ZmPHYLL gene, application thereof and method for improving resistance of corn plants to southern corn rust

Abstract

The invention relates to the technical field of gene engineering, and provides a ZmPHYLL gene, the full-length nucleotide sequence of the ZmPHYLL gene is shown as SEQ ID NO: 1, and the nucleotide sequence of a coding region is shown as SEQ ID NO: 2. The invention also provides application of the ZmPHYLL gene in inhibiting germination of puccinia polypoda spores and improving resistance of corn plants to southern corn rust. Meanwhile, the invention provides a method for improving the resistance of corn plants to the southern corn rust disease. The ZmPHYLL gene is screened in the research, and the expression protein of the gene can effectively inhibit the spore germination of puccinia polypoda, so that the resistance of corn to the corn southern rust disease can be improved by regulating and controlling the overexpression of the gene; according to the invention, a new gene resource is provided for creation of breeding materials, and an important scientific basis is provided for breeding of a corn variety with high resistance to southern corn rust.

Description

Technical Field

[0001] The invention relates to the technical field of gene engineering, and in particular to a ZmPHYLL gene and an application thereof, and a method for improving the resistance of corn plants to southern corn rust. Background Art

[0002] Corn southern rust is a highly destructive airborne fungal disease caused by Puccinia multiflora. The pathogen mainly infects corn leaves, and in severe cases will further infect the sheaths, bracts and other parts, causing the leaves to dry up, the photosynthesis function to be lost, the kernels to shrivel, and the yield to drop significantly. Currently, corn southern rust is common in warm temperate and tropical regions, and is a major problem facing corn producers, seriously threatening the safety of corn production.

[0003] In order to solve the problem of resistance to southern rust of corn, the predecessors have discovered Rpp-type disease resistance genes (NLR family) such as Rpp1, Rpp2, RppK and RppM in resistant germplasm, and bred a series of disease-resistant varieties by using these genes alone or in combination. However, as the pathogen race changes, the resistance conferred by these NLR family genes is usually not persistent in the field.

[0004] Based on this, the current focus is to study and screen out new disease-resistant genes related to southern corn rust. This will provide new genetic resources for the creation of breeding materials and provide an important scientific basis for the selection and breeding of corn varieties with high resistance to southern corn rust. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a ZmPHYLL gene and its application and a method for improving the resistance of corn plants to southern corn rust.

[0006] The present invention adopts the following technical solutions to solve the above technical problems:

[0007] A ZmPHYLL gene, the full-length nucleotide sequence of which is shown in SEQ ID NO: 1, and the coding region nucleotide sequence 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, there is a positive correlation between the expression level of the ZmPHYLL gene and the resistance of the corn plant to southern corn rust.

[0010] An application of the ZmPHYLL gene in inhibiting the germination of spores of Puccinia multiflora and improving the resistance of corn plants to southern corn rust.

[0011] A method for improving the resistance of corn plants to southern corn rust, comprising overexpressing the ZmPHYLL gene in the corn plants through a genetic engineering method; 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, and the gene expression level is positively correlated with the resistance of the corn plants to southern corn rust.

[0012] As one of the preferred embodiments of the present invention, the expression protein of the ZmPHYLL gene improves the resistance of corn plants to southern corn rust by inhibiting the spore germination of Puccinia multifiliis.

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

[0014] (1) In the present invention, a ZmPHYLL gene with high resistance to southern corn rust was screened; the study found that the expression protein of the ZmPHYLL gene can effectively inhibit the spore germination and growth of multiple piles of rust fungi, and thus, the resistance of corn plants to southern corn rust can be improved by regulating the overexpression of the ZmPHYLL gene; the discovery of the ZmPHYLL gene of the present invention provides a new gene resource for the creation of breeding materials and provides an important scientific basis for the breeding of corn varieties with high resistance to southern corn rust.

[0015] (2) The existing Rpp-type genes (NLR family) drive cell death after rust infection, making it impossible for the multi-pile rust to complete its life cycle. When this type of gene is used to screen and cultivate corn varieties, the effector genes in the multi-pile rust will recognize the corn Rpp-type genes and cause allergic cell death, so that individuals without this effector gene in the rust fungus are screened out, that is, they are subject to evolutionary pressure, resulting in high variation of effectors and NLR proteins, gene loss, and non-persistent resistance. However, the ZmPHYLL gene of the present invention, after research, does not belong to the NLR family. Its function is different from that of the Rpp-type genes, and it can effectively avoid the problem of non-persistent resistance of the existing NLR family genes.

[0016] (3) The present invention uses big data analysis and qRT-PCR technology to find that the expression of the ZmPHYLL gene is significantly upregulated after infection with Puccinia multiflora, and the protein produced by its expression has an inhibitory effect on the germination and growth of Puccinia multiflora spores, thereby inhibiting the outbreak and damage of southern corn rust. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 is the volcano plot of differential gene expression in Example 1;

[0018] Figure 2 The expression results of the ZmPHYLL gene at different time points after different plant samples were infected by multiple piles of Puccinia in Example 2;

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

[0020] Figure 4 This is a graph showing the results of the ZmPHYLL gene expression protein inhibiting the spore germination of multiple piles of Puccinia odorifera in Example 5 (in the graph, 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 DESCRIPTION

[0021] The embodiments of the present invention are described in detail below. The present embodiments are implemented under the premise of the technical solution of the present invention, and detailed implementation methods and specific operation processes are given, but the protection scope of the present invention is not limited to the following embodiments. At the same time, the reagents and materials used in the following embodiments are commercially available reagents and materials conventional in the art unless otherwise specified. The experimental methods used in the following embodiments are conventional methods in the art unless otherwise specified, and will not be repeated.

[0022] Example 1, ZmPHYLL gene screening:

[0023] Southern rust of corn was observed in the field, and rust-infected and rust-resistant plants were screened based on the observation results, and then transcriptome sequencing was performed. Differential expression analysis, enrichment analysis, expression pattern analysis, etc. were performed by combining transcriptome data and BSA-Seq data. Figure 1 , and finally the candidate gene ZmPHYLL was screened out for subsequent experiments.

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

[0025] Example 2: Analysis of ZmPHYLL gene expression after infection with Puccinia multiflora in corn:

[0026] The non-rust-resistant "Corn B73" (existing variety) and the rust-resistant "Corn Yanzi 7016" (existing variety, seed number: 20231002628) were selected as plant samples, and were marked as non-rust-resistant plants SS and rust-resistant plants RS, respectively; leaves of the samples were infected with multiple piles of stem rust fungi, and the expression levels of the ZmPHYLL gene in the corresponding plants at different time points were determined by fluorescent quantitative PCR. The specific operations are as follows:

[0027] Primers (upstream primer-F1, downstream primer-R1) were designed using Primer Primier 5.0 software and synthesized by Shanghai Shenggong Company. At the same time, RNA was extracted from plant leaves using a plant RNA extraction kit (Chengdu Biofit Technology Co., Ltd., catalog number RN33050). One-Step gDNA Removal and cDNA Synthesis SuperMix Kit was used to reverse transcribe and synthesize cDNA.

[0028] Reagents were added according to the system in Table 1, three biological replicates were set, and the reaction was carried out according to the reaction conditions in Table 1. The relative expression level was calculated using the 2-ΔΔCt method.

[0029] Table 1. Fluorescence quantitative PCR reaction system and reaction conditions

[0030]

[0031]

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

[0033] Example 3, cloning of ZmPHYLL gene:

[0034] RNA from leaves of corn B73 was extracted using a plant RNA extraction kit; RNA from leaves of corn B73 was extracted using a reverse transcription kit (Full-length Gold One-Step gDNARemoval and cDNASynthesis SuperMix) was used to reverse transcribe RNA into cDNA according to the instructions, and the coding region sequence of the maize ZmPHYLL gene (SEQ ID NO: 2) was obtained according to the maize genome (V5.0); the corresponding 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 product was detected by electrophoresis, and the target fragment was recovered and purified. The gel-recovered product containing the target gene was mixed with pMD18 TM-T Vector was used for ligation, and the competent E. coli DH5α was transformed. Single clones were selected for sequencing.

[0039] Example 4, purification of ZmPHYLL gene expression product:

[0040] According to the coding region sequence of ZmPHYLL (SEQ ID NO: 2) and the restriction site of the vector pCold-TF sequence, Primer Premier 5.0 software was used to detect the restriction site of the target gene, and primers with restriction 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 connected by homologous recombination, transformed into Escherichia coli competent DH5α, single clones were selected for colony PCR verification, and sequencing was performed to confirm the correctness of the sequence, and the recombinant plasmid was extracted from the strain confirmed by sequencing.

[0044] The homologous recombination construct pCold-TF-ZmPHYLL plasmid was introduced into BL21 (DE3) competent cells, and then the bacterial culture was expanded. OD 600 At 0.6-0.8, 30 μL 1 mol / L IPTG was added to induce E. coli protein, and the mixture was placed on a shaker at 16°C and 220 r / min for 18 h.

[0045] Afterwards, the cells were centrifuged at 12000 r / min for 10 min at 4°C, and the cells were resuspended in 35 mL of pre-cooled 1× PBS buffer and centrifuged in a refrigerated centrifuge (6000 r / min) for 2 times. Afterwards, the cells were ultrasonically treated (ultrasonic power ratio 30%, breaking for 2 sec, interval 3 sec, breaking for 20 min), and the protein solution after breaking was centrifuged at 4°C, 6000 r / min for 10 min, and the supernatant was collected to obtain the crude protein of pCold-TF-ZmPHYLL.

[0046] The protein was purified by the traditional nickel column method, and the purified recombinant protein was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The formulas of the concentrated gel and the separation gel are shown in Table 4. Figure 3 shown.

[0047] Table 4. SDS-PAGE gel formula

[0048]

[0049]

[0050] Example 5: ZmPHYLL gene expression product inhibits the germination and growth of multiple piles of Puccinia spores:

[0051] The pCold-TF-ZmPHYLL purified protein obtained in the above example was co-cultured with multiple piles of Puccinia spores, and the effect of the pCold-TF-ZmPHYLL protein on the germination of multiple piles of Puccinia spores was observed and analyzed by microscopy. At the same time, sterile water treatment and protein buffer (buffer containing 100 mM imidazole) treatment were used as controls.

[0052] The results are as follows Figure 4 The results showed that sterile water ( Figure 4 A) and protein buffer ( Figure 4 B) After treatment, the spores of Puccinia multipilosa can germinate normally; however, His-ZmPHYLL protein treatment can inhibit the germination of Puccinia multipilosa ( Figure 4 C).

[0053] Based on this, the resistance of corn plants to southern corn rust can be improved by regulating the overexpression of the ZmPHYLL gene in corn plants, which will provide an important scientific basis for the breeding of corn varieties with high resistance to southern corn 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 in the protection scope of the present invention.

Claims

1. A ZmPHYLL gene, characterized in that: The full-length nucleotide sequence is shown in SEQ ID NO: 1, and the coding region nucleotide sequence is shown in SEQ ID NO:

2.

2. The ZmPHYLL gene according to claim 1, characterized in that The amino acid sequence of the protein encoded by the ZmPHYLL gene is shown in SEQ ID NO:

3.

3. The ZmPHYLL gene according to claim 1, characterized in that There is a positive correlation between the expression level of the ZmPHYLL gene and the resistance of the corn plant to southern corn rust.

4. Use of the ZmPHYLL gene according to any one of claims 1 to 3 in inhibiting the spore germination of Puccinia multiflora and improving the resistance of corn plants to southern corn rust.

5. A method for improving the resistance of corn plants to southern corn rust, characterized in that: The ZmPHYLL gene in corn plants is overexpressed by genetic engineering methods; 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, and the gene expression level is positively correlated with the resistance of corn plants to southern corn rust.

6. The method for improving corn plant resistance to southern corn rust according to claim 5, characterized in that: The expression protein of the ZmPHYLL gene improves the resistance of corn plants to southern corn rust by inhibiting the spore germination of Puccinia multispinosa.

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