Application of ZmMIK2 protein in regulation and control of drought resistance and salt resistance of plants

Abstract

The invention relates to application of a ZmMIK2 protein in regulation and control of drought resistance and salt resistance of plants. The amino acid sequence of the ZmMIK2 protein disclosed by the invention is as shown in SEQ ID No. 1, and the nucleotide sequence of the ZmMIK2 gene for coding the ZmMIK2 protein is as shown in SEQ ID No. 2. By knocking out the ZmMIK2 gene, the drought resistance and the salt resistance of a mutant plant are improved. Gene resources are provided for cultivation and improvement of new varieties of stress-resistant plants, and a theoretical basis is provided for clarification of molecular mechanisms of the ZmMIK2 protein in drought resistance and salt resistance of plants.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering breeding technology, specifically the application of ZmMIK2 protein in regulating plant drought resistance and salt tolerance. Background Technology

[0002] With global climate change and a significant increase in greenhouse gas emissions, coupled with improper fertilization and irrigation practices in agricultural production, extreme weather events are becoming more frequent, leading to a deterioration of the agricultural ecological environment. Environmental stresses such as soil salinization, drought, and extreme temperature changes have become the most significant factors limiting crop yield and quality. Arid and semi-arid agricultural areas account for approximately 52.5% of my country's land area, of which nearly 800 million mu (approximately 53 million hectares) of arable land lack irrigation, representing about 51% of the country's total arable land, and are constantly threatened by drought. Simultaneously, nearly a quarter of my country's arable land suffers from varying degrees of salinization, with 1.2-1.5 billion mu (approximately 100-120 million hectares) of saline-alkali land, of which 150 million mu (approximately 10 million hectares) have exploitable potential. Identifying superior genes and loci in crops that enhance their tolerance to abiotic stresses and elucidating the molecular mechanisms underlying these traits is a crucial foundation for the genetic improvement of crop stress resistance.

[0003] Maize (Zea mays L.) is one of the three major staple foods. Due to its tall stature, high water requirement, and sensitivity to environmental stress, research on the genetic improvement of maize's stress resistance is particularly important. Traditional breeding techniques suffer from problems such as long cycles, high degree of uncertainty, and large workload. Improving crop stress resistance by introducing stress-related genes into plants or knocking out related genes through gene engineering techniques has become increasingly mature, providing possible genetic resources and theoretical basis for the genetic improvement of maize's stress resistance and stable yield. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide an application of the ZmMIK2 protein in regulating plant drought resistance and salt tolerance. This invention improves the drought resistance and salt tolerance of maize plants by knocking out the ZmMIK2 gene.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] Applications of the ZmMIK2 gene and biomaterials containing the ZmMIK2 gene in the following areas:

[0007] Regulate plant drought resistance and salt tolerance;

[0008] Genetic breeding of drought-resistant and salt-tolerant plants;

[0009] Improvement of plant germplasm resources for drought-resistant and salt-tolerant plants;

[0010] The coding sequence of the ZmMIK2 gene is as follows:

[0011] (1) As shown in SEQ ID No. 2; the gene sequence encodes the protein shown in SEQ ID No. 1;

[0012] (2) A nucleotide sequence of the nucleotide sequence shown in SEQ ID No. 2 that has been substituted, deleted and / or added with one or more nucleotides and expresses a protein with the same function as the protein shown in SEQ ID No. 1;

[0013] The biomaterials containing the ZmMIK2 gene include:

[0014] B1) The nucleic acid molecules described in items (1) and (2) above;

[0015] B2) An expression cassette containing the nucleic acid molecule described in B1);

[0016] B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0017] B4) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3);

[0018] B5) A transgenic plant cell line containing the nucleic acid molecule described in B1), or a transgenic plant cell line containing the expression cassette described in B2), or a transgenic plant cell line containing the recombinant vector described in B3);

[0019] B6) Transgenic plant tissue containing the nucleic acid molecules described in B1), or transgenic plant tissue containing the expression cassette described in B2), or transgenic plant tissue containing the recombinant vector described in B3);

[0020] B7) A transgenic plant organ containing the nucleic acid molecule described in B1), or a transgenic plant organ containing the expression cassette described in B2), or a transgenic plant organ containing the recombinant vector described in B3);

[0021] B8) Nucleic acid molecules that inhibit or reduce the expression of the coding gene of the ZmMIK2 gene or nucleic acid molecules that inhibit or reduce the activity of the protein encoded by the coding gene of the ZmMIK2 gene.

[0022] B9) Expression cassettes, recombinant vectors, recombinant microorganisms, or transgenic plant cell lines containing the nucleic acid molecules described in B8).

[0023] Specifically, the above applications are: knocking out the ZmMIK2 gene in plants to improve plant drought resistance and salt tolerance; breeding drought-resistant and salt-tolerant plants; and improving germplasm resources of drought-resistant and salt-tolerant plants.

[0024] Applications of ZmMIK2 protein in the following areas:

[0025] Regulate plant drought resistance and salt tolerance;

[0026] Genetic breeding of drought-resistant and salt-tolerant plants;

[0027] Improvement of plant germplasm resources for drought-resistant and salt-tolerant plants;

[0028] The application is characterized as follows:

[0029] To reduce or inhibit the level of the ZmMIK2 protein in plants to improve plant drought resistance and salt tolerance; to breed drought-resistant and salt-tolerant plants; and to improve germplasm resources of drought-resistant and salt-tolerant plants.

[0030] The ZmMIK2 protein includes:

[0031] (1) The amino acid sequence shown in SEQ ID No. 1;

[0032] (2) Proteins with the same biological function obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID No.1.

[0033] Primers used to amplify the ZmMIK2 gene described above are characterized by:

[0034] The sequences of the primers are shown in SEQ ID No. 3 and SEQ ID No. 4.

[0035] A gRNA target, characterized in that the sequence of the gRNA target is shown in SEQ ID No. 13; the gRNA target is used for:

[0036] Inhibit or reduce the activity of the aforementioned ZmMIK2 protein;

[0037] Inhibit or reduce the expression of the ZmMIK2 gene mentioned above.

[0038] A ZmMIK2 gene mutant, characterized in that: the sequence of the ZmMIK2 gene mutant is shown in SEQ ID No. 18, and the ZmMIK2 gene mutant encodes the ZmMIK2-1 protein shown in SEQ ID No. 19.

[0039] A ZmMIK2 gene mutant, characterized in that: the sequence of the ZmMIK2 gene mutant is shown in SEQ ID No. 20, and the ZmMIK2 gene mutant encodes the ZmMIK2-2 protein shown in SEQ ID No. 21.

[0040] In this invention, when the recipient is a plant, the recombinant expression vector contains the Zmubi1 promoter, which initiates transcription of the encoding gene. More specifically, the recombinant vector is a recombinant plasmid obtained by inserting the encoding gene of the ZmMIK2 protein into the multiple cloning site (such as XcmI, located downstream of the Zmubi1 promoter) of the pCUNm vector.

[0041] In the above method, the recombinant expression vector is introduced into the recipient plant, specifically by transforming plant cells or tissues using conventional biological methods such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, electroporation, or Agrobacterium-mediated transformation, and then culturing the transformed plant tissues into plants. The transformed cells, tissues, or plants are understood to include not only the final product of the transformation process but also its transgenic progeny.

[0042] In the aforementioned biological materials, B4) the recombinant microorganisms may specifically be yeast, bacteria, algae, and fungi; B6) the plant tissues may be derived from roots, stems, leaves, flowers, fruits, seeds, pollen, embryos, and anthers; B7) the transgenic plant organs may be the roots, stems, leaves, flowers, fruits, and seeds of transgenic plants; the transgenic plant cell lines, transgenic plant tissues, and transgenic plant organs may or may not include propagation material.

[0043] In the above applications, the substance regulating gene expression can be a substance that performs at least one of the following six types of regulation: 1) regulation at the gene transcription level; 2) post-transcriptional regulation of the gene (i.e., regulation of splicing or processing of the primary transcript of the gene); 3) regulation of RNA transport of the gene (i.e., regulation of mRNA transport of the gene from the nucleus to the cytoplasm); 4) regulation of gene translation; 5) regulation of mRNA degradation of the gene; and 6) post-translational regulation of the gene (i.e., regulation of the activity of the protein translated from the gene).

[0044] In the above applications, the substance regulating gene expression can be a reagent that inhibits or reduces the expression of the gene. The reagent that inhibits or reduces the expression of the gene can be a gene knockout reagent, such as a reagent that knocks out the gene through homologous recombination or a reagent that knocks out the gene through CRISPR-Cas9. The reagent that inhibits or reduces the expression of the gene can contain a polynucleotide that targets the gene, such as siRNA, shRNA, sgRNA, miRNA, or antisense RNA.

[0045] The primer pair for amplifying the ZmMIK2 gene has the nucleotide sequences shown in SEQ ID No. 3 and SEQ ID No. 4.

[0046] In the above applications, the plant can be any of the following:

[0047] P1) Monocotyledons,

[0048] P2) Plants of the order Poales,

[0049] P3) Gramineae plants,

[0050] P4) Plants of the genus *Zea*.

[0051] P5) Corn.

[0052] The application of the ZmMIK2 protein described in this invention in regulating plant drought resistance and salt tolerance has the following beneficial effects:

[0053] 1. This invention experimentally verifies that the ZmMIK2 protein has a regulatory function on plant drought resistance and salt tolerance, and that the ZmMIK2 protein and its related biomaterials can be applied to the regulation of plant drought resistance and salt tolerance.

[0054] 2. This invention provides a method for regulating plant drought resistance and salt tolerance. Compared with traditional breeding methods, it has a shorter breeding time, stronger purpose, significantly shortens the cycle of drought resistance breeding, and improves the efficiency of crop stress resistance breeding.

[0055] 3. This invention uses transgenic mutation technology to obtain gene-edited homozygous lines with improved drought resistance and salt tolerance, and transgenic homozygous lines with reduced drought resistance and salt tolerance, providing gene resources for breeding and improving new varieties of stress-resistant plants.

[0056] 4. This invention provides a theoretical basis for elucidating the molecular mechanism of ZmMIK2 protein in plant drought resistance and salt tolerance. Attached Figure Description

[0057] The present invention includes the following figures:

[0058] Figure 1 The results show the identification of ZmMIK2 overexpression and mutant materials. A represents the qRT-PCR results of the ZmMIK2 overexpression material; B represents the identification results of the mutant material.

[0059] Figure 2 The ZmMIK2 gene negatively regulates drought resistance in maize. A shows photographs of WT, ZmMIK2 OE#1, and ZmMIK2 OE#2 before and after drought and rehydration, with a scale bar of 10cm; B shows the drought survival rate statistics for WT, ZmMIK2 OE#1, and ZmMIK2 OE#2; C shows photographs of WT, Zmmik2#1, and Zmmik2#1 before and after drought and rehydration, with a scale bar of 10cm; D shows the drought survival rate statistics for WT, Zmmik2#1, and Zmmik2#1.

[0060] Figure 3 The ZmMIK2 gene negatively regulates salt tolerance in maize. A shows photographs of the WT, ZmMIK2 OE#1, and ZmMIK2 OE#2 salt stress treatments (80 mM NaCl) and the control treatment (0 mM NaCl), with a scale bar of 10 cm; B shows the percentage reduction in biomass after WT, ZmMIK2 OE#1, and ZmMIK2 OE#2 salt stress treatments; C shows the NaCl levels in the underground parts under the WT, ZmMIK2 OE#1, and ZmMIK2 OE#2 control treatments and salt stress treatments. + Ion content; D represents the Na+ content in the aboveground parts under the control treatments (WT, ZmMIK2 OE#1, and ZmMIK2 OE#2) and salt treatment conditions. + Ion content; E represents photographs of WT, Zmmik2#1, and Zmmik2#1 under salt stress treatment and the control group, with a scale bar of 10 cm; F represents the percentage reduction in biomass after salt treatment of WT, Zmmik2#1, and Zmmik2#1; G represents the Na+ content in the underground parts of WT, Zmmik2#1, and Zmmik2#1 under control and salt stress conditions. + Ion content; H is the Na content in the aboveground parts under the conditions of WT, Zmmik2#1 and Zmmik2#1 control treatments and salt treatment. + Ion content. Detailed Implementation

[0061] The preferred embodiments of the present invention will be described below with reference to the accompanying drawings. It should be understood that the following embodiments are given for illustrative purposes only and are not intended to limit the scope of the present invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0062] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0063] The maize transgenic overexpression vector pBCXUN was modified from the pCXUN (NCBI GenBank: FJ905215) vector. By replacing the selection marker gene Hyg with the Bar gene at the XhoI site, this vector was kindly provided by Professor Chen Qijun of China Agricultural University.

[0064] The maize transgenic recipient material LH244 (PI 612589) was obtained from GRIN-Global (https: / / npgsweb.ars-grin.gov / gringlobal / search).

[0065] The maize gene editing vector pBUE411 was kindly provided by Professor Qijun Chen of China Agricultural University and was disclosed in the literature "Hui-LiXing,Li Dong,Zhi-Ping Wang,Hai-Yan Zhang,Chun-Yan Han,Bing Liu,Xue-Chen Wangand Qi-Jun Chen (2014).A CRISPR / Cas9 toolkit for multiplex genome editing inplants.BMC Plant Biology 2014,14:327." (in the literature, it is named pBUE411(Bar)).

[0066] In this invention, a two-tailed t-test is used to determine statistical significance. * indicates a significant difference (P < 0.05), and ** indicates an extremely significant difference (P < 0.01).

[0067] Example 1: Obtaining the ZmMIK2 protein and its encoding gene

[0068] 1. Cloning of ZmMIK2 protein and its encoding gene

[0069] Seeds of the sensitive maize inbred line B73 were germinated at 24℃ for 5 days. Germinated seeds were then transferred to a nutrient solution and cultured for one week. Whole seedlings were then flash-frozen in liquid nitrogen, ground, and total RNA was extracted. Reverse transcription was performed to obtain cDNA. Using this cDNA as a template, PCR amplification was performed with primers F1 and R1. The amplified product was subjected to 1% agarose gel electrophoresis, yielding a 3672 bp PCR amplification product (SEQ ID No. 2).

[0070] Sequencing revealed that the PCR product derived from the sensitive maize inbred line B73 has the nucleotide sequence shown in positions 136-1137 of SEQ ID No. 2 (this reference sequence can be obtained by searching Zm00001eb046220 on https: / / www.maizegdb.org / / ).

[0071] The primer sequences mentioned above are as follows:

[0072] F1: 5'-ATGCGGAAGCCACCACTCG-3'; (SEQ ID No. 3)

[0073] R1: 5'-TCACTTCTGGTAGTCCGTTAGCTTG-3' (SEQ ID No. 4)

[0074] 2. Construction of the recombinant vector pBCXUN-ZmMIK2

[0075] The coding sequence of the ZmMIK2 gene (SEQ ID No. 2) was modified by adding a homologous arm to the XcmI restriction enzyme site (downstream of the ubi promoter). The vector was then digested with XcmI, and the digestion product was ligated with a similarly digested pBCXUN vector fragment using T4 ligase to obtain a recombinant vector containing the ZmMIK2 coding sequence, named pBCXUN-ZmMIK2. pBCXUN-ZmMIK2 is obtained by inserting a DNA molecule with the nucleotide sequence shown in SEQ ID No. 2 between the restriction endonuclease XcmI sites of the pBCXUN vector, while keeping the other nucleotide sequences of the pBCXUN vector unchanged. The promoter for initiating the ZmMIK2 gene in the recombinant vector pBCXUN-ZmMIK2 is Zmubiquitin1.

[0076] The primers for amplification with homologous arms are F2 and R2, with the following sequences:

[0077] F2: 5'-ccaagcttATGCGGAAGCCACCACTCG-3' (SEQ ID No. 5);

[0078] R2: 5'-ggactagtCTTCTGGTAGTCCGTTAGCTTGC-3' (SEQ ID No. 6).

[0079] 3. Obtaining recombinant Agrobacterium

[0080] The recombinant vector pBCXUN-ZmMIK2 was transformed into Agrobacterium EHA105 to obtain recombinant Agrobacterium EHA105 / pBCXUN-ZmMIK2 containing the recombinant vector pBCXUN-ZmMIK2 (after colony PCR, plasmid was extracted by shaking and sequencing to verify that the recombinant Agrobacterium was a positive clone).

[0081] 4. Obtaining transgenic homozygous ZmMIK2 maize

[0082] Recombinant Agrobacterium EHA105 / pBCXUN-ZmMIK2 was used to infect the immature embryos of wild-type maize LH244, yielding T1 generation seeds. Whole-genome DNA was extracted from the T1 generation transgenic plants, and PCR was performed to identify transgenic positivity using primers F3 and R3. T2 generation seeds were harvested from the positive plants, and genomic DNA was extracted from at least 24 seeds after germination. PCR was then performed using primers F2 and R2. If at least 24 seeds from a package showed positive PCR results, the package was considered likely to be homozygous for the transgenic ZmMIK2. RNA was extracted from the seed plants and reverse transcribed to obtain cDNA. The maize gene ZmUbi2 (Zm00001d053838) was used as an internal control, with primers QF1 and QR1. The expression level of the ZmMIK2 gene was detected using specific primers QF2 and QR2, with wild-type LH244 as a control.

[0083] The sequences of the primers mentioned above are as follows:

[0084] F3: 5'-CGTGACATCACCGTCAACAAC-3' (SEQ ID No. 7)

[0085] R3: 5'-AGACCGGCAACAGGATTCAATC-3' (SEQ ID No. 8)

[0086] QF1: 5'-TGGTTGTGGCTTCGTTGGTT-3' (SEQ ID No. 9)

[0087] QR1: 5'-GCTGCAGAAGAGTTTTGGGTACA-3' (SEQ ID No.10)

[0088] QF2: 5'-CGTGACATCACCGTCAACAAC-3' (SEQ ID No. 11)

[0089] QR2: 5'-AGCAACAGGTCTTCTTCTCCG-3' (SEQ ID No.12)

[0090] T2 generation homozygous seeds or seeds produced by self-pollination of homozygous T2 generation plants (T3 generation) are used for experiments such as drought phenotype. T1 represents the seeds and plants produced by the current generation of the transformation recipient plant; T2 generation represents the seeds and plants produced by self-pollination of T1 generation; T3 generation represents the seeds and plants produced by self-pollination of T2 generation, and so on.

[0091] Total RNA was extracted from T3 generation ZmMIK2 transgenic maize lines and wild-type LH244 (control) and reverse transcribed to obtain cDNA. Using the maize gene ZmUbi2 (Zm00001d053838) as an internal control, real-time quantitative PCR (RT-qPCR) analysis was performed using specific primers QF2 and QR2. -△△CT Method (Livak KJ,Schmittgen TD.2001.Analysis of relativegene expression data using real-time quantitative PCR and the 2 -△△CT The expression level of the ZmMIK2 gene was analyzed using the method (Methods. 25:402-408). The results are as follows: Figure 1 As shown in Figure A, the relative expression levels of the T3 generation ZmMIK2 transgenic maize lines named OE#1 and OE#2 are more than 10 times higher than those of wild-type maize, indicating that the T3 generation ZmMIK2 transgenic maize lines OE#1 and OE#2 are positive transgenic maizes.

[0092] Example 2: Functional study of ZmMIK2 protein and its encoding gene

[0093] 1. Obtaining the ZmMIK2 gene mutant in maize

[0094] A gRNA target (5'-GACTCAGGGACATGCACCTTGG-3') (SEQ ID No. 13) was designed on the third exon of ZmMIK2 using CRISPR-P (http: / / crispr.hzau.edu.cn / CRISPR2 / ). The target sequence was constructed into the pBUE411 vector. After identifying positive clones by colony PCR, plasmids were extracted and sequenced. The correctly sequenced plasmids were transformed into Agrobacterium EHA105. Agrobacterium colony PCR-positive bacteria infected maize LH244 embryos to obtain T0 generation plants. T0 generation plants were self-crossed to obtain T1 generation seeds. T1 generation seeds were germinated, planted, and maize cotyledons were harvested. Genomic DNA was extracted, and genotyping was performed using F4 and R4 primer pairs, respectively. Self-crossing yielded T2 generation seeds of two edited types of maize ZmMIK2 gene mutants. The materials of these two mutant genotypes were CRISPR Cas9 free using F5 and R5 cells, with wild-type LH244 as a control. PCR-negative plants were CRISPR Cas9 free. Seeds of the T2 generation homozygous edited lines without Cas9, obtained from self-crossing of the two edited maize ZmMIK2 gene mutants, were named Zmmik2#1 and Zmmik2#2. Figure 1 As shown in B, it is used for subsequent experiments.

[0095] The sequences of the primers mentioned above are as follows:

[0096] F4: 5'-GCTGAGTGAACTGCCAAAG-3' (SEQ ID No. 14)

[0097] R4: 5'-TGAGATTGTTGCTGAAGAGG-3' (SEQ ID No. 15)

[0098] F5: 5'-GACAGGCGTCTTCTACTGGTGCTAC-3' (SEQ ID No. 16)

[0099] R5: 5'-TATTCACTAGCTCGGGATAGTTGGC-3' (SEQ ID No. 17)

[0100] like Figure 1 The mutant Zmmik2#1 shown in B, compared with wild-type maize LH244, has the following mutation in the ZmMIK2 gene in the maize genome on both homologous chromosomes: a deletion of 5'-TGCACCTTGGCGGCAACAACCT-3' (bases 824-845 of SEQ ID No. 2), resulting in a premature stop codon in the coding sequence of the ZmMIK2 gene, thus knocking out the ZmMIK2 gene (wild-type). This mutated gene is named the ZmMIK2-1 gene (SEQ ID No. 18); the coding sequence (CDS) of the ZmMIK2-1 gene is obtained by deleting positions 824-845 of the nucleotide molecule shown in SEQ ID No. 2, while keeping the other nucleotide sequences of SEQ ID No. 2 unchanged; it encodes a protein ZmMIK2-1 composed of 288 amino acid residues, the amino acid sequence of which is shown in SEQ ID No. 19 of the sequence listing. Compared to wild-type maize LH244, the mutant Zmmik2#2 has an insertion of one base (A) at position 828 of the coding sequence of the ZmMIK2 gene on both homologous chromosomes, resulting in a premature stop codon in the ZmMIK2 coding sequence, thus knocking out the ZmMIK2 gene. This mutated gene is named ZmMIK2-2 (SEQ ID No. 20). The coding sequence (CDS) of the ZmMIK2-2 gene is obtained by inserting one base (A) at position 828 of the nucleotide molecule shown in SEQ ID No. 2, while keeping the other nucleotide sequences of SEQ ID No. 2 unchanged. It encodes a protein ZmMIK2-2 composed of 299 amino acid residues, the amino acid sequence of which is shown in SEQ ID No. 21 of the sequence listing.

[0101] 2. Phenotypic analysis of ZmMIK2's role in drought resistance in maize

[0102] T3 generation ZmMIK2 maize lines (OE#1 and OE#2) and wild-type maize LH244 (WT) plants, sown 5 days prior, were transferred to white boxes containing 2 kg of nutrient soil: vermiculite: imported soil in a 1:1:1 ratio. After 7 days of growth under normal conditions, a drought treatment (i.e., cessation of watering) was applied for 25 days, followed by rewatering. Five days after rewatering, the survival rate of each line was recorded (plants with normal leaf color and normal growth were defined as surviving plants, while those with scorched leaves and abnormal growth were defined as dead plants; the survival rate was the percentage of surviving plants in each line out of the total number of plants). The experiment was repeated three times, with at least 12 plants from each line in each replicate. The average value was used for statistical analysis.

[0103] The results are as follows Figure 2 China A and Figure 2 As shown in B, Figure 2 Photo A shows WT, ZmMIK2 OE#1, and ZmMIK2 OE#2 before drought and after rehydration, with a scale bar of 10cm. Figure 2 Figure B shows the drought survival rates of WT, ZmMIK2 OE#1, and ZmMIK2 OE#2. The figure indicates that after drought treatment, the leaf shriveling of T3 generation ZmMIK2 transgenic maize (OE#1 and OE#2) was more severe than that of wild-type maize LH244(WT), and their survival rates were significantly lower.

[0104] The survival rates of WT, Zmmik2#1, and Zmmik2#1 were statistically analyzed using the same method as described above. The experiment was conducted in triplicate, with at least 12 plants from each line in each replicate. The average value was used for statistical analysis. The results are as follows: Figure 2 C and Figure 2 As shown in D; Figure 2 C represents photos of WT, Zmmik2#1, and Zmmik2#1 before drought and after re-watering, with a scale bar of 10cm. Figure 2 Figure D shows the drought survival rate statistics for WT, Zmmik2#1, and Zmmik2#1. The results indicate that after drought treatment, the leaf dries of the ZmMIK2 mutants Zmmik2#1 and Zmmik2#1 were less severe than those of wild-type maize WT, and their survival rate was significantly higher than that of wild-type maize WT.

[0105] 3. Phenotypic analysis of ZmMIK2 participation in maize salt tolerance

[0106] Experimental materials: T3 generation ZmMIK2 transgenic maize lines (OE#1 and OE#2) and wild-type maize LH244 (WT) plants

[0107] The experimental procedure for identifying salt stress phenotypes is as follows:

[0108] 1) Filling with soil: Sift the nutrient soil and mix it well. Take an equal amount of soil and fill it into a 12cm x 12cm black round plastic pot, and compact it.

[0109] b. Pouring salt solution: Add 80mM NaCl solution to the bottom of the pot and let the soil in the pot fully absorb it until the surface soil absorbs the salt water.

[0110] c. Sowing: Select plump seeds and sow 10 seeds in each pot. Cover the seeds with an equal amount of sand (a mixture of nutrient soil and sand in equal proportions). After the surface soil absorbs the salt water, discard the excess salt water at the bottom.

[0111] d. Salt phenotype identification: After growing in the culture room for about 12 days, the salt phenotype of the maize material was determined based on the seedling size and leaf chlorosis, and the Na content in the aboveground (shoot) and underground (root) parts was tested. + Ion content was confirmed; incubation conditions: temperature 24±1℃, humidity approximately 40%, 14h light, 10h darkness. The experiment was repeated in triplicate, and the average value was used for statistical analysis.

[0112] The results are as follows Figure 3 As shown in AD, Figure 3 Image A shows photographs of the salt stress treatments (80 mM NaCl) and control treatment (0 mM NaCl) of WT, ZmMIK2 OE#1 and ZmMIK2OE#2, with a scale bar of 10 cm. Figure 3 In the figure, B represents the percentage reduction in biomass after salt stress treatments of WT, ZmMIK2 OE#1, and ZmMIK2 OE#2. Figure 3 In the context of the control treatments (WT, ZmMIK2 OE#1, and ZmMIK2 OE#2) and salt stress treatment, the Na content in the underground parts was... + Ion content. Figure 3 Under the conditions of WT, ZmMIK2 OE#1 and ZmMIK2 OE#2 control treatments and salt treatment, the Na content in the aboveground parts was... + Ion content. Among them... Figure 3 The significance analysis for C and D in section 3 was performed using one-way ANOVA combined with Turkey's post-hoc test. Different letters indicate significance at the P < 0.05 level. The figure shows that after salt stress treatment, T3 generation ZmMIK2 transgenic maize plants (OE#1 and OE#2) were smaller and had greater biomass reduction than wild-type maize LH244 (WT), with higher Na+ levels in the aboveground parts. + The ion content was significantly higher than that of wild-type maize LH244(WT).

[0113] The salt stress phenotype of WT, Zmmik2#1, and Zmmik2#1 was identified using the same method described above, and the results are as follows: Figure 3 As shown in EH; Figure 3 Photographs of WT, Zmmik2#1 and Zmmik2#1 salt stress treatment and control group are shown in the image, with a scale bar of 10cm. Figure 3 F represents the percentage reduction in biomass after salt treatment with WT, Zmmik2#1, and Zmmik2#1. Figure 3 Under the conditions of WT, Zmmik2#1 and Zmmik2#1 control treatments and salt stress treatment, the Na content in the underground part was... + Ion content. Figure 3 Under the conditions of WT, Zmmik2#1 and Zmmik2#1 control treatments and salt treatment, the Na+ content in the aboveground parts was... + Ion content. Among them... Figure 3 The significance analysis of G and 3H was performed using one-way ANOVA combined with Turkey's post-hoc test, with different letters indicating significance at the P < 0.05 level. The results showed that after salt stress treatment, the ZmMIK2 mutants Zmmik2#1 and Zmmik2#1 plants were larger and had less biomass reduction than wild-type maize LH244(WT), and the Na+ content in the aboveground parts was lower. + The ion content was significantly lower than that of wild-type maize LH244(WT).

[0114] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

[0115] The contents not described in detail in this specification are existing technologies known to those skilled in the art.

Claims

1. Applications of the ZmMIK2 gene and biomaterials containing the ZmMIK2 gene in the following areas: Regulate plant drought resistance and salt tolerance; Genetic breeding of drought-resistant and salt-tolerant plants; Improvement of plant germplasm resources for drought-resistant and salt-tolerant plants; The coding sequence of the ZmMIK2 gene is as follows: (1) As shown in SEQ ID No. 2; the gene sequence encodes the protein shown in SEQ ID No. 1; (2) A nucleotide sequence of the nucleotide sequence shown in SEQ ID No. 2 that has been substituted, deleted and / or added with one or more nucleotides and expresses a protein with the same function as the protein shown in SEQ ID No. 1; The biomaterials containing the ZmMIK2 gene include: B1) The nucleic acid molecules described in items (1) and (2) above; B2) An expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3); B5) A transgenic plant cell line containing the nucleic acid molecule described in B1), or a transgenic plant cell line containing the expression cassette described in B2), or a transgenic plant cell line containing the recombinant vector described in B3); B6) Transgenic plant tissue containing the nucleic acid molecules described in B1), or transgenic plant tissue containing the expression cassette described in B2), or transgenic plant tissue containing the recombinant vector described in B3); B7) A transgenic plant organ containing the nucleic acid molecule described in B1), or a transgenic plant organ containing the expression cassette described in B2), or a transgenic plant organ containing the recombinant vector described in B3); B8) Nucleic acid molecules that inhibit or reduce the expression of the coding gene of the ZmMIK2 gene or nucleic acid molecules that inhibit or reduce the activity of the protein encoded by the coding gene of the ZmMIK2 gene. B9) Expression cassettes, recombinant vectors, recombinant microorganisms, or transgenic plant cell lines containing the nucleic acid molecules described in B8).

2. The application according to claim 1, characterized in that, The application is as follows: knocking out the ZmMIK2 gene in plants to improve plant drought resistance and salt tolerance; breeding drought-resistant and salt-tolerant plants; and improving germplasm resources of drought-resistant and salt-tolerant plants.

3. Applications of ZmMIK2 protein in the following areas: Regulate plant drought resistance and salt tolerance; Genetic breeding of drought-resistant and salt-tolerant plants; Improvement of plant germplasm resources for drought-resistant and salt-tolerant plants; Its features are, The application is as follows: To reduce or inhibit the level of the ZmMIK2 protein in plants to improve drought resistance and salt tolerance; and to breed drought-resistant and salt-tolerant plants. Improve drought-resistant and salt-tolerant plant germplasm resources; The ZmMIK2 protein includes: (1) The amino acid sequence shown in SEQ ID No. 1; (2) Proteins with the same biological function obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID No.

1.

4. Primers for amplifying the ZmMIK2 gene as described in claim 1, characterized in that: The sequences of the primers are shown in SEQ ID No. 3 and SEQ ID No.

4.

5. A gRNA target, characterized in that, The sequence of the gRNA target is shown in SEQ ID No. 13; this gRNA target is used for: Inhibit or reduce the activity of the ZmMIK2 protein as described in claim 1; Inhibit or reduce the expression of the ZmMIK2 gene as described in claim 1.

6. A ZmMIK2 gene mutant, characterized in that: The sequence of the ZmMIK2 gene mutant is shown in SEQ ID No. 18, and the ZmMIK2 gene mutant encodes the ZmMIK2-1 protein shown in SEQ ID No.

19.

7. A ZmMIK2 gene mutant, characterized in that: The sequence of the ZmMIK2 gene mutant is shown in SEQ ID No. 20, and the ZmMIK2 gene mutant encodes the ZmMIK2-2 protein shown in SEQ ID No. 21.

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