Oswrky41 gene, method for improving rice blast resistance and application thereof

Abstract

The application relates to the field of biotechnology, in particular to an OsWRKY41 gene and a method and application for improving rice blast resistance, a new broad-spectrum rice blast resistance gene OsWRKY41 is cloned through the expression research of a miRNA target gene for the negative regulation of rice disease resistance, the OsWRKY41 gene is induced to express in rice plants under the invasion of rice blast fungus, and the rice plants with overexpressed OsWRKY41 genes are endowed with broad-spectrum rice blast resistance by enhancing active oxygen accumulation. The research provides a new thought and a new method for the identification of rice blast resistance genes, and provides a new gene resource for the improvement of rice variety resistance and the breeding of a new variety with broad-spectrum disease resistance.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a method and application of using the OsWRKY41 gene to enhance rice blast resistance. Background Technology

[0002] Rice (Oryza sativa L.) is one of the most important food crops in my country and the world. Rice blast is a fungal disease caused by the ascomycete Magnaporthe oryzae. Currently, breeding and promoting resistant varieties is the most economical, effective, and environmentally friendly strategy for controlling rice blast. However, the complex structure and rapid mutation of the rice blast fungus population lead to the easy loss of resistance in existing varieties. Therefore, discovering and utilizing new broad-spectrum and durable resistance genes and elucidating their molecular mechanisms are of great significance for rice disease resistance breeding.

[0003] To date, researchers have identified over 140 rice blast resistance (R) genes, 38 of which have been cloned. Most of the cloned R genes encode nucleotide-binding and leucine-rich repeat receptor (NLR) proteins. Some of these resistance genes have been applied in rice disease resistance breeding practices, playing a crucial role in the green control of rice blast. For example, the Pb1 gene, discovered in the indica rice variety 'Modan,' encodes a CC-NBS-LRR protein containing a coiled helical structure. This gene has been introduced into several mainstream superior varieties in Japan, and no resistance decline has been observed in nearly 30 years of field application. The team of Chinese scientist He Zuhua identified the broad-spectrum and durable rice blast resistance locus Pigm from farmyard rice breeding materials. This locus is a gene cluster containing multiple NBS-LRR type resistance genes; rice varieties improved based on the Pigm locus possess both broad-spectrum and durable rice blast resistance without negatively impacting yield traits.

[0004] We identified a novel miRNA, miR9664, that negatively regulates rice blast resistance. Its predicted target genes are mainly those encoding NBS-LRR proteins. The expression level of the predicted target gene LOC_Os11g45924 (https: / / www.ricedata.cn / gene / ) was significantly downregulated in transgenic rice overexpressing miR9664, but significantly upregulated in transgenic rice with miR9664 knocked out. It is speculated that miR9664 is likely a major epigenetically regulated gene for rice blast resistance and has important potential application value in the control of rice blast. Summary of the Invention

[0005] The purpose of this invention is to provide a method and application for improving rice blast resistance using the OsWRKY41 gene.

[0006] To achieve the above objectives, the technical solution adopted in this invention is as follows: This invention takes the predicted target gene LOC_Os11g45924 of rice miR9664 as the object, extracts total RNA from rice seed 44 and reverse transcribes it into cDNA, and uses PCR technology to amplify the CDS region sequence of the LOC_Os11g45924 gene of 1566bp (as shown in SEQ ID No. 1), which encodes a disease resistance protein containing two NB-ARC domains, which we name OsWRKY41.

[0007] The gene sequence is shown in SQE ID No. 1, and the encoded amino acid sequence is shown in SQE ID No. 2. An OsWRKY41 overexpression vector was constructed and introduced into susceptible rice TP309 using Agrobacterium-mediated genetic transformation to obtain OsWRKY41 overexpressing plants. The OsWRKY41 overexpressing rice lines significantly enhanced resistance to rice blast fungus. T1 generation positive transgenic lines were inoculated with 1.5 × 10⁻⁶ conidial suspensions of rice blast fungus Guy11, 70-15, ZB15, Y28-2, and LP11 at the tillering stage after detached leaf injury. 5 The average lesion length and fungal spore count of transgenic lines were significantly lower than those of the non-transgenic TP309 control. This indicates that OsWRKY41 can significantly enhance the resistance of rice to different pathogenic rice blast strains. DAB (3,3'Diaminobenzidine-HCl) staining for H2O2 accumulation in rice leaf cells showed that rice plants overexpressing the OsWRKY41 gene had significantly higher H2O2 accumulation in leaf cells 48 h after infection with rice blast fungus than the non-transgenic TP309 control.

[0008] The above results indicate that OsWRKY41 confers broad-spectrum rice blast resistance by enhancing H2O2 accumulation in rice plants and activating the expression of defense-related genes. This application seeks protection for the application of the OsWRKY41 gene shown in SEQ ID No. 1 in improving rice resistance to rice blast.

[0009] The second objective of this invention is to provide the application of the amino acid sequence encoded by the OsWRKY41 gene, as shown in SEQ ID No. 2, in improving rice blast resistance.

[0010] For those skilled in the art, using the OsWRKY41 gene sequence shown in SEQ ID No. 1 for the breeding of new rice varieties also falls within the scope of protection of this invention.

[0011] This method overexpresses the OsWRKY41 target gene in rice variety TP309, thereby improving resistance. Therefore, this method can also be used for rice improvement or new variety breeding and is within the scope of protection of this invention.

[0012] A third objective of this invention is to provide a recombinant vector for improving resistance to rice blast disease, wherein the recombinant vector contains the CDS sequence of the OsWRKY41 gene as shown in SEQ ID No. 1.

[0013] The fourth objective of this invention is to provide a method for improving rice resistance to rice blast by utilizing the OsWRKY41 gene or its encoded amino acids as shown in SEQ ID No. 1, the steps of which include:

[0014] (1) Total RNA was extracted from wild-type rice seed 44 and then reverse transcribed into cDNA;

[0015] (2) The target gene OsWRKY41 was amplified by PCR technology. The sequence of the target gene OsWRKY41 is shown in SEQ ID No.1.

[0016] (3) The OsWRKY41 gene fragment was ligated into the expression vector to obtain the plasmid pCAMBIA1300-OsWRKY41 that overexpresses the OsWRKY41 target gene;

[0017] (4) Plasmid pCAMBIA1300-OsWRKY41 was introduced into Agrobacterium;

[0018] (5) Rice lines with positive disease resistance can be obtained by co-culturing Agrobacterium containing plasmid pCAMBIA1300-OsWRKY41 with rice strains.

[0019] Furthermore, in step (2), the primer sequences for amplifying the target gene OsWRKY41 are OsWRKY41-F and OsWRKY41-R:

[0020] OsWRKY41-F:5'-ATTCGAGCTCGGTAATGGAGTCCACCTGTC-3' (SEQ ID No. 3);

[0021] OsWRKY41-R:5'-AGGATCCCCGGGTATCAGCTCACCAAGGCT-3' (SEQ ID No. 4).

[0022] The beneficial technical effects of this invention are as follows: This invention provides a method for studying the expression of miRNA target genes for rice blast resistance through negative regulation. A novel broad-spectrum rice blast resistance gene, OsWRKY41, was cloned. The OsWRKY41 gene is induced to express in rice plants by infection with rice blast fungus. Rice plants overexpressing the OsWRKY41 gene exhibit enhanced accumulation of reactive oxygen species (H2O2), thus conferring broad-spectrum rice blast resistance. This research provides new ideas and methods for identifying rice blast resistance genes and offers new gene resources for improving rice variety resistance and breeding new broad-spectrum resistant varieties. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art 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.

[0024] Figure 1 OsWRKY41 protein structure prediction diagram.

[0025] Figure 2 Identification of homozygous rice lines overexpressing OsWRKY41 (the figure shows the positive identification results of OX-OsWRKY41 T1).

[0026] Figure 3 Seedling resistance identification results of transgenic rice lines overexpressing OsWRKY41 (Figure A shows disease symptoms after inoculation; Figure B shows the average disease severity statistics).

[0027] Figure 4 OsWRKY41 overexpression transgenic rice resistance identification at the tillering stage after inoculation with different rice blast strains (Figure A shows disease symptoms after inoculation, B shows the statistical diagram of rice blast lesion length, and C shows the statistical diagram of fungal biomass).

[0028] Figure 5 : Detection of reactive oxygen species accumulation in leaves of control and transgenic lines after infection with rice blast fungus (DAB staining, OsRboh gene expression) (Figure: A is the DAB staining result; B is the OsRboh gene expression level detection result).

[0029] Figure 6 Subcellular localization of OsWRKY41 in Nicotiana benthamiana cells. Detailed Implementation

[0030] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] Example 1

[0032] Construction of OsWRKY41 gene overexpression vector, including the following steps:

[0033] 1. Total RNA was extracted from the leaves of four-leaf stage rice seedlings (Ziyu 44) using the MiniBEST Plant RNA Extraction Kit (TaKaRa, Dalian, China) according to the kit instructions. RNA concentration and quality were determined using a NanoDrop 2000 UV-Vis spectrophotometer (Thermo Fisher Scientific, Waltham, Massachusetts, USA), and integrity distribution was checked by 2% agarose gel electrophoresis.

[0034] 2. RNA was transcribed into cDNA using reverse transcription PCR.

[0035] 3. Using amplification primers OsWRKY41-F / R (as shown in SEQ ID No. 3-4), the CDS sequence of the OsWRKY41 gene with restriction sites was amplified by PCR technology (as shown in SEQ ID No. 1, and the encoded amino acid series is shown in SEQ ID No. 2).

[0036] 4. Use 1% agarose gel electrophoresis at 120V for 23 minutes. Cut out the 1566bp electrophoretic fragment under UV light and place it in a system for sol-gel recovery. Refer to the specific manufacturer's kit instructions for the recovery procedure. Dissolve and recover the DNA with 30μL of water.

[0037] 5. The T4 connection method was used to construct the carrier.

[0038] The plasmid used to construct the OsWRKY41 overexpression vector is pCAMBIA1300, which contains two strong 35S promoters: one to promote the target gene and the other to promote the hygromycin gene (for screening and identification of positive transgenic plants). The vector also contains a kanamycin resistance gene, used to screen for positive bacterial clones during vector construction.

[0039] 6. Transform the pCAMBIA1300 plasmid into Escherichia coli (DH5α) (see the standard method for competent transformation of E. coli) and incubate overnight at 37°C and 200 rpm. Extract the plasmid the next day. Then, digest the pCAMBIA1300 plasmid with the restriction endonuclease KpnI.

[0040] 7. Recombinate the target gene with the plasmid. Add the ligation product to *E. coli* (DH5α) and incubate overnight at 37°C. Pick single colonies and place them on LB liquid medium containing KAN resistance in a constant temperature shaker (37°C, 200 rpm / h) for 12 h. Then, perform plaque PCR identification using specific primers (OsWRKY41-F / R). Perform plasmid mini-extraction on the correctly sequenced positive colonies to obtain the purified plasmid.

[0041] 8. The plasmid was then transferred to Agrobacterium tumefaciens EHA105, placed on a Kan+hygromycin antibiotic plate, and incubated upside down overnight at 28°C. Single colonies were picked the next day for PCR colony identification.

[0042] 9. Rice callus induction

[0043] (1) Wash the seeds with 75% alcohol for 10 seconds, then wash them three times with sterile water.

[0044] (2) Soak the seeds in 1.5% sodium hypochlorite, shake them in a clean bench for 10 minutes, discard the sodium hypochlorite, and then rinse with sterile water until there is no odor.

[0045] (3) Use sterile filter paper to absorb the liquid on the surface of the seeds. Use sterile tweezers to evenly place the dried seeds on N6 medium. Seal the plate with sealing film and place it in a 30℃ incubator for dark culture for 7-10 days until pale yellow callus tissue grows.

[0046] 10. Infection and Co-culture

[0047] (1) Streak the Agrobacterium strain with transformation vector on LB (with added Kan and Rif antibiotics) plates and incubate at 28°C for activation.

[0048] (2) Pick a single colony into 2 mL of liquid LB (with Kan and Rif antibiotics), shake overnight at 200 rpm at 28℃. The next day, take 1 mL of bacterial solution into 50 mL of TY medium, shake for 1 hour at 200 rpm at 28℃, and then measure the concentration. Adjust the OD600 value to 0.05-0.1.

[0049] (3) In a clean bench, soak the rice callus tissue that has been peeled off in the bacterial solution and gently shake for 25 minutes (to avoid contamination).

[0050] (4) Use sterile filter paper to absorb the bacterial solution on the surface of the callus, and blow it in the clean bench for 30 minutes until the surface of the callus is dry.

[0051] (5) Transfer the dried callus to a co-culture medium and incubate in the dark at 22°C for 3 days.

[0052] 11. Screening, Differentiation and Rooting Culture

[0053] (1) Place the callus tissue from the co-culture medium onto the screening medium with sterile forceps and culture at 30°C for about a month under light. Replace the screening medium every 10 days during this period.

[0054] (2) Transfer the selected surviving callus tissue to the initial differentiation medium with a wide spacing and culture at 30°C for about 14 days.

[0055] (3) Transfer the callus that has undergone initial differentiation to the redifferentiation medium until the callus differentiates into green shoots.

[0056] (4) Once the green shoots have grown strong, transfer the entire tissue to a rooting medium.

[0057] 12. When the tissue in the rooting medium has grown strong roots and the leaves have grown to about 10cm (at which point it has become a seedling), remove the seedling from the medium, wash off the medium from the roots, soak it in tap water for 3-5 days, and then transplant the seedlings that are still healthy into the soil.

[0058] Example 2

[0059] Identification of OsWRKY41 overexpression positive rice lines

[0060] DNA was extracted from transgenic rice using the CTAB method, and PCR amplification was performed using gene-specific primers OsWRKY41-PI-F / R. Fifteen T1 generation positive transgenic plants were identified. Figure 2 A).

[0061] OsWRKY41-PI-F: 5'-TCCTTCGCAAGACCCTTCCTC-3' (SEQ ID No. 5);

[0062] OsWRKY41-PI-R: 5'- ACAGATTCGGTCGCACCAATC-3' (SEQ ID No. 6).

[0063] Example 3

[0064] OsWRKY41 overexpression in rice seedling resistance identification

[0065] Using non-transgenic TP309 as a control, T1 generation positive transgenic seedlings at the three-leaf-one-heart stage were sprayed with a suspension of Guy11 spores (1.5 × 10⁻⁶). 5 (number of cells / mL), and investigate the incidence of disease after 7 days.

[0066] The results showed that the OsWRKY41 overexpression lines were significantly more resistant to rice blast, and the lesion area was reduced by 40% compared with the control (Figure 3).

[0067] Example 4

[0068] Identification of resistance at tillering stage in OsWRKY41 overexpression transgenic rice

[0069] T1 generation positive plants at the tillering stage were inoculated in vitro with Guy11, 70-15, ZB15, LP11, and Y28-2 rice blast fungus strains. Resistance was identified and fungal biomass was measured after 7 days.

[0070] The results showed that the disease severity of the overexpressing plants was significantly lower than that of the control, with significantly shorter lesion length and significantly reduced fungal biomass. Figure 4 This demonstrates that OsWRKY41 confers broad-spectrum resistance to rice blast.

[0071] Example 5

[0072] Response of rice seedling leaves to OsWRKY41 overexpression in response to rice blast fungus infection

[0073] 48 h after inoculation with rice blast fungus Guy11, rice leaves were stained with DAB to detect H2O2 accumulation.

[0074] The results showed that the brown staining area of ​​the leaves of the transgenic plants was significantly larger than that of the control, and the accumulation of H2O2 was significantly increased; the expression level of the reactive oxygen species synthesis gene OsRboh was significantly higher than that of TP309 (Figure 5), confirming that the H2O2 burst is an important way for OsWRKY41 to enhance disease resistance.

[0075] Example 6

[0076] Subcellular localization of OsWRKY41 in Nicotiana benthamiana cells

[0077] The full-length CDS of OsWRKY41 was fused to the N-terminus of the GFP protein to construct the 35S promoter-driven pCAMBIA1300-OsWRKY41-GFP vector. Using pCAMBIA1300-GFP as a control, Agrobacterium was used to transiently transform tobacco leaves.

[0078] The results showed that the green fluorescence of OsWRKY41-GFP was concentrated in the cell nucleus ( Figure 6This confirms that OsWRKY41 is specifically located in the cell nucleus.

[0079] Finally, it should be noted that the above embodiments are only used to illustrate and not limit the technical solutions of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the present invention without departing from the spirit and scope of the present invention. Any modifications or partial substitutions should be covered within the scope of the claims of the present invention.

Claims

1. The application of the OsWRKY41 gene CDS sequence as shown in SEQ ID No. 1 in improving rice blast resistance, characterized in that, Overexpression of the OsWRKY41 gene improved rice blast resistance.

2. Application of the amino acid sequence encoded by the OsWRKY41 gene as shown in SEQ ID No.2 in improving rice blast resistance.

3. A recombinant vector for enhancing rice blast resistance, characterized in that, The recombinant vector contains the OsWRKY41 gene CDS sequence as shown in SEQ ID No.

1.

4. The recombinant vector according to claim 3, characterized in that, The original vector for the recombinant vector was the expression vector pCAMBIA1300-OXHLL.

5. A method for improving rice blast resistance using the CDS sequence of the OsWRKY41 gene as shown in SEQ ID No. 1 or the amino acid sequence encoded by the OsWRKY41 gene as shown in SEQ ID No. 2, characterized in that the steps are as follows: include: (1) Total RNA was extracted from wild-type rice seed 44 and then reverse transcribed into cDNA; (2) The target gene OsWRKY41 was amplified by PCR using amplification primers OsWRKY41-F and OsWRKY41-R, as shown in SEQ ID No.1, with a full length of 1566bp; (3) The OsWRKY41 gene fragment was ligated into the expression vector to obtain the plasmid pCAMBIA1300-OsWRKY41 that overexpresses the OsWRKY41 target gene; (4) Plasmid pCAMBIA1300-OsWRKY41 was introduced into Agrobacterium; (5) Rice lines with positively correlated disease resistance can be obtained by co-culturing Agrobacterium containing plasmid pCAMBIA1300-OsWRKY41 with rice strains.

6. The method according to claim 5, characterized in that, In step (2), the primer sequences for amplifying the target gene OsWRKY41 are OsWRKY41-F and OsWRKY41-R: OsWRKY41-F:5'-ATTCGAGCTCGGTAATGGAGTCCACCTGTC-3', OsWRKY41-R:5'-AGGATCCCCGGGTATCAGCTCACCAAGGCT-3'.

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