Application of ZmMAPK16 gene in forward regulation of ear rot resistance and / or yield of corn

Abstract

The invention discloses application of a ZmMAPK16 gene in forward regulation and control of ear rot resistance and / or yield of corn, and belongs to the field of gene engineering. Through overexpression of the ZmMAPK16 gene in corn, it is found that the gene can positively regulate and control the ear rot resistance of the corn, and meanwhile the yield of the corn is increased. The ZmMAPK16 gene provided by the invention can improve the resistance and yield of corn ear rot at the same time, and the same gene balances the yield and resistance of corn, so that the ZmMAPK16 gene has important value for the development of new varieties of corn, lays a foundation for disease-resistant high-yield genes with independent intellectual property rights in China, and improves the safety of seed industry.

Description

Technical Field

[0001] The invention relates to the field of genetic engineering, and in particular to the application of ZmMAPK16 gene in positively regulating ear rot resistance and / or yield of corn. Background Art

[0002] Corn ear rot is mainly caused by the plant pathogen Fusarium spp., which not only causes direct yield reduction by causing ear rot, but also inhibits germination or causes mass death of seedlings in the field through infected seeds, resulting in serious losses. In addition, Fusarium produces mycotoxins during the infection process, which causes the infected grains to be unable to be used as food or feed, and lose their economic value. In 2022, Feng et al. used maize inbred lines B73 and Zheng58 to cross with two different teosinte species, Z. diploperennis and Z. parviglumis, and identified four reliable QTL loci for ear rot resistance on chromosomes 4, 5 and 10, respectively. Zila et al. used 1,687 public inbred lines to identify seven SNPs associated with improved resistance to Fusarium ear rot (FER). At the same time, Liu et al. discovered the ZmFER1 gene located on chromosome 3 of maize through the homologous Fhb1 resistance gene in wheat, and proved that the ZmFER1 mutant can improve the resistance of maize to ear rot. Mitogen-activated protein kinase MAPK and mitogen-activated protein kinase kinase (MAPKK) and mitogen-activated protein kinase kinase kinase (MAPKKK) constitute the MAPKKK-MAPKK-MAPK cascade signal transduction pathway (MAPK cascade) through step-by-step phosphorylation. The plant MAPK cascade pathway can respond to a variety of biological stresses (such as microbial pathogen infection and damage), using cell surface and intercellular receptors to sense pathogen infection and initiate immune signals. Both flg22 and elf18 can trigger strong but short-term activation of MAPKs including MAPK3, MAPK6, MAPK4 and MAPK11 in Arabidopsis. Liu et al. found that after Arabidopsis thaliana senses pathogen infection, MAPK3 and MAPK6 phosphorylate ACS2 / ACS6 to stabilize ACS proteins in vivo, leading to enhanced cellular ACS activity and increased ethylene production, thereby enhancing its disease resistance. Becker et al. found that treating plants with benzothiadiazole (BTH), a functional analog of SA, would lead to the activation of MAPK3 and MAPK6, which play a core role in plant defense signal transduction in local parts of the plant through their downstream component NPR1 and three redundant transcription factors TGA2, TGA5 and TGA6. At the same time, the MKK4 / 5-MAPK3 / 6 cascade is also involved in regulating ACC synthase activity and inducing ethylene biosynthesis to resist pathogen attack. The MAPK cascade can regulate plant defense responses through the synthesis and signal transduction of defense hormones. In the MAPK cascade reaction pathway, MAPK is the most direct member acting on the target gene, so it plays a vital role in the MAPK signal transduction pathway.

[0003] The research on the resistance and susceptibility mechanism of corn ear rot is very weak internationally. Although it has been widely reported that the MAPK cascade pathway is involved in plant disease resistance or growth and development, there are not many in-depth studies on its role in crop-Fusarium interactions. The most widely studied MAPKs are MAPK3, MAPK4 and MAPK6, which are widely involved in the regulation of plant development and signal transduction pathways by hormone signals (ethylene, IAA, ABA), as well as the regulation of plant development (pollen development, stomatal development, aging). 20 MAPK genes have been found in corn, but their functions have not been widely studied. Therefore, it is of great significance to explore more ear rot resistance genes with independent intellectual property rights; and the present invention has discovered a gene that simultaneously balances plant yield and resistance through corn MAPK gene mining. There are few reports in the prior art that balance plant yield and resistance through one gene, indicating that the gene resources mined by the present invention are very precious and have high production and utilization value. Summary of the invention

[0004] The purpose of the present invention is to provide an application of the ZmMAPK16 gene in positively regulating the ear rot resistance and / or yield of corn, so as to solve the problems existing in the above-mentioned prior art. The ZmMAPK16 gene can be overexpressed in corn to positively regulate the ear rot resistance of corn and increase the yield of corn.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides the use of ZmMAPK16 gene in any of the following:

[0007] (1) Application in positively regulating corn ear rot resistance and / or yield;

[0008] (2) Application in the breeding of corn resistant to ear rot.

[0009] Optionally, the CDS nucleotide sequence of the ZmMAPK16 gene is as shown in SEQ ID NO: 2. The ZmMAPK16 gene may also be a gene having more than 80% homology with the above CDS nucleotide sequence.

[0010] Optionally, positive regulation of corn ear rot is not limited to specifically expressing the ZmMAPK16 gene in corn ears to achieve this function, but can also be achieved by specifically expressing the ZmMAPK16 gene in other corn tissues to achieve the same function.

[0011] Optionally, the present invention is not limited to positively regulating corn ear rot, but can also be a scheme for achieving ear rot resistance by expressing the ZmMAPK16 gene in other crops.

[0012] Optionally, the present invention is not limited to affecting the expression level of the ZmMAPK16 gene by expression, but may also affect the expression level of the ZmMAPK16 gene by other means such as gene editing, use of activators, etc.

[0013] The present invention also provides the use of the protein encoded by the ZmMAPK16 gene in any of the following:

[0014] (1) Application in positively regulating corn ear rot resistance and / or yield;

[0015] (2) Application in breeding of corn resistant to ear rot;

[0016] Wherein, the amino acid sequence of the protein is shown in SEQ ID NO:3.

[0017] The present invention also provides the use of a recombinant vector containing the ZmMAPK16 gene in any of the following:

[0018] (1) Application in positively regulating corn ear rot resistance and / or yield;

[0019] (2) Application in the breeding of corn resistant to ear rot.

[0020] The present invention also provides the use of a host bacterium containing the recombinant vector in any of the following:

[0021] (1) Application in positively regulating corn ear rot resistance and / or yield;

[0022] (2) Application in the breeding of corn resistant to ear rot.

[0023] The present invention also provides a method for improving ear rot resistance and / or yield of corn, comprising the step of overexpressing the ZmMAPK16 gene in corn.

[0024] The present invention also provides a method for breeding corn resistant to ear rot, comprising the step of overexpressing the ZmMAPK16 gene in corn.

[0025] Optionally, the CDS nucleotide sequence of the ZmMAPK16 gene is as shown in SEQ ID NO:2.

[0026] The present invention discloses the following technical effects:

[0027] The present invention overexpresses ZmMAPK16 in corn and finds that the area of ​​transgenic corn plant ears infected with ear rot is significantly smaller than that of the wild type, and the disease resistance is significantly enhanced. Therefore, the overexpression of ZmMAPK16 can positively regulate corn ear rot. In addition, it is found that overexpression of the gene can also increase the yield of corn under normal conditions. The same gene balances the yield and resistance of corn, which is of great value for the development of new corn varieties. The discovery of the ZmMAPK16 gene and its related functions excavated by the present invention will enable my country to have disease-resistant and high-yield genes with independent intellectual property rights, and provide a scientific basis for screening new disease-resistant and high-yield crop varieties, contributing to the safety of the seed industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0029] Figure 1 is the map of plasmid pLANT-cFlag;

[0030] Figure 2 The effects of overexpression or knockout of the ZmMAPK16 gene in corn on corn ear rot and yield; A: The upper row of the figure shows the phenotype of uninoculated materials, and the lower row of the figure shows the phenotype of materials inoculated with Pseudomonas verticillium XY-1; B: Statistics of ear weight in the uninoculated group; C: Statistics of diseased area of ​​ears in the inoculated group; KN5585 is a wild-type receptor material, KO1-KO2 are materials with gene function loss, and OE1-OE3 are materials with high gene expression. DETAILED DESCRIPTION

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

[0032] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0033] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0034] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

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

[0036] DNA and protein characteristics of ZmMAPK16 involved in the present invention:

[0037] The ZmMAPK16 gene is derived from corn, and its universal gene number is Zm00001d006790_T006 (reference genome information is B73 Ref_v4). Its cDNA, CDS and protein sequences are shown in SEQ ID NO: 1-3, respectively. The present invention utilizes its CDS to fuse with the Flag tag sequence for transgenic expression.

[0038] Example 1

[0039] 1. Cloning and transgenic expression of ZmMAPK16

[0040] The present invention uses the leaves of corn inbred line CIMBL145 to extract total RNA, then generates cDNA through reverse transcription, then amplifies the gene protein coding region through PCR, and finally clones it into the expression plasmid pLNAT-cFlag through homologous recombination. The RNA extraction, cDNA reverse transcription, PCR amplification technology and homologous recombination cloning technology used in the present invention are all conventional technologies and can be modified and adjusted.

[0041] 1.1 RNA extraction

[0042] Plant total RNA isolation kit (RE-05014, FOREGENE) was used. All the kit equipment was cleaned with DEPC water and sterilized at 121℃ for 1h. The samples were kept at 4℃ during RNA extraction. The specific steps were as follows:

[0043] (1) Grind the fresh sample thoroughly under liquid nitrogen and take about 200 mg into a 1.5 mL centrifuge tube.

[0044] (2) Take 50 μL of PSL1 reagent and 10 μL of β-mercaptoethanol from the kit and mix well for later use.

[0045] (3) Add the PSL1 mixture prepared in the previous step to the centrifuge tube sample, quickly shake to mix, and then let it stand for 5 minutes.

[0046] (4) Add 100 μL of reagent PS to the sample in the previous step, invert to mix, and centrifuge at 12,000 rpm for 3 min.

[0047] (5) Transfer the supernatant from the previous step to a DNA cleaning column and centrifuge at 12,000 rpm for 2 min before discarding the adsorption column.

[0048] (6) Add 1.5 times the volume of PSL2 reagent to the liquid in the collection tube, mix gently and transfer the liquid to the RNAonly column. Centrifuge at 12000 rpm for 2 min and discard the supernatant.

[0049] (7) Add 500 μL of reagent PRW1 to the RNAonly column, centrifuge at 12,000 rpm for 2 min, and discard the supernatant.

[0050] (8) Add 700 μL of anhydrous ethanol to the RNAonly column, centrifuge at 12,000 rpm for 2 min, and discard the supernatant.

[0051] (9) Add 700 μL PRW2 to the RNA only column, centrifuge at 12,000 rpm for 2 min, and discard the supernatant. Repeat this step once.

[0052] (10) Centrifuge the RNAonly column tube at 12000 rpm for 3 min to remove residual reagent.

[0053] (11) Transfer the RNA only column to a new 1.5 mL centrifuge tube, add 50 μL of RNase-free water preheated at 65°C to the center of the membrane, centrifuge at 12,000 rpm for 3 min, discard the adsorption column, and collect the RNA at the bottom of the tube.

[0054] 1.2 Reverse transcription of cDNA

[0055] Reverse transcription was performed using PrimeScript from TAKARA TM RT reagent Kit (RR047A) is completed. The specific steps are as follows:

[0056] The genomic DNA was removed (reaction system: 5×gDNAEraser Buffer 2 μL, gDNAEraser 1 μL, TotalRN A 1 μg, supplemented with RNase-free deionized water to a volume of 10 μL), and placed in a PCR instrument for amplification at 42°C for 2 min.

[0057] Reverse transcription reaction (reaction system: 10 μL of DNA-removed RNA liquid, 1 μL of PrimeScript RT Enzyme Mix I, 4 μL of reverse transcription primer Oligo dT, 24 μL of 5× PrimeScript Buffer, 4 μL of RNase-free deionized water), mix well and react at 37°C for 20 min; treat at 85°C for 10 sec and store at -20°C.

[0058] 1.3 PCR amplification

[0059] The primers used were: 7351F (SEQ ID NO: 4): 5'-CAGCACCTTCAGAAATGGATT-3'; 7351R (SEQ ID NO: 5): 5'-AGATCTACCAATGGCCGCCG-3'.

[0060] Reaction system: 2×PCR buffer 10μL, 2mM dNTPs 4μL, 10μM forward and reverse primers 0.3μL each, cDNA template 0.4μL, KOD FX Neo DNApolymerase 0.4μL, add deionized water to 20μL.

[0061] Amplification parameters: pre-denaturation at 94°C for 2 min, melting at 98°C for 30 sec, extension at 68°C for 15 sec, and amplification for 38 cycles.

[0062] 1.4 Construction of transgenic expression plasmid

[0063] The ZmMAPK16 protein coding region was ligated to the plant transgenic binary expression plasmid pLNAT-c Flag (see the plasmid map for information) by homologous recombination cloning. Figure 1 ).

[0064] Homologous recombination fragment PCR amplification primers: T7351F-flag (SEQ ID NO: 6): 5'-tctgcaggtcgactctagaATGGA TTTTTTCACAGAGTA-3'; T7351R-flag (SEQ ID NO: 7): 5'-ggtctttgtagtccatttgCCAATGGCCG CCGCG-3'. PCR amplification parameters and reaction system are the same as 1.3.

[0065] Enzyme digestion of the vector: The binary expression plasmid pLNAT-cFlag was digested using the BamHI restriction site. The linearized plasmid was then recovered by agarose gel electrophoresis and purified using the The homologous recombination reaction was carried out according to the instructions of MultiS One Step Cloning Kit (C115). The constructed expression plasmid was confirmed to be correct by sequencing and transformed into Agrobacterium EHA105. Jiangsu Weimi Biotechnology Co., Ltd. was commissioned to carry out corn transformation (overexpression and knockout of ZmMAPK16), and the transgenic receptor material was KN5585.

[0066] 2. ZmMAPK16 positively regulates corn ear rot resistance

[0067] The company was entrusted to carry out corn transformation and screening to obtain homozygous plants with ZmMAPK16 gene knockout, homozygous plants with gene overexpression, and wild-type plants isolated from transgenic materials. Two homozygous knockout lines KO1 and KO2, three homozygous overexpression lines OE1, OE2, OE3, and the isolated wild-type plant KN5585 were selected for field planting. 14 days after pollination, the materials were artificially inoculated with Fusarium spp. XY-1 (provided by the State Key Laboratory of Exploration and Utilization of Southwest Crop Gene Resources of Sichuan Agricultural University; inoculation with other Fusarium spp. strains had the same effect, and there was no restriction on this pathogen). After the seeds were fully mature, they were harvested and the phenotype was investigated. The results showed that the yield of ZmMAPK16 knockout mutant corn plants decreased significantly, and the resistance to ear rot was also significantly reduced; compared with the wild type, the ZmMAPK16 overexpression lines had significantly enhanced resistance to ear rot and did not reduce the yield under normal conditions ( Figure 2 B, C). These results indicate that ZmMAPK16 is involved in positively improving maize ear rot resistance and is a gene essential for maize yield formation.

[0068] >Zm00001d006790_T006 CDNA(SEQ ID NO:1)

[0069]

[0070] >Zm00001d006790_T006 CDS(SEQ ID NO:2)

[0071]

[0072] >Protein expressed by the coding region of Zm00001d006790_T006 (SEQ ID NO: 3)

[0073] .

[0074] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Application of ZmMAPK16 gene in any of the following: (1) Application in positively regulating corn ear rot resistance and / or yield; (2) Application in breeding of corn resistant to ear rot.

2. The use according to claim 1, characterized in that The CDS nucleotide sequence of the ZmMAPK16 gene is shown in SEQ ID NO:

2.

3. Use of the protein encoded by the ZmMAPK16 gene according to claim 1 or 2 in any of the following: (1) Application in positively regulating corn ear rot resistance and / or yield; (2) Application in breeding of corn resistant to ear rot; in, The amino acid sequence of the protein is shown in SEQ ID NO:

3.

4. Use of the recombinant vector containing the ZmMAPK16 gene according to claim 1 or 2 in any of the following: (1) Application in positively regulating corn ear rot resistance and / or yield; (2) Application in breeding of corn resistant to ear rot.

5. Use of a host bacterium containing the recombinant vector according to claim 4 in any of the following: (1) Application in positively regulating corn ear rot resistance and / or yield; (2) Application in the breeding of corn resistant to ear rot.

6. A method for improving corn ear rot resistance and / or yield, characterized in that: The method comprises the steps of overexpressing the ZmMAPK16 gene in corn.

7. A method for breeding corn resistant to ear rot, characterized in that: The method comprises the steps of overexpressing the ZmMAPK16 gene in corn.

8. The method according to claim 6 or 7, characterized in that The CDS nucleotide sequence of the ZmMAPK16 gene is shown in SEQ ID NO:2.

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