Application of a biological material for knocking out ZmMPK8 gene in improving drought tolerance of corn
By knocking out the ZmMPK8 gene in maize using CRISPR-Cas9 gene editing technology, the problem of insufficient drought resistance in maize was solved, the drought resistance of maize was improved, and the growth performance was enhanced.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- CHINA AGRI UNIV
- Filing Date
- 2025-07-10
- Publication Date
- 2026-07-14
AI Technical Summary
Existing technologies are insufficient to effectively improve the drought resistance of maize. Drought stress leads to limited maize growth, reduced photosynthetic efficiency, and insufficient grain filling, affecting yield and quality.
The ZmMPK8 gene in maize was knocked out using CRISPR-Cas9 gene editing technology to construct a ZmMPK8 gene knockout mutant. Maize was then transformed using recombinant Agrobacterium-mediated transformation to obtain transgenic plants with improved drought resistance.
It significantly improved the drought resistance of maize, and the mutant materials showed resistance to drought, reduced leaf water loss rate, and improved growth performance.
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Figure CN120775865B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of plant genetic engineering technology, and in particular to the application of a biomaterial with the ZmMPK8 gene knocked out in improving the drought resistance of maize. Background Technology
[0002] As a sessile organism, maize is inevitably affected by various environmental factors throughout its growth and development, with drought stress being particularly prominent. Drought is one of the greatest abiotic stresses facing agricultural production and a major factor limiting maize yield. Due to intensified global climate change and uneven water resource distribution, drought problems are becoming increasingly serious, posing a significant threat to agricultural production. According to relevant research statistics, the annual economic losses caused by drought exceed the total losses from all other abiotic stresses combined. Drought stress easily leads to problems such as limited maize plant growth, reduced photosynthetic efficiency, and insufficient grain filling, thus significantly affecting maize yield and quality. To address drought stress, improving the drought resistance of maize has become one of the important topics in agricultural scientific research.
[0003] As a crop with strong environmental adaptability, maize's genetic diversity and complex gene regulatory network provide abundant resources for discovering drought-resistant genes. Studies have shown that maize has developed various stress resistance mechanisms during its long evolutionary process. These mechanisms involve multi-level regulation, including transcriptional regulation, protein modification, signal transduction, and metabolic pathway adjustment. In-depth analysis of the molecular mechanisms of maize drought resistance can effectively screen and identify key regulatory genes, providing important technical support for the development of new drought-resistant maize varieties.
[0004] This invention aims to identify key genes regulating drought resistance in maize, elucidate their mechanisms of action, and apply these genes to the breeding of new drought-resistant maize varieties. Against this backdrop, the invention strives to reveal the regulatory mechanisms of drought resistance in maize at the molecular level, providing a theoretical basis and technical support for the development of novel drought-resistant maize varieties. Summary of the Invention
[0005] The purpose of this invention is to provide an application of ZmMPK8 gene knockout biomaterials in improving drought resistance in maize, thereby addressing the problems existing in the prior art. This invention has found that the ZmMPK8 gene negatively regulates drought resistance in maize; knocking out the ZmMPK8 gene in maize can improve the drought resistance of maize plants.
[0006] To achieve the above objectives, the present invention provides the following solution:
[0007] This invention provides the application of a biomaterial with the ZmMPK8 gene knocked out in improving the drought resistance of maize, wherein the nucleotide sequence of the ZmMPK8 gene is shown in SEQ ID NO.1.
[0008] Furthermore, the biomaterial is any one of (1)-(2):
[0009] (1) CRISPR-Cas9 gene knockout vector;
[0010] (2) Non-plant host cells containing the CRISPR-Cas9 gene knockout vector.
[0011] Furthermore, the CRISPR-Cas9 gene knockout vector is constructed by ligating a DNA fragment encoding sgRNA into a vector carrying CRISPR-Cas9;
[0012] The nucleotide sequence of the sgRNA is shown in SEQ ID NO.5.
[0013] Furthermore, the non-plant host cell is recombinant Agrobacterium.
[0014] The present invention also provides a biomaterial for improving the drought resistance of maize, wherein the biomaterial is any one of (1)-(2):
[0015] (1) CRISPR-Cas9 gene knockout vector for knocking out the ZmMPK8 gene;
[0016] (2) Non-plant host cells containing the CRISPR-Cas9 gene knockout vector;
[0017] The nucleotide sequence of the ZmMPK8 gene is shown in SEQ ID NO.1.
[0018] Furthermore, the CRISPR-Cas9 gene knockout vector is constructed by ligating a DNA fragment encoding sgRNA into a vector carrying CRISPR-Cas9;
[0019] The nucleotide sequence of the sgRNA is shown in SEQ ID NO.5.
[0020] Furthermore, the non-plant host cell is recombinant Agrobacterium.
[0021] The present invention also provides a method for improving the drought resistance of maize, including the step of knocking out the ZmMPK8 gene of maize to construct a transgenic maize plant with the ZmMPK8 gene knocked out;
[0022] The nucleotide sequence of the ZmMPK8 gene is shown in SEQ ID NO.1.
[0023] Furthermore, the ZmMPK8 gene in maize was knocked out using the CRISPR-Cas9 gene knockout method.
[0024] Furthermore, the nucleotide sequence of the sgRNA used in the CRISPR-Cas9 gene knockout method is shown in SEQ ID NO. 5.
[0025] The present invention discloses the following technical effects:
[0026] This invention cloned the ZmMPK8 gene from maize and obtained overexpression materials and gene knockout mutants of the maize ZmMPK8 gene using plant genetic engineering techniques. Drought experiments showed that the overexpression materials were sensitive to drought, while the gene knockout mutants exhibited a drought-tolerant phenotype. These results indicate that the ZmMPK8 gene negatively regulates drought tolerance in maize. Furthermore, in-depth analysis of the drought tolerance mechanism revealed that the drought sensitivity of the ZmMPK8 overexpression materials and the drought-tolerant phenotype of the mutant materials may be related to the rate of water loss from leaves. This invention provides a new gene resource for regulating drought resistance in maize and offers theoretical basis and technical support for developing novel drought-tolerant maize varieties. Attached Figure Description
[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0028] Figure 1 A schematic diagram illustrating the construction of the ZmMPK8 gene knockout mutant;
[0029] Figure 2 This is a sequence alignment diagram of maize mutant materials zmmpk8 c1 and zmmpk8 c2 and wild-type material B73-329; red sequences indicate protein sequences translated after the target site, and * indicates the protein translation termination position;
[0030] Figure 3 Phenotypic identification results for maize overexpression materials ZmMPK8-OE1 and ZmMPK8-OE2, mutant materials zmmpk8 c1 and zmmpk8c2, and wild-type material B73-329;
[0031] Figure 4 A statistical chart showing the fresh weight of the aboveground parts of different corn varieties;
[0032] Figure 5 A statistical chart showing the aboveground dry weight of different corn varieties;
[0033] Figure 6The figure shows the results of identifying the expression levels of the target gene in maize overexpression materials ZmMPK8-OE1 and ZmMPK8-OE2 and wild-type material B73-329;
[0034] Figure 7 Infrared imaging and statistical results of maize overexpression materials ZmMPK8-OE1 and ZmMPK8-OE2 and wild-type material B73-329;
[0035] Figure 8 Infrared imaging and statistical results of maize mutant materials zmmpk8 c1 and zmmpk8 c2 and wild-type material B73-329;
[0036] Figure 9 This is a schematic diagram of the blade cutting position in Example 5;
[0037] Figure 10 Image showing the stomata on the upper and lower epidermis of ZmMPK8 overexpressing and mutant plants;
[0038] Figure 11 A statistical graph showing the stomatal density of ZmMPK8 overexpression and mutant plants. Detailed Implementation
[0039] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.
[0040] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.
[0041] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.
[0042] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.
[0043] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.
[0044] Example 1: Preparation of maize ZmMPK8 gene overexpression plants
[0045] The CDS sequence of the maize ZmMPK8 gene (nucleotide sequence shown in SEQ ID NO.1) was obtained from the NCBI database. The CDS sequence is 1110 bp in length and encodes a protein containing 369 amino acids (SEQ ID NO.2). Gene-specific primers were designed based on the CDS sequence, with NdeⅠ and EcoRI restriction enzyme sites added to both ends. The primer sequences are as follows:
[0046] ZmMPK8-F: 5'-CATATGGCGATGATGGTGGATCC-3' (SEQ ID NO. 3);
[0047] ZmMPK8-R: 5'-GAATTCTCACATGCTGATTCTCGTGAGG-3' (SEQ ID NO. 4).
[0048] The ZmMPK8 gene was amplified by PCR. The 30.0 μL amplification system consisted of: 3.0 μL of 10×LA Taq Buffer II, 4.8 μL of dNTPs, 0.8 μL of primer ZmMPK8-F (10 μmol / mL), 0.8 μL of primer ZmMPK8-R (10 μmol / mL), 1.0 μL of template DNA, 0.6 μL of TaKaRa LATaq, and 19.0 μL of ddH2O.
[0049] The PCR program was as follows: 94℃ pre-denaturation for 1 min; 98℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 1 min 15 s (TaKaRa LATaq 1Kb / 1 min), 34 cycles; 72℃ post-extension for 10 min; storage at 4℃.
[0050] Subsequently, 30 μL of the PCR product was subjected to 1% agarose gel electrophoresis, the target band was excised, and the gel was recovered using the Zhuangmeng Biogel Recovery Kit.
[0051] The PCR product was ligated to the vector. The T4 DNA Ligase used for ligation was purchased from NEB. The 10.0 μL ligation system consisted of 1 μL of T4 DNA ligase, 10.4 μL of pCXUN-Vector, 8.0 μL of PCR product, and 0.6 μL of T4 DNA Ligase. After the sample was added, the mixture was mixed, briefly centrifuged, and ligated overnight at 16°C. After ligation, the sample was transformed into E. coli DH5α to obtain the pUbi::ZmMPK8 recombinant plasmid. After the sequencing was confirmed to be correct, the sample was handed over to the maize transgenic platform of China Agricultural University for overexpression transformation of maize materials (wild-type maize material B73-329 was selected). The pCXUN-Vector1 vector was provided by the maize functional platform of China Agricultural University and has been disclosed in the literature "Chen S, Songkumarn P, Liu J, & Wang GL (2009) A versatile zero background T-vector system for gene cloning and functional genomics. Plant physiology 150(3):1111-1121".
[0052] Identification of homozygous ZmMPK8 gene overexpression lines: After transformation, the materials were germinated, and samples were taken when they reached the three-leaf stage. Genomic DNA was extracted for PCR identification. Maize genomic DNA was extracted using the CTAB method. The reagents were purchased from Beijing Solarbio Science & Technology Co., Ltd. Reagent name: 2×CTAB extraction buffer; catalog number: LS00066. Specific experimental methods were followed according to the manufacturer's instructions. Two ZmMPK8 gene overexpression lines were obtained: ZmMPK8-OE1 and ZmMPK8-OE2. The target gene expression levels of maize overexpression lines ZmMPK8-OE1 and ZmMPK8-OE2, as well as the wild-type material B73-329, are shown in the table below. Figure 6 .
[0053] Example 2: Obtaining and Identifying ZmMPK8 Gene-Edited Transgenic Maize Plants
[0054] Using conventional CRISPR-Cas9 technology, the ZmMPK8 gene in wild-type maize B73-329 was knocked out. After transformation and identification, maize mutant materials zmmpk8 c1 and zmmpk8 c2 were obtained (see schematic diagram). Figure 1 The CRISPR / Cas9 gene knockout method is as follows:
[0055] Using the ZmMPK8 gene as a target, a CRISPR-Cas9-based sgRNA sequence was designed. A DNA fragment containing the encoding sgRNA sequence was ligated into a vector carrying CRISPR-Cas9. Maize was transformed using Agrobacterium-mediated transformation (wild-type maize material B73-329 was selected), and maize mutant materials with loss of gene function were identified.
[0056] sgRNA: 5'-AATGATGCCTGCGCATAGA-3' (SEQ ID NO. 5).
[0057] Compared to the wild type, the mutant material zmmpk8 c1 has a two-base TA deletion in the ZmMPK8 gene sequence, resulting in a frameshift mutation at the amino acid level. Specific sequence differences are as follows: Figure 2 As shown; compared with the wild type, the mutant material zmmpk8c2 has an insertion of 1 T base in the ZmMPK8 gene sequence, resulting in a frameshift mutation. The specific sequence differences are as follows. Figure 2 As shown.
[0058] Example 3 Phenotypic Identification of Overexpression Materials and Mutant Materials
[0059] The following is an experiment on soil drought during the corn seedling stage:
[0060] 1) Sowing: Mix the nutrient soil evenly according to the specified ratio. Sow 4 seeds of transgenic maize and 4 seeds of wild-type B73-329 maize separately in three seedling trays containing 200g of nutrient soil each. Cover each seedling tray with 100g of nutrient soil and place them in the same tray. Water appropriately. The cultivation conditions are: temperature 25℃, photoperiod of 14h light / 10h dark, and humidity of 50%-60%.
[0061] 2) Seedling establishment: When the seeds germinate and grow to 2-3cm above the ground, remove any seedlings that are growing unevenly.
[0062] 3) Drought treatment: When the seedlings reach the two-leaf-one-heart stage, fill the tray with water. After the soil has fully absorbed the water (3-6 hours), pour out the remaining water and begin the drought treatment. At the same time, use normally growing plants that have not undergone drought treatment as a control.
[0063] 4) To reduce the impact of location effects on drought phenotypes, the seedling pots in each tray and the trays are swapped daily.
[0064] 5) Observe the drought-resistant phenotype approximately 5-7 days after drought treatment. Take photos when there is a significant difference in phenotype between wild-type and transgenic plants. Figure 3The sensitivity and drought resistance phenotypes were statistically analyzed. The results showed that the overexpression materials ZmMPK8-OE1 and ZmMPK8-OE2 were sensitive to drought, while the mutant materials zmmpk8 c1 and zmmpk8 c2 had drought-resistant phenotypes.
[0065] 6) After phenotypic observation, the dry and fresh weights of transgenic materials under normal growth and drought treatment, as well as wild-type plants, were measured. The results of the aboveground fresh and dry weight measurements of maize overexpression materials ZmMPK8-OE1 and ZmMPK8-OE2, mutant materials zmmpk8c1 and zmmpk8c2, and wild-type material B73-329 are as follows: Figure 4 and Figure 5 As shown.
[0066] Example 4: Infrared camera measurement of corn leaf surface temperature
[0067] Plant leaves transpire through stomata, resulting in water loss along with heat loss. Under identical external conditions, the amount of water lost depends on the stomatal opening; a larger opening indicates stronger transpiration, leading to greater heat loss. The greater the heat loss, the lower the leaf surface temperature. Therefore, leaf surface temperature can indirectly reflect stomatal diameter. Transgenic maize and wild-type B73-329 plants, cultured under normal conditions and exhibiting consistent growth, were photographed using an infrared camera (FLIR System). The average leaf surface temperature was then quantitatively analyzed using IRBIS3 software. Figures 7-8 As shown, the leaf surface temperature of ZmMPK8 overexpressing plants was lower than that of B73-329, indicating that the transpiration rate of ZmMPK8 overexpressing plants was higher than that of wild-type plants; conversely, the leaf surface temperature of ZmMPK8 mutant plants was higher than that of B73-329, indicating that the transpiration rate of ZmMPK8 mutant plants was lower than that of wild-type plants. The experimental results suggest that the drought sensitivity of ZmMPK8 overexpressing plants and the drought resistance phenotype of mutant plants may be related to the rate of leaf water loss.
[0068] Example 5: Statistics on stomatal density of maize leaves
[0069] Stomata are crucial for maintaining water and CO2 balance within plants. To cope with changes in the external environment, plants can respond to stress signals by regulating stomatal movement and density. Unlike the stomata of dicotyledonous plants, the stomatal complex in maize consists of a pair of dumbbell-shaped guard cells and a pair of subsidiary cells on the flanks. To further investigate how the ZmMPK8 gene regulates drought tolerance in maize plants, this invention first observed whether the stomatal density in the leaves of overexpressing plants and CRISPR / Cas9 plants differed from that of the wild type. The specific implementation steps are as follows:
[0070] 1) Sowing: In the greenhouse, genetically modified corn seeds and B73-329 wild-type corn seeds were sown in two seedling beds filled with nutrient soil, with 4 seeds sown in each pot and watered appropriately.
[0071] 2) After about 14 days of normal seedling growth, when the third leaf is fully unfolded, select healthy seedlings of uniform size and cut off the third leaf at 3 / 4 of the way down the leaf (see diagram for location). Figure 9 Cut a 3cm section from about 1 / 4 of the way down from the leaf tip. Apply nail polish to the lower epidermis of the leaf and let it dry for about 1 minute. Use transparent tape to attach the lower epidermis of the leaf and place it flat on a glass slide.
[0072] 3) Observe the density of stomata under a 10x optical microscope. Take pictures starting from the fourth row of stomata from the leaf edge, and take one picture above and below each leaf vein. Take 6 leaves for each strain and repeat 3 times.
[0073] 4) Statistical analysis of stomatal density: Stomatal density = number of stomata / field of view area.
[0074] The statistical results of pore density are shown in Figures 10-11 .from Figures 10-11 It can be seen that there is no significant difference in stomatal density between the overexpressed materials ZmMPK8-OE1 and ZmMPK8-OE2, the mutant materials zmmpk8 c1 and zmmpk8 c2, and the wild-type material B73-329, indicating that the ZmMPK8 gene does not respond to drought stress by regulating stomatal density.
[0075] The sequence information involved in this invention is as follows:
[0076] SEQ ID NO.1(GRMZM2G048455_Genomic DNA)
[0077]
[0078] SEQ ID NO.2(GRMZM2G048455_P01):
[0079] MAMMVDPPNGMASQGKHYYTMWQTLFEIDTKYVPIKPIGRGAYGIVCSSVNRETNEKVAIKKINNVFDNRVDALRTLRELKLLRHLRHENVIALKDIMMPAHRRSFKDVYLVYELMDTDL HQIIKSSQPLSNDHCQYFLFQLLRGLKYLHSAGILHRDLKPGNLLVNANCDLKICDFGLARTNNTKGQFMTEYVVTRWYRAPELLLCCDNYGTSIDVWSVGCIFAELLGRKPIFPGTECL NQLKLIVNVLGTMGEADLAFIDNPKARKYIKSLPYAPGAPFTGMYPQAHPLAIDLLQKMLVFDPSKRISVTEALEHPYMSPLYDPSANPPAQVPIDLDIDENLGVDMIREMMWQEMIHYHPEVLTRISM.
[0080] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.
Claims
1. A knockout ZmMPK8 The application of gene-based biomaterials in improving drought resistance in maize is characterized by, The ZmMPK8 The nucleotide sequence of the gene is shown in SEQ ID NO.1; The biomaterial is any one of (1)-(2): (1) CRISPR-Cas9 gene knockout vector; (2) Non-plant host cells containing the CRISPR-Cas9 gene knockout vector.
2. The application according to claim 1, characterized in that, The CRISPR-Cas9 gene knockout vector is constructed by ligating a DNA fragment encoding sgRNA into a vector carrying CRISPR-Cas9. The nucleotide sequence of the sgRNA is shown in SEQ ID NO.
5.
3. The application according to claim 1, characterized in that, The non-plant host cell is recombinant Agrobacterium.
4. A biomaterial for improving the drought resistance of maize, characterized in that, The biomaterial is any one of (1)-(2): (1) Used for knockout ZmMPK8 CRISPR-Cas9 gene knockout vector; (2) Non-plant host cells containing the CRISPR-Cas9 gene knockout vector; The ZmMPK8 The nucleotide sequence of the gene is shown in SEQ ID NO.
1.
5. The biomaterial according to claim 4, characterized in that, The CRISPR-Cas9 gene knockout vector is constructed by ligating a DNA fragment encoding sgRNA into a vector carrying CRISPR-Cas9. The nucleotide sequence of the sgRNA is shown in SEQ ID NO.
5.
6. The biomaterial according to claim 4, characterized in that, The non-plant host cell is recombinant Agrobacterium.
7. A method for improving the drought resistance of maize, characterized in that, Including corn ZmMPK8 Gene knockout, to construct ZmMPK8 Steps for producing gene-knockout transgenic maize plants; The ZmMPK8 The nucleotide sequence of the gene is shown in SEQ ID NO.
1.
8. The method according to claim 7, characterized in that, Using the CRISPR-Cas9 gene knockout method to knock out maize ZmMPK8 Gene knockout.
9. The method according to claim 7, characterized in that, The nucleotide sequence of the sgRNA used in the CRISPR-Cas9 gene knockout method is shown in SEQ ID NO.5.