RNAi target gene for inhibiting root-knot nematode disease and application thereof

Abstract

The application discloses an RNAi target gene for inhibiting root-knot nematode disease and application thereof, and belongs to the technical field of biotechnology and agricultural application. The nucleotide sequence of the RNAi is shown in SEQ ID No. 3, and the obtaining steps are as follows: (1) total RNA of southern root-knot nematode is extracted, and cDNA is obtained through reverse transcription operation; (2) the cDNA obtained in the step (1) is taken as a template, the sequences shown in SEQ ID No. 1 and SEQ ID No. 2 are taken as primers, and the full-length sequence of MiMSP30 is obtained through PCR amplification; (3) the full-length sequence of MiMSP30 is taken as a template, the sequences shown in SEQ ID No. 4 and SEQ ID No. 5 or the sequences shown in SEQ ID No. 6 and SEQ ID No. 7 are taken as primers respectively, and the DNA sequence which can be used as a dsRNA positive and negative chain synthesis template is obtained through PCR reaction amplification. The MiMSP30 silencing efficiency of the dsRNA-MSP30 fed to the free 2nd instar larvae of the root-knot nematode reaches 70.02 %, the infestation insect number of the root-knot nematode is significantly reduced, the root development process appears obvious lag, and the root knot and the egg mass number are significantly reduced.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and agricultural applications, specifically relating to an RNAi target gene for inhibiting root-knot nematode disease and its application. Background Technology

[0002] Root-knot nematodes (Meloidogyne spp.) are obligate endoparasites on plant roots, distributed worldwide. They are major pathogens threatening agricultural production and are subject to quarantine by many countries. Currently, chemical pesticides are the primary means of controlling root-knot nematodes in production. However, chemical nematicides not only cause serious environmental pollution but are also unsafe for humans and livestock. With the banning of a series of chemical nematicides such as methyl bromide, aldicarb, and chlorpyrifos, biological control technologies that are effective in controlling the disease and environmentally friendly have gradually become a research hotspot.

[0003] The southern root-knot nematode putative esophageal gland cell secretory protein 30 (MSP30) weakens plant defenses against root-knot nematodes by regulating the transcriptional levels of plant cell wall and immune-related genes, promoting the infection and development of root-knot nematodes, and thus positively regulates the pathogenicity of root-knot nematodes. It is one of the key proteins in the pathogenesis of root-knot nematodes, and its encoding gene can serve as a new target for the prevention and control of root-knot nematode diseases.

[0004] RNA interference (RNAi) is a conserved process in eukaryotes. When exogenous or endogenous double-stranded RNA (dsRNA) or small interfering RNA (siRNA) exists in eukaryotic cells, their target mRNA is cleaved by Dicer and AGO proteins, thus interfering with the expression of the gene encoding the target mRNA. Silencing specific genes can be achieved by synthesizing dsRNA in eukaryotic cells or by introducing in vitro synthesized dsRNA or siRNA into eukaryotic cells. Currently, RNAi has become a commonly used method for gene silencing.

[0005] Host-induced gene silencing (HIGS) refers to the production of RNA molecules by plants that are complementary to specific gene sequences of pathogens or insects. These RNA molecules, after processing by Dicer proteins in plant cells, specifically recognize and silence the pathogen's gene sequence, thereby interfering with the pathogen's development or pathogenic process and inhibiting the occurrence and development of diseases or pests. Currently, HIGS technology has been applied to the control of root-knot nematodes and insects in many important crops. Summary of the Invention

[0006] The purpose of this invention is to provide an RNAi target gene for inhibiting root-knot nematode disease and its application, so as to solve the problems mentioned in the background art.

[0007] To achieve the above objectives, in a first aspect, the present invention provides an RNAi target gene for inhibiting root-knot nematode disease, the nucleotide sequence of which is shown in SEQ ID No. 3.

[0008] The in vitro synthesis method of this RNAi target gene includes the following steps:

[0009] (1) Total RNA was extracted from Southern Root-knot Nematode and cDNA was obtained by reverse transcription.

[0010] (2) Using the cDNA obtained in step (1) as a template, and using the sequences shown in SEQ ID No.1 and SEQ ID No.2 as primers, the full-length MiMSP30 sequence was amplified by PCR.

[0011] A second aspect of the invention provides the application of RNAi targeting the said target gene in the control of root-knot nematode disease.

[0012] The in vitro synthesis method of dsRNA with RNAi effect on the target gene MiMSP30 includes the following steps:

[0013] (1) Using the full-length sequence of MiMSP30 as a template, and using the sequences shown in SEQ ID No.4 and SEQ ID No.5, or the sequences shown in SEQ ID No.6 and SEQ ID No.7, as primers, the DNA sequence that can be used as a template for the synthesis of dsRNA positive and negative strands was amplified by PCR reaction.

[0014] (2) Using the PCR amplification product recovered in step (1) as a template, the positive and reverse strands of dsRNA are transcribed and synthesized respectively, and annealed to allow the positive and reverse strands to hybridize and form dsRNA.

[0015] (3) The residual single-stranded RNA (ssRNA) and DNA are removed by enzymatic hydrolysis, and the hydrolysis products are recovered and purified to obtain dsRNA (dsRNA-MSP30) that has RNAi effect on the target gene MiMSP30.

[0016] The HIGS method, which targets the MiMSP30 gene and has RNAi effects, includes the following steps:

[0017] (1) Using the full-length sequence of MiMSP30 as a template, and the sequences shown in SEQ ID No. 14 and SEQ ID No. 15 as primers, the MiMSP30 sequence was amplified by PCR reaction and inserted into the HIGS vector sequence to obtain a HIGS vector that carries both the MiMSP30 sequence and the reverse complementary sequence of MiMSP30.

[0018] (2) Transform the HIGS vector obtained in step (1) into Agrobacterium GV3101;

[0019] (3) Using the Agrobacterium-mediated plant transgenic system, the HIGS vector sequence obtained in step (1) carrying both the MiMSP30 sequence and the MiMSP30 reverse complementary sequence was transformed into a plant to obtain a transgenic plant that can synthesize dsRNA-MSP30 in vivo.

[0020] A third aspect of the present invention is to provide a biological product containing the aforementioned RNAi target gene.

[0021] Furthermore, the biological product is a feed-type insecticide or bait.

[0022] Compared with the prior art, the beneficial effects of the present invention are:

[0023] Using dsRNA-GFP (dsRNA-GFP), which has an RNAi effect on the green fluorescent protein gene GFP, as a control, free second-instar root-knot nematode larvae were fed dsRNA-MSP30. The number of infected root-knot nematodes, root development progress, number of root knots, number of egg masses, and dsRNA gene silencing efficiency were recorded after dsRNA ingestion. The results showed that feeding dsRNA-MSP30 resulted in a MiMSP30 silencing efficiency of 70.02%, a significant decrease in the number of infected root-knot nematodes, a marked lag in root development, and a significant reduction in both the number of root knots and egg masses.

[0024] Using wild-type plants as a control, the number of root knots, egg masses, and dsRNA gene silencing efficiency of HIGS transgenic plants capable of synthesizing dsRNA-MSP30 in vivo were statistically analyzed. The results showed that after wild-type root-knot nematodes infected HIGS-MSP30 transgenic plants, the MiMSP30 silencing efficiency reached 46.32%, and both the number of root knots and egg masses were significantly reduced. Attached Figure Description

[0025] Figure 1 The results of the relative expression level detection of MiMSP30 after feeding second-instar larvae of Southern Root-knot Nematode with dsRNA-GFP and dsRNA-MSP30 for 4 hours in Example 3 are shown.

[0026] Figure 2 This is the statistical result of the number of successfully infected southern root-knot nematodes 7 days after being fed with dsRNA-GFP and dsRNA-MSP30 respectively in Example 3.

[0027] Figure 3 This is a statistical result of the developmental progress of the southern root-knot nematode in the roots 14 days after being fed with dsRNA-GFP and dsRNA-MSP30 respectively, as described in Example 3.

[0028] Figure 4 This is the statistical result of the number of root knots in tomatoes 45 days after being fed with dsRNA-GFP and dsRNA-MSP30, respectively, in Example 4.

[0029] Figure 5 The results of the egg mass count of Southern Root-knot Nematode inoculated with tomatoes 45 days after feeding with dsRNA-GFP and dsRNA-MSP30 respectively, are from Example 4.

[0030] Figure 6 The results of the relative expression level detection of MiMSP30 after infecting HIGS-MSP30 transgenic plants with second-instar larvae of wild-type southern root-knot nematodes in Example 6.

[0031] Figure 7 The results show the number of root knots in HIGS-MSP30 transgenic plants 45 days after being infected with second-instar larvae of wild-type southern root-knot nematodes in Example 6.

[0032] Figure 8 The results show the number of egg masses in HIGS-MSP30 transgenic plants 45 days after being infected with second-instar larvae of wild-type southern root-knot nematodes in Example 6. Detailed Implementation

[0033] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0034] Example 1

[0035] 1. Rearing of Southern Root-knot Nematodes

[0036] Tomato roots infected with southern root-knot nematodes for 45-60 days were gently washed, and mature egg masses were collected. The egg masses were then isolated using a filter and immersed in sterile water at 28°C. The sterile water was collected every 3 days, and the egg masses were immersed in fresh sterile water. This process was repeated until no new nematodes hatched. The collected sterile water containing hatched nematodes is called the nematode suspension.

[0037] 2. Total RNA extraction from Southern Root-knot Nematode

[0038] The steps for extracting total RNA from free second-instar larvae of *Southern Root-Knot Nematode* are as follows:

[0039] ① After concentrating the nematode suspension (containing 20,000 nematodes) to a volume of 100 μL, it was repeatedly frozen and thawed 5 times with liquid nitrogen.

[0040] ② After adding 1 mL of TRIzol (Invitrogen), vortex the sample to mix it, and then use an ultrasonic homogenizer (Nanjing Emmanuel Instrument Equipment Co., Ltd.) to homogenize for 10 min.

[0041] ③ Add 200 μL of chloroform (Shanghai test) and vortex for 3 min, then centrifuge at 12,000 rpm at 4℃ for 15 min. Collect the supernatant and transfer it to a clean RNase-free 1.5 mL centrifuge tube.

[0042] ④ Add 2.2 times the volume of anhydrous ethanol (Shanghai test) to the supernatant, mix by inversion, and incubate overnight at -20℃.

[0043] ⑤ Centrifuge at 12,000 rpm at 4℃ for 15 min to remove the supernatant, and wash the precipitate twice with RNase-free 70% ethanol solution.

[0044] ⑥ Centrifuge at 12,000 rpm at 4℃ to remove the ethanol solution, then leave at room temperature for 5-10 minutes after opening the lid.

[0045] ⑦ Add 20-40 μL of RNase-free deionized water, incubate on ice for 30 min to dissolve the precipitate, then aspirate and mix thoroughly to obtain total RNA from Southern Root-knot Nematodes, and store at -80℃.

[0046] 3. cDNA synthesis in Southern Root-knot Nematode

[0047] according to The instructions for using III RT SuperMix for qPCR (+gDNA Remover) (Qingke Biotechnology) to synthesize cDNA from Southern Root-Knot Nematode are as follows:

[0048] ① After dissolving the RNA sample and reaction components of *Strombus heterophylla* on ice, prepare the genomic de-radicalization reaction system (10 ng - 2 μg total RNA, 2 μL 5×g DNA Romover Mix, and RNase-free deionized water to make up to 10 μL). After mixing with a pipette, incubate at 42°C for 2 min, incubate at 60°C for 1 min, and then place on ice.

[0049] ② Prepare the reverse transcription reaction system (10 μL of the reaction product in ①, 4 μL of SynScript III RTSuperMix, and 20 μL of RNase-free deionized water). After mixing with a pipette, incubate at 50℃ for 15 min and at 85℃ for 5 s to obtain Southern Root-knot Nematode cDNA, which is then stored at -20℃.

[0050] 4. Full sequence amplification of the MiMSP30 encoding gene

[0051] According to I-5 TM The full sequence of the MiMSP30 encoding gene was amplified using 2×High-Fidelity Master Mix (Qingke Biotechnology) according to the instructions. A 10-fold diluted cDNA of *Strombus haematobium* (Southern Root-knot Nematode) was used as the amplification template. The pre- and post-primer sequences are shown in Table 1. The PCR amplification reaction system was prepared (12.5 μL I-5). TM 2× High-Fidelity Master Mix, 1 μL 10 μM front primer, 1 μL 10 μM back primer, 1 μL template, and deionized water to a final volume of 25 μL. PCR reaction program: 98℃, 2 min; 98℃, 10 s, 55℃, 15 s; 72℃, 15 s, 35 cycles; 72℃, 2 min. Sequencing analysis of the PCR products; if the sequence is correct, the complete sequence of the MiMSP30 encoding gene is obtained, i.e., sequence SEQ ID NO.3.

[0052] Table 1 Primer sequences for amplifying the full sequence of the MiMSP30 encoding gene.

[0053]

[0054]

[0055] Example 2

[0056] 1. Amplification of dsRNA-MSP30 and dsRNA-GFP templates

[0057] dsRNA synthesis template amplification was performed using the T7 RNAi Transcription Kit (Novizan) and I-5. TMThe procedure was performed using 2× High-Fidelity Master Mix (Qingke Biotechnology) according to the instructions. Using the full-length MiMSP30 encoding gene sequence and pSAT5-EGFP-N1 as templates, PCR amplification was performed by adding T7 promoter sequences to the 5' ends of the sense and antisense strands of the MiMSP30 and GFP target amplification fragments, respectively, to obtain dsRNA forward and reverse strand templates. The primer sequences for the PCR amplification reaction are shown in Table 2—SEQ ID No. 4 and SEQ ID No. 5 were used as primers to amplify the dsRNA-MSP30 forward strand template; SEQ ID No. 6 and SEQ ID No. 7 were used as primers to amplify the dsRNA-MSP30 reverse strand template; SEQ ID No. 8 and SEQ ID No. 9 were used as primers to amplify the dsRNA-GFP forward strand template; and SEQ ID No. 10 and SEQ ID No. 11 were used as primers to amplify the dsRNA-GFP reverse strand template. The PCR amplification reaction system was prepared as follows (12.5 μL I-5). TM 2× High-Fidelity Master Mix, 1 μL 10 μM front primer, 1 μL 10 μM back primer, 1 μL template, and deionized water to a final volume of 25 μL. PCR reaction program: 98℃, 2 min; 98℃, 10 s, 55℃, 15 s; 72℃, 15 s, 35 cycles; 72℃, 2 min. After product recovery, dsRNA-MSP30 and dsRNA-GFP templates were obtained.

[0058] Table 2. Primer sequences for dsRNA synthesis template amplification

[0059]

[0060] 2. Synthesis of dsRNA-MSP30 and dsRNA-GFP

[0061] Following the instructions of the T7 RNAi Transcription Kit (Novizan), dsRNA-MSP30 and dsRNA-GFP were synthesized in vitro. The reaction system was prepared as follows: (8 μL NTP Mix, 2 μL 10×Transcription Buffer, 2 μL T7 Enzyme Mix, 0.5 μg positive strand template, 0.5 μg reverse strand template, and RNase-free deionized water to a final volume of 20 μL). After gentle pipetting and mixing, the mixture was incubated at 37°C for 2 h and then allowed to cool naturally to anneal and form dsRNA.

[0062] 3. dsRNA double enzyme digestion and purification

[0063] Double enzyme digestion and purification of dsRNA-MSP30 and dsRNA-GFP were performed according to the T7 RNAi Transcription Kit (Novizan) instructions. The specific steps are as follows:

[0064] ① Prepare a dual-enzyme digestion system (20 μL transcription product, 17 μL RNase-free deionized water, 1 μL LDNase I, 2 μL 10 U / μL RNase T1), gently pipette and mix well, then incubate at 37℃ for 30 min.

[0065] ② Add 80 μL of magnetic bead solution that has been equilibrated at room temperature for 30 min to the digestion product, gently mix with a pipette, and incubate at room temperature for 8 min.

[0066] ③ Place the sample on the magnetic rack for about 5 minutes until the solution becomes clear, and remove the supernatant without disturbing the magnetic beads.

[0067] ④ Keep the sample on the magnetic rack, add 200 μL of freshly prepared 80% ethanol without disturbing the magnetic beads, incubate at room temperature for 30 seconds, and then carefully remove the supernatant. Repeat this step once.

[0068] ⑤ Allow the sample to stand at room temperature for 5-10 minutes to allow any residual ethanol to evaporate.

[0069] ⑥ Remove the sample from the magnetic rack, add 40 μL of RNase-free deionized water, use a pipette to remove the magnetic beads from the tube wall, mix the deionized water and magnetic beads thoroughly, and incubate at room temperature for 3 min.

[0070] ⑦ Place the sample back on the magnetic rack. After the solution clarifies, transfer the supernatant to a new RNase-free EP tube without disturbing the magnetic beads to obtain purified dsRNA.

[0071] Example 3

[0072] Using dsRNA-GFP as a control, approximately 20,000 freshly hatched second-instar nematodes were collected in 1.5 mL centrifuge tubes without RNase. The nematodes were washed twice with RNase-free deionized water, and the supernatant was carefully removed using a pipette. 50 μL of dsRNA-MSP30 was added to each 1.5 mL centrifuge tube to achieve a final dsRNA concentration of 2 μg / μL, and the tubes were incubated at 25°C for 4 hours, with the samples being rotated continuously during incubation. After incubation, the dsRNA-treated nematodes were divided into two groups. One group was used to determine the silencing efficiency of dsRNA feeding, and the other group was used for statistics on the number of infected root-knot nematodes and the developmental progress within the roots.

[0073] 1. Detection of silencing efficiency after dsRNA feeding

[0074] Total RNA was extracted from nematodes fed with dsRNA-MSP30 and dsRNA-GFP, respectively, and reverse transcribed into cDNA. The total RNA extraction and cDNA synthesis steps were the same as in Example 1. Using MiGAPDH as an internal control gene and cDNA diluted 10-fold as a template, the silencing efficiency against dsRNA-MSP30 feeding treatment was detected by quantitative real-time PCR (qPCR). The detection primers are shown in Table 3, and the detection system was a 7500 Real-Time PCR system (ABI). The specific steps are as follows: Following 2× According to the Master qPCR Mix (SYBR Green I) (Qingke Biotechnology) instructions, prepare the qPCR reaction system (10μL 2× The Master qPCR Mix consisted of 0.8 μL of 10 μM pre-primer, 0.8 μL of 10 μM post-primer, 2 μL of cDNA template, 0.4 μL of 50×ROX Reference Dye II, and deionized water to a final volume of 20 μL. The qPCR reaction program was as follows: 95℃, 1 min; 95℃, 10 s; 60℃, 30 s, 40 cycles (fluorescence signal was acquired at 60℃); 95℃, 15 s; 60℃, 1 min; 95℃, 15 s. Data analysis was performed using the ΔΔCT method with three biological replicates. Differences were analyzed using t-tests.

[0075] Table 3. Specific primer sequences for qPCR detection

[0076]

[0077] Experimental results showed that the silencing efficiency of MiMSP30 in root-knot nematodes was 70.02% after dsRNA-MSP30 feeding treatment.

[0078] 2. Statistics on the number of root-knot nematodes 7 days after infection

[0079] Nematodes fed with dsRNA-GFP were used as a control. The supernatant of a nematode suspension fed with dsRNA-MSP30 was removed by sedimentation and mixed with 23% Pluronic F-127 (Sigma) pre-cooled to 4°C. Arabidopsis Col-0 seedlings cultured on Murashige and Skoog (MS) medium for two weeks were carefully picked up with forceps and their roots were immersed in the nematode mixture. Each seedling was inoculated with 200 nematodes. The nematode mixture containing the Arabidopsis seedlings was placed in a 25°C plant culture incubator (10h light, 14h dark) for 7 days. Diseased root tissues were rinsed several times with distilled water to remove residual colloid. The diseased root tissues were stained with sodium hypochlorite-acid fuchsin, and the number of nematodes in the roots was counted. This experiment was performed in triplicate, and the t-test was used for difference analysis.

[0080] The experimental results showed that the average number of root-knot nematodes infected after feeding with dsRNA-MSP30 (7 days) was 19.86 nematodes / plant; the average number of root-knot nematodes infected in the control group (7 days) was 37.64 nematodes / plant. The number of root-knot nematodes infected after feeding with dsRNA-MSP30 was significantly lower than that in the control group, with a reduction of 47.24%.

[0081] 3. Statistics on the developmental process of root-knot nematodes within the roots 14 days after infection

[0082] Nematodes fed with dsRNA-GFP were used as a control. A nematode suspension fed with dsRNA-MSP30 was mixed with an equal volume of 1% sodium carboxymethyl cellulose solution and applied as a root drenching treatment to the base of the stems of 6-week-old Arabidopsis thaliana Col-0 seedlings cultured in sandy soil. Each seedling was inoculated with 500 nematodes and placed in a 25℃ plant culture incubator (10h light, 14h dark). Diseased root tissues were rinsed several times with distilled water to remove residual sand. The root tissues were stained with sodium hypochlorite-acid fuchsin, and the number of nematodes at different instars within the roots was counted. The proportion of nematodes in the total nematode population was analyzed to assess the nematode developmental process within the roots. This experiment was performed in triplicate, and the t-test was used for difference analysis.

[0083] The experimental results showed that after feeding with dsRNA-MSP30, the percentages of different larval stages of root-knot nematodes (after 14 days) were: 74.02% second-instar larvae, 15.01% third-instar larvae, and 10.79% fourth-instar larvae; while in the control group, the percentages were: 63.67% second-instar larvae, 17.67% third-instar larvae, and 18.18% fourth-instar larvae. Compared with the control group, the developmental rate of root-knot nematodes was significantly slowed down after feeding with dsRNA-MSP30.

[0084] Example 4

[0085] dsRNA-GFP was used as a control. Tomato seedlings aged 4 weeks were selected and inoculated with root-knot nematodes after being fed with dsRNA. The nematode feeding method for dsRNA-MSP30 and dsRNA-GFP was the same as that for nematode inoculation in Example 3. The inoculated tomato seedlings were placed in a greenhouse at 25℃ (14h light, 10h darkness) for 45 days. The diseased root tissues were rinsed several times with distilled water to remove residual soil from the tissues, and the number of root knots and egg masses on each seedling was counted.

[0086] The experimental results showed that after feeding with dsRNA-MSP30, the number of root knots in infected tomato plants was 246.67 per plant, and the number of egg masses was 23.91 per plant; while the number of root knots in the control group was 330.50 per plant, and the number of egg masses was 50.82 per plant. The number of root knots and egg masses caused by root-knot nematode infection in tomatoes after feeding with dsRNA-MSP30 was significantly lower than that in the control group, with a 25.36% reduction in root knots and a 52.95% reduction in egg masses.

[0087] Example 5

[0088] 1. Construction of HIGS vectors targeting MiMSP30 and GFP

[0089] The amplification of the target fragments of MiMSP30 and GFP was performed using the Nimble Cloning kit (Yita Biotechnology) and I-5. TM The procedure was performed using 2× High-Fidelity Master Mix (Qingke Biotechnology). Using the full-length MiMSP30 encoding gene sequence and pSAT5-EGFP-N1 as templates, PCR amplification was performed to add 20bp universal adapter sequences to the 5' and 3' ends of the target amplified fragments of MiMSP30 and GFP, respectively. The primer sequences for the pre- and post-PCR amplification reactions are shown in Table 4—SEQ ID No. 14 and SEQ ID No. 15 were used as primers to amplify the MSP30 target amplified fragment with universal adapter sequences added to the 5' and 3' ends; SEQ ID No. 16 and SEQ ID No. 17 were used as primers to amplify the GFP target amplified fragment with universal adapter sequences added to the 5' and 3' ends. The reaction system and amplification procedure for the target fragment amplification were the same as in Example 2. After recovery of the amplification products, the MiMSP30 and GFP target amplified fragments with universal adapter sequences at the 5' and 3' ends were obtained, respectively.

[0090] The construction of HIGS vectors targeting MiMSP30 and GFP was performed according to the Nimble Cloning kit (Yita Biotechnology) instructions. The selected HIGS vector was pNC-Cam1304-RNAi (Yita Biotechnology). The NC cloning reaction mixture was prepared as follows: 10-80 ng target amplification fragment, 20-120 ng HIGS expression vector, 5 μL Nimble Mix, and sterile deionized water to a final volume of 10 μL. The mixture was pipetted and incubated at 50°C for 30-60 min. Then, 2-5 μL of the reaction product was transformed into 100 μL of a 100 μL transformation solution with a transformation efficiency greater than 10%. 8 Escherichia coli DH5α competent cells were plated on Luria Bertani (LB) plates containing 50 μg / mL kanamycin and 5 μg / mL chloramphenicol and incubated overnight at 37°C.

[0091] Single colonies grown on the plates were selected for PCR identification. The primers for identification are shown in Table 4—two pairs of primers, SEQ ID No. 18 and SEQ ID No. 20 and SEQ ID No. 19 and SEQ ID No. 21, were used to identify the single colonies. Single colonies were picked and resuspended in 20-30 μL of sterile deionized water as PCR templates. The PCR identification reaction system was prepared according to the 2×T5 Super PCR Mix (Qingke Biotechnology) instructions (12.5 μL 2×T5 Super PCR Mix, 1 μL 10 μM front primer, 1 μL 10 μM back primer, 3 μL template, and sterile deionized water to a final volume of 25 μL); the reaction program was: 98℃, 5 min; 98℃, 10 s, 55℃, 15 s; 72℃, 15 s, 35 cycles; 72℃, 2 min. After electrophoresis of the PCR products, positive clones were selected for sequencing verification. If both inserted sequences were correctly sequenced, the positive clones were mixed with an equal volume of 80% glycerol and stored at -80℃.

[0092] Table 4 Primer sequences for monoclonal PCR detection

[0093]

[0094] 2. Construction of HIGS Agrobacterium targeting MiMSP30 and GFP

[0095] HIGS vectors targeting MiMSP30 and GFP were extracted from positive clones. Agrobacterium LBA4404 transfected competent cells (Qingke Biotechnology) were placed on ice. 2-5 μL of HIGS vector was added to the undried competent cells, and the cells were incubated at 37°C for 5 min. Then, the cells were gently transferred to ice and incubated for another 2-5 min. 1 mL of LB liquid medium was added to the ice-bathed mixture of competent cells and vectors, and the bacterial suspension was incubated at 28°C with shaking at 200 rpm for 2-4 h. 100 μL of the shaken bacterial suspension stock solution and 100 μL of the concentrated solution (centrifuged at 5000 rpm for 1 min at room temperature and the supernatant removed) were plated onto LB agar plates containing 50 μg / mL rifampin, 50 μg / mL kanamycin, and 5 μg / mL chloramphenicol, and incubated at 28°C for 2 days. Single clones grown on the plates were selected for PCR identification. The primers, reaction system, and reaction procedure were the same as in point 1 of Example 5.

[0096] 3. Construction of HIGS transgenic plants targeting MiMSP30 and GFP

[0097] The HIGS transgenic plants were constructed using Agrobacterium-mediated leaf disc transformation, with the tomato variety Money Maker as the plant material. The specific steps were modified from those described by Cao et al. (2008) and Liang et al. (2009). The detailed steps are as follows:

[0098] ① Disinfect tomato seeds with 75% ethanol solution for 1 min, soak in 2% sodium hypochlorite solution for 15 min, and wash seeds 6-10 times with sterile water. Sow seeds at appropriate intervals on MS solid medium, and incubate in the dark in a 24-26℃ light incubator for 3-4 days. Then transfer to a 16h light / 8h dark environment for incubation until the cotyledons are fully expanded (approximately 2 days of seedling age).

[0099] ② When the tomato seedlings are 2 days old, Agrobacterium is activated on LB plates using the streak plate method. After being incubated upside down in a 28°C incubator for 24 hours, Agrobacterium is inoculated into LB liquid medium and cultured at 28°C and 200 rpm for nearly 24 hours with shaking.

[0100] ③ Take fully extended tomato cotyledons, remove the front and back sections, leaving the middle 0.5cm leaf. Place it with the adaxial surface facing up on the pre-medium (MS solid medium with added auxin at a final concentration of 0.2mg / L and zeatin at a final concentration of 2mg / L) and incubate in the dark at 24-26℃ for 2 days.

[0101] ④ Centrifuge at 5000 rpm to enrich Agrobacterium, resuspend Agrobacterium in MS medium and adjust the bacterial concentration to OD. 600 =0.4-0.6, place in a 5mL centrifuge tube, add acetyl syringone to a final concentration of 100μM / L, and let stand in the dark for 30min.

[0102] After soaking tomato explants in bacterial suspension for 25-30 minutes, use sterile filter paper to blot off any remaining bacterial suspension from the tissue blocks, and place them adaxially upwards on a new pre-culture medium. Co-culture at 24-26℃ in the dark for 2 days.

[0103] ⑤ After dark culture, remove the explants and rinse them with sterile water 6-10 times, 30 min each time. Finally, rinse the explants with a carbenicillin solution with a final concentration of 250 mg / L for 30 min. After blotting off any remaining moisture with sterile filter paper, place them adaxially upwards on a new pre-culture medium and incubate them in a 24-26℃ light incubator (16 h light / 8 h dark) for 3 days.

[0104] ⑥ Transfer the explants adaxially upward to differentiation medium (MS solid medium with added auxin at a final concentration of 0.2 mg / L, zeatin at a final concentration of 2 mg / L, carbenicillin at a final concentration of 400 mg / L, and hygromycin at different concentrations), and subculture every 2 weeks until resistant shoots differentiate.

[0105] ⑦ When the resistant buds grow to about 1 cm in length, cut off the resistant buds and insert the base of the stem into the rooting medium (MS solid medium with added auxin at a final concentration of 0.1 mg / L and carbenicillin at a final concentration of 400 mg / L). Cultivate until a large number of roots grow out, wash off the culture medium from the roots and transplant them into the soil. Cultivate at 24-26℃ under 16h light / 8h dark conditions.

[0106] ⑧ Extract DNA from resistant plants according to the instructions of the Rapid DNA Extraction and Detection Kit (TIANGEN) and use it as a template for PCR amplification. The primers used for amplification are the same as those in point 1 of Example 5. The PCR amplification reaction system (10 μL 2×Det PCRMaster Mix, 0.5 μL 10 μM front primer, 0.5 μL 10 μM back primer, 1 μL template DNA, and deionized water to make up to 20 μL) was as follows: 94℃, 3 min; 94℃, 30 s, 55℃, 30 s; 72℃, 1 min, 35 cycles; 72℃, 5 min. The PCR amplification results were detected by electrophoresis.

[0107] Example 6

[0108] HIGS-GFP transgenic tomato seedlings were used as a control. Four-week-old HIGS transgenic tomato seedlings were inoculated with wild-type root-knot nematodes, using the same inoculation method as in Example 3. The HIGS-MSP30 gene silencing efficiency was tested seven days after nematode inoculation, and the number of root knots and egg masses was counted forty-five days after inoculation.

[0109] 1. Detection of HIGS silencing efficiency

[0110] HIGS transgenic tomato seedlings inoculated with root-knot nematodes for 7 days were used to extract total RNA from the mixed tomato tissue and the infected root-knot nematodes. The extraction procedure was performed according to the instructions of the RNAsimple Total RNA Extraction Kit (TIANGEN). Detailed steps are as follows:

[0111] ① Grind tomato root tissue infected with root-knot nematodes with liquid nitrogen. Add 1 mL of lysis buffer RZ to every 100 mg of tissue, vortex for 1 min, and then let the sample stand at room temperature for 5 min.

[0112] ② Add chloroform to the sample in a volume equal to 0.2 times that of the lysis buffer RZ, vortex for 1 min, and then let stand at room temperature for 3 min.

[0113] ③ Centrifuge the sample at 4℃ and 12000rpm for 10min, and transfer the upper aqueous phase to a new RNase-free centrifuge tube.

[0114] ④ Add anhydrous ethanol to the aqueous phase at a volume of 0.5 times that of the aqueous phase, mix thoroughly by suction and blow, then transfer to the adsorption column CR3, centrifuge at 12000 rpm for 30 s at 4℃, and remove the filtrate.

[0115] ⑤ Add 500 μL of protein removal solution RD to the adsorption column, centrifuge at 12000 rpm for 30 s at 4℃, and remove the filtrate.

[0116] ⑥ Add 700 μL of protein removal buffer RW to the adsorption column, centrifuge at 12000 rpm for 30 s at 4℃, and remove the filtrate. Repeat this step once.

[0117] ⑦ Transfer the adsorption column to a new RNase-free centrifuge tube, add 40 μL of RNase-free deionized water to the adsorption membrane, incubate at room temperature for 2 min, centrifuge at 12000 rpm for 2 min at 4℃, collect the eluent to obtain total RNA, and store at -80℃.

[0118] The cDNA synthesis steps were the same as in Example 1. The silencing efficiency of HIGS transgenic tomatoes against root-knot nematodes was detected by quantitative real-time PCR (qPCR), with the reaction system, reaction procedure, and primers used the same as in Example 3.

[0119] The experimental results showed that after wild-type second-instar larvae of root-knot nematodes infected HIGS-MSP30 transgenic plants, the silencing efficiency of MiMSP30 in the root-knot nematodes was 46.32%.

[0120] 2. Statistics on the number of root knots and egg masses

[0121] The number of root knots and egg masses in HIGS-GFP and HIGS-MSP30 transgenic tomato seedlings 45 days after inoculation with nematodes was calculated using the same method as in Example 4. The results showed that after inoculation with HIGS-MSP30 by wild-type nematodes, the number of root knots was 299.92 / plant and the number of egg masses was 60.17 / plant; while in the control group, the number of root knots was 389.50 / plant and the number of egg masses was 83.17 / plant. The number of root knots and egg masses in HIGS-MSP30 transgenic tomato seedlings infected with wild-type nematodes was significantly lower than that in the control group, with a 23.00% reduction in root knots and a 27.65% reduction in egg masses.

[0122] As can be seen from Examples 3, 4 and 6, both implementation methods can achieve the silencing effect on the MiMSP30 gene, which has a significant inhibitory effect on the infectivity of root-knot nematodes and the development process in roots, and effectively reduces the incidence of root-knot nematode disease, showing great application potential.

[0123] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0124] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A dsRNA for inhibiting root-knot nematode disease, characterized in that, The method for preparing the dsRNA includes the following steps: Using the nucleotide shown in SEQ ID No. 3 as a template, and the sequences shown in SEQ ID No. 4 and SEQ ID No. 5, or the sequences shown in SEQ ID No. 6 and SEQ ID No. 7, respectively, PCR amplification was performed to obtain PCR amplification products. Using the aforementioned recovered PCR amplification products as templates, the positive and reverse strands of dsRNA were transcribed and synthesized, and then annealed to allow the positive and reverse strands to hybridize and form dsRNA.

2. The dsRNA for inhibiting root-knot nematode disease according to claim 1, characterized in that, The method for preparing the dsRNA includes the following steps: (1) Total RNA was extracted from Southern Root-knot Nematode and cDNA was obtained by reverse transcription. (2) Using the cDNA obtained in step (1) as a template, and the sequences shown in SEQ ID No. 1 and SEQ ID No. 2 as primers, PCR amplification was performed to obtain... MiMSP30 Full-length sequence; (3) with MiMSP30 Using the full-length sequence as a template, and using the sequences shown in SEQ ID No. 4 and SEQ ID No. 5, or the sequences shown in SEQ ID No. 6 and SEQ ID No. 7, as primers, the DNA sequence that can be used as a template for the synthesis of dsRNA positive and negative strands is amplified by PCR reaction; (4) Using the PCR amplification product recovered in step (3) as a template, the positive and reverse strands of dsRNA are transcribed and synthesized respectively, and annealed to allow the positive and reverse strands to hybridize and form dsRNA. (5) Residual single-stranded RNA and DNA are removed by enzymatic hydrolysis, and the hydrolysis products are recovered and purified to obtain the target gene. MiMSP30 dsRNA with RNAi effects.

3. A method for constructing a transgenic tomato plant, characterized in that, Includes the following steps: (1) with MiMSP30 Using the full-length sequence as a template, and the sequences shown in Seq ID No. 14 and Seq ID No. 15 as primers, the amplification was achieved through PCR. MiMSP30 The sequence was inserted into the HIGS vector sequence to obtain a sequence that simultaneously carries... MiMSP30 Sequence and MiMSP30 HIGS vector with reverse complementary sequence; (2) Transform the HIGS vector obtained in step (1) into Agrobacterium GV3101; (3) Using an Agrobacterium-mediated plant transgenic system, the transgenic material obtained in step (1) simultaneously carries... MiMSP30 Sequence and MiMSP30 The reverse complementary HIGS vector sequence was transformed into tomatoes to obtain dsRNA that can be synthesized in vivo. MSP30 Genetically modified tomato plants.

4. The application of the dsRNA according to claim 1 or 2 in the suppression of root-knot nematode disease.

5. A biological product comprising the dsRNA as described in claim 1 or 2.

6. The biological product according to claim 5, characterized in that, The biological product is a feed-type insecticide or bait.

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