Gene mets2 for regulating brown planthopper resistance trait of rice and application thereof
By identifying and overexpressing the METS2 gene, constructing a recombinant vector, and transforming it into the rice genome, the problem of limited number of brown planthopper resistance genes and single resistance in existing technologies has been solved. This significantly enhances rice resistance to brown planthopper, provides new breeding resources and technical support, reduces the use of chemical pesticides, and ensures food security.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- GUANGXI ZHUANG AUTONOMOUS REGION ACAD OF AGRI SCI
- Filing Date
- 2026-05-07
- Publication Date
- 2026-06-05
AI Technical Summary
The number of genes resistant to brown planthoppers in existing technologies is limited, and some genes have only one type of resistance, which can be easily overcome by brown planthopper populations, resulting in serious losses in rice yields, frequent use of chemical pesticides, and impacting food security.
By identifying and overexpressing the METS2 gene, the resistance of rice to brown planthopper was improved. A recombinant vector was constructed and transformed into the rice genome to enhance the expression level of the METS2 gene and to cultivate transgenic rice resistant to brown planthopper.
It significantly enhances rice's resistance to brown planthoppers, provides new genetic resources and technical support, offers a theoretical basis and germplasm materials for rice breeding, reduces the use of chemical pesticides, and ensures food security.
Smart Images
Figure CN122146729A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of genetic engineering technology, and in particular to a gene that regulates the brown planthopper resistance trait in rice. METS2 And its applications. Background Technology
[0002] Brown planthoppers are major migratory pests in rice production, feeding on rice sap by piercing and sucking its moisture. Severe infestations can cause rice to lodging and die, and the planthoppers also spread viral diseases, resulting in significant yield losses. Abundant rice resources contain many genes that influence brown planthopper resistance. Discovering and identifying superior brown planthopper-resistant genes can broaden the gene pool and enrich the genetic background for insect-resistant rice breeding, thereby enabling the development of new brown planthopper-resistant rice varieties, reducing the use of chemical pesticides, and ensuring food security. Currently, the number of identified brown planthopper-resistant genes is limited, and some genes exhibit single-type resistance or are easily overcome by brown planthopper populations. Therefore, discovering and utilizing new brown planthopper-resistant genes in rice is a crucial and pressing issue that needs to be addressed in the field of rice breeding. Summary of the Invention
[0003] The purpose of this invention is to provide a gene that regulates the brown planthopper resistance trait in rice. METS2 Its application provides new genetic resources and technical support for rice breeding resistant to brown planthoppers.
[0004] To achieve the above objectives, this invention provides a gene for regulating the brown planthopper resistance trait in rice. METS2 , METS2 The CDS sequence of the gene is shown in SEQ ID NO.3.
[0005] On the other hand, the present invention also provides the aforementioned gene. METS2 Application in regulating the brown planthopper resistance trait in rice.
[0006] Preferably, through overexpression METS2 Genes enhance rice's resistance to brown planthoppers.
[0007] On the other hand, the present invention also provides a protein that regulates rice resistance to brown planthoppers, derived from the above-mentioned... METS2 Gene encoding.
[0008] On the other hand, the present invention also provides the application of the above-mentioned protein in regulating the brown planthopper resistance trait in rice.
[0009] On the other hand, the present invention also provides a recombinant vector containing the above-mentioned... METS2 Gene.
[0010] On the other hand, the present invention also provides a biomaterial containing the above-mentioned recombinant vector.
[0011] Preferably, the biological material is recombinant microorganism, transgenic plant cell line, or transgenic plant tissue; The recombinant microorganisms are selected from bacteria, yeast, algae, or fungi; The bacteria are one of Escherichia coli, Erwinia coli, Agrobacterium tumefaciens, Flavobacterium, Alcaligenes, Pseudomonas, or Bacillus.
[0012] On the other hand, the present invention also provides the application of the above-mentioned recombinant vector or biological material in regulating rice resistance to brown planthopper.
[0013] On the other hand, the present invention also provides a method for breeding transgenic rice resistant to brown planthoppers by using transgenic technology to encode... METS2 The nucleotide sequence of the gene was transferred into recipient rice, increasing the quality of the recipient rice. METS2 Gene expression levels were measured to obtain transgenic rice resistant to brown planthoppers.
[0014] Therefore, this invention provides a gene for regulating the brown planthopper resistance trait in rice. METS2 Its application has the following beneficial effects: (1) This invention is the first to discover and identify the coding gene associated with rice resistance to brown planthopper. METS2 Experimental verification shows that when METS2 Enhanced gene function or increased expression levels can significantly improve rice resistance to brown planthoppers, demonstrating that this gene and its encoded protein play a key role in regulating brown planthopper resistance in rice.
[0015] (2) This invention not only provides an important theoretical basis for further elucidating the molecular mechanism of rice resistance to brown planthopper, but also provides new gene resources and technical support for rice breeding for resistance to brown planthopper.
[0016] (3) The invention obtained METS2 Transgenic rice with overexpressed genes can serve as a new rice germplasm material for in-depth research on the mechanism of rice resistance to brown planthoppers and the discovery of more genes that regulate resistance to brown planthoppers. It has important application value for effectively improving the brown planthopper resistance trait of rice by utilizing this gene resource through genetic breeding and genetic engineering methods.
[0017] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description
[0018] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0019] Picture 1 for METS2 PCR detection results of the hyg fragment in transgenic plants; Picture 2 for METS2 -OE1、 METS2 Schematic diagram of the relative expression levels of the gene overexpressed in OE2 and the parental rice Guanghui 998; Picture 3 Encoding genes related to rice resistance to brown planthopper METS2 Schematic diagram of the resistance phenotype of transgenic rice to brown planthopper overexpression; Picture 4 Encoding genes related to rice resistance to brown planthopper METS2 A schematic diagram showing the seedling mortality rate of transgenic rice after inoculation with brown planthoppers. Detailed Implementation
[0020] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.
[0021] To make the objectives, technical solutions, and advantages of this application clearer, more thorough, and more complete, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings and embodiments. The following detailed descriptions are all illustrations of embodiments, intended to provide further detailed explanation of the present invention. Unless otherwise specified, all technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.
[0022] The instruments, equipment, reagents, and materials used in the examples were all obtained commercially.
[0023] Example 1 Construction of overexpression vector for rice brown planthopper resistance genes: (1) METS2 Acquisition of genes: Common wild rice Y11 ( Oryza rufipogon Using Griff.'s DNA as a template, the target gene was obtained by PCR amplification using the following primers, primer1 and primer2: primer1: 5'-atggcgtctcacattgttgg-3', SEQ ID NO.1; primer2: 5'-tcacttcgcgctagcgag-3', SEQ ID NO.2.
[0024] The PCR amplification product was purified by agarose gel electrophoresis and ligated into the Zero sequencing vector (purchased from Beijing TransGen Biotech Co., Ltd.). This vector was then transformed into *E. coli* DH5α competent cells, and positive clones were screened by plating on antibiotic-resistant plates. Sequencing verification of the positive clones was performed. The sequencing results showed that the PCR product sequence, as shown in SEQ ID NO.3, was 2301 bp in length and encoded 766 amino acids. This gene was named... METS2 (Methionine synthase 2, LOC_Os12g42884 )Gene.
[0025] SEQ ID NO.3: (2) Overexpression vector PMDC32-OE- METS2 Construction: S1. Amplify wild rice Y11 cDNA using primer1 and primer2 to obtain... METS2 The gene sequence was extracted and ligated into the Zero vector to obtain recombinant Zero- METS2 Positive clones were obtained by digesting the recombinant vector Zero- with restriction endonucleases KpnI and PacI. METS2 Obtain OE- METS2 Excerpt.
[0026] S2. The expression vector PMDC32 was digested with restriction endonucleases Kpn I and PacI to obtain the linear expression vector PMDC32, and the linear fragment was recovered. The OE- fragment obtained in S1 was then processed. METS2 The homologous recombination-directed cloning method was used to integrate the product into the linear expression vector PMDC32 (for specific methods, please refer to the PMDC32 instruction manual), resulting in homologous recombination product 1 (i.e., PMDC32-OE-). METS2 , for genes METS2 (Overexpression vector), and then the homologous recombination product 1 was transformed into DH5α competent cells and cultured overnight at 37°C.
[0027] S3, the recombinant vector PMDC32-OE- obtained from S2 METS2 Sequencing results showed that the recombinant vector inserted the nucleotide sequence shown in SEQ ID NO.3 into the Kpn I restriction site of the expression vector PMDC32 in the forward direction, that is, successfully replaced the DNA sequence between the Kpn I and PacI recognition sites (recognition sequence) of PMDC32 with the DNA sequence shown in SEQ ID NO.3.
[0028] (3) Transformation: Heat shock transformation of Escherichia coli: Take 5 μL of recombinant vector PMDC32-OE- METS2 For the heat shock transformation of E. coli, please refer to the Tiangen Transformation Kit for specific steps.
[0029] (4) PCR verification of bacterial culture.
[0030] Single colonies were picked from the clean bench and used as templates for PCR amplification, employing the Taq DNA polymerase PCR amplification system and procedure.
[0031] (5) Plasmid extraction: Plasmids were extracted and their concentrations were detected according to the instructions of the Tiangen plasmid mini-prep kit to obtain the recombinant expression vector PMDC32-OE- METS2 .
[0032] Example 2 METS2Cultivation and identification of transgenic rice plants overexpressing the gene.
[0033] (1) Cultivation METS2 Gene overexpression transgenic plants.
[0034] Using the Agrobacterium tumefaciens EHA105-mediated method, the PMDC32-OE- constructed in Example 1 was... METS2 The overexpression vector was used to transform the recipient rice variety Guanghui 998 (GH998) indica rice. The specific method is as follows: S1, Plasmid transformation of Agrobacterium tumefaciens: PMDC32-OE- METS2 The recombinant plasmid was introduced into *Agrobacterium tumefaciens* EHA105 competent cells via heat shock to obtain recombinant *Agrobacterium tumefaciens* EHA105 containing the recombinant vector. The recombinant *Agrobacterium tumefaciens* was inoculated into LB liquid medium containing the corresponding antibiotic and cultured at 28°C with shaking at 200 rpm for 16 h. The cells were then collected by centrifugation. The cells were diluted to OD using N6 liquid medium (Sigma, catalog number C1416) containing 100 μM acetylsyleugenol. 600 ≈0.5, to obtain the infected bacterial solution.
[0035] S2. Callus Infection: Embryogenic callus induced by mature embryos of GH998 indica rice cultured for about 30 days was selected and mixed with the above-mentioned infecting bacterial solution and soaked for 30 minutes, gently shaking several times during the process. The bacterial solution on the surface of the callus was blotted dry with sterile filter paper, and then transferred to N6 solid co-culture medium and incubated in the dark at 24℃ for 3 days to obtain the co-cultured callus.
[0036] S3, Screening of resistant callus: First screening: The co-cultured callus was inoculated into N6 solid screening medium containing 150 mg / L hygromycin (150 mg / L hygromycin was added to N6 solid medium) and cultured in the dark at 24°C for 16 days.
[0037] Second screening: Healthy, dense callus tissue was selected and transferred to N6 solid screening medium containing 200 mg / L hygromycin. It was cultured in the dark at 24°C and subcultured every 15 days for a total of 1 subculture to obtain resistant callus tissue.
[0038] S4. Differentiation and Hardening Transplanting: Resistant callus tissue was selected and transferred to differentiation medium containing 150 mg / L hygromycin (formula: 6-BA 2 mg / L, NAA 0.2 mg / L, N6 medium powder 4 g / L, hydrolyzed casein 1 g / L, inositol 0.1 g / L, sucrose 25 g / L, sorbitol 2.4 g / L, agar powder 7 g / L, diluted to 1 L with deionized water, pH adjusted to 5.8). The medium was cultured at 24℃ under 16 h light / 8 h dark conditions for 45 days (plant height approximately 15 cm above ground). The culture bottles were opened for hardening for 3 days, followed by transplanting to a greenhouse for cultivation to obtain T0 generation PMDC32-OE-. METS2 Plants. Positive plants from different transformation events (transformation into different callus tissues) were selected and named accordingly. METS2 -OE1、 METS2 -OE2.
[0039] (2) METS2 PCR identification of transgenic plants: Extract the above METS2 -OE1、 METS2 Genomic DNA was extracted from leaves of T0 generation seedlings of the -OE2 plant and seedlings of the recipient parent rice GH998. Positive seedlings were identified by PCR using primers hyg-F and hyg-R. PCR verification confirmed the presence of a hyg-positive band in individual plants, as mentioned above. METS2 -OE1、 METS2 -OE2 was identified as a transgenic single plant.
[0040] hyg-F: 5'-AAAAGTTCGACAGCGTCTCCGACC-3', SEQ ID NO.4; hyg-R: 5'-TCTACACAGCCATCGGTCCAGACG-3', SEQ ID NO.5.
[0041] PCR amplification system (10 μL): 2×EasyTaq® PCR SuperMix (+dye) 5 μL, 10 μmol / L hyg-F 1 μL, 10 μmol / L hyg-R 1 μL, genomic DNA (50 ng / μL) 1 μL, ddH2O 2 μL.
[0042] PCR amplification program: 94℃ pre-denaturation for 2 min; 98℃ denaturation for 10 sec, 55℃ annealing for 30 sec, 68℃ extension for 1 min / kb, 25-40 cycles; 68℃ final extension for 5 min; 4℃ incubation.
[0043] The results of PCR product detection by agarose gel electrophoresis are as follows: Picture 1 As shown, the results are as follows: METS2-OE1、 METS2 -OE2 plants all amplified specific hyg positive bands, while the parental GH998 did not show a corresponding band, indicating that METS2 The gene has been successfully integrated into the rice genome, resulting in a positive transgenic plant.
[0044] (3) METS2 In transgenic plants METS2 Identification of gene expression levels: Extract the above METS2 -OE1、 METS2 RNA was extracted from leaves of OE2 generation T0 seedlings and the recipient parent rice GH998 plants. Actin was set as the internal control, and the internal control primers Actin-F and Actin-R were used. METS2 Gene-specific quantitative primers METS2 -qRT-F and METS2 -qRT-R was used to perform quantitative real-time PCR to detect different transgenic plants. METS2 Changes in gene expression levels.
[0045] The primer sequences are as follows: METS2 -qRT-F: 5'-GCGCTGAGGATTTGGAGAAG-3', SEQ ID NO.6; METS2 -qRT-R: 5'-ATAGCAGGGACAGTGGCATT-3', SEQ ID NO.7; Actin-F: 5'-ATTTGGCACCACACATTCTAC-3', SEQ ID NO.8; Actin-R: 5'-ATAACCTTCGTAGATTGGGACT-3', SEQ ID NO. 9.
[0046] qRT-PCR results are as follows Picture 2 As shown, METS2 -OE1、 METS2 -OE2 plants METS2 The relative expression levels of the genes were significantly higher than those of the parent GH998, indicating that... METS2 The gene was successfully overexpressed in the transgenic plants.
[0047] (4) METS2 Phenotypic identification of transgenic plants.
[0048] The parent rice varieties GH998 and transgenic rice were selected respectively. METS2 -OE1、 METS2-OE2 seeds were sterilized and germinated before being sown in identification pots, with 20 seedlings per line. When the plants reached the two-leaf-one-heart stage, healthy 1st-2nd instar brown planthopper nymphs were inoculated, with 8 nymphs per plant. After inoculation, the plants were placed in an artificial climate chamber (temperature 26±1℃, relative humidity 70%~80%, photoperiod 16h light: 8h darkness) and isolated with nylon netting. The experiment was stopped when the mortality rate of the control line (GH998) reached 80%, and the mortality rate of each line was recorded.
[0049] Observation results as follows Picture 3 and Picture 4 As shown, the parent variety Guanghui 998 (GH998) was used as a control. METS2 Overexpression lines ( METS2 -OE1、 METS2 Statistical analysis showed that the seedling mortality rate of plants overexpressing OE2 (using PMDC32 overexpression vector) was significantly lower than that of the control plants; this result indicates that... METS2 Overexpression of the gene can significantly enhance the resistance of rice to brown planthopper.
[0050] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.
Claims
1. A gene regulating the brown planthopper resistance trait in rice. METS2 Its features are: METS2 The CDS sequence of the gene is shown in SEQ ID NO.
3.
2. The gene as described in claim 1 METS2 Application in regulating the brown planthopper resistance trait in rice.
3. The application according to claim 2, characterized in that: Through overexpression METS2 Genes enhance rice's resistance to brown planthoppers.
4. A protein that regulates resistance to brown planthopper in rice, characterized in that: As described in claim 1 METS2 Gene encoding.
5. The application of the protein as described in claim 4 in regulating the brown planthopper resistance trait in rice.
6. A recombinant vector, characterized in that: Containing the contents of claim 1 METS2 Gene.
7. A biomaterial, characterized in that: Contains the recombinant vector as described in claim 6.
8. The biomaterial according to claim 7, characterized in that: Biological materials include recombinant microorganisms, transgenic plant cell lines, or transgenic plant tissues; The recombinant microorganisms are selected from bacteria, yeast, algae, or fungi; The bacteria are one of Escherichia coli, Erwinia coli, Agrobacterium tumefaciens, Flavobacterium, Alcaligenes, Pseudomonas, or Bacillus.
9. The application of the recombinant vector as described in claim 6 or the biomaterial as described in claim 7 in regulating rice resistance to brown planthopper.
10. A method for breeding transgenic rice resistant to brown planthoppers, characterized in that: Using genetic engineering to encode METS2 The nucleotide sequence of the gene was transferred into recipient rice, increasing the quality of the recipient rice. METS2 Gene expression levels were measured to obtain transgenic rice resistant to brown planthoppers.