Application of OsTMS5 gene in regulating the ability of rice to resist rice sawtoothed stunt virus

Abstract

This invention relates to the field of genetic engineering, specifically to... OsTMS5 Application of genes in regulating rice resistance to rice spur dwarf virus. Previous research in this invention found that... OsTMS5 The mutant material exhibited higher susceptibility levels, stronger virus accumulation, and more severe dwarfing symptoms under RRSV infection conditions; conversely, the mutant material showed increased susceptibility. OsTMS5 After expression level was reached, adverse phenotypes caused by RRSV infection were alleviated, and viral accumulation was reduced. Further studies have shown that... OsTMS5 Encoded RNase Z S1 The protein can degrade specific RRSV single-stranded RNA fragments, indicating that... OsTMS5 It is not only related to fertility regulation, but can also participate in the antiviral defense process of rice against RRSV.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering, specifically to the application of the OsTMS5 gene in regulating the resistance of rice to rice dwarf virus. Background Technology

[0002] Rice ragged dwarf disease is an important viral disease of rice caused by rice ragged dwarf virus (RRSV) and mainly transmitted by brown planthoppers. RRSV infection leads to significantly stunted plants, twisted and wrinkled leaves with characteristic serrated notches, and severely inhibits overall plant growth and development. In severe cases, it can also cause plants to fail to head or significantly reduce the grain-filling rate after heading, resulting in severe yield reduction. Because RRSV can be spread between different rice-growing areas via migratory brown planthoppers, and early symptoms after infection are difficult to accurately identify, this disease is characterized by its insidious transmission, high risk of epidemic spread, and difficulty in field control.

[0003] Currently, the control of RRSV in production mainly relies on measures such as vector control, disease monitoring, and field management. For example, chemical control of brown planthoppers, reduction of infected insect sources, and removal of diseased plants are used to reduce the risk of disease transmission. However, these measures primarily target the disease transmission stage, and their effectiveness is easily affected by factors such as insect population density, the proportion of infected insects, climatic conditions, and field management levels, making it difficult to fundamentally improve the resistance of rice materials to RRSV. Especially under conditions of persistent brown planthopper infestation or a high proportion of infected insects, relying solely on field control measures is still insufficient to effectively prevent the damage caused by RRSV infection. Therefore, breeding rice materials with stable RRSV resistance is an important direction for achieving sustainable, green, and efficient control of this disease. At present, research on RRSV resistance mainly focuses on disease phenotypic surveys, virus detection, germplasm resource screening, and the analysis of some disease susceptibility regulatory mechanisms. Although germplasm screening can obtain some materials with relatively mild symptoms under RRSV infection, the genetic basis and key regulatory genes for resistance formation are still unclear, making it difficult to directly use them for molecular breeding or the precise creation of resistant materials. In particular, there is currently a lack of rice endogenous functional genes that have been clearly proven to participate in restricting RRSV infection and can be further transformed into breeding targets.

[0004] On the other hand, existing studies have attempted to directly suppress RRSV virus gene expression using transgenic or RNA silencing strategies. For example, by constructing exogenous nucleic acid expression elements that target specific viral sequences, plants can produce corresponding small RNAs, thereby reducing viral RNA accumulation and alleviating disease symptoms. While this type of technology can improve the resistance of materials to RRSV to some extent, it essentially uses the viral sequence as the direct intervention target, belonging to a resistance construction strategy targeting the pathogen. Compared with conventional breeding or disease resistance improvement based on endogenous host genes, this technical approach has limitations such as a narrower breeding application path, reliance on exogenous component design, and high requirements for viral sequence matching. A universal breeding method based on enhancing rice's own antiviral capabilities has not yet been developed.

[0005] Oryza sativa thermosensitive male sterile 5 ( OsTMS5 ) is the core functional gene for the formation of temperature-sensitive male sterility in rice, encoding RNase Z S1 Proteins. Current research and applications mainly revolve around... OsTMS5 The role of this research in the creation of male-sterile lines, fertility regulation, and detection of related molecular markers in two-line hybrid rice was explored. OsTMS5 Thermosensitive two-line male sterile lines with functional deficiencies have important application value in current two-line hybrid rice production. However, current technologies mainly focus on the effects of OsTMS5 on rice fertility, without recognizing the functional link between OsTMS5 and rice resistance to RRSV, and without applying OsTMS5 as a molecular target for the creation of RRSV-resistant materials, disease resistance screening, and breeding improvement. Summary of the Invention

[0006] at present OsTMS5 Existing research mainly focuses on temperature-sensitive male sterility in rice and two-line hybridization breeding, while its role in RRSV resistance has not yet been explored and utilized. Therefore, this invention provides a... OsTMS5 Application of genes in regulating rice resistance to rice dwarf virus.

[0007] The specific technical solution is as follows: The present invention provides in a first aspect OsTMS5 The use of genes or related biological materials in at least one of the following: A1) Regulate the ability of rice to resist rice spur dwarf virus or prepare products that regulate the ability of rice to resist rice spur dwarf virus. A2) Cultivate rice varieties with enhanced resistance to rice spur dwarf virus or prepare products that enhance rice's resistance to rice spur dwarf virus; A3) Preparation of transgenic rice; The OsTMS5 The nucleotide sequence encoding the gene is shown in SEQ ID NO.1.

[0008] Furthermore, the biomaterial includes one or more of the following: The OsTMS5 The gene-encoded protein and / or containing the above OsTMS5 Gene recombinant vectors, recombinant microorganisms, or transgenic rice cell lines; The OsTMS5 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.2.

[0009] Furthermore, the regulation of rice's resistance to rice dwarf virus includes: by upregulating... OsTMS5 Gene expression to enhance rice's resistance to rice dwarf virus; Or, by lowering OsTMS5 Gene expression is used to reduce rice's resistance to rice dwarf virus.

[0010] The present invention provides an upward adjustment in a second aspect. OsTMS5 Application of gene-expressing biological materials in the cultivation of rice with enhanced resistance to rice spur leaf dwarf virus; OsTMS5 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0011] Further, the biomaterial includes: OsTMS5 The gene encodes a protein and / or contains upregulated proteins. OsTMS5 Recombinant vectors for gene expression, recombinant microorganisms, or transgenic rice cell lines.

[0012] Furthermore, the recombinant vector includes a base vector and OsTMS5 Gene.

[0013] Furthermore, the base carrier is the pCUbi1390 carrier.

[0014] Furthermore, the recombinant microorganism includes Agrobacterium.

[0015] Furthermore, the recombinant microorganism is Agrobacterium tumefaciens EHA105.

[0016] In a third aspect, the present invention provides a method for improving the resistance of rice to rice dwarf virus, comprising: Will be raised OsTMS5 Gene-expressing biological materials were transferred into rice; OsTMS5 The coding nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0017] In a fourth aspect, this invention provides a method for cultivating rice with enhanced resistance to rice spur dwarf virus, comprising: Will be raised OsTMS5Gene-expressing biological materials were transferred into rice; OsTMS5 The coding nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0018] The present invention provides, in a fifth aspect, a rice variety with enhanced resistance to rice spur dwarf virus, wherein the rice comprises upregulating... OsTMS5 Biological materials for gene expression; The biomaterial includes: OsTMS5 The gene encodes a protein and / or contains upregulated proteins. OsTMS5 Recombinant vectors for gene expression, recombinant microorganisms, or transgenic rice cell lines; OsTMS5 The coding nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0019] Compared with the prior art, the present invention has the following beneficial effects: This invention revolves around OsTMS5 This invention establishes a complete technical solution for regulating the resistance of rice to rice spur dwarf virus, encompassing functional discovery, mechanism analysis, creation of disease-resistant materials, and breeding applications. This invention is the first to clearly define... OsTMS5 It is an important host functional gene involved in rice resistance to RRSV, and it was revealed that OsTMS5 encodes the protein RNase Z. S1 An antiviral mechanism that selectively degrades RRSV single-stranded RNA. Furthermore, this invention establishes a feasible technical approach for creating RRSV-resistant rice materials by enhancing OsTMS5 expression, which can be used for RRSV resistance improvement in rice materials with different genetic backgrounds. Attached Figure Description

[0020] Figure 1 This diagram illustrates the mechanism of action of OsTMS5 in regulating rice resistance to RRSV and the creation of related materials. In this diagram, A represents the susceptibility of rice to RRSV due to the loss of OsTMS5 function; B represents the ability of normal OsTMS5 expression to limit RRSV accumulation to a certain extent; and C represents the ability of OsTMS5 overexpression to enhance resistance to RRSV.

[0021] Figure 2 The results of a field survey on RRSV infection status; among them, Figure 2 In the figure, A represents the comparison of RRSV infection rates among wild-type JXB, mutant JXS, wild-type DR610, and mutant DR610S, with each biological replicate consisting of 30 plants. Figure 2 B represents the comparison of plant heights of wild-type JXB, mutant JXS, wild-type DR610, and mutant DR610S under infected and uninfected conditions; statistical analysis was performed using Student's t-test combined with one-way ANOVA, and p<0.05 indicated significant differences.

[0022] Figure 3 The results of experiments involving artificial inoculation of OsTMS5 mutant and wild-type plants with RRSV in a greenhouse; among them, Figure 3 In the figure, A represents the comparison of RRSV infection rates between wild-type JXB and mutant JXS, and between wild-type DR610 and mutant DR610S, with 30 plants per biological replicate. Figure 3 In this context, B represents the comparison of the relative toxicity levels between wild-type JXB and mutant JXS, as well as between wild-type DR610 and mutant DR610S.

[0023] Figure 4 For RNase Z S1 Figure showing the in vitro degradation results of RRSV S6 and S9 single-stranded RNA; where, Figure 4 In this context, A represents different amounts of RNase Z. S1 Detection results after processing RRSV S6 single-stranded RNA; Figure 4 In this context, B represents different dosages of RNase Z. S1 Detection results after processing RRSV S9 single-stranded RNA.

[0024] Figure 5 This is a schematic diagram of the pCUbi1390-FLAG-OsTMS5 overexpression vector.

[0025] Figure 6 The image shows the results of functional identification of OsTMS5 overexpression lines against RRSV in the DR610 background; among them, Figure 6 In Figure A, the RRSV susceptibility rate of wild-type, OsTMS5 mutant, and two OsTMS5 overexpression lines is shown under artificial inoculation and natural disease conditions. Each scatter plot represents the statistical results of 30 rice plants. Figure 6 B in the figure shows the relative height of infected plants relative to healthy plants under artificial inoculation and natural infection conditions. Each scatter point represents the statistical results of 30 rice plants. Figure 6 C in the figure shows the relative expression level of OsTMS5 in the OsTMS5 overexpression line and the wild type; Figure 6 The results of FLAG tag protein detection in wild-type and OsTMS5 overexpression lines are shown in D, with Actin as an internal control; Figure 6 E in the figure shows the comparison of plant height between OsTMS5 overexpression homozygous lines, wild type and OsTMS5 mutant under normal conditions and 14 days after RRSV inoculation.

[0026] Figure 7 The image shows the results of functional identification of OsTMS5 overexpression lines against RRSV in the JXB background; among them, Figure 7In Figure A, the RRSV susceptibility rate of wild-type JXB, OsTMS5 mutant JXS, and two JXB background OsTMS5 overexpression lines is shown under artificial inoculation and natural disease conditions. Each scatter plot represents the statistical results of 30 rice plants. Figure 7 B in the figure shows the relative height of infected plants relative to healthy plants under artificial inoculation and natural infection conditions. Each scatter point represents the statistical results of 30 rice plants. Figure 7 C in the figure shows the relative expression level of OsTMS5 in the background OsTMS5 overexpression line of JXB and wild-type JXB; Figure 7 The D in the figure shows the detection results of FLAG tag protein in wild-type JXB and JXB background OsTMS5 overexpression lines, with Actin as an internal control. Detailed Implementation

[0027] Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. The experimental materials used in the embodiments of this invention are all conventional experimental materials in the art and are commercially available. Experimental methods not specifying detailed conditions are performed according to conventional experimental methods or the supplier's recommended operating instructions.

[0028] In the following examples, the rice varieties were selected from japonica rice JXB and indica rice DR610.

[0029] In the following embodiments, OsTMS5 The nucleotide sequence encoding the gene is shown in SEQ ID NO.1; the amino acid sequence of the OsTMS5 protein is shown in SEQ ID NO.2.

[0030] SEQ ID NO.1: ATGGCGAACAGCGGCAAGTCATCGCCGGCGGCGACCTCCACCACCGCGCCGCCACCGGGTCGGCCGAAGGCGAAGGCGCCGCCCCTCACCGTCGAGGGCTACCCCGTGGAGGGCATCTCCATCGGCGGGCAGGAGACCTGCGTCATCTTCCCGACGCTGAGCGCCGCCTTCGACATCGGCCGGTGCCCGCAGCGCGCCGTCTCGCAGGAGTTCCTCTTCATCTCCCACGCCCACCTCGACCACATCGGCGGCCTCCCCATGTACGTCGCCACCCGGGGCCTCTACCGGCAGCGCCCGCCCACCATCTTCATCCCCGCCTGCCTCAGGGACCCCGTGGAGCGCCTCTTCGAGCTCCACCGCTCCATGGACCAGTCCGAGCTCAGCCACAACCTCGTCCCCCTCGAGATTGGTCAGGAGCACGAGCTCAGGAGGGACCTCAAGGTGAAGGCCTTCAAGACCTACCACGCCATTCCCAGCCAGGGGTATGTGATATACACGGTGAAGCAAAAGCTCAAGCCAGAGTATCTTGGCCTCCCTGGGAGCGAGATCAAGCAGCTGAAGCTGTCAGGTGTGGAGATTACGAATACATTGACGGTGCCTGAGATTGCTTTTACCGGAGATACGATGGCAGATTTCATTCTTGATCCTGATAATGCGGATGTTTTGAAGGCGAAAATTCTTGTAGTGGAGAGTACTTTTGTTGATGACTCTGTTACAATTGAGCATGCAAGAGAATATGGGCACACCCATCTGTTTGAGATACTGAATCAGTGTGACAAACTTGAAAACAAAGCTATTCTGCTAATCCACTTTTCTGCTCGTTATACCGCAGAGGAAATTGATATAGCAATCAATAAGTTGCCACCCTCTTTCAGAAGTAGAGTTCATGCATTGAAGGAAGGTTTCTGA。

[0031] SEQ ID NO.2: MANSGKSSPAATSTTAPPPGRPKAKAPPLTVEGYPVEGISIGGQETCVIFPTLSAAFDIGRCPQRAVSQEFLFISHAHLDHIGGLPMYVATRGLYRQRPPTIFIPACLRDPVERLFELHRSMDQSELSHNLVPLEIGQEHELRRDLKVKAF KTYHAIPSQGYVIYTVKQKLKPEYLGLPGSEIKQLKLSGVEITNTLTVPEIAFTGDTMADFILDPDNADVLKAKILVVESTFVDDSVTIEHAREYGHTHLFEILNQCDKLENKAILLIHFSARYTAEEIDIAINKLPPSFRSRVHALKEGF.

[0032] In the following examples, the nucleotide sequences used for protein expression are shown in SEQ ID NO.13~SEQ ID NO.14; the nucleotide sequences used for overexpression are shown in SEQ ID NO.15~SEQ ID NO.16; the nucleotide sequences used for overexpression identification are shown in SEQ ID NO.17~SEQ ID NO.18; and the nucleotide sequences used for homozygous identification are shown in SEQ ID NO.19~SEQ ID NO.24.

[0033] pET-OsTMS5-F (SEQ ID NO. 13): TAAGAAGGAGATATACATATGATGGCGAACAGCGGCAAG; pET-OsTMS5-R (SEQ ID NO. 14): TTCCTGCAGAAGCTTGGATCCGAAACCTTCCTTCAATGCATGAA; pCUbi1390-OsTMS5-F (SEQ ID NO. 15): TACCTGCAGGTCGACGGATCCATGGACTACAAAGACCATGACGGTGATTATAAAGATCATGATATCGATTACAAGGATGACGATGACAAGATGGCGAACAGCGGCAAG; pCUbi1390-OsTMS5-R (SEQ ID NO. 16): GTGGCTAGCGTAACACTAGTTCAGAAACCTTCCTTCAATGCA; pCUbi1390-JD-F (SEQ ID NO. 17): CGATGCTCACCCTGTTGTTTGG; pCUbi1390-JD-R (SEQ ID NO. 18): AAGACCGGCAACAGGATTCA; DR610-OE-9-P1-F (SEQ ID NO. 19): TGTGACCAAGTTCACAAGAATA; DR610-OE-9-P2-R (SEQ ID NO. 20): TGTGCCTCGCTAATTAACGG; DR610-OE-9-P3-F (SEQ ID NO.21): ATCAATAAGTTGCCACCCTCT; DR610-OE-10-P1-F (SEQ ID NO. 22): TGCAGGCGATATTGAATCCACAC; DR610-OE-10-P2-R (SEQ ID NO. 23): GAAAACACATGAAGGCACCACAAC; DR610-OE-10-P3-F (SEQ ID NO. 24): AGCAATCAATAAGTTGCCACC.

[0034] Example 1 OsTMS5 Materials lacking functionality exhibited higher susceptibility to RRSV infection under natural field conditions. 1.1 Experimental Objective To verify whether the loss of OsTMS5 function leads to an increase in the susceptibility of rice to RRSV infection under natural field conditions, and to analyze its effect on the degree of growth inhibition of infected plants.

[0035] 1.2 Experimental Materials This experiment selected two groups of rice materials with corresponding genetic background relationships: 1) Japonica rice background material: JXB is a wild-type material with normal OsTMS5 function; JXS is an OsTMS5 loss-of-function mutant obtained from the JXB background.

[0036] 2) Indica rice background material: DR610 is a wild-type material with normal OsTMS5 function; DR610S is an OsTMS5 loss-of-function mutant obtained from the DR610 background.

[0037] 1.3 Experimental Methods (1) Field natural infection experiment The above materials were planted in field environments where RRSV naturally occurs and brown planthopper infestations are relatively severe, thus exposing them to natural contact with the toxic insect. All materials were cultivated and managed using the same methods to ensure comparability between different genotypes.

[0038] (2) Detection of RRSV infection status Rice leaf samples were collected at typical disease occurrence stages, total RNA was extracted and reverse transcribed, and RT-PCR was performed using RRSV-specific primer S8 to determine the proportion of RRSV-positive plants in each material and calculate the susceptibility rate.

[0039] RRSV S8-F (SEQ ID NO.3): CTGAATACAACCGATACCGT; RRSV S8-R (SEQ ID NO. 4): GACCACTGTTACTGCCTTTA.

[0040] (3) Plant phenotypic survey After RRSV infection, the plant height of different materials was measured and compared with the corresponding wild type. OsTMS5 Differences in the degree of dwarfing among mutants.

[0041] 1.4 Experimental Results The results are as follows Figure 2 As shown in Figure A, the RRSV susceptibility rate of JXS is 18.9% higher than that of JXB; the RRSV susceptibility rate of DR610S is 25.6% higher than that of DR610. Figure 2 As shown in B, after infection with RRSV, JXS showed more significant dwarfing than JXB. In terms of the degree of plant height reduction, the plant height of JXS decreased by about 29.6% after infection, while the plant height of JXB decreased by about 17.1% after infection.

[0042] This example demonstrates that under natural RRSV infection conditions in the field, OsTMS5 Loss of function significantly increases susceptibility to RRSV in rice and exacerbates the dwarfing phenotype caused by viral infection. Therefore, OsTMS5 It is an important endogenous functional gene that affects the susceptibility of rice to RRSV, and its functional integrity is closely related to the rice's resistance to RRSV.

[0043] Example 2 OsTMS5 Loss-of-function materials exhibit stronger RRSV susceptibility under artificial inoculation conditions. 2.1 Experimental Objective Further verification under indoor artificial virus exposure conditions OsTMS5 Effects of loss of function on RRSV susceptibility, viral accumulation, and susceptible phenotype in rice.

[0044] 2.2 Experimental Materials The materials are the same as in Example 1, including JXB and JXS, DR610 and DR610S.

[0045] 2.3 Experimental Methods (1) Preparation of RRSV-carrying brown planthoppers A stable source of RRSV-infected brown planthoppers was established and maintained. Adult brown planthoppers of uniform morphology were selected before inoculation for artificial virus transfer to different rice materials.

[0046] (2) Artificial poisoning treatment Select rice seedlings that are 14 days old and have uniform growth. For different materials, use the same number of insects (30 seedlings to 40 seedlings) and the same 2-day inoculation time to ensure the reliability of the comparison results.

[0047] (3) RRSV detection Leaf samples were collected after inoculation, and the proportion of RRSV-positive plants was detected by RT-PCR using the RRSV S8 primers from Example 1.

[0048] (4) Detection of viral protein accumulation Use RRSV S10 primers, and with OsActin As internal control primers, real-time quantitative PCR was used to detect the relative accumulation level of RRSV RNA in different materials to further verify the differences in viral amplification in materials with different genotypes.

[0049] RRSV S10-F (SEQ ID NO.5):TTCTCCACTGCGCTGTCTTA; RRSV S10-R (SEQ ID NO. 6): ACGAGTGATGATCCTCCAA; OsActin-F (SEQ ID NO.7): CAGCACATCCAGCAGAT; OsActin-R (SEQ ID NO. 8): GGCTTAGCATTCTTGGGT.

[0050] 2.4 Experimental Results The results are as follows Figure 3 As shown in Figure A, under artificial inoculation conditions, the RRSV susceptibility rate of JXS reached 93.33%, approximately 19.1% higher than that of JXB; the susceptibility rate of DR610S was approximately 13.3% higher than that of DR610. Real-time quantitative PCR results showed that... OsTMS5 The accumulation of RRSV RNA in mutant materials was higher than that in the corresponding wild type. Figure 3 (B in the example). This example further proves that... OsTMS5 Functional deficiencies not only increase the risk of RRSV infection under natural field conditions, but also lead to an increase in the infection rate of materials under uniform artificial transmission conditions. OsTMS5 The absence of this substance promotes the accumulation of RRSV in rice, resulting in elevated viral RNA levels. OsTMS5 It can play a positive defensive role in rice resistance to RRSV. This result provides a basis for further enhancement... OsTMS5 This provides direct genetic evidence that the function enhances rice's resistance to RRSV.

[0051] Example 3 OsTMS5 Encoding protein RNase Z S1 Selectively degrades specific RRSV single-stranded RNA 3.1 Experimental Objective Validation of OsTMS5 encoded RNase Z S1 To determine whether the protein can directly act on RRSV single-stranded RNA and to elucidate the molecular mechanism by which it limits viral accumulation.

[0052] 3.2 Experimental Materials (1) RNase Z obtained through recombinant expression and purification S1 protein; (2) RRSV single-stranded RNA substrates prepared by in vitro transcription include: RRSV S6 fragment single-stranded RNA; RRSV S9 fragment single-stranded RNA; and negative control GFP single-stranded RNA.

[0053] 3.3 Experimental Methods (1) RNase Z S1 Protein preparation The CDS coding sequence of OsTMS5 (SEQ ID NO.1) was constructed into a protein expression vector, and recombinant RNase Z was obtained after induction of expression. S1 The protein was purified using his-tag affinity to obtain the target protein that can be used for in vitro reactions.

[0054] (2) In vitro transcription of RRSV single-stranded RNA In vitro transcription templates were constructed based on the target sequences of RRSV S6 and S9 fragments to prepare the corresponding single-stranded RNA substrates. The RNA was then purified, and its integrity was assessed.

[0055] RRSV S6-F (SEQ ID NO.9): TAATACGACTCACTATAGGGTTCAGCCTGGGAGCGTTACA; RRSV S6-R (SEQ ID NO. 10): CATGCTCCGCTTAGGCTTGA; RRSV S9-F (SEQ ID NO. 11): TAATACGACTCACTATAGGGATGCCTTTCGTGCAATTCC; RRSV S9-R (SEQ ID NO. 12): CTACTCTGCGTCATCACCAAA.

[0056] (3) In vitro RNA degradation reaction Purified RNase Z S1 The protein was incubated with either RRSV S6 ssRNA or S9 ssRNA, respectively. To determine whether degradation was dose-dependent, different protein dosage gradients were set up, including 0, 0.125, 0.25, 0.5, and 1 µg, and an option was set without the addition of RNase Z. S1 Protein was expressed as a control by adding 1 µg of blank vector to express purified protein.

[0057] (4) Detection of degradation products After the reaction, RNA bands were analyzed by urea PAGE gel to compare the degree of degradation of the target RNA under different treatments.

[0058] 3.4 Experimental Results Experimental results are as follows Figure 4 As shown, RNase Z S1 The protein can significantly degrade the RRSV S6 fragment of single-stranded RNA; with RNase Z S1 As the amount of protein added increased, the intact band of S6 ssRNA gradually weakened, indicating increased degradation, and significant small degraded bands appeared. Figure 4 (The red box in section A). Under the same reaction conditions, RNase Z S1 The protein did not show significant degradation activity against the RRSV S9 fragment single-stranded RNA. Figure 4 (B in the text); indicating RNase Z S1 OsTMS5 does not indiscriminately degrade RRSV RNA; rather, it exhibits substrate selectivity for different viral RNA fragments. This example demonstrates that OsTMS5 encodes RNase Z. S1 The protein has the ability to directly act on specific RRSV single-stranded RNAs. Therefore, the OsTMS5-mediated anti-RRSV effect is not simply caused indirectly by a general disease resistance response, but is at least partially mediated by RNase Z. S1 This mechanism achieves its effect by selectively degrading viral RNA and reducing viral accumulation. This provides direct molecular evidence for OsTMS5 as a breeding target for resistance to RRSV.

[0059] Example 4: Construction of pCUbi1390-FLAG-OsTMS5 overexpression vector and creation of OsTMS5 overexpression rice materials 4.1 Experimental Objective OsTMS5 overexpression vectors were used to create OsTMS5 enhanced expression materials in indica and japonica rice genetic backgrounds, providing a material basis for subsequent RRSV resistance evaluation and breeding applications.

[0060] 4.2 Construction of OsTMS5 overexpression vector (1) Target gene The CDS coding sequence of OsTMS5 was used as the target gene, as in Example 3.

[0061] (2) Plant expression vectors The pCUbi1390 plant overexpression vector was used as the backbone. The vector map is shown below. Figure 5 As shown.

[0062] (3) Expression box design The coding sequence of OsTMS5 was ligated into the pCUbi1390 vector, and enhanced expression was achieved under the drive of the maize ubiquitin promoter Ubi. Simultaneously, a FLAG tag was fused to the OsTMS5 protein terminus for subsequent protein expression detection using an anti-FLAG antibody. The resulting overexpression vector is denoted as: pCUbi1390-FLAG-OsTMS5.

[0063] 4.3. Genetic transformation of rice The constructed pCUbi1390-FLAG-OsTMS5 vector was transformed into Agrobacterium tumefaciens ( ). Agrobacterium tumefaciens The EHA105 strain was introduced into JXB and DR610 using Agrobacterium-mediated rice genetic transformation. After tissue culture, resistance screening, and regeneration, the corresponding OsTMS5 overexpressing transformed plants were obtained.

[0064] 4.4 Molecular identification of overexpression materials (1) Preliminary identification of positive plants Genomic DNA was extracted from the regenerated plants, and vector-specific fragments were detected by PCR to screen for positive transformed plants.

[0065] (2) Expression level detection RNA was extracted from positive materials, and cDNA was obtained by reverse transcription. The expression level of OsTMS5 was detected by real-time quantitative PCR, and overexpression lines with expression levels higher than the corresponding wild-type background were screened.

[0066] (3) Protein expression detection Since the overexpressed protein fusion has a FLAG tag, the expression of the OsTMS5-FLAG fusion protein can be detected by immunoblotting using an anti-FLAG antibody.

[0067] 4.5. Insertion site analysis and identification of T1 generation homozygotes Resequencing analysis was performed on the obtained overexpression transformation events to identify the T-DNA insertion site. Genotyping primers were designed based on the insertion site information, and genotyping was conducted in the T1 generation progeny to screen for homozygotes.

[0068] Overexpression events involving insertions at both ends of DR610-OsTMS5-OE-9 and DR610-OsTMS5-OE-10 were obtained and analyzed in the DR610 background. Homozygous OsTMS5 overexpression lines were identified in the T1 generation through insertion site analysis and molecular marker typing, as shown in Table 1. Positive overexpression lines with insertions at both ends of JXB-OsTMS5-OE-3 and JXB-OsTMS5-OE-19 were obtained in the JXB background. At present, the heterozygous overexpression lines have been used to conduct preliminary evaluation of RRSV resistance.

[0069] Table 1. Criteria for identifying homozygous OsTMS5 overexpression in DR610 background DR610-OsTMS5-OE-9 P1 + P2 (WT strip) P3 + P2 (transgenic band) Judgment basis Wild type (WT) have none Only 152 bp band Homozygous (HM) none have Only 494 bp band Hybrid (HT) have have Double stripe DR610-OsTMS5-OE-10 P1 + P2 (WT strip) P3 + P2 (transgenic band) Judgment basis Wild type (WT) have none Only 195 bp band Homozygous (HM) none have Only 626 bp bands Hybrid (HT) have have Double stripe In this example, the pCUbi1390-FLAG-OsTMS5 expression vector was constructed to obtain OsTMS5 overexpressing materials in different rice genetic backgrounds. Stable homozygous materials were further screened through resequencing and progeny genotyping. This material provides a direct experimental basis for verifying whether enhanced OsTMS5 expression can improve resistance to RRSV.

[0070] Example 5: Evaluation of the resistance to RRSV in DR610 background OsTMS5 overexpression lines 5.1 Experimental Objective To evaluate the disease resistance performance of OsTMS5 overexpressing lines in indica rice under DR610 background under RRSV artificial inoculation conditions.

[0071] 5.2 Experimental Materials Overexpressing plants of DR610-OsTMS5-OE-9 and DR610-OsTMS5-OE-10 and homozygous OsTMS5 overexpressing lines of the T2 generation of DR610 background that have been identified; corresponding wild-type control material DR610 and mutant material DR610S.

[0072] 5.3 Experimental Methods (1) Artificial inoculation Similar to Example 2, homozygous overexpression lines of similar age and growth, wild-type DR610, and DR610S control materials were selected, and artificial virus transmission was performed using brown planthoppers carrying RRSV. Throughout the experiment, the number of planthoppers, the inoculation time, and subsequent culture conditions were standardized.

[0073] (2) RRSV susceptibility detection Leaf samples were collected after inoculation, and the plants were tested for RRSV-specific RT-PCR to determine whether they were infected with the virus. The susceptibility rate of different materials was calculated as follows: susceptibility rate = (number of RRSV-positive plants / total number of plants tested) × 100%.

[0074] (3) Plant height survey after infection Record the plant height of RRSV-overexpressing materials and wild-type control plants after inoculation to evaluate the degree of growth inhibition caused by virus infection.

[0075] 5.4 Experimental Results like Figure 6 As shown in Figure A, the RRSV susceptibility rate of OsTMS5 overexpressing plants in the DR610 background was lower than that of the wild-type DR610 control; after RRSV infection, the plant height of OsTMS5 overexpressing homozygous plants decreased less than that of wild-type DR610; the overall growth of the overexpressing materials was better than that of the wild-type control, indicating that increasing OsTMS5 expression can alleviate the growth inhibition caused by RRSV infection. Figure 6 (B) This example demonstrates that, in the context of DR610 indica rice, OsTMS5 overexpression can stably enhance the material's resistance to RRSV. Whether the overexpressed material was unidentified as homozygous or identified as homozygous, under artificial RRSV inoculation conditions, it exhibited: reduced susceptibility rate; and lessened plant height loss after infection. Therefore, the OsTMS5 overexpression strategy proposed in this invention can be used for the creation of RRSV-resistant indica rice materials and subsequent breeding applications.

[0076] Example 6: Evaluation of the resistance to RRSV in JXB background OsTMS5 overexpression lines 6.1 Experimental Objective To evaluate the disease resistance performance of OsTMS5 overexpressing positive lines in Japonica rice under the JXB background under RRSV inoculation conditions, and to verify the applicability of this technical approach in the Japonica rice background.

[0077] 6.2 Experimental Materials JXB-OsTMS5-OE-3, JXB-OsTMS5-OE-19, and other OsTMS5 overexpression positive lines; corresponding to the wild-type control material JXB and the mutant material JXS. Currently, the JXB background overexpression materials used for RRSV resistance evaluation are heterozygous overexpression lines, and systematic resistance evaluation of their homozygous progeny has not yet been conducted.

[0078] 6.3 Experimental Methods (1) Evaluation of artificial RRSV exposure Brown planthoppers carrying RRSV were used to artificially inoculate JXB background OsTMS5 overexpressing lines, wild-type JXB, and mutant JXS.

[0079] (2) RRSV positive rate statistics After inoculation, RRSV molecular detection was performed on the plants, and the RRSV susceptibility rate in different treatments was statistically analyzed.

[0080] (3) Analysis of plant height differences Record the plant height after infection and calculate the relative plant height of infected plants compared to healthy plants.

[0081] (4) Auxiliary evaluation under natural infection conditions Under natural RRSV occurrence conditions, the natural susceptibility rate of overexpression lines was statistically analyzed compared with that of wild-type and mutant lines to verify their performance in the field disease context.

[0082] 6.4 Experimental Results like Figure 7 As shown in Figure A, under artificial inoculation conditions, the RRSV susceptibility rate of the JXB background OsTMS5 overexpression lines was approximately 43.33%–56.67%, lower than the 78.89% of the wild-type JXB control; under natural infection conditions, the RRSV susceptibility rate of the JXB background OsTMS5 overexpression lines was approximately 10.00%–15.56%; after infection, the relative plant height of the JXB background OsTMS5 overexpression lines was approximately 0.86–0.89 cm, indicating that their plant growth was relatively less inhibited by RRSV. Figure 7 (B in the text) Compared with wild-type JXB, OsTMS5 overexpression materials showed lower RRSV susceptibility and a milder tendency for growth damage.

[0083] This example demonstrates that even when using heterozygous OsTMS5 overexpression lines in the JXB background for evaluation at this stage, the following observations were still made: a significant decrease in RRSV susceptibility; reduced plant height impairment after infection; and a clear trend towards improved RRSV resistance. Therefore, increasing OsTMS5 expression also has the potential to enhance RRSV resistance in the Japonica rice background. This result corroborates the results from homozygous overexpression lines in the DR610 background, supporting the cross-genetic background application value of the OsTMS5 regulation strategy proposed in this invention.

Claims

1. OsTMS5 The use of genes or related biological materials in at least one of the following: A1) Regulates the ability of rice to resist rice serrated leaf dwarf virus; A2) Prepare products that regulate the resistance of rice to rice spur dwarf virus; A3) Breed rice varieties with enhanced resistance to rice spur dwarf virus; A4) Prepare products that enhance the resistance of rice to rice spur dwarf virus; The ability to regulate rice resistance to rice spur dwarf virus includes: By raising OsTMS5 Gene expression to enhance rice's resistance to rice dwarf virus; The OsTMS5 The coding nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The application as described in claim 1, characterized in that, The biomaterials include one or more of the following: The OsTMS5 The gene-encoded protein and / or containing the above OsTMS5 Gene recombinant vectors, recombinant microorganisms, or transgenic rice cell lines; The OsTMS5 The amino acid sequence of the gene-encoded protein is shown in SEQ ID NO.

2.

3. Upward adjustment OsTMS5 Application of gene-expressing biological materials in the cultivation of rice with enhanced resistance to rice spur leaf dwarf virus; OsTMS5 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

4. The application as described in claim 3, characterized in that, The biomaterial includes: OsTMS5 Gene-encoded proteins and / or those containing upregulated OsTMS5 Recombinant vectors for gene expression, recombinant microorganisms, or transgenic rice cell lines.

5. A method for improving the resistance of rice to rice dwarf virus, characterized in that, include: Will be raised OsTMS5 Gene-expressing biological materials were transferred into rice; The OsTMS5 The coding nucleotide sequence of the gene is shown in SEQ ID NO.

1.

6. A method for cultivating rice with enhanced resistance to rice spur dwarf virus, characterized in that, include: Will be raised OsTMS5 Gene-expressing biological materials were transferred into rice; The OsTMS5 The coding nucleotide sequence of the gene is shown in SEQ ID NO.1.

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