injection of dsRNA of the Molting Gene LsMH Gene into Laodelphax striatellus

[0055] (1) Take 4th instar nymphs of Laodelphax striatellus, about 100 nymphs in each group, for a total of 6 groups, place them on ice for 10 minutes, and wait for injection.

[0056] (2) The glass capillary tubes required for microinjection were pulled to the appropriate size using a needle puller (NARISHIGE, PC-10). The needle pulling parameters were as follows: STEP 1, HEATER 62°C.

[0057] (3) After adding the dsRNA of the molting gene LsMH of the gray leafhopper into a glass capillary using an Eppendorf micropipette tip, the glass capillary was mounted on a microinjection instrument (FemtoJet 4x, Eppendorf) and the dsRNA was injected into the body of the gray leafhopper under a microscope. The injection parameters were as follows: pi = 1000pah; ti [s]: 0.3s; pc = 10pah. Three groups of gray leafhopper nymphs were injected, with about 100 nymphs in each group.

[0058] (4) Using the dsRNA of the jellyfish fluorescent protein gene (GFP, Aequorea Victoria green fluorescent protein) as the control group, the same method was used to inject three groups of gray leafhopper nymphs, with about 100 nymphs in each group.

[0059] (5) After waking up, the nymphs of the gray leafhopper injected with dsRNA of the molting gene LsMH and dsGFP were moved to rice and reared normally under the following rearing conditions: temperature 26±0.5°C, light intensity 16:8h (day:night), and humidity 50±5%.

[0060] (6) After the nymphs of the gray leafhopper were injected with dsRNA of the molting gene LsMH and dsGFP, the nymph emergence was observed daily. Figure 3 As shown; the emerged gray leafhoppers were removed every day, and the number of emerged insects was counted every day. The emergence rate of gray leafhopper nymphs injected with dsRNA of the gray leafhopper molting gene LsMH and dsGFP was counted. The results are shown in Figure 4 shown.

[0061] from Figure 3 It can be seen that during the molting process, the nymphs of the gray leafhopper injected with dsRNA of the molting gene LsMH of the gray leafhopper had bent or incomplete wings, shrunken abdomen, incomplete molting, and death. During the molting process, the nymphs of the gray leafhopper injected with dsGFP had complete and flat wings, a plump and shiny abdomen, and successfully emerged into adults.

[0062] from Figure 4 It can be seen that after the gray leafhopper nymphs were injected with dsRNA of the gray leafhopper molting gene LsMH and dsGFP, the emergence rate of the gray leafhopper nymphs injected with dsRNA of the gray leafhopper molting gene LsMH was significantly different from that of the control group injected with dsGFP (P<0.05), indicating that the dsRNA of the gray leafhopper molting gene LsMH caused the death of the gray leafhopper by affecting the abnormal molting of the gray leafhopper, indicating that the dsRNA of the LsMH gene has potential application value in the control of gray leafhopper.

[0063] In summary, the present invention provides a gray planthopper molting gene LsMH that plays a key role in the emergence process of gray planthoppers, and prepares the corresponding dsRNA based on the gray planthopper molting gene LsMH. After the dsRNA is injected into the gray planthopper to interfere with the LsMH gene, it can significantly inhibit the emergence of gray planthoppers into adults, causing the death of gray planthoppers, thereby controlling the gray planthopper population, reducing the harm of gray planthoppers to food crops, and providing a new strategy for the green prevention and control of gray planthoppers.

[0064] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present application. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine different embodiments or examples described in this specification and the features of different embodiments or examples, unless they are contradictory.

[0065] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include at least one of the features. In the description of this application, the meaning of "plurality" is two or more, unless otherwise clearly and specifically defined.

[0066] The above is only a specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any technician familiar with the technical field can easily think of various changes or substitutions within the technical scope disclosed in the present application, which should be included in the protection scope of the present application. Therefore, the protection scope of the present application should be based on the protection scope of the claims.

Claims

1. A molting gene LsMH of Laodelphax striatellus, characterized in that: The nucleotide sequence of the molting gene LsMH of the Laodelphax striatellus is shown in SEQ ID NO.

1.

2. The molting gene LsMH of the Laodelphax striatellus according to claim 1, characterized in that: The amino acid sequence encoded by the molting gene LsMH of the brown planthopper is shown in SEQ ID NO.

2.

3. A primer set for amplifying the molting gene LsMH of the Laodelphax striatellus according to claim 1, characterized in that: It includes an upstream primer SEQ ID NO.3 and a downstream primer SEQ ID NO.

4.

4. A dsRNA, characterized in that The dsRNA targets and silences the molting gene LsMH of the Laodelphax striatellus.

5. A dsRNA according to claim 4, characterized in that The nucleotide sequence of the dsRNA is shown in SEQ ID NO.

5.

6. The method for preparing dsRNA according to claim 4 or 5, characterized in that: The nucleotide sequence SEQ ID NO.5 of the molting gene LsMH of the small brown planthopper was used as a template and transcribed using a T7 High Yield RNA transcription kit to prepare the product.

7. The method for preparing dsRNA according to claim 6, characterized in that: The reaction system for transcription preparation is: 10x Reaction Buffer 2μl, Enzyme mix 2μl, ATP solution 2μl, UTP solution 2μl, CTP solution 2μl, GTP solution 2μl, 1μg DNA template, add water to a total volume of 20μl; mix well, incubate at 37°C overnight, add DNase and mix well, then treat at 37°C for 15min and 65°C for 5min to obtain dsRNA of the molting gene LsMH of the gray leafhopper.

8. Use of the molting gene LsMH of the small brown planthopper according to any one of claims 1 to 3 or the dsRNA according to any one of claims 4 to 7 in controlling small brown planthopper.

9. A Laodelphax striatellus molting gene LsMH interference reagent, characterized in that: The interfering agent comprises the dsRNA according to any one of claims 4 to 7; or comprises a gene fragment transcribed from the molting gene LsMH of the Laodelphax striatellus according to any one of claims 1 to 3.

10. The interference agent for the molting gene LsMH of the Laodelphax striatellus according to claim 9, characterized in that: The Laodelphax striatellus molting gene LsMH interference reagent acts on the Laodelphax striatellus by injection.

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