A two-spotted spider mite feeding-related gene SSGP2970 and its application

Abstract

The present invention belongs to the technical field of transgenic drugs, and discloses a two-spotted spider mite feeding-related gene and its corresponding dsRNA sequence and application, wherein the two-spotted spider mite feeding-related gene is SSGP2970; the nucleotide sequence and amino acid sequence of the two-spotted spider mite SSGP2970; the present invention also includes a dsRNA sequence targeting the two-spotted spider mite SSGP2970 gene, and the sequence has a good silencing effect on the two-spotted spider mite SSGP2970 gene. Feeding the dsRNA sequence can significantly reduce the survival rate of the two-spotted spider mite. The SSGP2970 gene and the lethal dsRNA sequence provided by the present invention can be used for the development of transgenic anti-mite plants or new acaricides.

Description

Technical Field

[0001] The invention belongs to the technical field of transgenic drugs, and in particular relates to a two-spotted spider mite feeding-related gene and a corresponding dsRNA sequence and application thereof. Background Art

[0002] The two-spotted spider mite, Tetranychus urticae, is an important agricultural pest mite widely distributed throughout the world. It can harm more than 1,000 host plants, especially causing serious economic losses to cash crops such as vegetables and flowers in my country. Due to the small size of the two-spotted spider mite, its short development period, strong reproductive capacity, and the unscientific use of pesticides, its resistance develops rapidly, its population grows sharply, and it is very easy to cause a short-term outbreak during the crop production season, making its prevention and control increasingly difficult. At present, the prevention and control of the two-spotted spider mite is still mainly based on chemical agents, which has caused more serious environmental pressure and drug resistance problems. Therefore, it is urgent to explore and develop new green control technologies for spider mites.

[0003] RNA interference (RNAi) is a conservative post-transcriptional specific gene silencing mechanism, which is mainly caused by mRNA degradation triggered by endogenous or exogenous double-stranded RNA, resulting in the phenomenon of specific obstruction of target gene expression. RNAi has the advantages of high efficiency, specificity, no pollution and a large number of target genes to choose from. It can be effectively used in the prevention and control of pest mites and is also considered to be a new generation of insecticides. By silencing spider mite-specific target genes and reducing the survival rate of pest mites, the purpose of controlling pest mites can be achieved. Among them, the targets that can achieve RNAi control of pest mites include the following: lethal genes, growth and development genes, resistance and immunity genes, and feeding and digestion genes. Most lethal, resistance and growth and development genes are highly conservative in insects or mites and may also cause harm to beneficial insects. The unannotated genes related to spider mite feeding are spider mite-specific and have extremely low gene homology with other beneficial insects with different feeding habits. They are important natural selective RNAi targets. At present, many piercing-sucking mouthparts insects have interfered with feeding-related genes through RNAi technology, which can significantly increase the mortality rate of pests and reduce the population size. At present, the number of specific RNAi targets for spider mites is extremely limited. In summary, discovering target genes related to the feeding of two-spotted spider mites can effectively expand RNAi targets, and at the same time, it can be further applied to the prevention and control of pest mites through transgenic technology. Summary of the invention

[0004] In view of the problems existing in the prior art, the present invention provides a two-spotted spider mite feeding-related gene and its corresponding dsRNA sequence and application.

[0005] The present invention screens an unannotated gene tetur08g02970 (secreted salivary gland peptide, SSGP2970) related to the feeding of two-spotted spider mites through spider mite transcriptome data, and the gene only exists in spider mites, and has no homologous genes in other species such as natural enemy predatory mites, and is a natural spider mite-specific RNAi target. The expression level of SSGP2970 in the feeding stage of two-spotted spider mites is significantly higher than that in the egg stage and the resting stage, and is related to the type and duration of feeding host plants. The dsRNA of the target gene SSGP2970 is synthesized in vitro, and the two-spotted spider mite dsRNA is fed through an artificial device, which can effectively silence the SSGP2970 gene of the two-spotted spider mite. In the process of transferring the two-spotted spider mite female adult mites to the host plant for feeding, the feeding amount and survival rate are significantly reduced. The invention solves the problem of the lack of efficient acaricidal dsRNA that is effective against spider mites but ineffective against beneficial insects, and has important application value for the research and development of new RNAi acaricidal pesticides and the cultivation of mite-resistant plants.

[0006] Furthermore, the nucleotide sequence of the two-spotted spider mite feeding-related gene SSGP2970 is:

[0007] ATGAAGATTCTCTTAGCCTTATTTACAATCTCCTTTTGTTTCATTACATTGAATGCTGACAAAAAGAAGTCGCCGAAACCAACTAGTGAAGATATAACTGAAAAAAACGCAACCGCATGTGAAATAACTTTGAATCAAACGGCAACTCTCATTGAACCAGCTTTACTTGTTGGATATCAAGAATGGCCATCTACACTGGAAGAGATTGATAAACATTGCGATCTTGGAGCAAAGGTCTATAAAAGCATTAGACGCTATGGTCGATGTACAACTGGATTATCTCGTCAATCACTGAACATCTTTTTGCGTGGACTGAAAAAATTCATGCAAAACATTTGTTCATCCAACAAGAGGAAACAAGACACGGCTGATATATTGAAATGTGCCTCCAATGGAACCAACGCAGGCTGGAGTAAATGCCTGAAAGAAACTAATTCACGACTTGATATTACCTCGACAAATGCCAGTGACCCGGCCATGGTTGGTAATATTTGCTGTGCTTATTCCTCGTTAGTTAATTGTATCAACACCAATACCGGTACAAAATCTGAATGTGATAAAAGCAAAGATACAACGTTTTTCCTCCAATCATCCATGGCACTGGTTATAAAAGATTTAACAGACTTGCTGTGTACCAATTATGAATCTAAGGAATCCTGTGAGATTCATGATTCACAAAGATCTGACATTTTACAATCAATTGCCGAAGGTAAACATGTTTTTAAAGGAGCTTTCTTAATGACACCTTTAATAAAAGCAGCAAGTCAACTGGGTAAAATCCATGCTGATGATATTTGA。

[0008] Furthermore, the amino acid sequence of the Tetranychus urticae feeding-related gene SSGP2970:

[0009] MKILLALFTISFCFITLNADKKKSPKPTSEDITEKNATACEITLNQTATLIEPALLVGYQEWPSTLEEIDKHCDLGAKVYKSIRRYGRCTTGLSRQSLNIFLRGLKKFMQNICSSNKRKQDTADILKCASNGT NAGWSKCLKETNSRLDITSTNASDPAMVGNICCAYSSLVNCINTNTGTKSECDKSKDTTFFLQSSMALVIKDLTDLCTNYESKESCEIHDSQRSDILQSIAEGKHVFKGAFLMTPLIKAASQLGKIHADDI.

[0010] Furthermore, the sequence of the full-length primer for cloning the two-spotted spider mite feeding-related gene SSGP2970 is:

[0011] SSGP2970-F: ATGAAGATTCTCTTAGCCTTA

[0012] SSGP2970-R: TCAAATATCATCAGCATGG.

[0013] Furthermore, the sequence of the interference primer of the two-spotted spider mite feeding-related gene SSGP2970 is:

[0014] dsSSGP2970-F:TAATACGACTCACTATAGGG

[0015] ATTGCGATCTTGGAGCAAAGG dsSSGP2970-R:TAATACGACTCACTATAGGGCCCAGTTGACTTGCTGCTTTT.

[0016] Furthermore, the sequence of the quantitative primer of the two-spotted spider mite feeding-related gene SSGP2970 is:

[0017] qSSGP2970-F:AGAAGTCGCCGAAAACCAACT

[0018] qSSGP2970-R:ACCTTTTGCTCCAAGATCGCA.

[0019] Another object of the present invention is to provide a dsRNA sequence corresponding to the two-spotted spider mite feeding-related gene SSGP2970, wherein a fragment of the dsRNA sequence is:

[0020] .

[0021] Another object of the present invention is to provide a method for reducing the survival rate of two-spotted spider mites.

[0022] Feeding the dsRNA sequence. Pick the adult female two-spotted spider mites to a sandwich device simulating leaves and feed them with in vitro synthesized dsRNA (a 100-mesh nylon mesh is placed between two layers of sealing film, and the dsRNA diffuses in the pores of the nylon mesh). Specific steps: spread a square nylon mesh with a side length of 1.5 cm on a layer of square sealing film with a side length of 2.5 cm, add 45 μL of 1000 ng / μL (1% blue food coloring is added) dsRNA on the nylon mesh, and spread the sealing film that is stretched four times larger on the nylon mesh to make a sandwich device. There should be no bubbles in the device. After the adult female two-spotted spider mites are picked to feed on the device for 24 hours, the two-spotted spider mites that have fed on the dsRNA are transferred to soybean leaves for feeding and observation. The results show that the survival rate of the two-spotted spider mites is significantly reduced.

[0023] Another object of the present invention is to provide a preparation for transgenic anti-mite plants, wherein the preparation for transgenic anti-mite plants comprises the dsRNA sequence.

[0024] Another object of the present invention is to provide a miticide, which comprises the dsRNA sequence.

[0025] In combination with the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solutions to be protected by the present invention are as follows:

[0026] First, at present, RNAi technology has great application potential in pest control, and there are also reports of successful pest control in the field. However, the research on RNAi control technology of spider mites is relatively lagging, lacking targets that are effective in killing harmful mites and ineffective against beneficial insects. The genes related to spider mite feeding are natural RNAi control targets because of the large species differences, rapid evolution, and low homology with genes of other species such as natural enemies. The present invention provides a two-spotted spider mite and feeding-related gene SSGP2970 and its corresponding dsRNA sequence and application. The nucleotide sequence and amino acid sequence of the two-spotted spider mite SSGP2970 have homologous genes only in individual spider mites, and have extremely low homology with genes of other species, especially with natural enemies and other beneficial insects. The present invention also includes a dsRNA sequence targeting the two-spotted spider mite SSGP2970 gene, which has a good silencing effect of 48% on the two-spotted spider mite SSGP2970 gene. Feeding the dsRNA sequence can significantly reduce the survival rate of the two-spotted spider mite by more than 50%. An unannotated gene SSGP2970 related to spider mite feeding discovered in the present invention plays an important role in the spider mite feeding process and has great potential for field application. At the same time, it also enriches the spider mite-specific RNAi target library.

[0027] Second, the SSGP2970 gene and its lethal dsRNA sequence provided by the present invention have a high lethal effect on female adult spider mites, can effectively control the population of spider mites, reduce the field damage of spider mites, and can also be used for the development of transgenic anti-mite plants or new acaricides. This technical solution can not only improve the anti-mite ability of plants, but also effectively reduce the use of chemical acaricides. Since this technical solution has no effect on natural enemies, it can also be combined with natural enemies to develop green and new joint prevention and control technologies for pest mites, which has an important positive impact on the environment and human health. Compared with traditional chemical acaricides, the anti-mite plants or new acaricides developed based on the gene and its lethal dsRNA sequence of the present invention have high efficiency and specificity. It can specifically fight against specific types of mites without affecting other beneficial insects and organisms. At the same time, it also has higher environmental protection. The technical solution of the present invention can improve the plant defense ability through genetic engineering means, thereby reducing dependence on external chemicals, thereby reducing the negative impact on non-target organisms and ecosystems. The technical solution of the present invention has a wide range of application prospects and reference value, and can be used to develop anti-mite products for different types of mites and different plants.

[0028] Third, the expected benefits and commercial value of the technical solution of the present invention after transformation are: the anti-mite plants or new acaricides developed using the gene of the present invention and its lethal dsRNA sequence to control the reproduction and distribution of mites will bring economic benefits to agricultural production, and help reduce dependence on chemical acaricides and reduce environmental pollution. The development of transgenic anti-mite crops using the gene of the present invention and its lethal dsRNA sequence will have huge market potential and commercial value. The technical solution of the present invention can be authorized to other companies for use or transferred to related enterprises for production and sales, which will bring business opportunities to enterprises, promote the sustainable development of agricultural production, and also help promote the development and innovation of related industries.

[0029] The present invention develops anti-mite plants or novel acaricides by utilizing the SSGP2970 gene and its lethal dsRNA sequence, which have the characteristics of lethal effect on specific mites, sustainability, high efficiency, innovation, etc., and have no effect on beneficial insects such as natural enemies, thus filling the gap of lack of specific RNAi targets for spider mites at home and abroad. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 is a schematic diagram of the expression pattern of SSGP2970 in different developmental stages of two-spotted spider mites provided by an embodiment of the present invention;

[0031] Figure 2 Schematic diagram of the expression pattern of SSGP2970 at different time points of feeding by two-spotted spider mites provided by an embodiment of the present invention;

[0032] Figure 3 It is a schematic diagram of the silencing efficiency of SSGP2970 provided in an embodiment of the present invention;

[0033] Figure 4 It is a schematic diagram of the survival curve of two-spotted spider mites on soybean leaf discs after being fed with dsSSGP2970 provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0034] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0035] The nucleotide sequence of Tetranychus urticae SSGP2970 provided in the embodiment of the present invention is:

[0036] ATGAAGATTCTCTTAGCCTTATTTACAATCTCCTTTTGTTTCATTACATTGAATGCTGACAAAAAGAAGTCGCCGAAACCAACTAGTGAAGATATAACTGAAAAAAACGCAACCGCATGTGAAATAACTTTGAATCAAACGGCAACTCTCATTGAACCAGCTTTACTTGTTGGATATCAAGAATGGCCATCTACACTGGAAGAGATTGATAAACATTGCGATCTTGGAGCAAAGGTCTATAAAAGCATTAGACGCTATGGTCGATGTACAACTGGATTATCTCGTCAATCACTGAACATCTTTTTGCGTGGACTGAAAAAATTCATGCAAAACATTTGTTCATCCAACAAGAGGAAACAAGACACGGCTGATATATTGAAATGTGCCTCCAATGGAACCAACGCAGGCTGGAGTAAATGCCTGAAAGAAACTAATTCACGACTTGATATTACCTCGACAAATGCCAGTGACCCGGCCATGGTTGGTAATATTTGCTGTGCTTATTCCTCGTTAGTTAATTGTATCAACACCAATACCGGTACAAAATCTGAATGTGATAAAAGCAAAGATACAACGTTTTTCCTCCAATCATCCATGGCACTGGTTATAAAAGATTTAACAGACTTGCTGTGTACCAATTATGAATCTAAGGAATCCTGTGAGATTCATGATTCACAAAGATCTGACATTTTACAATCAATTGCCGAAGGTAAACATGTTTTTAAAGGAGCTTTCTTAATGACACCTTTAATAAAAGCAGCAAGTCAACTGGGTAAAATCCATGCTGATGATATTTGA In an embodiment of the present invention, the amino acid sequence of Tetranychus urticae SSGP2970: >SSGP2970

[0037] MKILLALFTISFCFITLNADKKKSPKPTSEDITEKNATACEITLNQTATLIEPALLVGYQEWPSTLEEIDKHCDLGAKVYKSIRRYGRCTTGLSRQSLNIFLRGLKKFMQNICSSNKRKQDTADILKCASNG TNAGWSKCLKETNSRLDITSTNASDPAMVGNICCAYSSLVNCINTNTGTKSECDKSKDTTFFLQSSMALVIKDLTDLCTNYESKESCEIHDSQRSDILQSIAEGKHVFKGAFLMTPLIKAASQLGKIHADDI

[0038] In the present embodiment, the full-length primers for cloning SSGP2970

[0039] SSGP2970-F: ATGAAGATTCTCTTAGCCTTA

[0040] SSGP2970-R: TCAAATATCATCAGCATGG

[0041] In an embodiment of the present invention, the interfering primer is:

[0042] dsSSGP2970-F:TAATACGACTCACTATAGGG

[0043] ATTGCGATCTTGGAGCAAAGG

[0044] dsSSGP2970-R:TAATACGACTCACTATAGGG

[0045] CCCAGTTGACTTGCTGCTTTT

[0046] In this embodiment of the present invention, the quantitative primers are:

[0047] qSSGP2970-F:AGAAGTCGCCGAAAACCAACT

[0048] qSSGP2970-R:ACCTTTTGCTCCAAGATCGCA

[0049] In this embodiment of the present invention, the dsRNA fragment:

[0050] ATTGCGATCTTGGAGCAAAGGTCTATAAAAGCATTAGACGCTATGGTCGATGTACAACTGGATTATCTCGTCAATCACTGAACATCTTTTTGCGTGGACTGAAAAAATTCATGCAAAACATTTGTTCATCCAACAAGAG GAAACAAGACACGGCTGATATATTGAAATGTGCCTCCAATGGAACCAACGCAGGCTGGAGTAAATGCCTGAAAGAAACTAATTCACGACTTGATATTACCTCGACAAATGCCAGTGACCCGGCCATGGTTGGTAATATTT GCTGTGCTTATTCCTCGTTAGTTAATTGTATCAACACCAATACCGGTACAAAATCTGAATGTGATAAAAGCAAAGATACAACGTTTTCCTCCAATCATCCATGGCACTGGTTATAAAAGATTTAACAGACTTGCTGTGT ACCAATTATGAATCTAAGGAATCCTGTGAGATTCATGATTCACAAAGATCTGACATTTTACAATCAATTGCCGAAGGTAAACATGTTTTTAAAGGAGCTTTCTTAATGACACCTTTAATAAAAGCAGCAAGTCAACTGGG

[0051] Development of transgenic mite-resistant plants: Obtain the lethal dsRNA sequence of the SSGP2970 gene from related organisms through gene cloning technology. Insert the lethal dsRNA sequence of the SSGP2970 gene into a plant expression vector to construct a recombinant plasmid. Use gene transformation technology to introduce the recombinant plasmid into plant cells to achieve stable integration and expression of the gene. Screen and identify transgenic plants through molecular biological methods to confirm the correct expression of the lethal dsRNA sequence of the SSGP2970 gene in transgenic plants. Identify the anti-mite performance and yield of transgenic mite-resistant plants through indoor tests and field tests.

[0052] Development of new acaricides: Use chemical synthesis methods to prepare large quantities of lethal dsRNA. Combine lethal dsRNA with appropriate carriers to prepare new acaricides. Test the biological activity of new acaricides and their effects on non-target organisms through indoor tests and field test sprays. Based on the test results, optimize and improve the production process to improve the production efficiency and efficacy of acaricides. Commercialize and apply the optimized new acaricides for mite control in agricultural production.

[0053] Samples of the two-spotted spider mite cowpea strain at different developmental time points were collected. First, the female adult mites (population) on the cowpea plant were transferred to new cowpea leaf discs to lay eggs, 3 to 4 leaf discs each time, 5 to 6 leaves on each leaf disc, 40 female adult mites and 5 male adult mites on each leaf. Test mite samples of 3-day-old eggs (800 per replicate), young mites (500), young mites entering the dormant period for 8h (500), pre-nymph mites (300), pre-nymph mites entering the dormant period for 8h (300), post-nymph mites (150 to 200), post-nymph mites in the dormant period for 8h (150 to 200), and adult mites (60 to 100) were collected, with 4 replicates at each time point. qPCR technology was used to detect the expression level of two-spotted spider mite SSGP2970. Figure 1 As shown, the real-time fluorescence quantification results showed that SSGP2970 was expressed in different developmental stages of the two-spotted spider mite, among which the expression level of SSGP2970 in the feeding stage of the two-spotted spider mite tended to be higher than that in the egg and resting stages, among which the larval mite stage and the adult mite stage were significantly higher than other stages, indicating that this gene is closely related to the feeding of the two-spotted spider mite.

[0054] Data are expressed as mean ± standard error and analyzed by one-way ANOVA in SPSS with Turkey HSD and LSD as post hoc tests at a significance level of 0.05.

[0055] Samples of two-spotted spider mite cowpea / soybean strains at different feeding time points were collected. First, the female adult mites (population) on the cowpea / soybean plants were transferred to new cowpea / soybean plants for feeding. 70 female adult mites were collected for each seedling. Samples of test mites were collected at 6 time points (1h, 3h, 6h, 9h, 12h, and 24h), and each time point contained 4 replicates. The expression level of two-spotted spider mite SSGP2970 was detected by qPCR technology. Figure 2 As shown in the figure, after the soybean strain and cowpea strain test mites of the two-spotted spider mite were transferred to the corresponding fresh plants, the expression of SSGP2970 at different time points when the two-spotted spider mite fed on the host was examined. The real-time fluorescence quantitative results showed that SSGP2970 was induced to up-regulate expression when the two-spotted spider mite fed for 3 hours, with the highest expression level at 6 hours, and then decreased and stabilized, indicating that this gene is closely related to the early stage of the two-spotted spider mite feeding on the host.

[0056] Data are expressed as mean ± standard error and analyzed by one-way ANOVA in SPSS with Turkey HSD and LSD as post hoc tests at a significance level of 0.05.

[0057] The two-spotted spider mites that had been feeding on dsRNA for 24 h were transferred to fresh plant leaves for feeding. The two-spotted spider mites that had been feeding on fresh plant leaves for 24 h were collected, and four biological replicates were used for each treatment. The expression level of the two-spotted spider mite SSGP was detected by qPCR technology, that is, the silencing efficiency of the two-spotted spider mite SSGP2970 was as follows: Figure 3 As shown, the real-time fluorescence quantitative results showed that 24 hours after the two-spotted spider mite fed on dsRNA, the expression of SSGP2970 at the mRNA level was significantly downregulated by 48.67%, indicating that the gene was effectively interfered.

[0058] "dsSSGP2970" indicates the treatment group fed with SSGP2970 gene dsRNA; "dsGFP" indicates the control group fed with GFP gene dsRNA. Independent t-test, "**" indicates p<0.01.

[0059] The two-spotted spider mites that had been feeding on dsRNA for 24 h were transferred to fresh plant leaves for feeding observation. There were 60 female adult mites on each leaf. The survival status of the two-spotted spider mites was observed every 24 h for 6 consecutive days. Figure 4 As shown, the survival curve results all show that the mortality rate of spider mites fed with SSGP2970 gene dsRNA is significantly higher than that of the control group (dsGFP). In addition, the present invention also observed that the spider mites fed with SSGP2970 gene dsRNA have a peak mortality period of 3 to 4 days.

[0060] "dsSSGP2970" indicates the treatment group fed with SSGP2970 gene dsRNA; "dsGFP" indicates the control group fed with GFP gene dsRNA, and "***" indicates p<0.001. Sample size: dsGFP, N=61, dsSSGP2970: N=60.

[0061] The above results indicate that feeding dsRNA targeting SSGP2970 can significantly inhibit the expression of SSGP2970 and effectively kill adult two-spotted spider mites.

[0062] The above description is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by any technician familiar with the technical field within the technical scope disclosed by the present invention and within the spirit and principle of the present invention should be covered by the protection scope of the present invention.

Claims

1. A method for reducing the survival rate of two-spotted spider mites, characterized in that: The method comprises feeding dsRNA; the sequence of the dsRNA is: .

2. The method for reducing the survival rate of two-spotted spider mites according to claim 1, characterized in that: Female adult spider mites were selected and placed in a sandwich device simulating leaves, and fed with dsRNA synthesized in vitro. A 100-mesh nylon mesh was placed between the two layers of sealing film, and the dsRNA diffused in the pores of the nylon mesh.

3. The method for reducing the survival rate of two-spotted spider mites according to claim 2, characterized in that: The specific steps of the method are as follows: a nylon mesh is spread on a layer of sealing film, 45 μL of 1000 ng / μL dsRNA is added to the nylon mesh, and a sealing film that is stretched four times larger is spread on the nylon mesh to form a sandwich device without bubbles; female adult two-spotted spider mites are picked up on the device to feed for 24 hours, and then the two-spotted spider mites that have fed on the dsRNA are transferred to soybean leaves for feeding and observation.

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