A pathogen-induced promoter Mig6p and its application in improving disease resistance in rice.

Abstract

This invention discloses a pathogen-induced promoter Mig6p and its application in improving disease resistance in rice, belonging to the fields of plant pathology and genetic engineering. This invention screened a rice promoter Mig6p (nucleotide sequence shown in SEQ ID NO.1) induced by multiple pathogens; Mig6p exhibits very low basal expression activity and is not induced by various abiotic stresses. Furthermore, a dominant-negative regulatory mutant of the Mig6p promoter-induced lesion gene Lrd6-6, Lrd6-6, was constructed. E315Q The expression vector was obtained by transforming rice to obtain Mig6p:Lrd6-6 E315Q Rice, verified, Mig6p:Lrd6-6 E315Q The broad-spectrum disease resistance of rice was significantly improved, while agronomic traits were not significantly affected. This invention provides important guidance for improving crop disease resistance using lesion-like genes and also provides a reference for improving other similar traits.

Description

Technical Field

[0001] This invention relates to the fields of plant pathology and genetic engineering, and in particular to a pathogen-induced promoter Mig6p and its application in improving disease resistance in rice. Background Technology

[0002] Rice blast, sheath blight, and bacterial leaf blight, among other diseases, severely threaten rice production. By utilizing disease resistance or resistance-related genes, combined with modern genetic engineering techniques, resistant rice materials can be rapidly created, achieving green and efficient disease control. Domestic and international researchers have conducted research on plant disease resistance, cloning a series of disease resistance or resistance-related genes (such as Pigm, Pib, rod1, bsr-d1, and lrd6-6), elucidating the molecular regulatory mechanisms of disease resistance, and establishing and continuously improving molecular models of plant disease resistance. Applying disease resistance genes and molecular theories to the improvement of resistance in crops such as rice has successfully bred a number of new crop varieties with excellent resistance, making a positive contribution to ensuring food security.

[0003] Currently, most genes used for improving crop disease resistance are R genes. These genes-mediated disease resistance are race-specific and have a narrow spectrum. At the same time, due to the rapid evolution and frequent replacement of dominant pathogen populations, disease-resistant varieties bred using R genes usually lose their resistance after 3 to 5 years of planting, and the resistance is not long-lasting.

[0004] In recent years, a class of genes called lesion mimic mutants (LMMs) has attracted attention due to the broad-spectrum and durable disease resistance they mediate. However, these genes often require recessive homozygous mutations to activate resistance, and sustained activation of resistance can negatively impact crop growth and yield, making it difficult to improve crop disease resistance using lesion mimic genes. Therefore, there is an urgent need to develop methods for improving crop disease resistance using lesion mimic genes. Summary of the Invention

[0005] The purpose of this invention is to provide a pathogen-induced promoter Mig6p and its application in improving disease resistance in rice, thereby solving the problems existing in the prior art. The nucleotide sequence of the promoter Mig6p is shown in SEQ ID NO.1, and it can induce the lesion-like gene Lrd6-6. E315Q This expression improves the disease resistance of rice.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] The present invention provides a pathogen-induced promoter Mig6p, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0008] Optionally, the pathogens include Magnaphalthe oryzae, Rhizoctonia solani, and Xanthomonas oryzae pv. oryzae.

[0009] The present invention also provides an expression vector comprising the aforementioned promoter Mig6p.

[0010] Optionally, the expression vector further comprises the lesion-like gene Lrd6-6. E315Q and the terminator Mig6t;

[0011] The Lrd6-6 E315Q The nucleotide sequence is shown in SEQ ID NO.15, and the nucleotide sequence of the terminator Mig6t is shown in SEQ ID NO.2.

[0012] The present invention also provides a method for constructing the expression vector, including the use of promoters Mig6p and Lrd6-6. E315Q The steps are as follows: the terminator Mig6t is sequentially ligated into the vector, and the expression vector is obtained by transforming E. coli and extracting plasmids.

[0013] The expression vector expresses the lesion-like gene Lrd6-6 under pathogen induction. E315Q .

[0014] The present invention also provides the application of the aforementioned promoter Mig6p in the improvement of disease resistance in rice.

[0015] Alternatively, a plant expression vector can be constructed by fusing the promoter Mig6p with a lesion-like gene and then transferred into rice to improve the disease resistance of rice.

[0016] Optionally, the lesion-like gene includes Lrd6-6. E315Q ;

[0017] The Lrd6-6 E315Q The nucleotide sequence is shown in SEQ ID NO.15;

[0018] The disease resistance includes resistance to rice blast, sheath blight, and bacterial blight.

[0019] The present invention also provides a method for improving the disease resistance of rice, comprising the steps of fusing the promoter Mig6p with a lesion-like gene to construct a plant expression vector and transferring it into rice to improve the disease resistance of rice.

[0020] Optionally, the lesion-like gene includes Lrd6-6. E315Q ;

[0021] The Lrd6-6 E315Q The nucleotide sequence is shown in SEQ ID NO.15;

[0022] The disease resistance includes resistance to rice blast, sheath blight, and bacterial blight.

[0023] The present invention discloses the following technical effects:

[0024] This invention screened a rice promoter, Mig6p, that is induced by multiple pathogens; Mig6p exhibits very low basal expression activity and is not induced by various abiotic stresses. This invention also constructed a dominant-negative regulatory mutant of the Mig6p promoter-induced lesion gene Lrd6-6, Lrd6-6. E315Q The expression vector was obtained, and Mig6p:Lrd6-6 was obtained by transforming rice TP309. E315Q Rice, verified, Mig6p:Lrd6-6 E315Q The broad-spectrum disease resistance of rice was significantly improved, while agronomic traits were not significantly affected. This invention provides important guidance for improving crop disease resistance using lesion-like genes and also provides a reference for improving other similar traits. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments 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.

[0026] Figure 1 The expression activity of promoter Mig6p in rice at different growth and development stages (three-leaf stage, six-leaf stage, tillering stage, heading stage, booting stage, grain-filling stage and maturity stage);

[0027] Figure 2 The expression activity of promoter Mig6p under infection with *Oryza sativa* (A), *Bacillus thuringiensis* strain P2 (B), and *Rhizoctonia solani* strain AG1-IA (C) was determined.

[0028] Figure 3 The expression activity of the promoter Mig6p under high temperature (A), simulated drought (B), and high salt (C) stress was evaluated.

[0029] Figure 4 The results of the identification of the disease-resistant genetically engineered rice constructed in this invention and its disease resistance are shown in Figure A; A represents the induced expression of the lesion-like gene Lrd6-6 and its dominant-negative regulatory mutant Lrd6-6. E315QA schematic diagram of the component construction; B represents wild-type TP309, the control transgenic line Mig6p:Lrd6-6, and the disease-resistant genetically engineered rice Mig6p:Lrd6-6. E315Q A) Plant images; B) Leaf phenotype and lesion density statistics for identifying rice blast resistance using spray inoculation; C) Leaf phenotype and lesion length statistics for identifying rice blast resistance using puncture inoculation; D) Leaf phenotype and lesion length statistics for identifying bacterial blight resistance; E) Leaf phenotype and lesion length statistics for identifying sheath blight resistance.

[0030] Figure 5 The results of the investigation of the main agronomic traits of the disease-resistant genetically engineered rice constructed in this invention are shown in Figure A. The results of the investigation of agronomic traits such as plant height, number of tillers, panicle length, thousand-grain weight and seed setting rate are shown in Figure B. The appearance of the polished rice of each rice variety is shown in Figure C. The statistical charts of the grain chalky degree (CD) and the percentage of chalky grain (PCG) of each rice variety are shown in Figure C. Detailed Implementation

[0031] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0032] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0033] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0034] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0035] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0036] Both Nipponbare rice and TP309 rice are commonly used rice varieties in this field. The Nipponbare rice and TP309 rice used in the following examples were provided by Sichuan Agricultural University.

[0037] The physiological race Zhong10-8-14 of Magnaphalthe oryzae has been published in the literature Li, W., Wang, K., Chern, M., Liu, Y., Zhu, Z., Liu, J., Zhu, X., Yin, J., Ran, L., Xiong, J., He, K., Xu, L., He, M., Wang, J., Liu, J., Bi, Y., Qing, H., Li, M., Hu, K., Song, L., Wang, L., Qi, T., Hou, Q., Chen, W., Li, Y., Wang, W., and Chen, X. (2020). Sclerenchyma cell thickening through enhanced lignification induced by OsMYB30 prevents fungal penetration of riceleaves. New Phytologist 226, 1850-1863;

[0038] Rhizoctonia solani solani) AG1-IA has been disclosed in the literature Zheng, A., Lin, R., Zhang, D., Qin, P., Xu, L., Ai, P., Ding, L., Wang, Y., Chen, Y., Liu, Y., Sun, Z. ,Feng,H.,Liang,X.,Fu,R.,Tang,C.,Li,Q.,Zhang,J.,Xie,Z.,Deng,Q.,Li,S.,Wang,S.,Zhu,J.,Wang,L.,Liu,H.,and Li, P. (2013). The evolution and pathogenic mechanisms of therice sheath blight pathogen. Nature Communications 4, 1424;

[0039] Xanthomonas oryzae pv. oryzae has been published in the literature Han, J., Xia, Z., Liu, P., Li, C., Wang, Y., Guo, L., Jiang, G., and Zhai, W. (2020). TALEN-based editing of TFIIAy5 changes rice response to Xanthomonas oryzae pv. Oryzae. Scientific Reports 10, 2036.

[0040] Example 1 clarifies the activity characteristics of the promoter Mig6p

[0041] 1. Analysis of the background activity characteristics of the Mig6p promoter

[0042] Tissue samples were collected from rice at the three-leaf stage, six-leaf stage, tillering stage, heading stage, booting stage, grain-filling stage, and maturity stage. Total RNA was extracted from each sample using the Trizol method. RNA reverse transcription was performed using the Vazyme HiScript IV 1st StrandcDNA Synthesis Kit with gDNA Eraser (R412-01). Quantitative real-time PCR was performed using specific primers for the Mig6 promoter gene Mig6: Mig6-qF: 5'-GCTGGAGTTCTTCTTCGGC-3' (SEQ ID NO.3) and Mig6-qR: 5'-CGAAGTTGAGCGTGCACAT-3' (SEQ ID NO.4) to determine the background activity characteristics of the Mig6p promoter.

[0043] Quantitative real-time PCR was performed using a Bio-Rad CFX96 Real-Time System coupled with a C1000 Thermal Cycler (Bio-Rad, USA). The internal reference gene was Ubq5, and the primers were Ubq5-Qf: 5'-AACCAGCTGAGGCCCAAGA-3' (SEQ ID NO.5) and Ubq5-Qr: 5'-ACGATTGATTTAACCAGTCCATGA-3' (SEQ ID NO.6).

[0044] The Trizol method for extracting total RNA from rice is as follows: First, pour alcohol into a mortar and ignite it to remove RNase from the mortar using high temperature. After the mortar cools, add liquid nitrogen to freeze-grind the sample. Take 100 mg of the ground sample powder and put it into a 1.5 mL EP tube, add 1 mL of Trizol reagent. After the sample powder and Trizol solution are thoroughly mixed, let it stand at room temperature for 5 min to allow the nucleoprotein complex to be completely separated. Add 0.2 mL of chloroform, vortex vigorously for 20 seconds, and incubate at room temperature for 3 minutes. Centrifuge at 12000 g and 4°C for 15 minutes until the RNA is completely dissolved in the upper aqueous phase. Transfer the upper aqueous phase to a new 1.5 mL EP tube (approximately 0.6 mL). Add an equal volume of chloroform again, vortex for 20 seconds, centrifuge at 12000 g and 4°C for 15 minutes, and transfer the upper aqueous phase to a new 1.5 mL EP tube. Add an equal volume of isopropanol, incubate at room temperature for 10 minutes, and centrifuge at 12000 g and 4°C for 10 minutes to precipitate the RNA. Remove the supernatant; the RNA will adhere to the bottom of the tube. Add 0.75 mL of 70% ethanol to wash the precipitate, gently tapping it to ensure thorough washing. Centrifuge at 7000 g and 4°C for 5 minutes and remove the supernatant. Repeat this step. Place the EP tube in a fume hood to allow the alcohol to evaporate completely, until the precipitate is transparent. Add 30 μL of RNAase-free water and incubate at 4°C to dissolve completely.

[0045] 2. Mig6p promoter activity is induced by a variety of pathogens.

[0046] The activity of the Mig6p promoter was analyzed under infection by *Bacillus oryzae*, *Rhizoctonia solani*, or *Bacillus thuringiensis*. The specific methods are as follows:

[0047] For the activity analysis of the Mig6p promoter induced by *Magnaporthe oryzae*, conidia of the physiological race Zhong10-8-14 of *Magnaporthe oryzae* (concentration 5 × 10⁻⁶) were used. 5Three-week-old TP309 rice plants were spray-inoculated with spores / mL, and samples were taken at 0, 6, 12 and 24 h after inoculation. A 0.2% Tween treatment was used as a control. The expression of the Mig6 gene was analyzed using the method described in Section 1, Analysis of the Background Activity Characteristics of the Mig6p Promoter, to reflect the activity of the Mig6p promoter.

[0048] For the activity analysis of the Mig6p promoter induced by *Rhizoctonia solani*, TP309 rice leaves at the tillering stage were placed on sterile filter paper moistened with 6-BA solution (1 mg / L, pH = 7.0), and mycelial blocks of *Rhizoctonia solani* strain AG1-IA with the same area were inoculated onto the leaves. After inoculation, rice leaf samples near the mycelial blocks were collected at 0, 6, 12, and 24 h, respectively. A blank culture block was used as a control. The method described in Section 1, "Analysis of the Background Activity Characteristics of the Mig6p Promoter," was used to analyze the expression of the Mig6p gene to reflect the activity of the Mig6p promoter.

[0049] To analyze the activity of the Mig6p promoter induced by Bacillus subtilis, TP309 rice leaves at the tillering stage were cut into segments of approximately 1 cm and immersed in OD245 solution. 600 Samples were taken from the bacterial culture of *Bacillus subtilis* strain P2 at 0, 6, 12, and 24 h, with sterile treatment as a control. The expression of the Mig6 gene was analyzed using the method described in section 1, "Analysis of the Background Activity Characteristics of the Mig6p Promoter," to reflect the activity of the Mig6p promoter.

[0050] 3. The activity of the Mig6p promoter is not induced by various abiotic stresses.

[0051] The activity of the Mig6p promoter under high temperature, drought, or high salinity conditions was analyzed. The specific methods are as follows:

[0052] For the activity analysis of the Mig6p promoter under high temperature stress, 3-week-old rice seedlings were transferred from a 26℃ incubator to a 40℃ incubator, and samples were taken at 0, 6, 12, 24, 36, 48, 60, and 84 hours. Seedlings cultured at 26℃ were used as controls. The method described in section 1, "Analysis of the Background Activity Characteristics of the Mig6p Promoter," was used to analyze the expression of the Mig6 gene to reflect the activity of the Mig6p promoter.

[0053] For the analysis of Mig6p promoter activity under drought stress, PEG6000 was used to simulate drought. Rice seedlings that had grown normally to 3 weeks old in Yoshida (Coolaber, NS1040) hydroponic solution were transferred to a new Yoshida hydroponic solution containing 15% PEG6000 (Yeasen, 60366ES76). Leaf samples were taken at 0, 1, 2, and 4 days. With normal Yoshida hydroponic solution treatment as a control, the method in Section 1, Analysis of the Background Activity Characteristics of Mig6p Promoter, was used to analyze Mig6 gene expression to reflect Mig6p promoter activity.

[0054] To analyze the activity of the Mig6p promoter under salt stress, rice seedlings that had grown normally to 3 weeks old in Yoshida hydroponic solution were transferred to a new Yoshida hydroponic solution containing 0.5% NaCl. Leaf samples were taken at 0, 1, 2, and 4 days. With normal Yoshida hydroponic solution treatment as a control, the method described in Section 1, Analysis of the Background Activity Characteristics of the Mig6p Promoter, was used to analyze the expression of the Mig6p gene to reflect the activity of the Mig6p promoter.

[0055] 4. Results

[0056] 4.1 Expression activity of promoter Mig6p at different growth and development stages of rice

[0057] like Figure 1 As shown, Mig6p activity was highest in the stems during the heading stage, but it was only about three-thousandths of the activity of the internal reference gene promoter, indicating that the background expression activity of Mig6p was very low.

[0058] 4.2 Expression activity of promoter Mig6p under different pathogen infections

[0059] like Figure 2 As shown, Mig6p activity increases rapidly under different pathogen infections, indicating that Mig6p activity is rapidly induced by pathogens.

[0060] 4.3 Expression activity of promoter Mig6p under different abiotic stresses

[0061] like Figure 3 As shown, Mig6p activity generally decreased under high temperature, simulated drought, and high salt stress, indicating that Mig6p activity is not induced by various abiotic stresses.

[0062] Example 2: Construction of the lesion-like gene Lrd6-6 E315Q carrier

[0063] 1. Isolate and clone the Mig6p DNA fragment

[0064] Total DNA was extracted from rice using the CTAB method: Fresh rice leaves, about 5 cm long, were cut into small pieces and placed in a 2 mL EP tube. Steel beads were added, and the tube was flash-frozen in liquid nitrogen. The mixture was then ground into powder using a tissue homogenizer for 40 seconds. 600 μL of CTAB extraction buffer (2 L: CTAB 40 g, NaCl 163.6 g, EDTA 14.88 g, Tris-100 g) was added. 24.2g (adjusted to pH 8.0 with ddH2O), mixed thoroughly, and placed in a 65℃ oven for 30-60 min, shaking every 10 min during this period; add an equal volume of chloroform-isoamyl alcohol (chloroform to isoamyl alcohol volume ratio 24:1), mix thoroughly, let stand at room temperature for 10 min, then centrifuge at 12000 r / min for 6 min; transfer 350 μL of the supernatant to 500 μL of pre-cooled anhydrous ethanol at -20℃, mix well, and let stand for a few minutes until white flocculent precipitate appears in the tube; centrifuge at 12000 r / min for 2 min to precipitate DNA; discard the liquid, wash the precipitate with 1 mL of 75% alcohol; discard the liquid, air dry in a fume hood, dissolve in 200 μL of sterile water, and store at -20℃ for later use.

[0065] The Mig6 promoter Mig6p and terminator Mig6t were cloned from total rice DNA using Vazyme high-fidelity PCR polymerase 2×Phanta Max Master Mix (P525-01). The primer sequences were: Mig6p-F: 5'-GGAGGTGCCACTTGTTTAACTTACT-3' (SEQ ID NO.7) and Mig6p-R: 5'-GACGATATACGAAATTCGCCGAAGA-3' (SEQ ID NO.8), Mig6t-F: 5'-GACGTCATTGTTAGCTTTGCAGCTG-3' (SEQ ID NO.9) and Mig6t-R: 5'-TTCCATGGACCAACTAACTTTGCCT-3' (SEQ ID NO.10). The specific PCR operation steps were performed according to the instructions provided with the kit.

[0066] After amplification, the PCR products were separated by 1.0% agarose gel electrophoresis. A piece of agarose gel the size of the target fragment was cut off under UV light using a clean scalpel. The DNA was recovered using an agarose gel DNA recovery kit (Thermo Fisher) to obtain the isolated and purified Mig6 promoter Mig6p and terminator Mig6t.

[0067] The recovered products obtained above were sent to a sequencing company for sequencing, and the nucleotide sequences of the promoter Mig6p and terminator Mig6t were obtained as shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0068] SEQ ID NO.1 (promoter Mig6p):

[0069]

[0070] SEQ ID NO.2 (Terminator Mig6t):

[0071]

[0072] 2. Pathogen-induced expression of lesion-like gene Lrd6-6 E315Q Carrier construction

[0073] Inducible expression of the lesion-like gene Lrd6-6 and its dominant-negative regulatory mutant Lrd6-6 E315Q The component construction diagram is as follows Figure 4 As shown in Figure A.

[0074] Using the aforementioned promoter Mig6p and terminator Mig6t, and the previously reported (Zhu X, Yin J, Liang S, et al. The multivesicular bodies (mvbs)-localized aaa atpase lrd6-6 inhibits immunity and cell death likely through regulating mvbs-mediated vesicular trafficking in rice. PLOS Genetics, 2016, 12:e1006311) Lrd6-6-containing... E315Q The gene plasmid was used as a PCR template. The promoters Mig6p and Lrd6-6 were amplified using Vazyme's 2×Phanta Max Master Mix (P525-01) high-fidelity PCR enzyme. E315Q The gene and terminator Mig6t were extracted (specific procedures were performed according to the instructions provided with the kit), and the above three elements were progressively recombined into the plasmid pDTmar-hyg (Leng C, Sun B, Liu Z, et al. An optimized double t-DNA binary vector system for improved production of marker-free transgenic tobacco plants. Biotechnology Letters, 2020, 42: 641-655) using the Vazyme ClonExpress II One Step Cloning Kit to obtain the final expression vector pDTmar-Mig6p-Lrd6-6. E315Q .

[0075] Specifically, the Mig6p sequence containing the homologous sequence was first amplified using primer pairs pDTmar-Mig6p-F: 5'-tgcatgcctgcaggtcgactctagaGGAGGTGCCACTTGTTTAACTTACT-3' (SEQ ID NO.11, the lowercase sequence is the upstream homologous sequence that will recombine with the target vector) and pDTmar-Mig6p-R: 5'-tgattacgaattcgagctcggtaccGACGATATACGAAATTCGCCGAAGA-3' (SEQ ID NO.12, the lowercase sequence is the downstream homologous sequence that will recombine with the target vector). The plasmid vector pDTmar was then linearized by double digestion with the rapid digestive enzymes Xba I and Kpn I (purchased from Thermo Fisher, refer to the corresponding product instructions for specific usage and dosage). The obtained Mig6p sequence was then homologously recombinated with the double-digested vector using recombinase (Vazyme ClonExpress II One Step Cloning Kit). The recombinant product was transformed into Escherichia coli, and after colony PCR detection of positive clones and correct sequencing, the plasmid was extracted and amplified to obtain the pDTmar-Mig6p intermediate vector.

[0076] Subsequently, primer pairs pDTmar-Mig6p-Lrd6-6-F: 5'-tcttcggcgaatttcgtatatcgtcATGAGCTTCCTCCGCGCGCTCGCGG-3' (SEQ ID NO.13, the lowercase sequence is the upstream homologous sequence that recombines with the target vector) and pDTmar-Mig6p-Lrd6-6-R: 5'-tgattacgaattcgagctcggtaccTCAACTTGAACCAAATTCTTCGTTC-3' (SEQ ID NO.14, the lowercase sequence is the downstream homologous sequence that recombines with the target vector) were used to amplify Lrd6-6 containing homologous sequences (control, nucleotide sequence as shown in SEQ ID NO.15) and Lrd6-6, respectively. E315Q The sequence (nucleotide sequence as shown in SEQ ID NO.16) was obtained, and the plasmid vector pDTmar-Mig6p was linearized by single digestion with the rapid digestion enzyme Kpn I (purchased from Thermo Fisher, refer to the corresponding product instructions for specific usage and dosage); the obtained Lrd6-6 (control) and Lrd6-6 were then recombinantly digested using recombinase (Vazyme ClonExpress II One Step Cloning Kit). E315QThe sequences were subjected to homologous recombination with the digested vectors described above. The recombinant products were transformed into *E. coli*, and after colony PCR detection and confirmation of positive clones and sequencing, plasmids were extracted and amplified to obtain pDTmar-Mig6p-Lrd6-6 and pDTmar-Mig6p-Lrd6-6. E315Q Intermediate carrier.

[0077] Finally, the Mig6t sequence containing the homologous sequence was amplified using primer pairs pDTmar-Mig6p-Lrd6-6-Mig6t-F: 5'-gaacgaagaatttggttcaagttgaGACGTCATTGTTAGCTTTGCAGCTG-3' (SEQ ID NO.17, the lowercase sequence is the upstream homologous sequence that recombines with the target vector) and pDTmar-Mig6p-Lrd6-6-Mig6t-R: 5'-tgattacgaattcgagctcggtaccTTCCATGGACCAACTAACTTTGCCT-3' (SEQ ID NO.18, the lowercase sequence is the downstream homologous sequence that recombines with the target vector). The plasmid vectors pDTmar-Mig6p-Lrd6-6 and pDTmar-Mig6p-Lrd6-6 were then digested with the fast digester Kpn I (purchased from Thermo Fisher, refer to the corresponding product instructions for specific usage and dosage). E315Q Linearized plasmid vectors were prepared. The obtained Mig6t sequence was homologously recombinated with the digested vector using recombinase (Vazyme ClonExpress II One Step Cloning Kit). The recombinant products were transformed into *E. coli*, and positive clones were detected by colony PCR. After sequencing confirmation, plasmids were extracted and amplified to obtain pDTmar-Mig6p-Lrd6-6-Mig6t and pDTmar-Mig6p-Lrd6-6. E315Q -Mig6t vector.

[0078] SEQ ID NO.15 (Lrd6-6 nucleotide sequence):

[0079]

[0080] SEQ ID NO.16(Lrd6-6 E315Q Nucleotide sequence):

[0081]

[0082] Example 3: Pathogen-induced expression of lesion-like gene Lrd6-6 E315Q rice construction

[0083] The pDTmar-Mig6p-Lrd6-6-Mig6t and pDTmar-Mig6p-Lrd6-6 obtained in Example 2 E315Q The Mig6t vector was transformed into Agrobacterium EHA105 (purchased from Chengdu Qingke Biotechnology Co., Ltd., according to the instructions). Single clones were picked for colony PCR detection, and positive clones were stored at -80℃ for later use.

[0084] Agrobacterium-mediated genetic transformation was used to transform the two vectors mentioned above into rice TP309. The Agrobacterium-mediated genetic transformation method for rice is as follows:

[0085] 1) Inducing callus: Remove the seed coat, weigh about 24g of seeds, treat with 50mL of 75% alcohol for 2min, wash twice with sterile distilled water; disinfect with 2.5% sodium hypochlorite for 20min, then wash off as much sodium hypochlorite as possible from the seed surface with sterile distilled water, and absorb as much liquid as possible from the seed surface with sterile filter paper; place the disinfected seeds evenly on N6 medium with tweezers, and then place them in a 30℃ incubator for dark culture, which takes about 10 days. Then peel the callus off the seeds for the next step.

[0086] 2) Agrobacterium infection of rice callus: Agrobacterium carrying recombinant plasmids, taken from a -80℃ freezer, was streaked on YM solid medium (containing Kan / Rif / Gm) and cultured at 28℃ for 2 days. Then, a small amount of bacteria was picked and added to 3 mL of YM liquid medium (containing Kan / Rif / Gm) and cultured overnight at 28℃ and 200 rpm. 1 mL of bacterial solution was taken and added to 50 mL of infection medium and cultured at 8℃ and 200 rpm until the OD value did not exceed 0.1. The peeled callus was immersed in the infection solution and infected at 28℃ and 140 rpm for 30 min. The infection solution was discarded, and the callus was transferred to sterile filter paper. The surface of the callus was dried as much as possible. Then, the callus was transferred to co-culture medium and cultured in the dark at 22℃ for 3 days.

[0087] 3) Screening and differentiation: Transfer the callus to the screening medium and culture it in the dark at 30°C for at least 30 days, changing the medium once during the period; then transfer the callus to the predifferentiation medium and culture it in the light at 30°C for 15 days; after that, transfer the callus to the redifferentiation medium and culture it in the light at 30°C until green shoots emerge.

[0088] 4) Rooting: Transfer the differentiated buds to a rooting medium and culture them under 30°C light. When the seedlings grow to about 10cm, harden them off and then transplant them into the soil.

[0089] The formulations of the culture media obtained above are as follows:

[0090] Basic culture medium: MS and N6 powder (manufactured by Phytotechnology Lab).

[0091] Callus induction medium (1L): N6 4g, sucrose 30g, inositol 0.1g, acid-hydrolyzed casein 0.3g, proline 0.5g, glutamine 0.5g, 2,4-D 2mg and plant gel 4g, adjusted to pH=5.8.

[0092] Infection medium (1L): 3g yeast extract and 5g tryptone, autoclaved, then 40mg AS was added and the pH was adjusted to 5.5.

[0093] Co-culture medium (1L): MS 4.4g, sucrose 30g, 2,4-D 2mg, D-Sorbitol 50g and plant gel 4g, adjusted pH=5.8, autoclaved at high temperature and added 40mg AS.

[0094] Screening medium (1L): MS 4.4g, sucrose 30g, 2,4-D 2mg and plant gel 4g, adjust pH to 5.8, autoclave and then add 400mg carbenicillin and 50mg genimycin (G418).

[0095] Predifferentiation medium (1L): MS 4.4g, sucrose 30g, D-Sorbitol 50g, 6-BA 3mg, NAA 0.5mg and plant gel 4g, adjust pH=5.8, autoclave and add 200mg carbenicillin and 25mg G418.

[0096] Redifferentiation medium (1L): MS 4.4g, sucrose 30g, D-Sorbitol 50g, 6-BA 2mg, NAA 0.05mg and plant gel 4g, adjust pH to 5.8, autoclave and add 200mg carbenicillin and 25mg G418.

[0097] Rooting medium (1L): MS 2.2g, sucrose 30g and plant gel 4g, adjust pH to 5.8, autoclave and pour into tissue culture flask.

[0098] The obtained plants were amplified by PCR to carry the Hyg resistance gene in the plasmid to determine whether the plants were positive transformants.

[0099] Example 4: Identification of Disease Resistance in Rice

[0100] The wild-type rice cultivation materials and genetically transformed seedlings involved in this invention were all planted in rice cultivation greenhouses at the transgenic base of Sichuan Agricultural University. Three Mig6p:Lrd6-6 and three Mig6p:Lrd6-6 seedlings were selected from each. E315Q The strains were tested for resistance to rice blast, sheath blight, and bacterial blight. Among them, the rice blast resistance test referenced Li W, Wang K, Chern M, et al. Sclerenchyma cell thickening through enhanced lignification induced by osmyb30 prevents fungal penetration of rice leaves. New Phytologist, 2020, 226:1850-1863; the sheath blight resistance test referenced Feng Q, Wang H, Yang XM, et al. Osa-mir160aconfers broad-spectrum resistance to fungal and bacterial pathogens in rice. New Phytologist, 2022, 236:2216-2232; and the bacterial leaf blight resistance test referenced Zhou X, Wang J, Peng C, et al. Four receptor-like cytoplasmic kinases regulate development and immunity in rice. Plant, Cell & Environment, 2016, 39:1381-1392.

[0101] Results: Control transgenic rice Mig6p:Lrd6-6 and disease-resistant genetically engineered rice Mig6p:Lrd6-6 E315Q There was no significant difference in plant phenotype compared to wild-type TP309. Figure 4 In the middle B), rice strain Zhong10-8-14 of *Magnapordica oryzae* was inoculated using both spray inoculation and puncture inoculation methods. The resulting genetically engineered rice strain Mig6p:Lrd6-6 was resistant to the disease. E315Q The resistance to rice blast disease was significantly improved. Figure 4 C- Figure 4 (D); Identification of bacterial blight resistance and sheath blight resistance in vitro using *Bacillus thuringiensis* strain P2 and *Bacillus thuringiensis* strain AG1-IA both showed a significant increase in the corresponding disease resistance. Figure 4 China E- Figure 4 (F).

[0102] Example 5: Investigation of Major Agronomic Traits of Rice

[0103] For each strain selected for disease resistance testing in Example 4, a randomized block design was used in the rice cultivation greenhouse at the Sichuan Agricultural University transgenic base. Three experimental plots were planted, with four replicates per plot and ten plants per row. Water and fertilizer management was performed according to normal rice field management practices. After maturity, the middle four plants from each row were selected for analysis of traits such as plant height, number of tillers, panicle length, thousand-grain weight, seed setting rate, and chalkiness. Thousand-grain weight was measured using the Mini 1600 automatic analysis system from Chengdu Jielaimei Company, while chalky grain rate and chalkiness were measured using the WSeen SC-E type rice appearance quality detection analyzer from Hangzhou Wanshen Company.

[0104] Results: Control transgenic rice Mig6p:Lrd6-6 and disease-resistant genetically engineered rice Mig6p:Lrd6-6 E315Q There were no significant differences in plant height, tiller number, panicle length, thousand-grain weight, and seed setting rate compared to the wild-type TP309. Figure 5 (A); there was no significant difference in the appearance of the polished rice from each group. Figure 5 (B); as for the grain chalky degree (CD) and percentage of chalky grain (PCG) of the rice in each group, there were no significant differences. Figure 5 (C), the results show that Mig6p:Lrd6-6 E315Q While the broad-spectrum disease resistance of rice was significantly improved, its agronomic traits were not significantly affected.

[0105] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A pathogen-induced promoter Mig6p Its characteristics are, The promoter Mig6p The nucleotide sequence is shown in SEQ ID NO.

1.

2. The promoter as described in claim 1 Mig6p Its characteristics are, The pathogen is selected from rice blast fungus (… Magnaporthe oryzae Rhizoctonia solani ( ) Rhizoctonia solani ) and white leaf blight ( Xanthomonas oryzae pv. oryzae ).

3. An expression carrier, characterized in that, The expression vector comprises the promoter according to claim 1. Mig6p .

4. The expression vector as described in claim 3, characterized in that, The expression vector also contains a lesion-like gene. Lrd6- 6 E315Q and Termination Sub Mig6t ; The Lrd6-6 E315Q The nucleotide sequence is shown in SEQ ID NO.15, and the terminator... Mig6t The nucleotide sequence is shown in SEQ ID NO.

2.

5. The method for constructing the expression vector as described in claim 4, characterized in that, Including the promoter Mig6p , Lrd6- 6 E315Q and Termination Sub Mig6t The steps are as follows: sequentially ligating the expression vector into the vector, transforming E. coli, and extracting the plasmid to obtain the expression vector; The expression vector expresses a lesion-like gene under pathogen induction. Lrd6-6 E315Q .

6. The promoter as described in claim 1 Mig6p Its application in improving disease resistance in rice is characterized by, The disease resistance is selected from resistance to rice blast, sheath blight and bacterial blight.

7. The application as described in claim 6, characterized in that, By promoting the promoter Mig6p A plant expression vector was constructed by fusing the lesion-like gene with the lesion-like gene and then transferred into rice to improve the disease resistance of rice.

8. The application as described in claim 7, characterized in that, The plaque-like genes include Lrd6-6 E315Q ; The Lrd6-6 E315Q The nucleotide sequence is shown in SEQ ID NO.

15.

9. A method for improving the disease resistance of rice, characterized in that, Including the promoter described in claim 1 Mig6p The process involves fusing the lesion-like gene with a plant expression vector and then transferring it into rice to improve the disease resistance of rice. The disease resistance is selected from resistance to rice blast, sheath blight and bacterial blight.

10. The method as described in claim 9, characterized in that, The plaque-like genes include Lrd6-6 E315Q ; The Lrd6-6 E315Q The nucleotide sequence is shown in SEQ ID NO.15.

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