Application of avrHtn1 gene or its encoded protein in improving corn resistance to corn leaf blight

Abstract

This invention discloses the application of the AvrHtn1 gene or its encoded protein in improving maize resistance to maize leaf spot pathogen, belonging to the field of plant genetic engineering technology. This invention is the first to isolate and identify the non-toxic gene AvrHtn1 from maize leaf spot pathogen, successfully obtaining a transgenic maize family overexpressing AvrHtn1. Experiments show that the relative expression level of the AvrHtn1 gene in AvrHtn1-overexpressing transgenic maize is significantly higher than that in the non-transgenic control, and after inoculation with maize leaf spot pathogen, the lesion area of ​​the transgenic maize is significantly smaller than that of the control. Setosphaeria turcica The resulting maize leaf spot disease resistance is significantly enhanced. This invention provides important genetic resources for maize disease-resistant breeding, and is of great significance for ensuring high and stable maize yields, reducing the use of chemical pesticides, and promoting the sustainable development of green agriculture. It has extremely high scientific research value and promising prospects for industrial application.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, and in particular to the application of the AvrHtn1 gene or the protein it encodes in improving maize's resistance to maize leaf spot pathogen. Background Technology

[0002] Corn is my country's largest grain crop, accounting for 41.7% of total grain production. Its quality and yield significantly impact my country's food security and social stability. High and stable corn yields are of great importance to my country's agricultural development. This is due to the corn leaf blight fungus (…). Setosphaeria turcica (Luttr.) KJ Leonard & Suggs) causes maize leaf spot, a major fungal disease that seriously affects maize production in my country. In recent years, due to increased planting density and changes in agricultural farming systems, the occurrence of maize leaf spot has become increasingly serious, severely impacting my country's grain yield and quality.

[0003] In the long-term co-evolution of pathogens and plants, pathogens have evolved a series of effector factors, some of which are called avirulence genes. These avirulence genes can interact with the host's R genes through direct binding or indirect recognition, stimulating the ETI response. Currently, protein interaction verification experiments have confirmed that several R-Avr combinations can directly interact. For example, Pita in rice directly interacts with the avirulence gene AvrPita of rice blast fungus, and the avirulence protein AvrPi9 interacts with the resistance protein Pi9 in rice, altering the protein structure of Pi9 to activate the immune defense response; Arabidopsis RRS1 directly binds to PopP2 of Ralstonia solanacearum, and the avirulence gene AvrPm1a of wheat powdery mildew interacts with Pm1a to induce a necrosis response.

[0004] Maize leaf spot pathogens are characterized by a high mutation rate; their physiological races can undergo multiple mutations in a short period, making it easy for maize varieties with only single resistance to lose their resistance in a short time. Therefore, identifying avirulence-free genes is particularly necessary. Summary of the Invention

[0005] The purpose of this invention is to provide the application of the AvrHtn1 gene or its encoded protein in improving maize resistance to maize leaf spot pathogen, thereby solving the problems existing in the prior art.

[0006] To achieve the above objectives, the present invention provides the following solution: One of the technical solutions of this invention is the application of the AvrHtn1 gene or its encoded protein in improving maize's resistance to maize leaf spot pathogen.

[0007] The second technical solution of the present invention relates to the application of recombinant expression vectors, expression cassettes, or recombinant bacteria containing the AvrHtn1 gene in improving maize resistance to maize leaf spot pathogen.

[0008] The third technical solution of the present invention is a method for improving the resistance of maize to maize leaf spot pathogen, which includes the step of overexpressing the AvrHtn1 gene in maize or increasing the level of its encoded protein.

[0009] The fourth technical solution of this invention is the application of the AvrHtn1 gene or its encoded protein in maize disease resistance breeding. The AvrHtn1 gene is used as the target gene to transform maize immature embryos to obtain transgenic maize plants for breeding new maize varieties with enhanced resistance to maize leaf spot pathogen.

[0010] The fifth technical solution of this invention is a method for breeding new maize varieties with enhanced resistance to maize leaf spot pathogen, which involves overexpressing the AvrHtn1 gene or increasing the level of its encoded protein to improve maize's resistance to maize leaf spot pathogen.

[0011] Based on the above technical solution, the present invention has the following technical effects: This invention marks the first time that a virus-free gene, AvrHtn1, has been isolated and identified from *Setosphaeria turcica*, the causal agent of maize leaf spot disease. By constructing the overexpression vector pBWA(V)BU-OEAvrHtn1 and transforming it into maize, a transgenic maize family overexpressing AvrHtn1 was successfully obtained. Experiments demonstrated that the relative expression level of the AvrHtn1 gene in the transgenic maize overexpressing AvrHtn1 was significantly higher than that in the non-transgenic control. Furthermore, after inoculation with *Setosphaeria turcica*, the lesion area in the transgenic maize was significantly smaller than that in the control, indicating significantly enhanced resistance to maize leaf spot disease caused by *Setosphaeria turcica*. This invention provides important genetic resources for maize disease-resistant breeding, and is of great significance for ensuring high and stable maize yields, reducing the use of chemical pesticides, and promoting the sustainable development of green agriculture. It possesses extremely high scientific research value and promising prospects for industrial application. Attached Figure Description

[0012] Figure 1 A plot to identify the expression level of AvrHtn1 in transgenic plants (the expression level in WT is set to 0).

[0013] Figure 2 Photographs showing the resistance phenotype of AvrHtn1-overexpressing transgenic maize to the fungus Setosphaeria turcica.

[0014] Figure 3 Statistical photographs of lesion area after AvrHtn1 overexpressing transgenic maize was inoculated with the fungus Setosphaeria turcica. Detailed Implementation

[0015] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0016] This invention provides the application of the AvrHtn1 gene or its encoded protein in improving maize's resistance to maize leaf spot pathogen.

[0017] In some specific embodiments, the nucleotide sequence of the AvrHtn1 gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.2.

[0018] In some specific implementation schemes, overexpression of the AvrHtn1 gene or increased levels of its encoded protein can enhance maize's resistance to maize leaf spot pathogen.

[0019] This invention also provides the application of recombinant expression vectors, expression cassettes, or recombinant bacteria containing the AvrHtn1 gene in improving maize resistance to maize leaf spot pathogen.

[0020] In some specific embodiments, the nucleotide sequence of the AvrHtn1 gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.2; The recombinant expression vector, expression cassette, or recombinant bacteria overexpress the AvrHtn1 gene or increase the level of its encoded protein in maize.

[0021] This invention also provides a method for improving maize resistance to maize leaf spot pathogen, including the step of overexpressing the AvrHtn1 gene in maize or increasing the level of its encoded protein.

[0022] In some specific embodiments, the nucleotide sequence of the AvrHtn1 gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.2; The overexpression includes the following steps: (1) Construct an overexpression vector for the AvrHtn1 gene; (2) Transform the overexpression vector into Agrobacterium; (3) Use the Agrobacterium to transform maize embryos to obtain transgenic maize plants.

[0023] This invention also provides the application of the AvrHtn1 gene or its encoded protein in maize disease resistance breeding. The AvrHtn1 gene is used as the target gene to transform maize immature embryos to obtain transgenic maize plants for breeding new maize varieties with enhanced resistance to maize leaf spot fungus.

[0024] This invention also provides a method for breeding new maize varieties with enhanced resistance to maize leaf spot pathogens by overexpressing the AvrHtn1 gene or increasing the level of its encoded protein to improve maize resistance to maize leaf spot pathogens.

[0025] In some specific implementation schemes, the AvrHtn1 gene is overexpressed or its encoded protein level is increased using recombinant expression vectors, expression cassettes, or recombinant bacteria containing the AvrHtn1 gene.

[0026] Example 1 1. Cloning of the AvrHtn1 gene Primers were designed using the cDNA of *Cercospora maize* strain 1 (CGMCC NO: 9857, disclosed in patent CN105754863A) as a template, with reference to the gene of SETTUDRAFT_174518 (NCBI accession number: NW_007360310.1).

[0027] The gene sequence was cloned using polymerase chain reaction PCR with primers, and the nucleotide sequences of the primers are shown in SEQ ID NO.3~4.

[0028] SEQ ID NO.3: AvrHtn1F:ATGAAGTTCTCTACCATAGCCGCT; SEQ ID NO.4: AvrHtn1R: TCAAAAGAGACAAGTACATTGGGCC.

[0029] The amplification products were collected and sequenced. The nucleotide sequence of the gene AvrHtn1 was obtained based on the sequencing results, as shown in SEQ ID NO.1, and the sequence of the AvrHtn1-P protein it encodes is shown in SEQ ID NO.2.

[0030] SEQ ID NO.1: atgaagttctctaccatagccgctatcttcgtcagcctggctatcccagcatcctgtattgaatgttatgatgcaaaaccgtgttctagctt cccatgctttggttggtgtgatggagtctttcctgctacacgtataaagggtgttgaagtcccggcccagccaggccaatgtacttgtctcttttga; SEQ ID NO. 2: MKFSTIAAIFVSLAIPASCIECYDAKPCSSFPCFGWCDGVFPATRIKGVEVPAQPGQCTCLF*.

[0031] 2. Construction of the overexpression vector pBWA(V)BU-OEAvrHtn1 Primers were designed based on SETTUDRAFT_174518 (NCBI accession number: NW_007360310.1): pBWA(V)BU-OEAvrHtn1F (SEQ ID NO.5): GGTGTTACTTCTGTTGCAACatgaagttctctaccatagccgctatcttc; pBWA(V)BU-OEAvrHtn1R (SEQ ID NO. 6): TCACCGTTAATCAAACCCATaaagagacaagtacattggcctggctg.

[0032] The AvrHtn1 gene was amplified using 2 × Phanta Max Master Mix, and the PCR products were obtained. The reaction system is shown in Table 1 (total volume 25 μL): Table 1

[0033] The reaction procedure was as follows: pre-denaturation at 95℃ for 3 min; denaturation at 95℃ for 15 s, annealing at 56℃ for 15 s, extension at 72℃ for 30 s, 35 cycles; and final extension at 72℃ for 5 min.

[0034] The pBWA(V)BU vector was linearized using a single digestion with SapI / LguI.

[0035] The PCR products and vector digestion products were detected and recovered: the PCR products were detected by agarose gel electrophoresis, and the target fragment and linearized vector were recovered using the Omega Bio-tek Gel Extraction Kit.

[0036] Homologous recombination of the target fragment and the linearized vector, the reaction system is shown in Table 2 (20 μL).

[0037] Table 2

[0038] The reaction program was 37℃ for 30 min. The overexpression vector pBWA(V)BU-OEAvrHtn1 was constructed.

[0039] Transformation of recombinant plasmids: Thaw DH5α competent cells on ice for 5 min; add all the ligation solution and mix well, then incubate on ice for 20 min; heat shock in a metal bath for 60 s; incubate on ice for 5 min, then add 150 μL of LB liquid medium; shake at 37 ℃ and 200 rpm for 50 min; spread on a clean bench (Kan resistant solid medium), air dry, seal with sealing film, and incubate at 37 ℃ for 12 hours for plaque PCR identification.

[0040] Identification primers: pBWA(V)BU-F (SEQ ID NO.7): cctgccttcatacgctatttatttgcttgg; pBWA(V)BU-R (SEQ ID NO. 8):caagaccggcaacaggattcaatc.

[0041] Example 2 Maize genetic transformation The overexpression vector pBWA(V)BU-OEAvrHtn1 of AvrHtn1 was transformed into maize immature embryos to obtain transgenic plants. The specific steps are as follows: 1. Preparation of Agrobacterium Add 1 µL of pBWA(V)BU-OEAvrHtn1 plasmid to 50 µL of EHA105 Agrobacterium competent cells, mix thoroughly, and transfer to an electroporation cuvette. After electroporation, add 1 mL of LB liquid medium, mix thoroughly, and transfer to a 1.5 mL centrifuge tube. Incubate at 30°C and 180 rpm for 30 min on a shaker. Inoculate 50 µL of the activated Agrobacterium culture onto LB+Kan solid medium and incubate in the dark at 30°C for 48 h. Identify positive Agrobacterium clones using identification primers pBWA(V)BU-F and pBWA(V)BU-R.

[0042] 2. Maize genetic transformation 2.1 Disinfection and Embryo Retrieval Place the corn kernels in a container and sterilize. Take a sterilized 2mL EP tube, add the suspension to the tube, and collect the corn embryo.

[0043] 2.2 Agrobacterium infection and co-culture Agrobacterium was picked into the infection solution to prepare OD. 600 Prepare a 0.2 μL Agrobacterium resuspension, aspirate the liquid from the EP tube, add the prepared Agrobacterium culture, and infect the embryos. Add the infected embryos and culture medium together to a co-culture medium, then aspirate the culture medium dry. Incubate at 25°C in the dark for 2-3 days.

[0044] 2.3 Callus induction and screening After co-culture, the embryos were inoculated onto induction medium and cultured in the dark at 28°C for 7-10 days. The induced calluses were then inoculated onto selection medium (MS, sucrose 60 g / L, ZT 1.0 mg / L, TDZ 0.1 mg / L, 6-BA 1.0 mg / L, termethin 250 mg / L, dipropylamine 6 mg / L, plant gel 3 g / L, pH 5.8. The purpose of selection was to identify positive callus tissues transformed with plasmids). The calluses that survived the first selection were then used for a second selection.

[0045] 2.4 Differentiation and Rooting The selected embryogenic callus was inoculated onto a predifferentiation medium and cultured in the dark at 28°C for 10 days. The predifferentiated callus was then inoculated onto a differentiation medium and cultured under light at 25°C until seedlings differentiated. The differentiated seedlings were then transferred to a rooting medium and cultured under light at 25°C until the root system was fully developed. The developed seedlings were then hardened off and transplanted into the greenhouse substrate.

[0046] 2.5 Harvest and Substitution After growth and development to maturity, T0 generation overexpression transgenic seeds of AvrHtn1 were obtained, and T1 generation transgenic families were obtained through subsequent cultivation and planting for further use.

[0047] Example 3 Positive detection of AvrHtn1 overexpression transgenic maize Genotyping of T1 generation AvrHtn1-overexpressing transgenic maize was performed. T1-positive plants were identified by DNA detection of the overexpression vector insertion, and seeds were harvested and cultivated to obtain the T2 generation. PCR testing was then performed on the T2 plants to confirm that the target gene had not undergone segregation or loss, ultimately yielding three positive homozygous transgenic maize families with stable AvrHtn1 inheritance.

[0048] Positive plants of AvrHtn1-overexpressing transgenic maize were planted. Maize leaves were taken after three weeks of growth, and total RNA was extracted from the plants. The RNA was then reverse transcribed into cDNA using One-Step gDNA Removal and cDNA Synthesis SuperMix.

[0049] RT-PCR was performed using cDNA as a template. The ZmUBQ gene of maize (NM_001138130) was used as an internal control. The relative expression levels of the AvrHtn1-OE transgenic lines OE-AvrHtn1-1, OE-AvrHtn1-2, and OE-AvrHtn1-3, which overexpress the AvrHtn1 gene, were significantly higher than those of the control (B73), while the relative expression level of the control was 0 (…). Figure 1 The quantitative primers used are shown below: UBQ9-F (SEQ ID NO.9): TACAGTTCTACAAGGTGGACGAC; UBQ9-R (SEQ ID NO. 10): GCAGTAGTGGCGTCGAAGT; DLAVRHtn1F (SEQ ID NO.11):cttcgtcagcctggctatcc; DLAVRHtn1R (SEQ ID NO. 12):aagtacattggcctggctgg.

[0050] Example 4 AvrHtn1 transgenic maize resistance phenotype detection AvrHtn1 transgenic maize and control B73 maize were planted in a greenhouse. When the seedlings reached the 4-5 leaf stage, maize plants with good and uniform growth were selected for disease resistance phenotype identification. The plants were inoculated with a spore suspension of the first physiological race of maize leaf spot pathogen at a concentration of 1×10⁻⁶. 5 The spores were inoculated at a concentration of 300 µL per mL using a spore suspension. After inoculation, the plants were kept in a humid environment for 48 h, followed by normal management in the greenhouse. Ten days later, when typical lesion symptoms began to appear on the leaves, the area of ​​the lesions was investigated. Figure 2 and Figure 3 AvrHtn1 overexpression transgenic maize against fungi. Setosphaeria turcica The resulting corn leaf blight significantly enhanced resistance.

[0051] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. Application of the AvrHtn1 gene or its encoded protein in improving maize resistance to maize leaf spot pathogen.

2. The application according to claim 1, characterized in that, The nucleotide sequence of the AvrHtn1 gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.

2.

3. The application according to claim 2, characterized in that, Overexpression of the AvrHtn1 gene or increased levels of its encoded protein can enhance maize's resistance to maize leaf spot pathogen.

4. Application of recombinant expression vectors, expression cassettes, or recombinant bacteria containing the AvrHtn1 gene in improving maize resistance to maize leaf spot pathogen.

5. The application according to claim 4, characterized in that, The nucleotide sequence of the AvrHtn1 gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.2; The recombinant expression vector, expression cassette, or recombinant bacteria overexpress the AvrHtn1 gene or increase the level of its encoded protein in maize.

6. A method for improving the resistance of corn to corn leaf blight, characterized in that, This includes steps such as overexpressing the AvrHtn1 gene in maize or increasing the level of its encoded protein.

7. The method according to claim 6, characterized in that, The nucleotide sequence of the AvrHtn1 gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.2; The overexpression includes the following steps: (1) Construct an overexpression vector for the AvrHtn1 gene; (2) Transform the overexpression vector into Agrobacterium; (3) Use the Agrobacterium to transform maize embryos to obtain transgenic maize plants.

8. The application of the AvrHtn1 gene or its encoded protein in maize disease resistance breeding, characterized in that, The AvrHtn1 gene was used as the target gene to transform maize immature embryos, resulting in transgenic maize plants for breeding new maize varieties with enhanced resistance to maize leaf spot pathogen.

9. A method for breeding new maize varieties with enhanced resistance to maize leaf spot pathogen, characterized in that, Overexpression of the AvrHtn1 gene or increased levels of its encoded protein can enhance maize's resistance to maize leaf spot pathogen.

10. The method according to claim 9, characterized in that, The AvrHtn1 gene can be overexpressed or its encoded protein level can be increased using recombinant expression vectors, expression cassettes, or recombinant bacteria containing the AvrHtn1 gene.

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