Maize transcription factor gene ZmWRKY76, encoding protein thereof and application of biological material in high temperature stress resistance

Abstract

The invention discloses a corn transcription factor gene ZmWRKY76, an encoding protein thereof and an application of a biological material in high temperature stress resistance, and aims to solve the technical problem that high temperature resistance gene resources and germplasm resources of corn are deficient at present. Based on the discovery that a corn transcription factor ZmWRKY76 can respond to high temperature stress, a CRISPR-Cas9 technology is utilized to obtain a zmwrky76 knockout strain, and high temperature stress treatment is performed on the zmwrky76 knockout strain to find that the activity of superoxide dismutase in a zmwrky76 mutant is remarkably lower than that of a wild type, and the accumulation of hydrogen peroxide content is remarkably increased. According to the application, the function and the action mechanism of the corn transcription factor gene ZmWRKY76 and the encoded protein thereof for resisting high temperature stress are verified for the first time, and a solid theoretical basis is provided for high temperature stress damage coping in corn production and breeding of new high-temperature-resistant varieties.

Description

Technical Field

[0001] The present invention relates to the field of molecular breeding technology, and specifically to a corn transcription factor gene ZmWRKY76 , and the application of its encoded proteins and biomaterials in high temperature stress resistance. Background Art

[0002] corn( Zeamays L.) belongs to the genus Zea, Poaceae, native to Mexico. It is the third largest economic crop in my country after rice and wheat, and the crop with the highest total output in the world. The yield and quality of corn are particularly important to people's production and life. The Huanghuaihai region is the main dominant production area of ​​summer corn in my country, but high temperatures occur frequently in summer. During the critical growth and development period of corn, such as heading and filling stages, it is extremely susceptible to high temperature stress, resulting in low corn filling rate, causing a significant reduction in corn production, and posing a hidden danger to food security. Therefore, it is extremely important to improve the tolerance of corn under high temperature stress conditions.

[0003] The WRKY transcription factor family plays a key role in the process of plant response to environmental stress. As one of the largest transcription factor families in the plant kingdom, WRKY transcription factors are named after the highly conserved 7-amino acid sequence composed of WRKYGQK at their N-terminus. The basic structure of this transcription factor mainly consists of two parts: the N-terminal DNA binding domain and the transcription activation domain. The transcription activation domain includes multiple protein conserved sequences and plays a key role in transcription activation; while the N-terminal DNA binding domain has a variety of protein sequences and usually contains a zinc finger structure composed of C2H2 or C2HC type. WRKY transcription factors can specifically bind to the cis-acting element TTGAC (C / T) nucleotide sequence (i.e., W-box) in the promoter region of target genes through their N-terminal WRKY domain, thereby promoting or inhibiting the transcription of downstream genes and regulating the expression of these genes.

[0004] WRKY transcription factors play an important role in plant responses to a variety of abiotic stresses (such as cold, drought, UV-B, high temperature, salt and alkaline environment) and in regulating plant growth and development and defense mechanisms. In rapeseed, studies have shown that CPK (also known as CDPK) kinases can regulate the expression of WRKY transcription factor encoding gene WSR1, thereby regulating the homeostasis of reactive oxygen species (ROS). WRKY domains and their specifically bound sequences are usually present in the upstream regulatory regions of genes related to disease resistance, damage, aging, and salicylic acid-induced genes. In addition, WRKY genes are also expressed in processes such as fruit ripening, seed epidermal hair development, and sucrose and gibberellin signal transduction.

[0005] Although plants have evolved complex regulatory mechanisms to cope with stress, current research on the molecular mechanisms of plant responses to high temperature stress has mostly focused on model organisms such as Arabidopsis. In contrast, there are relatively few systematic studies on the molecular mechanisms of stress resistance in crops (such as corn). Compared with Arabidopsis, crops have more complex physiological structures and genetic backgrounds and may respond to high temperature stress through additional mechanisms. Therefore, exploring the heat stress response genes and mechanisms of different crop species is of great significance for improving crop heat tolerance and yield.

[0006] The information disclosed in this background technology section is only used to deepen the understanding of the background technology of the present disclosure, and should not be regarded as acknowledging or suggesting in any form that the information constitutes the prior art known to those skilled in the art. Summary of the invention

[0007] This application is based on the previous high temperature related omics research and found that there is a type of transcription factor in corn ZmWRKY76 Its transcriptional level is significantly induced by high temperature and may be involved in the high temperature stress response of maize, but there is currently a lack of ZmWRKY76 Study on the role of high temperature tolerance in corn. ZmWRKY76 The functions and mechanisms of action of corn in response to high temperature stress will provide gene resources for breeding a number of new high-quality corn varieties with outstanding comprehensive resistance and wide adaptability. ZmWRKY76 , and the application of their encoded proteins and biological materials in high temperature stress resistance to solve the current technical problem of the lack of high temperature resistance gene resources in corn.

[0008] According to one aspect of the present disclosure, the transcription factor gene ZmWRKY76 , which encode proteins or contain transcription factor genes ZmWRKY76 The biological material is used in regulating the high temperature resistance trait of corn or preparing an agent for regulating the high temperature resistance trait of corn.

[0009] According to a second aspect of the present disclosure, the transcription factor gene ZmWRKY76 , which encode proteins or contain transcription factor genes ZmWRKY76 The biomaterial is used to regulate the hydrogen peroxide content and / or superoxide dismutase activity of corn.

[0010] According to the third aspect of the present disclosure, the transcription factor gene ZmWRKY76 , which encode proteins or contain transcription factor genes ZmWRKY76 The biomaterials are used to remove active oxygen in plants.

[0011] According to a fourth aspect of the present disclosure, the transcription factor gene ZmWRKY76 , which encode proteins or contain transcription factor genes ZmWRKY76The biological material is used in preparing reagents for regulating the hydrogen peroxide content and / or superoxide dismutase activity of corn.

[0012] According to a fifth aspect of the present disclosure, the transcription factor gene ZmWRKY76 or contain transcription factor genes ZmWRKY76 The biological materials are used in the breeding / identification of corn varieties / lines with traits related to high temperature stress tolerance.

[0013] According to a sixth aspect of the present disclosure, the transcription factor gene ZmWRKY76 or contain transcription factor genes ZmWRKY76 The biomaterials are used in the construction / breeding of transgenic plants with high temperature stress resistance.

[0014] According to a seventh aspect of the present disclosure, the transcription factor gene ZmWRKY76 or contain transcription factor genes ZmWRKY76 Application of biomaterials in regulating heat-responsive genes ZmHSP82 and / or ZmHSP90 in the expression of.

[0015] In some embodiments of the present disclosure, the amino acid sequence of the encoded protein is shown as SEQ ID NO.2.

[0016] In some embodiments of the present disclosure, the transcription factor gene ZmWRKY76 The nucleotide sequence is shown in SEQ ID NO.1, and the transcription factor gene ZmWRKY76 The biological materials include recombinant DNA, expression cassettes, plasmid vectors or engineered bacteria.

[0017] One or more technical solutions provided in the embodiments of the present application have at least any of the following technical effects or advantages: Based on the previous high temperature-related omics research, this application found that there is a type of transcription factor ZmWRKY76 in corn, whose transcription level is significantly induced by high temperature and may be involved in the high temperature stress response of corn. ZmWRKY76 Further research revealed that it is a transcription factor localized in the cell nucleus and its expression level increases under high temperature stress, indicating that this transcription factor is involved in the plant's response to abiotic stress, especially high temperature stress. ZmWRKY76 function, using CRISPR-Cas9 technology to obtain ZmWRKY76 knockout strains and subjected them to high temperature stress at the seedling stage. It was found that under high temperature stress, ZmWRKY76The survival rate of the knockout strain was significantly lower than that of the control group, showing an extremely obvious heat-sensitive phenotype. Further analysis found that its superoxide dismutase (SOD) activity was significantly lower than that of the wild type, and the accumulation of reactive oxygen species (such as H2O2) was significantly increased. In addition, the heat-responsive gene ZmHSP82 and ZmHSP90 The expression level of was also significantly reduced, indicating that this transcription factor gene regulates the high temperature resistance of corn by regulating the activity of antioxidant protection enzymes such as SOD to maintain the homeostasis of reactive oxygen species and regulating the expression of heat response genes. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the subcellular localization analysis of the maize transcription factor ZmWRKY76 in one embodiment of the present application.

[0019] Figure 2 In one embodiment of the present application, high temperature stress treatment is applied to ZmWRKY76 Detection of the expression level.

[0020] Figure 3 In one embodiment of this application ZmWRKY76 Target design diagram.

[0021] Figure 4 In one embodiment of this application zmwrky76 Different knockout mutation forms of mutants and amino acid mutations caused by the loss of the gene ( zmwrky76 Both knockout forms result in premature termination of translation).

[0022] Figure 5 In one embodiment of this application zmwrky76 Identification of high temperature related phenotypes of mutants; A: zmwrky76 Knockout strains ( zmwrky76-c1 as well as zmwrky76-c2) Phenotypic comparison after 2 days of high temperature treatment; B: 2 days of high temperature treatment ZmWRKY76 Statistics of leaf injury area of ​​CRISPR-cas9 knockout lines, WT: wild type B104, Line 1: zmwrky76-c1 , Line 2: zmwrky76-c2 .

[0023] Figure 6 In one embodiment of this application zmwrky76 The activity of SOD and the content of H2O2 in the mutants; WT is the wild type, H is high temperature treatment, zmwrky76-c1, zmwrky76-c2 Represents mutants.

[0024] Figure 7 In one embodiment of this application zmwrky76 Detection of the expression of heat-responsive genes regulated by mutants under high temperature stress conditions; WT is the wild type, H is high temperature treatment, zmwrky76-c1, zmwrky76-c2 Represents mutants. DETAILED DESCRIPTION

[0025] The specific implementation methods of the present invention are described below in conjunction with examples, but the following examples are only used to illustrate the present application in detail and do not limit the scope of the present application in any way.

[0026] Unless otherwise specified, the instruments and equipment involved in the following embodiments are all conventional instruments and equipment; the reagents involved are all commercially available conventional reagents unless otherwise specified; the detection methods and preparation methods involved are all conventional methods unless otherwise specified.

[0027] Example 1: Analysis of the high temperature responsive transcription factor ZmWRKY76 in maize ZmWRKY76 ZmWRKY76 plays an indispensable role in regulating plant growth, development, and stress response, especially in response to adverse stresses including high temperature. As an important transcription factor involved in regulating different cell biological processes, ZmWRKY76 may respond to heat stress by regulating the variable transcription of downstream heat-responsive key genes. Therefore, in this case, the subcellular localization and expression level of ZmWRKY76 were analyzed.

[0028] 1. Subcellular localization analysis of maize transcription factor ZmWRKY76 Gene ZmWRKY76 The fusion protein was fused with the green fluorescent protein GFP driven by the 35S promoter, and the fusion protein was transferred into the maize protoplasts by PEG-mediated method. At the same time, the GFP empty vector was transferred as a control. The cells were cultured in the dark for 16 h and observed under a laser confocal microscope. Since the ZmWRKY76 protein is a transcription factor, the IMP4 protein, which is also localized in the nucleus, was used as a nuclear marker. The results are shown in Figure 2. Figure 1 As shown, the fluorescence signal of the ZmWRKY76 and GFP fusion protein overlaps with the IMP4-mCherry fluorescence signal, indicating that the ZmWRKY76 protein is localized in the cell nucleus.

[0029] 2. Effect of high temperature stress on ZmWRKY76 Detection of expression level Maize variety B104 was cultured to the three-leaf stage seedlings and subjected to 42°C high temperature treatment. Samples were taken at 0, 1, 2, 4, 6, 8, and 10 h of heat treatment, and total RNA was extracted from the leaves. ZmWRKY76 Relative expression level detection (see Figure 2 ), the results show ZmWRKY76 The expression of was significantly induced by high temperature, and after 10 h of high temperature treatment, ZmWRKY76The above results indicate that ZmWRKY76 is a transcription factor localized in the cell nucleus and is related to abiotic stress in plants, especially high temperature stress.

[0030] Embodiment 2: By ZmWRKY76 Identification of heat resistance in transgenic plants To further determine ZmWRKY76 Whether it is involved in the response of corn to high temperature, this case uses CRISPR-Cas9 technology to obtain ZmWRKY76 The knockout strains were subjected to high temperature stress (42°C) at the seedling stage (corn seedlings 13 days after breaking through the soil), and the treated seedlings were covered with water for 2 days to recover. Then the relevant physiological indicators were counted, so as to more intuitively and accurately determine the ZmWRKY76 Effects of high temperature stress on maize.

[0031] 1. CRISPR-Cas9 target site design First, download it from the Gramene website ZmWRKY76 The gene sequence was then used on the CRISPR-P website ZmWRKY76 The exon sequences were analyzed for targets, and targets with relatively high scores were selected for CDS comparison in the maize network to exclude the possibility of off-target, so as to select targets with better specificity. CRISPR construction primers (SEQ ID No.3~6) were designed, and the synthesized primers were diluted to a working concentration of 10×. 5 μL of each primer was taken, and the PCR program was set as follows: 95℃, 10min, (100%) Tm value; 5℃, 10 min (4%) 16℃, and finally small molecule single-strand annealing. At the same time, the vector PBUE411 was cut with Bsa Ⅰ, and the digestion was carried out in a gas bath at 37℃ for 6 h; denatured at 65℃ for 20 min, and then immediately placed on ice, and stored at 4℃ for use after stabilization for a few minutes. The ligase Solution I was used for ligation for 4-6 h, and heat shock transformation was performed. The whole bacteria were spread on the LB solid medium with kana resistance and inverted at 37℃. Colony PCR identification was performed using the universal primers OSU3-FD3 and TaU3-RD (SEQ ID No. 7~8) on the vector.

[0032] Select the positive clone with a size of 500 bp, shake the bacteria, and culture overnight at 37℃ and 180 rpm. The next day, extract the plasmid and sequence it with universal primers OSU3-FD3. At the same time, compare the sequencing results of the vector sequence and the target site, and keep the correct clone for subsequent experiments. The selected target sites and seedling cultivation conditions are shown in Figure 3 .

[0033] 2. zmwrky76 Identification of mutant forms Primers were designed 200 bp upstream and downstream of the knockout target site to amplify the wild-type B104 and mutant, respectively. Sequencing results showed that two different knockout forms were obtained using CRISPR-Cas9 technology. zmwrky76 The mutants were named zmwrky76-c1 as well as zmwrky76-c2 .in, zmwrky76-c1 as well as zmwrky76-c2 The mutation forms are all premature termination of translation ( Figure 4 ).

[0034] 3. zmwrky76 Identification of high temperature related phenotypes of mutants The mutant plants zmwrky76-c1 as well as zmwrky76-c2 The wild type WT was cultured to the three-leaf stage, and then subjected to high temperature stress treatment at 42℃ for 14 h during the day and 35℃ for 10 h at night. A group of normal treatment was set as a control to observe its phenotype. The phenotypic experimental results showed that ( Figure 5 ), under high temperature stress, the survival rate of the mutant strain was significantly lower than that of the control group, showing an extremely obvious heat-sensitive phenotype, which indicates that ZmWRKY76 Positively regulating the high temperature resistance of corn.

[0035] Example 3: Maize transcription factor ZmWRKY76 High temperature resistance test Plants accumulate a large amount of reactive oxygen species (ROS) during stress. Excessive ROS can cause oxidative damage to proteins, DNA, and lipids. The accumulation of hydrogen peroxide (H2O2) can directly or indirectly oxidize biological macromolecules such as nucleic acids and proteins in cells, and damage cell membranes, thereby accelerating cell aging and disintegration. Its content can not only be used as a direct reflection of the oxidative stress state in plant cells, but also reflect the degree of damage suffered by plants under adverse conditions. Therefore, the timely removal of ROS and H2O2 is crucial when plants respond to stress. During the stress process of plants, in order to effectively remove these harmful reactive oxygen species, plants rely on antioxidants such as superoxide dismutase (SOD).

[0036] 1. Under high temperature stress conditions zmwrky76 Effects of mutants on physiological parameters of maize The wild-type WT and zmwrky76 ( zmwrky76-c1 and zmwrky76-c2 ) mutants were treated with high temperature and the activity of SOD and the content of H2O2 were measured. Figure 6 As shown in Figure 2, under high temperature stress, zmwrky76 The superoxide dismutase (SOD) activity of the mutant was significantly lower than that of the wild type. Further observation showed that after high temperature stress, zmwrky76The increase in SOD activity in the mutant was also significantly lower than that in the wild type under normal conditions. zmwrky76 The accumulation of hydrogen peroxide H2O2 in the mutant increased significantly, and the content was significantly higher than that of the wild type. After being subjected to high temperature stress, the amount under normal conditions was also significantly higher than that of the wild type. ZmWRKY76 Response to heat by regulating ROS homeostasis.

[0037] 2. zmwrky76 Detection of heat response gene expression in mutants To further explore ZmWRKY76 Which heat response genes may be involved in regulating the expression of some known heat response genes (such as ZmHSP82 and ZmHSP90 The expression of ) was detected, and the results were as follows Figure 7 As shown in Figure 2, it was found that under high temperature stress, zmwrky76 In mutants, ZmHSP82 and ZmHSP90 The expression of genes was significantly reduced, indicating that ZmWRKY76 is involved in the heat response of maize by regulating the expression of heat-responsive genes.

[0038] In summary, ZmWRKY76 In corn, it is positively regulating the high temperature resistance of corn plants. It maintains the homeostasis of reactive oxygen by regulating the activity of antioxidant protection enzymes such as SOD, and regulates the expression of key heat response genes in the downstream high temperature signaling pathway, thereby regulating the high temperature stress resistance of corn.

[0039] Although some preferred embodiments of the present application have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present application.

[0040] Obviously, those skilled in the art can make various changes and modifications to the present application without departing from the spirit and scope of the inventive concept. Thus, if these modifications and variations of the present application fall within the scope of the claims of the present application and their equivalents, the present application is also intended to include these modifications and variations.

Claims

1. Transcription factor genes ZmWRKY76 , which encode proteins or contain transcription factor genes ZmWRKY76 The application of the biomaterial in regulating the high temperature resistance trait of corn or preparing an agent for regulating the high temperature resistance trait of corn.

2. Transcription factor genes ZmWRKY76 , which encode proteins or contain transcription factor genes ZmWRKY76 Application of biomaterials in regulating the hydrogen peroxide content and / or superoxide dismutase activity of corn.

3. Transcription factor genes ZmWRKY76 , which encode proteins or contain transcription factor genes ZmWRKY76 The invention discloses an application of the biomaterial in preparing a reagent for regulating the hydrogen peroxide content and / or superoxide dismutase activity of corn.

4. Transcription factor genes ZmWRKY76 , which encode proteins or contain transcription factor genes ZmWRKY76 Application of biomaterials in removing active oxygen in plants.

5. Transcription factor genes ZmWRKY76 or transcription factor gene ZmWRKY76 Application of biological materials in breeding / identification of maize varieties / lines with traits related to high temperature stress tolerance.

6. Transcription factor genes ZmWRKY76 or transcription factor gene ZmWRKY76 The application of biological materials in the construction / breeding of transgenic plants with high temperature stress resistance.

7. Transcription factor genes ZmWRKY76 or transcription factor gene ZmWRKY76 Biomaterials in regulating heat-responsive genes ZmHSP82 and / or ZmHSP90 Application in expression.

8. The use according to any one of claims 1 to 4, characterized in that: The amino acid sequence of the encoded protein is shown in SEQ ID NO.

2.

9. The use according to any one of claims 1 to 7, characterized in that: The transcription factor gene ZmWRKY76 The nucleotide sequence is shown in SEQ ID NO.1, and the transcription factor gene ZmWRKY76 The biological materials include recombinant DNA, expression cassettes, plasmid vectors or engineered bacteria.

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