Brown planthopper lethal gene Nlpfam fragment and application thereof in biological control

Abstract

According to the brown planthopper lethal gene Nlpfam and the application thereof in biological prevention and control, the brown planthopper lethal gene Nlpfam can cause brown planthopper death after being interfered, so that dsRNA or an interference reagent is prepared through transcription of the brown planthopper lethal gene Nlpfam, the Nlpfam gene can be effectively interfered after the dsRNA or the interference reagent is injected into the brown planthopper, the abdomen of the brown planthopper becomes black, and the brown planthopper death can be inhibited. The brown planthopper can be effectively killed, and the biological control on the brown planthopper is realized.

Description

Technical Field

[0001] The present application relates to the field of agricultural science and technology, and in particular to a fragment of a brown planthopper lethal gene Nlpfam and its application in biological control. Background Art

[0002] Brown planthoppers are the most important agricultural pests in my country and other Asian countries that grow rice. Brown planthoppers have the habit of seasonal and long-distance migration. They migrate from south to north on a large scale in spring and summer every year with the help of monsoons, and migrate back from north to south in autumn. Brown planthoppers are monophagous insects that feed on rice and wild rice. They mainly harm rice directly by feeding, and also indirectly harm rice by spreading a variety of rice viruses, which seriously affect the quality, stable yield and high yield of rice. Since the 1960s, brown planthoppers have broken out in various rice-growing areas in Asia. During the period of 2020-2023, major brown planthopper disasters broke out in southern rice-growing areas of my country, causing rice grains to be not full and dry weight to decrease. At the same time, they caused large areas of rice plants to dry up and fall, causing serious losses to my country's grain production and supply. In view of the severity of its harm, my country's Ministry of Agriculture and Rural Affairs has included brown planthoppers in the "List of Class I Crop Pests and Diseases" for many consecutive years.

[0003] The control of brown planthoppers mainly relies on chemical pesticides, but the continuous and unreasonable use of chemical pesticides in large quantities not only accelerates the development of brown planthoppers' resistance, aggravates environmental pollution and ecosystem damage, but also poses a serious threat to my country's agricultural modernization, food, ecological and economic security. Therefore, in agricultural production practice, pest control methods other than chemical pesticides are urgently needed. With the development of science and technology, biotechnology has gradually been developed and utilized as an important means of pest control. RNA interference is a gene silencing technology mediated by double-stranded RNA. By introducing small molecule double-stranded RNA into the target pest, it specifically binds to the pest target gene mRNA, degrades or inhibits the protein translation of the target gene, and causes teratogenicity or death to the pest. RNA interference technology has the characteristics of high specificity, good effect, and green environmental protection, and has been widely used in the prevention and control of various agricultural pests. Summary of the invention

[0004] The present application provides a fragment of the Nlpfam gene that causes brown planthopper death and its application in biological control 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 fragment of a brown planthopper lethal gene Nlpfam, the nucleotide sequence of the fragment of the brown planthopper lethal gene Nlpfam is shown in SEQ ID NO.1.

[0006] In one embodiment, the amino acid sequence encoded by the Nlpfam fragment of the brown planthopper lethal gene 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 brown planthopper lethal gene Nlpfam fragment, 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 of the brown planthopper lethal gene Nlpfam, wherein the dsRNA targets and silences the brown planthopper lethal gene Nlpfam.

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

[0010] In a fourth aspect, the embodiments of the present application provide a method for preparing the dsRNA of the above-mentioned brown planthopper lethal gene Nlpfam, which is prepared by transcription using the nucleotide sequence SEQ ID NO.5 of the brown planthopper lethal gene Nlpfam as a template 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 brown planthopper lethal gene Nlpfam.

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

[0013] In a sixth aspect, an embodiment of the present application provides an interference reagent for the brown planthopper lethal gene Nlpfam, wherein the interference reagent includes the dsRNA of the above-mentioned brown planthopper lethal gene Nlpfam; or includes a gene fragment transcribed from the above-mentioned brown planthopper lethal gene Nlpfam.

[0014] In one embodiment, the Nlpfam interference agent, a lethal gene of brown planthopper, acts on brown planthopper by injection.

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

[0016] The present application discloses a brown planthopper lethal gene Nlpfam, which plays a key role in the survival process of brown planthoppers. By targeted interference with the brown planthopper lethal gene Nlpfam and preventing its expression, the brown planthoppers can die. This helps to control brown planthoppers through biological means and reduce the harm of brown planthoppers to food crops; at the same time, compared with control by chemical pesticides, it is harmless to the environment and food, and improves environmental and food safety.

[0017] The present application discloses a dsRNA or interference agent of the brown planthopper lethal gene Nlpfam, which is prepared by transcribing the brown planthopper lethal gene Nlpfam. After being injected into the brown planthopper, it can effectively interfere with the expression of the brown planthopper lethal gene Nlpfam, causing the death of the brown planthopper, thereby controlling the brown planthopper population and providing a new strategy for the green prevention and control of the brown 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 The result of PCR amplification of the Nlpfam fragment of the brown planthopper lethal gene;

[0021] Figure 2 Synthesis of dsRNA for the brown planthopper lethal gene Nlpfam;

[0022] Figure 3 The comparison of the eclosion morphology of brown planthoppers after dsRNA of the lethal gene Nlpfam was introduced into brown planthoppers and that of the control group;

[0023] Figure 4 The emergence rate of brown planthoppers after dsRNA of the brown planthopper lethal gene Nlpfam was introduced into the brown planthoppers. 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 has been widely used in the prevention and control of various agricultural pests. The key lethal genes of pests are found, and the expression of lethal genes is interfered with by RNA to cause teratogenicity or lethality of pests, thereby achieving the effect of pest control. Therefore, the present application provides a fragment of the lethal gene Nlpfam of brown planthopper and its application in biological control.

[0026] Example 1 Cloning of the Nlpfam fragment of the brown planthopper lethal gene

[0027] 1. Amplification of the Nlpfam fragment of the brown planthopper lethal gene:

[0028] (1) Take the adult or nymph of brown planthopper, wash it three times with 1x PBS (2.68mM KCl, 37mM NaCl, 8.1mM Na2HPO4, 1.47mM KH2PO4, pH 7.4), and then 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 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.

[0029] (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 gDNARemover, 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 brown planthopper cDNA;

[0030] (3) Primers were designed based on SEQ ID NO.1 of the Nlpfam gene of brown planthopper and SEQ ID NO.2 of the amino acid encoded by the Nlpfam gene.

[0031] SEQ ID NO.1:

[0032]

[0033] SEQ ID NO.2:

[0034] GGDREVARERDSGRESGGRRNFKPSKASSNSNNDHQSTSLYKFKLSRPSGRWQYKTTPKPRIQIRRQDNDLENNHNAAQQQLTTTADQSTSSDLEGSNSEVGSAVDQLLDQAVNSGGQAAEPT VTAETIKVEISTPADFKDTYYEIQTIKSPYTFQVGTIKNTRFITVTSTVEKSLVEPTEAPQLRPSEPLTENILATATQNVYDNKEPPLDSSVATLPPIALAGDAETPPLETMTESFSTTQLML KTHLLPVVRAGNTSTYTLVQSYHVTRLVTAVKTLPPMEVYQFVPSKTLNEFNTRLDEAGSELHLELEFGDNNNNDDLPSAKAFPPDLDLANVGSEFDLSEMDKTKFPDMHLRLKKAHQNAPAQ QNQIQPVSTTQELATPSLSPEQLQQLALYRFLNPNAPLPPQLLTTSRPVLKVETIYESHVIPLFNGQSTLFSTISRPIATVSKTEYEVVTNSLALPPVQPPVNTINPFLQQPQPQFAVTSTP.

[0035] The primers are shown in SEQ ID NO.3 and SEQ ID NO.4,

[0036] SEQ ID NO.3: CACCTCCACCGTCGAGAAAA;

[0037] SEQ ID NO.4: GGAAAGCCTTAGCGGAAGGT;

[0038] (4) Using brown planthopper cDNA as a template, SEQ ID NO.3 and SEQ ID NO.4 primers were used to perform PCR amplification of the Nlpfam gene. The system was as follows: 5 μL 2x ApexHF Mix, 1 μL upstream primer Nlpfam-F (SEQ ID NO.3), 1 μL downstream primer Nlpfam-R (SEQ ID NO.4), 2 μL brown planthopper 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.

[0039] 2. Monocloning of the Nlpfam gene of brown planthopper:

[0040] (1) Separate the PCR products by 1% agarose gel electrophoresis. Figure 1 As shown; cut the band with a blade and recover the PCR product with a DNA agarose gel kit; obtain the purified brown planthopper Nlpfam gene DNA fragment; connect the brown planthopper Nlpfam gene DNA fragment to the pClone007 vector using a blunt-end cloning kit, and the reaction system is as follows: 2μL 10xBuffer, 2μL pClone007 Blunt simple vector, 2μL brown planthopper Nlpfam gene DNA fragment, 14μL ddH2O, and a total volume of 20μL. After the reagents are mixed, the ligation reaction is carried out at 25℃ for 5min.

[0041] (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.

[0042] (3) Colony PCR was used to verify the Nlpfam DNA fragment of brown planthopper. The reaction system was as follows: 5 μL 2×ApexHF Mix, 1 μL upstream primer Nlpfam (SEQ ID NO.3), 1 μL downstream primer Nlpfam-R (SEQ ID NO.4), 2 μL brown planthopper 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 brown planthopper Nlpfam gene was obtained as shown in SEQ ID NO.5;

[0043] SEQ ID NO.5:

[0044] CACCTCCACCGTCGAGAAAAGCCTAGTGGAACCCACAGAGGCACCCCAATTGCGCCCCTCAGAACCCCTTACCGAGAACATTCTCGCCACAGCCACCCAGAACGTCTATGACAACAAGGAACCCCCTCTAGATTCCAGTGTGGCCACCCTTCCACCCATTGCACTGGCTGGCGACGCTGAAACTCCACCCCTAGAGACCATGACTGAGTCTTTCAGTACTACACAGCTCATGTTGAAGACTCA TCTGTTACCTGTGGTGAGAGCTGGAAATACGTCAACATATACATTGGTGCAATCATACCATGTGACCAGGTTGGTAACAGCTGTGAAGACTCTACCTCCGATGGAAGTCTACCAGTTTGTC CCAAGTAAAACACTGAACGAGTTTAACACGAGGTTGGACGAGGCTGGATCAGAGTTGCATCTAGAACTAGAGTTTGGGGATAACAATAACAATGATGATCTACCTTCCGCTAAGGCTTTCC.

[0045] Example 2 Synthesis of dsRNA of the Brown Planthopper Lethal Gene Nlpfam

[0046] (1) PCR amplification of the Nlpfam gene fragment as shown in SEQ ID NO.5 using primers with T7 promoter. The bacterial solution containing the Nlpfam gene fragment of brown planthoppers 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 Nlpfam-F (SEQ ID NO.6), 10 μL downstream primer Nlpfam-R (SEQ ID NO.7), 20 μL bacterial solution containing the Nlpfam gene of brown planthoppers, 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.

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

[0048] TAATACGACTCACTATAGGGG CACCTCCACCGTCGAGAAAA;

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

[0050] TAATACGACTCACTATAGGGGGGAAAGCCTTAGCGGAAGGT.

[0051] (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 Nlpfam fragments of the brown planthopper lethal gene containing the T7 promoter.

[0052] (3) Synthesis and purification of dsRNA of Nlpfam, the lethal gene of brown planthoppers. The dsRNA of Nlpfam, the lethal gene of brown planthoppers, was synthesized using the T7 High Yield RNA Transcription kit as follows: the fragment of Nlpfam, the lethal gene of brown planthoppers, containing the T7 promoter was used as the DNA template, 10x Reaction Buffer 2μl, Enzyme mix 2μl, ATPsolution 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 mixed and incubated at 37°C overnight, DNase was added and mixed, and then incubated at 37°C for 15min and 65°C for 5min to obtain the dsRNA of Nlpfam, the lethal gene of brown planthoppers. The solubility of dsRNA of Nlpfam, the lethal gene of brown planthoppers, was determined, and the quality of dsRNA was determined by 1% agarose gel electrophoresis, as shown in FIG. Figure 2 shown.

[0053] Example 3 Injection of dsRNA of Nlpfam gene, a lethal gene of brown planthopper, into brown planthopper

[0054] (1) Take 3rd-4th instar nymphs of brown planthoppers, about 100 nymphs in each group, for a total of 6 groups, place them on ice and freeze for 10 minutes before injection.

[0055] (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.

[0056] (3) After adding the dsRNA of the Nlpfam lethal gene of brown planthoppers into the 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 brown planthoppers under a microscope. The injection parameters were as follows: pi = 1000 pah; ti [s]: 0.3 s; pc = 10 pah. Three groups of brown planthopper nymphs were injected, with about 100 nymphs in each group.

[0057] (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 brown planthopper nymphs, with about 100 nymphs in each group.

[0058] (5) After waking up, the nymphs of brown planthoppers injected with dsRNA of the brown planthopper lethal gene Nlpfam 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%.

[0059] (6) After the brown planthopper nymphs were injected with dsRNA and dsGFP of the brown planthopper lethal gene Nlpfam, the nymphs were observed daily. Figure 3 As shown; dead brown planthoppers were removed every day, and the number of dead insects was counted every day. The mortality rate of brown planthopper nymphs injected with dsRNA and dsGFP of the brown planthopper lethal gene Nlpfam was counted. The results are shown in Figure 4 shown.

[0060] from Figure 3 It can be seen that the abdomen of the brown planthopper injected with the lethal gene Nlpfam dsRNA turned black and died, while the abdomen of the brown planthopper injected with dsGFP in the control group was plump and shiny and grew normally.

[0061] from Figure 4 It can be seen that the survival rate of brown planthoppers injected with the lethal gene Nlpfam dsRNA was significantly different from that of the control group injected with dsGFP (P<0.0001), indicating that the dsRNA of the brown planthopper lethal gene Nlpfam affects the survival of brown planthoppers, indicating that the dsRNA of the Nlpfam gene has potential application value in the control of brown planthoppers.

[0062] In summary, the present application provides a fragment of the Nlpfam lethal gene of brown planthopper and its application in biological control. The corresponding dsRNA is prepared according to the Nlpfam lethal gene of brown planthopper. After the dsRNA is injected into the brown planthopper to interfere with the lethal gene Nlpfam, the abdomen of the brown planthopper turns black, which can effectively kill the brown planthopper. The control of the brown planthopper population is achieved, the harm of the brown planthopper to food crops is reduced, and a new strategy for the green control of the brown planthopper is provided.

[0063] 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.

[0064] 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.

[0065] 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 brown planthopper lethal gene Nlpfam, characterized in that: The nucleotide sequence of the brown planthopper lethal gene Nlpfam is shown in SEQ ID NO.

1.

2. The brown planthopper lethal gene Nlpfam according to claim 1, characterized in that: The amino acid sequence encoded by the Nlpfam fragment of the brown planthopper lethal gene is shown in SEQ ID NO.

2.

3. A primer set for amplifying the Nlpfam fragment of the brown planthopper lethal gene 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 of the brown planthopper lethal gene Nlpfam, characterized in that: The dsRNA targets and silences the brown planthopper lethal gene Nlpfam.

5. The dsRNA of the brown planthopper lethal gene Nlpfam 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 of the brown planthopper lethal gene Nlpfam according to claim 4 or 5, characterized in that: The nucleotide sequence SEQ ID NO.5 of the brown planthopper lethal gene Nlpfam 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 of the brown planthopper lethal gene Nlpfam 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 brown planthopper lethal gene Nlpfam.

8. Use of the brown planthopper lethal gene Nlpfam according to any one of claims 1 to 3 or the dsRNA of the brown planthopper lethal gene Nlpfam according to any one of claims 4 to 7 in the control of brown planthoppers.

9. A brown planthopper lethal gene Nlpfam interference agent, characterized in that: The interfering agent comprises the dsRNA of the brown planthopper lethal gene Nlpfam according to any one of claims 4-7; or comprises a gene fragment transcribed from the brown planthopper lethal gene Nlpfam according to any one of claims 1-3.

10. The Nlpfam interference agent for the brown planthopper lethal gene according to claim 9, characterized in that: The Nlpfam interference reagent, which is lethal gene to brown planthoppers, acts on brown planthoppers by injection.

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