A ZmPHYLL gene, its application, and methods to improve maize plant resistance to southern rust.

Abstract

This invention relates to the field of genetic engineering technology, and provides a ZmPHYLL gene, the full-length nucleotide sequence of which is shown in SEQ ID NO: 1, and the coding region nucleotide sequence of which is shown in SEQ ID NO: 2. This invention also provides the application of the above-mentioned ZmPHYLL gene in inhibiting the germination of *Russula multifiliis* spores and improving the resistance of maize plants to southern maize rust. Simultaneously, this invention provides a method for improving the resistance of maize plants to southern maize rust. In this study, a ZmPHYLL gene was screened, and the expressed protein of this gene can effectively inhibit the germination of *Russula multifiliis* spores. Therefore, by regulating the overexpression of this gene, the resistance of maize to southern maize rust can be improved. This invention provides new gene resources for the creation of breeding materials and provides important scientific basis for the breeding of maize varieties highly resistant to southern maize rust.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to a ZmPHYLL gene, its application, and a method for improving the resistance of maize plants to southern rust. Background Technology

[0002] Southern rust of maize is a highly destructive airborne fungal disease caused by *Russula multifiliis*. This pathogen primarily infects maize leaves, and in severe cases, it can further infect leaf sheaths and husks, leading to leaf withering, loss of photosynthetic function, shriveled kernels, and a significant reduction in yield. Currently, southern rust of maize is prevalent in warm temperate and tropical regions, posing a major problem for maize producers and seriously threatening maize production safety.

[0003] To address the resistance problem of southern rust in maize, previous researchers extracted Rpp-type resistance genes (NLR family), such as Rpp1, Rpp2, RppK, and RppM, from resistant germplasm and bred a series of resistant varieties by using these genes individually or in combination. However, with changes in pathogen species, the resistance conferred by these NLR family genes is usually not durable in the field.

[0004] Therefore, researching and screening new resistance genes related to southern rust of maize is currently a key focus. This will provide new gene resources for the creation of breeding materials and provide important scientific basis for the breeding of maize varieties with high resistance to southern rust. Summary of the Invention

[0005] The technical problem to be solved by this invention is to provide a ZmPHYLL gene and its application, and a method to improve the resistance of maize plants to southern rust.

[0006] The present invention solves the above-mentioned technical problems by adopting the following technical solutions:

[0007] A ZmPHYLL gene, the full-length nucleotide sequence of which is shown in SEQ ID NO: 1, and the coding region nucleotide sequence of which is shown in SEQ ID NO: 2.

[0008] As one of the preferred embodiments of the present invention, the amino acid sequence of the protein encoded by the ZmPHYLL gene is shown in SEQ ID NO: 3.

[0009] As one of the preferred embodiments of the present invention, the expression level of the ZmPHYLL gene is positively correlated with the resistance of maize plants to southern rust.

[0010] The application of the aforementioned ZmPHYLL gene in inhibiting the germination of *Russula multifiliis* spores and improving the resistance of maize plants to southern rust.

[0011] A method for improving the resistance of maize plants to southern rust involves overexpressing the ZmPHYLL gene in maize plants through genetic engineering. The full-length nucleotide sequence of the ZmPHYLL gene is shown in SEQ ID NO: 1, and the coding region nucleotide sequence is shown in SEQ ID NO: 2. The gene expression level is positively correlated with the resistance of maize plants to southern rust.

[0012] As one of the preferred embodiments of the present invention, the expression protein of the ZmPHYLL gene enhances the resistance of maize plants to southern maize rust by inhibiting the spore germination of *Symplocos rubrum*.

[0013] The advantages of this invention compared to the prior art are:

[0014] (1) In this study, a ZmPHYLL gene with high resistance to southern rust of maize was screened. The study found that the expression protein of the ZmPHYLL gene can effectively inhibit the germination and growth of spores of *Hemiberlesia lataniae*. Therefore, the resistance of maize plants to southern rust of maize can be improved by regulating the overexpression of the ZmPHYLL gene. The discovery of the ZmPHYLL gene in this invention provides new gene resources for the creation of breeding materials and provides important scientific basis for the breeding of maize varieties with high resistance to southern rust of maize.

[0015] (2) Existing Rpp genes (NLR family) drive cell death after rust fungus infection, preventing the multi-stalked rust fungus from completing its life cycle. When using these genes to screen and breed maize varieties, the effector genes in the multi-stalked rust fungus will recognize the maize Rpp genes and cause hypersensitive cell death, so that individuals without the effector gene in the rust fungus are selected. In other words, they are subjected to evolutionary pressure, resulting in high variation of effectors and NLR proteins, gene loss, and non-durable resistance. However, the ZmPHYLL gene of this invention, after research, does not belong to the NLR family and its function is different from that of Rpp genes, which can effectively avoid the problem of non-durable resistance of existing NLR family genes.

[0016] (3) This invention uses big data analysis and qRT-PCR technology to find that the ZmPHYLL gene is significantly upregulated after infection with *Russula multiflora* in maize. The protein produced by its expression has an inhibitory effect on the germination and growth of *Russula multiflora* spores, thereby inhibiting the outbreak and invasion of maize southern rust. Attached Figure Description

[0017] Figure 1 This is a volcano diagram of differential gene expression in Example 1;

[0018] Figure 2 These are the results of ZmPHYLL gene expression levels at different time points after different plant samples were infected with *Russula multifiliis* in Example 2.

[0019] Figure 3 This is a graph showing the purification results of pCold-TF-ZmPHYLL protein in Example 4 (in the graph, lane "M" is the 200kDa protein marker; lane "1" is the pCold-TF-ZmPHYLL protein);

[0020] Figure 4 This is a diagram showing the results of ZmPHYLL gene expression protein inhibiting the germination of *Russula multifiliis* spores in Example 5 (Figure A shows the results of sterile water treatment, Figure B shows the results of protein buffer treatment, and Figure C shows the results of pCold-TF-ZmPHYLL protein treatment). Detailed Implementation

[0021] The embodiments of the present invention are described in detail below. These embodiments are implemented based on the technical solution of the present invention, and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments. Furthermore, unless otherwise specified, the reagents and materials used in the following embodiments are all commercially available reagents and materials conventional in the art. Unless otherwise specified, the experimental methods used in the following embodiments are all conventional methods in the art and will not be described further.

[0022] Example 1: ZmPHYLL gene screening:

[0023] Southern rust disease was observed in maize fields, and rust-infected and rust-resistant plants were screened based on the observation results. Transcriptome sequencing was then performed. Differential expression analysis, enrichment analysis, and expression pattern analysis were conducted using transcriptome data and BSA-Seq data. (See below) Figure 1 Finally, the candidate gene ZmPHYLL was selected for subsequent experiments.

[0024] The ZmPHYLL gene that was screened has the following full-length nucleotide sequence as shown in SEQ ID NO: 1, the coding region nucleotide sequence as shown in SEQ ID NO: 2, and the amino acid sequence of the encoded protein as shown in SEQ ID NO: 3.

[0025] Example 2: Analysis of ZmPHYLL gene expression after infection with *Cercospora macrantha*:

[0026] Plant samples were selected from two varieties: the non-rust-resistant "Corn B73" (an existing variety) and the rust-resistant "Corn Yanzi 7016" (an existing variety, seed number: 20231002628), which were labeled as non-rust-resistant plants (SS) and rust-resistant plants (RS), respectively. Leaves of the samples were infected with *Pseudomonas aeruginosa*, and the expression level of the ZmPHYLL gene in the corresponding plants at different time points was determined using quantitative real-time PCR. The specific procedures are as follows:

[0027] Primers (upstream primer - F1, downstream primer - R1) were designed using Primer Primier 5.0 software and synthesized by Shanghai Sangon Biotech Co., Ltd. Simultaneously, RNA was extracted from plant leaves using a plant RNA extraction kit (Chengdu Biotech Co., Ltd., catalog number RN33050) and analyzed using full-spectrum RNA extraction. One-Step gDNA Removal and cDNA Synthesis SuperMix Kit for Reverse Transcription of cDNA.

[0028] Add reagents according to the system in Table 1, set up three biological replicates, and carry out the reaction according to the reaction conditions in Table 1. Calculate the relative expression level using the 2-ΔΔCt method.

[0029] Table 1. Reaction system and conditions for quantitative real-time PCR

[0030]

[0031]

[0032] The results of the expression level measurement are shown in Figure 2 The results showed that the expression level of the ZmPHYLL gene was significantly increased in the rust-resistant RS plants compared with the non-rust-resistant SS plants.

[0033] Example 3: Cloning of the ZmPHYLL gene:

[0034] RNA was extracted from leaves of “Maize B73” using a plant RNA extraction kit; RNA was then extracted using a reverse transcription kit (Total Metallurgical). The One-Step gDNA Removal and cDNA Synthesis SuperMix was used to reverse transcribe RNA into cDNA according to the instructions. The coding region sequence of the maize ZmPHYLL gene (SEQ ID NO: 2) was obtained based on the maize genome (V5.0). Appropriate amplification primers (upstream primer-F2, downstream primer-R2) were designed using Primer Premier 5.0 software, and PCR amplification was performed using cDNA as a template. The reaction system is shown in Table 2.

[0035] Table 2. PCR amplification reaction system and reaction conditions

[0036]

[0037]

[0038] After PCR amplification, the PCR products were detected by electrophoresis, and the target fragment was recovered and purified. The gel-recovered product containing the target gene was then mixed with pMD18. TM-T Vector was used for ligation, and E. coli competent cells DH5α were transformed. Single clones were selected and sequenced.

[0039] Example 4: Purification of ZmPHYLL gene expression product:

[0040] Based on the coding region sequence of ZmPHYLL (SEQ ID NO: 2) and the restriction enzyme sites of the vector pCold-TF, the restriction enzyme sites of the target gene were detected using Primer Premier 5.0 software, and primers with restriction enzyme sites were designed, namely upstream primer-F3 (SEQ ID NO: 8) and downstream primer-R3 (SEQ ID NO: 9). The double enzyme digestion system is shown in Table 3.

[0041] Table 3. Double enzyme digestion reaction system and reaction conditions

[0042]

[0043] The pCold-TF vector fragment and the target gene fragment were ligated by homologous recombination, transformed into competent E. coli DH5α cells, single clones were selected for colony PCR verification, and sequencing was used to confirm the correctness of the sequence. Recombinant plasmids were extracted from the sequencing-confirmed strains.

[0044] The pCold-TF-ZmPHYLL plasmid, constructed through homologous recombination, was introduced into BL21(DE3) competent cells, followed by large-scale culture. OD was measured. 600 At a concentration of 0.6–0.8, 30 μL of 1 mol / L IPTG was added to induce E. coli protein production. The mixture was then placed on a shaker at 16°C and 220 rpm for 18 hours.

[0045] Next, the bacterial cells were centrifuged at 12000 rpm for 10 min at 4°C, and the resuspended in 35 mL of pre-chilled 1×PBS buffer. The cells were then centrifuged at 6000 rpm using a refrigerated centrifuge, repeated twice. Following this, the cells were sonicated (30% sonication power, 2 sec interval, 3 sec interval, 20 min). The disrupted protein solution was centrifuged at 6000 rpm for 10 min at 4°C, and the supernatant was collected to obtain the crude pCold-TF-ZmPHYLL protein.

[0046] Protein purification was performed using a conventional nickel column method. The purified recombinant protein was then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The formulations of the stacking gel and separating gel are shown in Table 4 below. The purification results of pCold-TF-ZmPHYLL protein are as follows: Figure 3 As shown.

[0047] Table 4. SDS-PAGE Gel Formulation

[0048]

[0049]

[0050] Example 5: ZmPHYLL gene expression product inhibits the germination and growth of *Russula multifiliis* spores:

[0051] The pCold-TF-ZmPHYLL purified protein obtained in the above examples was co-cultured with *Russula multifiliis* spores, and the effect of pCold-TF-ZmPHYLL protein on the germination of *Russula multifiliis* spores was observed and analyzed under a microscope. Simultaneously, sterile water treatment and protein buffer treatment (containing 100 mM imidazole) were used as controls.

[0052] The results are as follows Figure 4 As shown. The results indicate that sterile water ( Figure 4 A) and protein buffer ( Figure 4 B) After treatment, the spores of *Rust hygroscopicus* could germinate normally; however, treatment with His-ZmPHYLL protein inhibited the germination of *Rust hygroscopicus*. Figure 4 C).

[0053] Therefore, it is possible to improve the resistance of maize plants to southern rust by regulating the overexpression of the ZmPHYLL gene in maize plants, which will provide an important scientific basis for the breeding of maize varieties with high resistance to southern rust.

[0054] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A kind ZmPHYLL The application of the gene's expressed protein in inhibiting the germination of *Rust hygroscopicus* spores, characterized by: The ZmPHYLL The amino acid sequence of the expressed protein of the gene is shown in SEQ ID NO:

3.

2. The application according to claim 1, characterized in that, The ZmPHYLL The full-length nucleotide sequence of the gene is shown in SEQ ID NO: 1, and the coding region nucleotide sequence is shown in SEQ ID NO: 2.

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