Method for creating broad-spectrum disease-resistant transgenic maize

Abstract

Provided is a method for creating broad-spectrum disease-resistant transgenic maize. Specifically provided is a method for preparing a transgenic plant having enhanced disease resistance and / or improved production performance. The method comprises the following steps: introducing a multifunctional gene or a multifunctional gene-related biomaterial into a receptor plant to obtain a transgenic plant having higher disease resistance and / or production performance higher than the receptor plant. The multifunctional gene is a DNA molecule as shown in SEQ ID NO: 1.

Description

A method for creating broad-spectrum disease-resistant transgenic corn Technical Field

[0001] The invention belongs to the field of plant genetic engineering and relates to a method for creating broad-spectrum disease-resistant transgenic corn. Background Art

[0002] Plant diseases have always been an important issue that cannot be ignored in agricultural production. They not only cause crop yield reduction, but also lead to a decline in the quality of agricultural products, causing significant losses to production. In severe cases, they can also cause food shortages and a series of social problems.

[0003] Corn (Zea mays L.) is a staple food crop in many parts of the world. During its breeding and production practices, it has been found that southern rust, small leaf spot, large leaf spot, and especially ear rot are the main diseases in its growth process. These diseases not only directly cause corn yield losses, but also affect corn quality and pose a threat to the safety of corn and its feed.

[0004] Corn ear rot, also known as fusarium smut or dry ear rot, affects both ears and kernels. The top or middle of the affected ear discolors, and a pink, blue-green, black-gray, dark brown, or yellow-brown mold appears. This mold, which represents the pathogen's fungi, conidiophores, and conidia, spreads to one-third to one-half of the ear. In rainy or humid conditions, it can spread to the entire ear. The kernels are dull, unfull, brittle, and hollow, often filled with interwoven hyphae. The bracts of the affected ear are often penetrated by dense hyphae, clinging to the ear and making them difficult to remove. Southern corn rust primarily affects leaves, but can also infect sheaths, stalks, and bracts. In severe cases, the entire leaf can be covered with rust-brown spots, causing it to wither and turn yellow. The bracts, ears, and male flowers can also be affected. Corn leaf spot, also known as corn spot, can occur throughout the corn's growth period, primarily affecting leaves, but also sheaths, bracts, and ears. Lesions on leaves are small and numerous. Under high temperature and humidity, a dense layer of gray mold grows on the surface of the lesions, representing the conidiophores and conidia of the pathogen. Corn leaf spot primarily affects leaves, but in severe cases, also affects sheaths and bracts. Lesions first appear on the lower leaves of the plant and then spread upward. Lesions are long, spindle-shaped, gray-brown or yellow-brown, or several lesions may connect to form large, irregular, dead spots. In severe cases, leaves may scorch.

[0005] Currently, using genetic engineering to enhance plant disease resistance has opened up a new avenue for disease-resistant breeding and has become a major strategy for mitigating pathogenic infestations. In recent years, genetically modified disease-, insect-, and herbicide-resistant crops such as soybeans, wheat, and cotton have been widely deployed and deployed, achieving significant economic and social benefits. However, due to the unpredictable effects of genetic modification, the same or similar genes can have different effects across species. A gene that exerts disease resistance in one species may have no effect in another, or even lead to negative side effects such as reduced yield. Therefore, researching and discovering genes with significant application potential or new functions of known genes, and leveraging these genes to successfully breed high-yield, multi-disease-resistant corn varieties, has become an urgent challenge in corn production. However, no reports have yet been released on the successful development of such genetically modified disease-resistant corn. SUMMARY OF THE INVENTION

[0006] The successful breeding of high-yield, multi-disease-resistant corn varieties has become a pressing challenge in corn production. To overcome the shortcomings of existing technologies, the present invention provides a method for creating broad-spectrum disease-resistant transgenic corn. Specifically, transgenic plants with enhanced disease resistance and / or improved production performance are prepared by introducing multifunctional genes or biomaterials related to these genes. This method has significant application and promotional value for the breeding of high-quality corn varieties. Technical Solutions

[0007] The present invention provides a method for creating broad-spectrum disease-resistant transgenic corn.

[0008] The present invention provides the use of multifunctional genes or multifunctional gene-related biological materials in preparing transgenic plants with enhanced disease resistance and / or improved production performance.

[0009] The present invention also provides a method for preparing transgenic plants with enhanced disease resistance and / or improved production performance, comprising the following steps: introducing a multifunctional gene or multifunctional gene-related biological material into a recipient plant to obtain a transgenic plant with enhanced disease resistance and / or production performance than the recipient plant.

[0010] Any of the above multifunctional genes is as follows (a1), (a2) or (a3):

[0011] (a1) a DNA molecule represented by SEQ ID NO: 1;

[0012] (a2) a DNA molecule that hybridizes to (a1) under stringent conditions;

[0013] (a3) A DNA molecule that is more than 99% identical to (a1).

[0014] The stringent conditions can be in a 0.1×SSPE (or 0.1×SSC), 0.1% SDS solution at 65 o Hybridize under C conditions and wash the membrane.

[0015] As used herein, the term "identity" refers to sequence similarity to a natural nucleic acid sequence. Identity can be assessed visually or using computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to assess the identity between related sequences.

[0016] The multifunctional gene-related biological material is an expression cassette having the multifunctional gene, a recombinant vector having the multifunctional gene, or a recombinant microorganism having the multifunctional gene.

[0017] Specifically, the recombinant vector can be constructed using a plant expression vector as a starting vector. Such plant expression vectors include binary Agrobacterium vectors and vectors suitable for plant microprojectile bombardment. The plant expression vector may also contain the 3′ untranslated region of the exogenous gene, i.e., a polyadenylation signal and any other DNA segments involved in mRNA processing or gene expression. When constructing a recombinant vector using the multifunctional gene of the present invention, enhancers, including translation enhancers or transcription enhancers, may also be used. To facilitate identification and screening of transgenic plant cells or plants, the plant expression vector may be modified to include genes encoding enzymes or luminescent compounds that can be expressed in plants and produce color changes, antibiotic marker genes, or chemical resistance marker genes. For safety reasons, the transgenic plants may be screened directly for transformed plants using stress without the addition of any selectable marker genes.

[0018] As an example, the recombinant vector is specifically shown as SEQ ID NO: 2.

[0019] Any of the above-mentioned enhanced disease resistance refers to enhanced disease resistance to ear rot and / or southern rust and / or small leaf spot and / or large leaf spot.

[0020] Specifically, the ear rot is corn ear rot.

[0021] Specifically, the pathogen of corn ear rot is Fusarium verticillioides.

[0022] Specifically, the southern rust is southern corn rust.

[0023] Specifically, the pathogen of the southern rust is Puccinia polysora Underw.

[0024] Specifically, the small leaf spot disease is corn small leaf spot disease.

[0025] Specifically, the pathogenic bacteria of the small leaf spot disease is Bipolaris maydis.

[0026] Specifically, the large leaf spot disease is corn large leaf spot disease.

[0027] Specifically, the pathogenic bacteria of the giant spot disease is Exserohlium turcicum.

[0028] Any of the above plants may be monocots or dicots.

[0029] Any of the above plants may be a grass plant.

[0030] Any of the above plants may be a plant of the genus Zea mays.

[0031] Any of the above-mentioned grass plants is corn.

[0032] Specifically, the corn may be corn PH4CV.

[0033] Any of the above-mentioned improvements in production performance refers to an increase in output.

[0034] The present invention also protects a DNA molecule, which is as follows (a1), (a2) or (a3):

[0035] (a1) a DNA molecule represented by SEQ ID NO: 1;

[0036] (a2) a DNA molecule that hybridizes to (a1) under stringent conditions;

[0037] (a3) A DNA molecule that is more than 99% identical to (a1).

[0038] The stringent conditions can be in a 0.1×SSPE (or 0.1×SSC), 0.1% SDS solution at 65 o Hybridize under C conditions and wash the membrane.

[0039] As used herein, the term "identity" refers to sequence similarity to a natural nucleic acid sequence. Identity can be assessed visually or using computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to assess the identity between related sequences.

[0040] Transgenic corn was obtained by overexpressing the multifunctional gene provided by the present invention in maize. Compared with non-transgenic wild-type control corn, the transgenic corn showed significantly improved disease resistance to ear rot, southern rust, small leaf spot, and large leaf spot, and significantly increased yield (yield increase of over 38%). Agronomic traits such as plant height, ear position, and plant architecture were not significantly different. Compared with the existing corn variety Chang 7-2, which has excellent comprehensive agronomic traits, the transgenic corn showed significantly improved disease resistance to ear rot, southern rust, small leaf spot, and large leaf spot, and significantly increased yield (yield increase of between 21.2% and 22.8%). Compared with the existing corn variety Zheng 58, which has excellent comprehensive agronomic traits, the transgenic corn showed significantly improved disease resistance to ear rot, southern rust, small leaf spot, and large leaf spot, and significantly increased yield (yield increase of between 14.7% and 16.2%). These results demonstrate that multifunctional genes can improve corn yield while enhancing broad-spectrum disease resistance. Beneficial effects

[0041] The invention has great application and promotion value for the breeding of excellent corn varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 is a schematic diagram of the elements of the recombinant plasmid.

[0043] FIG2 is an exemplary electrophoresis diagram of PCR-identified positive plants.

[0044] FIG3 shows exemplary phenotypes of transgenic corn plants and control corn plants after inoculation with ear rot pathogens.

[0045] FIG4 shows exemplary phenotypes of transgenic corn plants and control corn plants after inoculation with southern rust pathogens.

[0046] FIG5 shows exemplary phenotypes of transgenic corn plants and control corn plants after inoculation with the pathogenic bacteria of Southern leaf spot.

[0047] FIG6 shows exemplary phenotypes of transgenic corn plants and control corn plants after inoculation with the pathogenic bacteria of northern leaf spot. Modes for Carrying Out the Invention

[0048] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0049] The experimental methods in the following examples, unless otherwise specified, are all conventional methods and are carried out in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels. Unless otherwise specified, the quantitative tests in the following examples were repeated three times, and the results were averaged. Phytagar, i.e., plant gelatin. Corn PH4CV, i.e., corn inbred line PH4CV. Corn Chang 7-2, i.e., corn inbred line Chang 7-2. Corn Zheng 58, i.e., corn inbred line Zheng 58.

[0050] MS basal salt mixture, product name MS Plant Medium (MURASHIGE & SKOOG MEDIUM), Beijing Qiwei Yicheng Technology Co., Ltd., catalog number M0221. 1000× B5 vitamins, product name Gamborg Vitamin Solution (1000X), Beijing Ximeijie Technology Co., Ltd., catalog number G219. MS basal medium with vitamins, product name Murashige & Skoog Basal Medium with Vitamins, Beijing Ximeijie Technology Co., Ltd., catalog number M519. 1000× ERIKSSON vitamins, product name Eriksson Vitamin Solution (1000X), Beijing Ximeijie Technology Co., Ltd., catalog number E330. Picloram: Sangon Biotech (Shanghai) Co., Ltd., catalog number A610733.

[0051] Example 1: Preparation of transgenic corn plants

[0052] 1. Construction of plant expression vector

[0053] The broad-spectrum disease resistance gene is shown in SEQ ID NO: 1.

[0054] A recombinant plasmid expressing a broad-spectrum disease resistance gene was constructed. The recombinant plasmid is a circular double-stranded DNA molecule, as shown in SEQ ID NO: 2. In SEQ ID NO: 2, positions 425-1219 constitute the kanamycin resistance gene, positions 10536-11048 constitute the broad-spectrum disease resistance gene, and positions 11824-12375 constitute the Bar gene. A map of the recombinant plasmid is shown in Figure 1.

[0055] 2. Genetic Transformation of Maize

[0056] 1. Introduce the recombinant plasmid constructed in step 1 into Agrobacterium EHA105 to obtain recombinant Agrobacterium.

[0057] 2. Expand the recombinant Agrobacterium obtained in step 1, collect the cells, and resuspend them in an inoculation medium containing 200 μM acetosyringone to make the system OD 660nm A value of 0.5 indicates infection fluid.

[0058] Inoculation medium (pH 6.0): contains 2.16 g / L MS basal salt mixture, 1 mL / L 1000× B5 vitamins, 68.5 g / L sucrose, 36 g / L glucose, and 0.25 g / L L-proline, with the balance being water.

[0059] 3. Self-pollinate individual corn PH4CV plants and obtain immature ears approximately 11 days after pollination. Surface disinfect the ears with 70% ethanol solution for 1 minute, followed by 1% sodium hypochlorite solution (concentration based on available chlorine) for 20 minutes. Isolate and obtain immature embryos 1.5-2.0 mm in length. Collect the separated immature embryos into a 2 mL microcentrifuge tube containing inoculation medium within 30 minutes. Remove the inoculation medium with a pipette, and then add 1.5 mL of the infection solution prepared in step 2 to the tube. Mix by inverting 5-10 times, and let it stand for 5 minutes.

[0060] 4. After completing step 3, remove the immature embryos, place them with the scutellum facing up on the co-culture medium, and culture them in the dark at 21°C for 3 days.

[0061] Co-culture medium (pH 5.8): contains 2.16 g / L MS basal salt mixture, 1 mL / L 1000× B5 vitamins, 0.5 mg / L thiamine hydrochloride, 3 mg / L 2,4-D, 10 g / L glucose, 20 g / L sucrose, 0.25 g / L L-proline, 200 μM acetosyringone, 5 mg / L silver nitrate, and 8 g / L Phytagar, with the balance being water.

[0062] 5. After completing step 4, transfer the immature embryos to resting medium and culture in the dark at 28°C for 7 days.

[0063] Resting medium (pH 5.8): Contains 4.43 g / L MS basal medium with vitamins, 0.5 mg / L thiamine hydrochloride, 30 g / L sucrose, 0.5 g / L L-proline, 0.5 g / L casamino acids, 1 mg / L 2,4-D, 2.5 mg / L picloram, 5 mg / L silver nitrate, and 3 g / L Phytagar, with the balance being water.

[0064] 6. After completing step 5, transfer the immature embryos to selective medium and culture them in the dark at 28°C for 21 days. Then, subculture them to fresh selective medium and culture them in the dark at 28°C for 14 days. During this period, resistant callus can be observed developing on the scutellum surface of the zygotes.

[0065] Selective medium (pH 5.8): contains 4.43 g / L MS basal medium with vitamins, 0.5 mg / L thiamine hydrochloride, 30 g / L sucrose, 0.5 g / L L-proline, 0.5 g / L casamino acids, 1 mg / L 2,4-D, 2.5 mg / L picloram, 5 mg / L silver nitrate, 5 mg / mL bialaphos, and 3 g / L Phytagar, with the balance being water.

[0066] 7. After completing step 6, transfer the proliferated resistant callus to regeneration medium and culture for 14 days. Culture conditions: temperature 26°C, photoperiod 16 hours light (light intensity 100 μE) / 8 hours dark. During this period, green shoots can be observed.

[0067] Regeneration medium (pH 5.8): contains 4.33 g / L MS basal salt mixture, 1 mL / L 1000× ERIKSSON vitamins, 3.2 mg / L 6-benzyladenine, 30 g / L sucrose, 0.5 g / L L-proline, 0.5 g / L casamino acids, 200 mg / mL timentin, 3 mg / L bialaphos, and 3 g / L Phytagar, with the balance being water.

[0068] 8. After completing step 7, transfer healthy shoots to a culture tube containing rooting medium and culture for 14 days. The culture conditions are: temperature 26°C, photoperiod of 16 hours light (light intensity of 100 μE) / 8 hours dark.

[0069] Rooting medium (pH 5.8): contains 2.22 g / L MS basal medium with vitamins, 10 g / L glucose, 20 g / L maltose, 100 mg / L inositol, 0.2 mg / L indolebutyric acid, 200 mg / mL timentin, 3 mg / mL bialaphos, 3 g / L phytase and 3 g / L Phytagar, with the balance being water.

[0070] 9. After completing step 8, transfer the rooted plants to pots filled with peat moss, irrigate with Hoagland's nutrient solution, and incubate in a climatic chamber for 2 weeks. Culture conditions: 27°C, 70% humidity, and a photoperiod of 16 hours light (3000 lx) / 8 hours dark.

[0071] 10. After completing step 9, transplant the plants into pots filled with field soil and cultivate them in a climatic chamber under normal conditions. Cultivation conditions: 27°C, 70% humidity, and a photoperiod of 14 hours of light (3000 lx) / 10 hours of darkness. These plants are designated as the T0 generation.

[0072] 3. Screening and identification of genetically modified corn

[0073] 1. Initial screening of genetically modified corn

[0074] (1) When the T0 generation plants in step 2 grow 3 to 4 true leaves, the first round of screening is carried out.

[0075] Screening method: Apply a 1.0 g / 100 mL aqueous solution of glufosinate ammonium to the leaf surfaces of seedlings (apply one true leaf per seedling) and observe after five days. Some corn plants will show yellowing leaves after the application. These plants are symptomatic and all symptomatic plants are eliminated.

[0076] (2) After completing the observation of the first round of screening, the second round of screening will be carried out.

[0077] Screening method: Apply a 1.0 g / 100 mL aqueous solution of glufosinate ammonium to the leaf surfaces of seedlings (apply one true leaf per seedling, not the one used in the first round of screening). Observe the leaves five days later. Some corn plants with yellowing leaves were found to be symptomatic, and all symptomatic plants were eliminated.

[0078] (3) After completing the second round of screening observations, conduct the third round of screening.

[0079] Screening method: Apply a 1.0 g / 100 mL aqueous solution of glufosinate ammonium to the leaf surfaces of seedlings (apply one true leaf per seedling, not the one used for the first and second rounds of screening). Observe the seedlings five days later. Some corn plants with yellowing leaves were found to be symptomatic, and all symptomatic plants were eliminated.

[0080] 2. Molecular identification of genetically modified corn

[0081] Test plants: All remaining plants after completing the three rounds of screening in step 1.

[0082] Maize PH4CV plants were used as wild-type control plants for the test plants.

[0083] (1) Pick fresh young leaves from the test plants (or control plants) and extract total DNA using the CTAB method.

[0084] (2) Using the total DNA obtained in step (1) as a template, perform PCR amplification using the primer pair consisting of G4-F and G4-R. The target sequence is 602 bp. If the amplified product contains a 602 bp product, the PCR identification is positive.

[0085] G4-F: 5'-GCTTCCCCTGCTTCCTCTG-3';

[0086] G4-R: 5'-GCCCACATTATAGTGATTAGCATG-3'.

[0087] (3) The PCR amplification product of step (2) was subjected to 1.0% agarose gel electrophoresis.

[0088] An exemplary electrophoresis diagram of PCR-identified positive plants is shown in Figure 2. In Figure 2, lanes 1-16 correspond to different PCR-identified positive plants, and NC corresponds to the wild-type control plant.

[0089] The positive plants identified by PCR were T0 generation transgenic corn plants.

[0090] Three individual plants (i.e., PH4CV-3, PH4CV-11, and PH4CV-25) were randomly selected from the T0 generation transgenic corn plants for example 2.

[0091] Example 2: Identification of disease resistance and yield performance of transgenic corn plants

[0092] Seedling stage (i.e. before 6 leaves) culture conditions: temperature 26 ° C, humidity 60%; photoperiod 16 h (light intensity 200 μm / m 2 / s) / darkness 8h.

[0093] The culture conditions after the 6-leaf stage are as follows: temperature 26°C, humidity 60%; photoperiod 14 h (light intensity 400 μm / m 2 / s) / darkness 10h.

[0094] 1. Obtaining T1 transgenic plants

[0095] T1 seeds were obtained by self-pollination of individual PH4CV-3 plants. The T1 seeds were planted in an artificial climate chamber and cultivated and managed normally. Molecular identification was performed at the seedling stage (the molecular identification method was the same as step 2 of step 3 in Example 1). Plants that were positive in PCR identification were identified as PH4CV-3 T1 transgenic plants.

[0096] T1 seeds were obtained by self-pollination of individual PH4CV-11 plants. The T1 seeds were planted in an artificial climate chamber and cultivated and managed normally. Molecular identification was performed at the seedling stage (the molecular identification method was the same as step 2 of step 3 in Example 1). Plants that were positive in PCR identification were identified as PH4CV-11 T1 transgenic plants.

[0097] T1 seeds were obtained by self-pollination of individual PH4CV-25 plants. The T1 seeds were planted in an artificial climate chamber and cultivated and managed normally. Molecular identification was performed at the seedling stage (the molecular identification method was the same as step 2 of step 3 in Example 1). Plants that were positive in PCR identification were identified as PH4CV-25 T1 transgenic plants.

[0098] 2. Obtaining test plants for disease resistance testing

[0099] Disease resistance testing refers to steps three, four, five, and six.

[0100] Test plants: PH4CV-3 T1 transgenic plants, PH4CV-11 T1 transgenic plants, and PH4CV-25 T1 transgenic plants cultured in parallel in an artificial climate chamber. Maize PH4CV plants cultured under the same conditions at the same time served as wild-type controls. Maize Chang 7-2 and Zheng 58 plants cultured under the same conditions at the same time served as existing germplasm control plants.

[0101] 3. Ear rot resistance test

[0102] The causative agent of corn ear rot: Fusarium verticillioides.

[0103] When the test plants were cultured to the milky stage, the pathogen was inoculated using the conidia suspension inoculation method. The specific steps were as follows: after disinfecting the surface of the corn spore leaves, an awl was used to pierce the center of the ear axis from the upper oblique part of the middle of the female ear. After pulling out the awl, 2 mL of spore suspension (spore suspension: containing 1×10 6 The holes were inoculated with conidia of the pathogenic fungus of corn ear rot (1000 ng / mL) and 0.05% Tween-80 by volume, with the remainder being sterile water. The holes were then sealed with cotton soaked in sterile water. One female ear of each test plant was inoculated.

[0104] Thirty days after inoculation, corn ears emerging from the inoculated female ears were evaluated for resistance based on the diseased area within the ears. The resistance evaluation criteria are shown in Table 1. The resistance evaluation results are shown in Table 2 (10 plants were included for each corn accession). Exemplary photographs of susceptible and resistant corn ears are shown in Figure 3 (left: an ear from a wild-type control plant; right: an ear from a PH4CV-3 T1 transgenic plant).

[0105]

[0106]

[0107] The identification results showed that the transgenic corn had higher resistance to ear rot than the wild-type control and the existing corn germplasm control, indicating that the broad-spectrum disease-resistance gene shown in SEQ ID NO: 1 can significantly improve the disease resistance of corn to ear rot.

[0108] 4. Southern Rust Resistance Test

[0109] Southern rust pathogen: Puccinia polysora Underw.

[0110] When the test plants were cultured to the pollen shedding stage, spore suspension (spore suspension: containing 1×10 5 Spray inoculation was performed using a spore suspension containing 2 mL of spore suspension (containing spores of the pathogenic southern rust pathogen and 0.05% Tween-80 by volume, with the remainder being sterile water). One ear of each plant was sprayed (the volume of the spore suspension used for spraying was 2 mL).

[0111] Twenty days after inoculation, resistance was assessed by examining three leaves above the inoculated ear and three leaves below the inoculated ear. Resistance evaluation criteria are shown in Table 3. Resistance evaluation results are shown in Table 4 (10 plants were included for each corn accession). Exemplary photographs of susceptible and resistant corn leaves are shown in Figure 4 (left: leaf from a wild-type control plant; right: leaf from a PH4CV-3 T1 transgenic plant).

[0112]

[0113]

[0114] The identification results showed that the transgenic corn had better resistance to southern rust than the wild-type control and the existing corn germplasm control, indicating that the broad-spectrum disease-resistance gene shown in SEQ ID NO: 1 can significantly improve the resistance of corn to southern rust.

[0115] 5. Small spot disease resistance test

[0116] The causative agent of small leaf spot disease: Bipolaris maydis.

[0117] When the test plants were cultured to the trumpet stage, spore suspension (spore suspension: containing 1×10 5 The plants were spray-inoculated with conidia of the pathogenic bacteria of the leaf spot (conidia / mL) and 0.05% Tween-80 by volume, with the remainder being sterile water. One ear of each plant was sprayed (the volume of the spore suspension used for spraying was 2 mL).

[0118] Ten days after inoculation, resistance was assessed by examining three leaves above the inoculated ear and three leaves below the inoculated ear. Resistance evaluation criteria are shown in Table 5. Resistance evaluation results are shown in Table 6 (10 plants were included for each corn accession). Exemplary photographs of susceptible and resistant corn leaves are shown in Figure 5 (left: leaf from a wild-type control plant; right: leaf from a PH4CV-3 T1 transgenic plant).

[0119]

[0120]

[0121] The identification results showed that the transgenic corn had better resistance to southern leaf spot than the control and the existing corn germplasm control, indicating that the broad-spectrum disease-resistant gene shown in SEQ ID NO: 1 can significantly improve the resistance of corn to southern leaf spot.

[0122] 6. Disease resistance test of giant spot disease

[0123] The causative agent of the large spot disease is Exserohlium turcicum.

[0124] When the test plants were cultured to the trumpet stage, spore suspension (spore suspension: containing 1×10 5 Spray inoculation was performed using a spore suspension containing 2 mL of conidia of the pathogen causing northern leaf spot (Pseudomonas spp. / mL) and 0.05% Tween-80 by volume, with the remainder being sterile water. One ear of each plant was sprayed (the volume of the spore suspension used for spraying was 2 mL).

[0125] Fourteen days after inoculation, resistance was assessed by examining three leaves above the inoculated ear and three leaves below the inoculated ear. Resistance evaluation criteria are shown in Table 7. The results of resistance evaluation are shown in Table 8 (10 plants were included for each corn accession). Exemplary photographs of susceptible and resistant corn leaves are shown in Figure 6 (left: leaf from a wild-type control plant; right: leaf from a PH4CV-3 T1 transgenic plant).

[0126]

[0127]

[0128] The identification results showed that the transgenic corn had better resistance to northern leaf blight than the wild-type control and the existing corn germplasm control, indicating that the broad-spectrum disease-resistant gene shown in SEQ ID NO: 1 can significantly improve the resistance of corn to northern leaf blight.

[0129] 7. Yield Test

[0130] The transgenic maize (PH4CV-3 T1 transgenic plants, PH4CV-11 T1 transgenic plants, and PH4CV-25 T1 transgenic plants) identified by molecular analysis and control plants of the same period were transplanted to the experimental field of the Nangang base of Anhui Quanyin High-tech Seed Co., Ltd. Quanyin Agricultural Science Academy. The control plants refer to: maize PH4CV plants as wild-type control plants, maize Chang 7-2 plants and maize Zheng 58 plants as existing germplasm control plants. Each maize germplasm was planted in an area of ​​20m 2 The planting density was 4500 plants / mu and the cultivation and management were normal under parallel conditions.

[0131] Observe plant shape and ear position (cm). Measure and calculate plant height (cm) and yield per plant (g) at maturity. Yield per mu (kg) = yield per plant × 4500 ÷ 1000. Calculate the yield increase of transgenic corn relative to corn PH4CV, corn Chang 7-2, and corn Zheng 58.

[0132] Field observations of agronomic traits and yield are shown in Table 9. Compared to the wild-type control, maize PH4CV, the transgenic maize showed a significant yield increase (over 38%). Agronomic traits such as plant height, ear position, and plant type were not significantly different. Compared to the prior art maize variety Chang 7-2, which exhibits excellent overall agronomic traits, the transgenic maize showed a significant yield increase (between 21.2% and 22.8%). Compared to the prior art maize variety Zheng 58, which exhibits excellent overall agronomic traits, the transgenic maize showed a significant yield increase (between 14.7% and 16.2%). These results demonstrate that the broad-spectrum disease resistance gene represented by SEQ ID NO: 1 can significantly increase maize yield.

[0133]

[0134] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be practiced over a wide range of parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although specific embodiments have been given herein, it should be understood that further modifications may be made to the present invention. In summary, this application is intended to encompass any variations, uses, or improvements to the present invention, including those made by conventional techniques known in the art that depart from the scope of the present invention. Applications of the essential features may be made within the scope of the following claims.

[0135] CROSS-REFERENCE TO RELATED APPLICATIONS

[0136] This application claims priority to the Chinese patent application (application number: 202310879971.7) filed on July 18, 2023, the entire contents of which are incorporated herein by reference. Industrial Applicability

[0137] The present invention discloses a method for producing transgenic plants with enhanced disease resistance and / or improved productivity by introducing multifunctional genes or biomaterials related to these genes. The method can be used for plant germplasm improvement, particularly corn germplasm improvement. The method has significant application and promotion value for the selection and breeding of superior corn varieties.

Claims

1. Application of multifunctional genes or multifunctional gene-related biomaterials in the preparation of transgenic plants with enhanced disease resistance and / or improved production performance; The multifunctional gene is as follows (a1), (a2) or (a3): (a1) a DNA molecule represented by SEQ ID NO: 1; (a2) a DNA molecule that hybridizes with (a1) under stringent conditions; (a3) a DNA molecule having more than 99% identity with (a1); The multifunctional gene-related biological material is an expression cassette having the multifunctional gene, a recombinant vector having the multifunctional gene, or a recombinant microorganism having the multifunctional gene.

2. The use according to claim 1, characterized in that: The plant is a grass plant.

3. The use according to claim 2, characterized in that: The grass plant is corn.

4. The use according to any one of claims 1 to 3, characterized in that: The enhanced disease resistance refers to enhanced disease resistance to ear rot and / or southern rust and / or small leaf spot and / or large leaf spot.

5. The use according to any one of claims 1 to 3, characterized in that: The improved production performance refers to an improved yield.

6. A method for preparing a transgenic plant with enhanced disease resistance and / or improved production performance, comprising the following steps: introducing a multifunctional gene or multifunctional gene-related biological material into a recipient plant to obtain a transgenic plant with higher disease resistance and / or production performance than the recipient plant; The multifunctional gene is as follows (a1), (a2) or (a3): (a1) a DNA molecule represented by SEQ ID NO: 1; (a2) a DNA molecule that hybridizes with (a1) under stringent conditions; (a3) a DNA molecule having more than 99% identity with (a1); The multifunctional gene-related biological material is an expression cassette having the multifunctional gene, a recombinant vector having the multifunctional gene, or a recombinant microorganism having the multifunctional gene.

7. The method according to claim 6, characterized in that: The plant is a grass plant.

8. The method according to claim 6 or 7, characterized in that: The enhanced disease resistance refers to enhanced disease resistance to ear rot and / or southern rust and / or small leaf spot and / or large leaf spot.

9. The method according to claim 6 or 7, characterized in that: The improved production performance refers to an improved yield.

10. A DNA molecule, which is (a1), (a2) or (a3): (a1) a DNA molecule represented by SEQ ID NO: 1; (a2) a DNA molecule that hybridizes with (a1) under stringent conditions; (a3) A DNA molecule that is more than 99% identical to (a1).

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