Laodelphax striatellus juvenile hormone synthetic gene LsFAMeT and application thereof in biological control of Laodelphax striatellus

Abstract

According to the laodelphax striatellus juvenile hormone synthetic gene LsFAMeT and the application of the laodelphax striatellus juvenile hormone synthetic gene LsFAMeT in biological prevention and control of the laodelphax striatellus, the laodelphax striatellus juvenile hormone synthetic gene LsFAMeT is subjected to targeted inhibition, so that the oviposition of female adults can be remarkably inhibited, the population of the laodelphax striatellus is controlled, and the harm of the laodelphax striatellus to food crops is reduced. The dsRNA or interference reagent is prepared by transcription of the laodelphax striatellus juvenile hormone synthetic gene LsFAMeT, and after the dsRNA or interference reagent is injected into the laodelphax striatellus, the juvenile hormone synthetic gene LsFAMeT can be effectively inhibited, female adult laodelphax striatellus can be inhibited from laying eggs, and biological prevention and control of the laodelphax striatellus can be realized. 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.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of agricultural science and technology, and in particular to a Laodelphax striatellus juvenile hormone synthesis gene LsFAMeT and application thereof in biological control of L. striatellus. BACKGROUND

[0002] L. striatellus is an important agricultural pest in China and East Asia, which causes serious damage to major food crops such as rice and wheat through direct feeding and virus transmission. According to statistics, the pest can cause more than 30% reduction in crop yield per year, seriously threatening food production safety. At present, chemical control is still the main means to control L. striatellus, but long-term and large-scale use of chemical pesticides has led to multiple problems: first, the pest's resistance is rapidly increasing, second, the ecological environment is being polluted, and third, the quality and safety of agricultural products are at risk, which seriously hinders the sustainable development of agriculture.

[0003] Insect juvenile hormone, as an important endogenous hormone, plays a core role in the regulation of insect growth and development and reproduction. Studies have shown that juvenile hormone synthesis enzyme system is the key enzyme system for regulating JH biosynthesis, which directly affects important physiological processes such as molting, metamorphosis, and reproductive maturation of insects by fine-tuning the synthesis rate and titer of juvenile hormone. Specifically interfering with the key genes of the juvenile hormone synthesis and metabolism pathway can effectively break the hormone balance in the pest body, leading to abnormal development and reproductive disorders, which provides an ideal target for developing efficient and targeted new pest control technologies. SUMMARY

[0004] The present application provides a L. striatellus juvenile hormone synthesis gene LsFAMeT and application thereof in biological control of L. striatellus to solve the problems in the related art. The technical solution is as follows:

[0005] In a first aspect, the present application provides a L. striatellus juvenile hormone synthesis gene LsFAMeT, and the nucleotide sequence of the L. striatellus juvenile hormone synthesis gene LsFAMeT is shown in SEQ ID NO. 1.

[0006] In one embodiment, the amino acid sequence encoded by the L. striatellus juvenile hormone synthesis gene LsFAMeT is shown in SEQ ID NO. 2.

[0007] In a second aspect, the present application provides a primer set for amplifying the L. striatellus juvenile hormone synthesis gene LsFAMeT, which comprises an upstream primer SEQ ID NO. 3 and a downstream primer SEQ ID NO. 4.

[0008] In a third aspect, the present application provides a dsRNA, which targets the L. striatellus juvenile hormone synthesis gene LsFAMeT.

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

[0010] In a fourth aspect, the application provides a preparation method of the above-mentioned dsRNA, taking the nucleotide sequence of the LsFAMeT juvenile hormone synthesis gene of Laodelphax striatellus SEQ ID NO. 5 as a template, and using a T7 High Yield RNA transcription kit to prepare the dsRNA by transcription.

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

[0012] In a fifth aspect, the application provides the use of the above-mentioned LsFAMeT juvenile hormone synthesis gene of Laodelphax striatellus or the above-mentioned dsRNA in the control of Laodelphax striatellus.

[0013] In a sixth aspect, the application provides a LsFAMeT juvenile hormone synthesis gene of Laodelphax striatellus interference reagent, which comprises the above-mentioned dsRNA; or a gene fragment transcribed from the above-mentioned LsFAMeT juvenile hormone synthesis gene of Laodelphax striatellus.

[0014] In an embodiment, the LsFAMeT juvenile hormone synthesis gene of Laodelphax striatellus interference reagent is used on Laodelphax striatellus by injection.

[0015] The above-mentioned technical solutions have at least the following advantages or beneficial effects:

[0016] The application discloses a new LsFAMeT juvenile hormone synthesis gene of Laodelphax striatellus, which is a key juvenile hormone synthesis gene for the reproductive development of Laodelphax striatellus. By targeting the LsFAMeT juvenile hormone synthesis gene of Laodelphax striatellus for interference, the oogenesis of female adults of Laodelphax striatellus can be significantly inhibited. The biological method for controlling Laodelphax striatellus is safe and harmless to the environment and food, and provides a new strategy for the green control of Laodelphax striatellus.

[0017] The dsRNA or interference reagent of the present application is prepared by transcription of the LsFAMeT gene of the juvenile hormone synthesis gene of Laodelphax striatellus, and can effectively interfere with the LsFAMeT gene of the juvenile hormone synthesis gene of Laodelphax striatellus after being injected into the Laodelphax striatellus, can significantly inhibit the oviposition of female adults of the Laodelphax striatellus, and realizes the biological control of the Laodelphax striatellus.

[0018] The above summary is intended to illustrate only and is not intended to limit the application in any way. Further aspects, embodiments and features of the present application will be readily apparent from the following detailed description, taken in conjunction with the drawings and the following detailed description. BRIEF DESCRIPTION OF DRAWINGS

[0019] In the drawings, like numerals refer to like elements throughout the various drawings. The drawings are not necessarily to scale, the emphasis instead being placed on the relations between various parts and on the characteristics thereof. It should be understood that the drawings only depict some embodiments in accordance with the disclosure and should not be considered limiting of the scope of the disclosure.

[0020] Figure 1 PCR amplification results of the LsFAMeT gene fragment of the juvenile hormone synthesis gene of Laodelphax striatellus;

[0021] Figure 2 Synthesis of the dsRNA of the LsFAMeT gene of the juvenile hormone synthesis gene of Laodelphax striatellus;

[0022] Figure 3 Comparison of the morphology of the Laodelphax striatellus after being introduced with the dsRNA of the LsFAMeT gene of the juvenile hormone synthesis gene of Laodelphax striatellus and the control group;

[0023] Figure 4 Oviposition amount of the Laodelphax striatellus after being introduced with the dsRNA of the LsFAMeT gene of the juvenile hormone synthesis gene of Laodelphax striatellus. DETAILED DESCRIPTION

[0024] In the following, only some exemplary embodiments are simply described. As those skilled in the art can recognize, the described embodiments can be modified in various different ways without departing from the spirit or scope of the present application. Therefore, the drawings and the description are considered to be exemplary in nature and not limiting.

[0025] Example 1 Cloning of the LsFAMeT gene of the juvenile hormone synthesis gene of Laodelphax striatellus

[0026] 1. Amplification of the LsFAMeT gene fragment of the juvenile hormone synthesis gene of Laodelphax striatellus:

[0027] (1) Take 4-5 adult or nymphal planthoppers, wash them 3 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 and grind the planthoppers thoroughly; add 500μL chloroform and mix vigorously; centrifuge at 12000rpm at 4℃ for 15min; carefully transfer the supernatant to a new centrifuge tube; add an equal volume of isopropanol, mix well and let stand for 10min; centrifuge at 12000rpm at 4℃ for 10min and discard the supernatant; add 1mL 75% ethanol, centrifuge at 9600rpm and discard the supernatant; add RNase-free water to dissolve the precipitate and determine the RNA solubility.

[0028] (2) Using total RNA as a template, The One-Step gDNA Removal and cDNA Synthesis SuperMix Reverse Transcription Kit was used for reverse transcription of RNA: 5 μg total RNA, 1 μL LOligo(dT), 10 μL 2xTS Reaction Mix, 1 μL Enzyme Mix, 1 μL gDNA Remover, and RNase-free water were added to bring the total volume to 20 μL; after mixing, the mixture was incubated at 42°C for 30 min; denaturation was performed at 85°C for 5 s to inactivate the reverse transcriptase and obtain cDNA from the planthopper.

[0029] (3) Based on the open reading frame (SEQ ID NO.1) of the juvenile hormone synthesis gene LsFAMeT in the planthopper, primers SEQ ID NO.3 and SEQ ID NO.4 were designed;

[0030] SEQ ID NO.1:

[0031]

[0032] The primers are shown in SEQ ID NO.3 and SEQ ID NO.4;

[0033] SEQ ID NO.3(LsFAMeT-F):AAGTAACTGGCAGATTCAACC;

[0034] SEQ ID NO.4(LsFAMeT-R): CTCGAATAGATGTGCTGCAGG;

[0035] The amino acid encoded by the LsFAMeT gene, the juvenile hormone synthesis gene of the brown planthopper, is shown in SEQ ID NO.2;

[0036] SEQ ID NO.2:

[0037] MALEITTEDKLEYQFHEVSGGSLQFKVKAANDAHIALTTGPAESEPIYEIFIGGWSNAKSAIRKNKQKPDAALVETPNILNGDEQLAFWIRWNGGAIAVGRHGEAEPFISWEDPEPFVVTHYGICTGWGATGEWVIEAPIAQPGWAVDPAAE GAAPAGGAATWVPVAAGEPVPPSAIPAGLDTGGEQLYVGRGKHESGALIPGKVVPSHGVCYVPWGGEEHGIAEYEVLTNCEVTWMPSASGQVPSGALVAGETEDGETLYIGRASHEGVSSVGKVQGTHGVCYIPYGGAEIAYNDYEVLVAK.

[0038] (4) Using the cDNA of the planthopper as a template, the LsFAMeT 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 LsFAMeT-F (SEQ ID NO.3), 1 μL downstream primer LsFAMeT-R (SEQ ID NO.4), 2 μL planthopper cDNA, 1 μL ddH2O, total 10 μL. The PCR amplification reaction conditions were as follows: 95℃ for 2 min; 95℃ for 20 s, 55℃ for 20 s, 72℃ for 1 min, 35 cycles; extension at 72℃ for 10 min.

[0039] 2. Monoclonal strain of LsFAMeT gene from the planthopper:

[0040] (1) The PCR products were separated by 1% agarose gel electrophoresis, and the results are as follows: Figure 1As shown; the PCR product was recovered by cutting the band with a blade and recovering it using a DNA agarose gel electrophoresis kit; the purified LsFAMeT gene DNA fragment of the planthopper was obtained; the LsFAMeT gene DNA fragment of the planthopper 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 LsFAMeT gene DNA fragment of the planthopper, 14 μL ddH2O, total volume 20 μL. After mixing the reagents, the ligation reaction was carried out at 25℃ for 5 min.

[0041] (2) Transformation of ligation product into competent cells. The transformation procedure is as follows: Add ligation product to competent cells, gently tap the tube wall with your finger to mix, and incubate on ice for 30 min; after 90 s at 42℃, quickly incubate on ice for 2 min; transfer competent cells into antibiotic-free LB medium and incubate at 180 rpm for 1 h; take 150 μL of the revived bacterial solution and spread it on an LB medium plate containing ampicillin, and incubate overnight; pick a single colony and incubate it on LB liquid medium containing ampicillin, and incubate at 180 rpm for 6 h.

[0042] (3) Colony PCR verification of the LsFAMeT gene DNA fragment. The reaction system was as follows: 5 μL 2×ApexHF Mix, 1 μL upstream primer LsFAMeT-F (SEQ ID NO.3), 1 μL downstream primer LsFAMeT-R (SEQ ID NO.4), 2 μL planthopper cDNA, 1 μL ddH2O, total volume 10 μL; PCR amplification conditions were as follows: 95℃, 2 min; 95℃ 20 s, 55℃ 20 s, 72℃ 1 min, 35 cycles; extension at 72℃ for 10 min. 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 electrophoresis kit; the samples were sent to the company for sequencing, and the LsFAMeT gene sequence fragment of the planthopper was obtained as shown in SEQ ID NO.5;

[0043] SEQ ID NO.5:

[0044] CTATGGCATCTGCACCGGATGGGGTGCCACCGGCGAGTGGGTGATCGAAGCCCCAATCGCGCAGCCCGGCTGGGCCGTGGACCCGGCTGCCGAGGGCGCCGCACCGGCGGGAGGCGCGGCTACCTGGGT GCCAGTGGCGGCCGGCGAGCCGGTGCCGCCCTCAGCCATTCCGGCCGGCCTGGACACGGGCGGCGAGCAGCTGTATGTCGGCCGTGGCAAACACGAGAGTGGCGCCCTCATTCCGGGCAAGGTGGTGCC CTCGCACGGCGTCTGCTATGTGCCGTGGGGCGGTGAGGAGCACGGCATTGCTGAGTACGAGGTGCTGACCAATTGCGAAGTGACGTGGATGCCGTCAGCCAGTGGCCAGGTTCCGTCTGGCGCATTGGT CGCCGGTGAGACTGAAGACGGCGAGACGCTCTACATTGGCCGAGCCTCGCACGAGGGCGTATCTTCTGTTGGAAAGGTCCAAGGAACCCACGGAGTGTGCTACATACCATACGGCGGGAGCCGAAATAG.

[0045] Example 2: dsRNA synthesis of the LsFAMeT gene, a juvenile hormone synthesis gene in the gray planthopper.

[0046] (1) The LsFAMeT gene fragment shown in SEQ ID NO.5 was amplified by PCR using primers with the T7 promoter. Using bacterial culture containing the LsFAMeT gene for juvenile hormone synthesis in the planthopper (as shown in SEQ ID NO.5) as a template, the target gene was amplified by primers SEQ ID NO.6 and SEQ ID NO.7 with the T7 promoter sequence. The reaction system was as follows: 50 μL 2×ApexHF Mix, 10 μL upstream primer LsFAMeT-F (SEQ ID NO.6), 10 μL downstream primer LsFAMeT-R (SEQ ID NO.7), 20 μL bacterial culture containing the LsFAMeT gene in the planthopper, 10 μL ddH2O, for a total volume of 100 μL; the PCR amplification conditions were as follows: 95℃ for 2 min; 95℃ for 20 s, 55℃ for 20 s, 72℃ for 1 min, 35 cycles; extension at 72℃ for 10 min.

[0047] SEQ ID NO.6(dsLsFAMeT-F):

[0048] TAATACGACTCACTATAGGGGAAGTAACTGGCAGATTCAACC;

[0049] SEQ ID NO.7(dsLsFAMeT-R):

[0050] TAATACGACTCACTATAGGGGCTCGAATAGATGTGCTGCAGG.

[0051] (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 electrophoresis kit to obtain a large number of single fragments of the LsFAMeT gene containing the T7 promoter of the planthopper juvenile hormone synthesis gene.

[0052] (3) Synthesis and purification of LsFAMeT dsRNA from the planthopper. The LsFAMeT dsRNA from the planthopper was synthesized using the T7 High Yield RNA Transcription kit. The method was as follows: Using the LsFAMeT fragment containing the T7 promoter as the DNA template, 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, and 1 μg of DNA template were added, and water was added to a total volume of 20 μl. After mixing the reaction mixture, it was incubated overnight at 37°C. DNase was added and mixed well, then the mixture was incubated at 37°C for 15 min and 65°C for 5 min to obtain the dsRNA of the LsFAMeT juvenile hormone synthesis gene from the planthopper. The solubility of the LsFAMeT dsRNA was determined, and the mass of the dsRNA was determined by 1% agarose gel electrophoresis. Figure 2 As shown.

[0053] Example 3: Injection of dsRNA from the LsFAMeT gene (the gene for juvenile hormone synthesis in the planthopper) into the planthopper.

[0054] (1) Take about 100 nymphs of the third instar of the planthopper, and make 6 groups in total. Place them on ice and freeze for 10 minutes before injection.

[0055] (2) The glass capillary required for micro-injection is pulled to the appropriate size using a needle puller (NARISHIGE, PC-10). The needle puller parameters are as follows: STEP1, HEATER 62℃.

[0056] (3) Using an Eppendorf micropipe, the dsRNA of the juvenile hormone synthesis gene LsFAMeT was added to a glass capillary. A microinjection apparatus (FemtoJet 4x, Eppendorf) was then attached to the capillary, and the dsRNA was injected into the planthopper under a microscope. The injection parameters were as follows: pi = 1000 pah; ti[s]: 0.3 s; pc = 10 pah. Three groups of planthopper nymphs, approximately 100 nymphs per group, were injected.

[0057] (4) Using the dsRNA of the jellyfish fluorescent protein gene (GFP, Aequorea Victoria green fluorescent protein) as a control group, inject 3 groups of gray planthopper nymphs, about 100 in each group, using the same method.

[0058] (5) After the nymphs of the planthopper were injected with dsRNA and dsGFP of the LsFAMeT gene, which synthesizes the planthopper juvenile hormone, they were revived and then transferred to rice paddies for normal rearing. The rearing conditions were as follows: temperature 26±0.5℃, light intensity 16:8h (day:night), humidity 50±5%.

[0059] (6) After injecting LsFAMeT dsRNA and dsGFP into the nymphs of the planthopper, the emergence of nymphs was observed daily. The experimental results are as follows: Figure 3 As shown, female adult gray planthoppers injected with dsLsFAMeT exhibit blackening of the dorsal side of their abdomen. Female adult gray planthoppers injected with dsGFP have full, glossy abdomens without blackening.

[0060] Example 4: Statistics on egg production of planthoppers after microinjection

[0061] (1) After injecting dsLsFAMeT and dsGFP into the nymphs of the planthopper, the single female and single male that emerged on the same day were transferred to glass test tubes with sufficient fresh rice seedlings and allowed to mate and lay eggs.

[0062] (2) Three days later, the male and female adults were transferred to another glass test tube with plenty of fresh rice seedlings to mate and lay eggs; the number of eggs laid in the replaced rice seedlings was carefully counted under a dissecting microscope.

[0063] (3) Repeat step (2) until the female adult dies. If the male adult dies during the oviposition count, add male adults with the same treatment again.

[0064] (4) The oviposition rate of female adult gray planthoppers injected with dsLsFAMeT and dsGFP was statistically analyzed, and the results are as follows: Figure 4 As shown.

[0065] from Figure 4The results showed that the number of eggs laid by female planthoppers injected with dsLsFAMeT was significantly different from that of the control group injected with dsGFP (P = 0.0002). This indicates that the dsRNA of the planthopper juvenile hormone synthesis gene LsFAMeT inhibits planthopper egg laying by affecting the expression of the LsFAMeT gene, suggesting that the dsRNA of the LsFAMeT gene has potential application value for the control of planthoppers.

[0066] In summary, this invention provides a juvenile hormone synthesis gene, LsFAMeT, that plays a crucial role in the reproductive regulation of the planthopper. Based on the LsFAMeT gene, corresponding dsRNA was prepared. Injecting the dsRNA into the planthopper to interfere with the LsFAMeT gene silences its expression and significantly inhibits the oviposition capacity of female planthopper adults. This controls the planthopper population, reduces the damage to food crops, and provides a new strategy for the green control of planthoppers. The LsFAMeT juvenile hormone synthesis gene of this application provides a new target for the biological control of planthoppers and has significant application value.

[0067] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of this application. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of those different embodiments or examples.

[0068] 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 indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this application, "a plurality of" means two or more, unless otherwise explicitly specified.

[0069] The above description is merely a specific embodiment of this application, but the scope of protection of this application is not limited thereto. Any person skilled in the art can easily conceive of various variations or substitutions within the technical scope disclosed in this application, and these should all be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A gene for synthesizing juvenile hormone in the gray planthopper, LsFAMeT, characterized in that, The nucleotide sequence of the juvenile hormone synthesis gene LsFAMeT is shown in SEQ ID NO.

1.

2. The juvenile hormone synthesis gene LsFAMeT according to claim 1, characterized in that: The amino acid sequence encoded by the juvenile hormone synthesis gene LsFAMeT of the planthopper is shown in SEQ ID NO.

2.

3. A primer set for amplifying the juvenile hormone synthesis gene LsFAMeT of the planthopper according to claim 1, characterized in that: This includes the upstream primer SEQ ID NO.3 and the downstream primer SEQ ID NO.

4.

4. A dsRNA, characterized in that, The dsRNA targets the LsFAMeT gene, which synthesizes juvenile hormone in the gray planthopper.

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 gene was prepared by transcription using the nucleotide sequence SEQ ID NO.5 of the juvenile hormone synthesis gene LsFAMeT from the planthopper as a template and a T7 High Yield RNA transcription kit.

7. The method for preparing dsRNA according to claim 6, characterized in that, The reaction system for transcription preparation was as follows: 2 μL of 10x Reaction, 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 was added to a total volume of 20 μL. After mixing, the mixture was incubated at 37°C overnight. DNase was added and mixed well. Then, the mixture was treated at 37°C for 15 min and at 65°C for 5 min to obtain the dsRNA of the juvenile hormone synthesis gene LsFAMeT of the planthopper.

8. The application of the LsFAMeT gene for synthesizing juvenile hormones in the planthopper as described in any one of claims 1-3 or the dsRNA as described in any one of claims 4-7 in the control of planthoppers.

9. A reagent for interfering with the LsFAMeT gene of the juvenile hormone synthesis gene in the planthopper, characterized in that, The interference reagent includes the dsRNA according to any one of claims 4-7; or includes a gene fragment transcribed from the LsFAMeT gene, the juvenile hormone synthesis gene of the planthopper, according to any one of claims 1-3.

10. The LsFAMeT interference reagent for the juvenile hormone synthesis gene of the planthopper according to claim 9, characterized in that, The LsFAMeT interference reagent for the juvenile hormone synthesis gene of the planthopper was introduced into the planthopper via microinjection.

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