The Molting Gene LsMH of Laodelphax striatellus and Its Application in Biological Control of Laodelphax striatellus

Abstract

The present application proposes a molting gene LsMH of gray planthopper and its application in biological control of gray planthopper. As one of the key genes for molting of gray planthopper, targeted interference with the molting gene LsMH of gray planthopper can significantly inhibit the emergence of gray planthopper into adults, causing the death of gray planthopper, thereby controlling the gray planthopper population and reducing the damage of gray planthopper to food crops. Controlling gray planthoppers by biological means is safe and harmless to the environment, ecology and food, and provides a new strategy for green control of gray planthoppers. dsRNA or interference reagents are prepared by transcribing the molting gene LsMH of gray planthoppers, and can effectively interfere with the LsMH gene after being injected into gray planthoppers. During the molting process, the nymphs of gray planthoppers have bent or incomplete wings, shriveled abdomens, and incomplete molting, which inhibits the emergence of gray planthoppers into adults, causes the death of gray planthoppers, and achieves biological control of gray planthoppers.

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 small brown planthopper and its application in biological control of the small brown planthopper. Background Art

[0002] As a major pest in crop-growing areas in my country and other Asia, the gray planthopper not only directly harms rice, wheat, corn and other food crops by sucking plant sap and laying eggs, but also indirectly harms crops by spreading a variety of crop viruses, seriously affecting the high quality, stable and high yield of food.

[0003] Ecdysone is the primary metabolic enzyme for juvenile hormone in insects. It is synthesized by fat body cells and epithelial cells and distributed in the insect hemolymph, midgut, and fat body. Ecdysone is highly capable of recognizing and degrading bound or free juvenile hormone, balancing juvenile hormone levels in insects, and is involved in physiological and biochemical behaviors such as molting, pupation, and reproduction. Therefore, promoting or inhibiting ecdysone activity can disrupt juvenile hormone levels in insects, disrupting their development and reproduction, and achieving the goal of pest control. Ecdysone holds great potential for green pest control. Summary of the Invention

[0004] The present invention provides a molting gene LsMH of the small brown planthopper and its application in biological control of the small brown planthopper to solve the problems existing in the related art. The technical solution is as follows:

[0005] In a first aspect, the embodiments of the present application provide a molting gene LsMH of the small brown planthopper, the nucleotide sequence of the molting gene LsMH of the small brown planthopper 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, comprising 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 that 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 present invention provides 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: 2 μl of 10x Reaction Buffer, 2 μl of Enzyme mix, 2 μl of ATP solution, 2 μl of UTP solution, 2 μl of CTP solution, 2 μl of GTP solution, 1 μg of DNA template, and water is added to a total volume of 20 μl; the mixture is mixed and incubated at 37°C overnight, DNase is added and mixed, and then treated at 37°C for 15 minutes and 65°C for 5 minutes to obtain dsRNA of the molting gene LsMH of the small brown planthopper.

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

[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 Laodelphax striatellus molting gene LsMH interference reagent acts on the Laodelphax striatellus by injection.

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

[0016] This application discloses a novel molting gene, LsMH, in the small brown planthopper (Lady Stargazer). As one of the key genes involved in molting, targeted interference with LsMH significantly inhibits the emergence of small brown planthoppers into adults, leading to their death. This in turn controls the population and mitigates the damage these insects cause to food crops. This biological approach to controlling small brown planthoppers is safe for both the environment and food, providing a new strategy for green control of small brown planthoppers.

[0017] The present application discloses a dsRNA or interference agent, which 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 planthoppers from emerging into adults, causing the death of the gray planthoppers, thereby achieving biological control of the gray planthoppers.

[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 components or elements. These drawings are not necessarily drawn to scale. It should be understood that these drawings only depict some embodiments disclosed in this application and should not be construed as limiting the scope of this 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 with 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] Hereinafter, only certain exemplary embodiments are briefly described. As will be appreciated by those skilled in the art, the described embodiments may be modified in various ways without departing from the spirit or scope of the present application. Therefore, the drawings and description are to be regarded as illustrative in nature and not restrictive.

[0025] RNA interference (RNAi) technology involves introducing small double-stranded RNA molecules into target pests. The RNAi technology specifically binds to the target gene mRNA, degrading or inhibiting protein translation, disrupting the pest's normal physiological and biochemical behavior. This approach can control pest populations or reduce their ability to spread viruses. RNAi pest control offers advantages such as high specificity, effectiveness, and environmental friendliness, and has been widely used in the prevention and control of various agricultural pests.

[0026] Promoting or inhibiting the activity of molting hormones can disrupt juvenile hormone levels in insects, disrupting their development and reproduction, thereby achieving the goal of pest control. The Laodelphax striatellus molting gene, LsMH, and its application in biological control of Laodelphax striatellus are described in detail below using specific examples.

[0027] Example 1 Cloning of the Laodelphax striatellus molting gene LsMH

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

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

[0030] (2) Using total RNA as template, RNA was reverse transcribed using the One-Step gDNA Removal and cDNA Synthesis SuperMix Reverse Transcription Kit: 5 μg of total RNA, 1 μL of Oligo(dT), 10 μL of 2xTS Reaction Mix, 1 μL of Enzyme Mix, 1 μL of gDNA Remover, and RNase-free water to a total volume of 20 μL. Mix thoroughly and incubate at 42°C for 30 minutes. Denature at 85°C for 5 seconds to inactivate the reverse transcriptase and obtain Laodelphax striatellus cDNA.

[0031] (3) Primers were designed based on the SEQ ID NO.1 of the L. striatellus LsMH gene and the amino acid sequence SEQ ID NO.2 encoding the L. striatellus 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, the LsMH gene was amplified by PCR using primers SEQ ID NO.3 and SEQ ID NO.4. 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; 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.

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

[0041] (1) Separate the PCR products using 1% agarose gel electrophoresis. Figure 1The band was cut with a razor blade, and the PCR product was recovered using a DNA agarose gel kit. The purified L. striatellus LsMH gene DNA fragment was obtained. The L. striatellus LsMH gene DNA fragment was ligated into the pClone007 vector using a blunt-end cloning kit. The reaction system was as follows: 2 μL 10x Buffer, 2 μL pClone007 Blunt Simple vector, 2 μL L. striatellus LsMH gene DNA fragment, 14 μL ddH2O, for a total volume of 20 μL. After mixing, the ligation reaction was incubated 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 ice bath for 30 minutes; incubate at 42℃ for 90 seconds and then quickly incubate on ice for 2 minutes; transfer the competent cells into LB medium without antibiotics and incubate at 180 rpm for 1 hour; take 150 μL of the recovered bacterial solution and spread it on an LB medium plate containing ampicillin overnight; pick a single colony and transfer it to LB liquid medium containing ampicillin and incubate at 180 rpm for 6 hours for colony culture.

[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 using 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 SEQ ID NO.5.

[0044] SEQ ID NO.5:

[0045] AAGTAACTGGCAGATTCAACCGGTTCCAACCACTCCTCAGGAACAGGCCACTCCCTCACTAAGCCCTGAACAACTCCAACAGTTGGCGTTGTACAGGTTTTTTGAATCCCAACGCACCCCTACCACCACAATTGCTAACCACGTCCAGGCCAGTACTGAAAGTGGAGACCATCTACG AGAGTCATGTGATACCTCTGTTCAACGGACAAAGTACTCTGTTTAGTACCATCTCACGACCGATTGCAACTGTCAGCAAAACGGAATACGAAGTCGTGACCAATTCTTTGGCGCTACCTCCTGTACAACCTCCTGTAAATACTATCAACCCCTTCCTGCAGCACATCTATTCGAG.

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

[0047] (1) PCR amplification of the LsMH gene fragment shown in SEQ ID NO.5 using primers with a T7 promoter. Using a bacterial solution containing the LsMH fragment of the molting gene of the gray leafhopper shown in SEQ ID NO.5 as a template, primers with T7 promoter sequences (SEQ ID NO.6 and SEQ ID NO.7) were used to amplify the target gene. 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, total volume 100 μL; PCR amplification reaction conditions were as follows: 95°C for 2 min; 95°C for 20 s, 55°C for 20 s, 72°C for 1 min, 35 cycles; 72°C for 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 products were separated by 1% agarose gel electrophoresis, the target band was cut with a blade, and the PCR products were recovered using a DNA agarose gel kit to obtain a large number of single fragments of the molting gene LsMH of the Laodelphax striatellus 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, and the method was 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 were added to a total volume of 20μl. The reaction system was prepared and mixed, and incubated at 37℃ overnight. DNase was added and mixed, and then incubated at 37℃ for 15min and 65℃ for 5min 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, as shown in FIG. Figure 2 shown.

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

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

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

[0057] (3) dsRNA encoding the molting gene LsMH of the small brown planthopper was added to a glass capillary using an Eppendorf micropipette tip. The capillary was then mounted on a microinjector (FemtoJet 4x, Eppendorf) and the dsRNA was injected into the larvae under a microscope using the following injection parameters: pi = 1000 pah; ti [s]: 0.3 s; pc = 10 pah. Three groups of small brown planthopper nymphs were injected, with approximately 100 nymphs in each group.

[0058] (4) Using the same method, dsRNA of the jellyfish fluorescent protein gene (GFP, Aequorea Victoria green fluorescent protein) was injected into three groups of Laodelphax striatellus nymphs as a control group, with about 100 nymphs in each group.

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

[0060] (6) After the SBPH nymphs were injected with dsRNA and dsGFP of the SBPH molting gene LsMH, the nymphs were observed for emergence every day. Figure 3 As shown; the emerged SBPHs were removed every day and the number of emerged insects was counted every day. The emergence rate of SBPH nymphs injected with dsRNA of SBPH 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 larvae injected with dsRNA targeting the molting gene LsMH in the Laodelphax striatellus developed bent or incomplete wings, a shrunken abdomen, incomplete molting, and death. However, during the molting process, the larvae injected with dsGFP developed 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 affects the abnormal molting of the gray leafhopper, leading to the death of the gray leafhopper, indicating that the dsRNA of the LsMH gene has potential application value in the prevention and control of gray leafhopper.

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

[0064] In the description of this specification, the reference terms "one embodiment," "some embodiments," "example," "specific example," or "some examples" mean 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 appropriate manner in any one or more embodiments or examples. In addition, those skilled in the art may combine and combine different embodiments or examples described in this specification, as well as features of different embodiments or examples, unless they are mutually inconsistent.

[0065] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features being referred to. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. Throughout the description of this application, "plurality" means two or more, unless otherwise specifically defined.

[0066] The above description is merely a specific embodiment of the present application, but the scope of protection of the present application is not limited thereto. Any person skilled in the art can easily conceive of various modifications or substitutions within the technical scope disclosed in this application, and such modifications or substitutions should be included within the scope of protection of the present application. Therefore, the scope of protection of the present application should be based on the scope of protection of the claims.

Claims

1. A dsRNA, characterized in that The dsRNA targets and silences the molting gene LsMH of the small brown planthopper; the nucleotide sequence of the molting gene LsMH of the small brown planthopper is shown in SEQ ID NO.1; the nucleotide sequence of the dsRNA is shown in SEQ ID NO.

5.

2. The method for preparing dsRNA according to claim 1, wherein 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.

3. The method for preparing dsRNA according to claim 2, characterized in that: The reaction system for transcription preparation is as follows: 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 15 minutes and 65°C for 5 minutes to obtain dsRNA of the molting gene LsMH of the small brown planthopper.

4. Use of the dsRNA according to claim 1 in controlling Laodelphax striatellus.

5. A Laodelphax striatellus molting gene LsMH interference reagent, characterized in that: The interfering agent comprises the dsRNA according to claim 1.

6. The Laodelphax striatellus molting gene LsMH interfering agent according to claim 5, characterized in that The Laodelphax striatellus molting gene LsMH interference reagent acts on the Laodelphax striatellus by injection.

Images