Application of corn ZmTST1 gene in improvement of plant yield and stress resistance

Abstract

The invention discloses application of a corn ZmTST1 gene in improvement of plant yield and stress resistance. The nucleotide sequence of the corn ZmTST1 gene is as shown in SEQ ID NO. 1; the stress resistance is tolerance to salt stress and drought stress. Through sequence analysis and functional verification, after the ZmTST1 gene is over-expressed, the growth of plants can be promoted, the yield of the plants can be increased, meanwhile, the drought tolerance and the salt tolerance of ZmTST1 expressed plants are obviously higher than those of wild plants, and it is proved that the ZmTST1 gene has a remarkable effect in the aspects of high yield and stress resistance. By identifying the ZmTST1 gene, plants with high stress resistance can be screened, the cultivation period of the plants can be shortened, and a new method and thought are provided for plant breeding in the future.

Description

Technical Field

[0001] The invention relates to the technical field of plant bioengineering, and in particular to the application of corn ZmTST1 gene in improving plant yield and stress resistance. Background Art

[0002] Corn (Zea Mays L.) is an important raw material for feed production and industrial production. Its high and stable yield is of great significance in ensuring my country's feed supply, sustainable agricultural development and food security. Drought stress and soil salinization are the most serious environmental factors that harm crop growth, thus seriously affecting the yield and quality of corn. However, due to the narrow genetic basis of corn germplasm resources and the lack of innovation in inbred line breeding methods, the process of breeding high-yield and stress-resistant new corn varieties by traditional breeding methods is seriously hindered. The continuous development of genetic engineering technology and transgenic technology has provided new technical means for corn breeding, making up for the shortcomings of traditional corn breeding, such as long cycle, high contingency, low efficiency and difficulty in control, and making important contributions to corn genetic breeding. Therefore, the mining of effective genes for high yield, salt tolerance and drought tolerance of corn provides effective resources for breeding new corn varieties.

[0003] Tonoplast sugar transporters (TSTs) are located on the tonoplast membrane, transporting sugars in the cytoplasm to the vacuole for storage, mediating the transportation and distribution of photosynthetic products such as glucose, fructose, and sucrose from the "source" to the "sink" of the plant, playing an extremely important role in the distribution and accumulation of sugars in the source-sink organs, improving the efficiency of sugar loading at the source end and unloading at the sink end, and producing a positive feedback effect on photosynthesis, thereby increasing the yield of grain crops. Salt stress and drought stress can have many effects on plants, including water balance, seed setting, osmotic regulation, photosynthesis, absorption of mineral nutrients, metabolism, and growth and development. Plants have evolved many mechanisms to adapt to salt and drought stress environments. Ion balance is an important part of the homeostasis of the cell body, and rebuilding ion balance can improve the tolerance of plants to stress. Therefore, studying TST proteins is of great significance to improving grain yield and stress resistance. However, in corn, there are relatively few studies on the identification and function of TST.

[0004] Therefore, it is particularly important to develop a gene regulation strategy that can effectively balance the stress resistance and high yield of corn. It can not only significantly improve the adaptability of plants such as corn under salt stress and drought stress environments, but also further promote the sustainable development of agriculture and fill the gaps in existing technologies. Summary of the invention

[0005] In view of the defects in the prior art, the present invention proposes the application of the corn ZmTST1 gene in improving plant yield and stress resistance. The present invention innovatively clones the tonoplast sugar transporter ZmTST1 involved in sugar transport in corn B73, and conducts in-depth research on the corn ZmTST1 gene through gene expression analysis combined with functional verification.

[0006] The present invention provides the use of any one of (a) to (e) in improving plant yield and stress resistance:

[0007] (a) Maize ZmTST1 gene;

[0008] (b) a recombinant vector comprising the maize ZmTST1 gene;

[0009] (c) an expression cassette comprising the maize ZmTST1 gene;

[0010] (d) a cell comprising the maize ZmTST1 gene;

[0011] (e) a recombinant bacterium comprising the corn ZmTST1 gene;

[0012] The nucleotide sequence of the maize ZmTST1 gene is shown in SEQ ID NO.1; and the corresponding amino acid sequence is shown in SEQ ID NO.2.

[0013] In some embodiments, the stress resistance is tolerance to salt stress and drought stress.

[0014] The present invention analyzes the expression patterns of four ZmTSTs genes in corn and finds that salt stress and osmotic stress induce the expression of these four genes to a certain extent, among which ZmTST1 has the highest induced expression. At the same time, overexpression of the ZmTST1 gene significantly increases the grain size and yield of Arabidopsis thaliana, and enhances the resistance of transgenic Arabidopsis thaliana to salt stress and osmotic stress, indicating that the ZmTST1 gene may play an active regulatory role in corn yield, drought resistance and salt tolerance signaling pathways. This study deepens the understanding of the function of the plant ZmTST1 gene and lays an important foundation for further studying the molecular mechanism of corn stress and providing candidate target genes for improving corn yield and stress resistance.

[0015] In some embodiments, the plant is any one of corn, Arabidopsis, cucumber, tomato, wheat, oat, rice, corn, poplar, lawn grass, alfalfa, peanut, soybean, sorghum and millet; preferably corn or Arabidopsis.

[0016] A method for cultivating high-yield and stress-resistant plants, the method comprising: cloning the corn ZmTST1 gene into an expression vector, introducing the vector into plant cells, screening the plant cells carrying the expression vector, and cultivating the cells into plants to obtain high-yield and stress-resistant plants.

[0017] In some embodiments, the plant cell is a callus cell or an embryonic cell.

[0018] In some embodiments, the expression vector is a monocot expression vector.

[0019] In some embodiments, the stress resistance is tolerance to salt stress and drought stress.

[0020] Specifically, the ZmTST1 gene can be introduced into the target plant through the expression vector. In the method, the expression vector can be transformed into plant cells or tissues by conventional biological methods such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, electroporation, Agrobacterium-mediated, etc., and the transformed plant tissues can be cultivated into plants.

[0021] In some embodiments, the plant is any one of corn, Arabidopsis, cucumber, tomato, wheat, oat, rice, corn, poplar, turf grass, alfalfa, peanut, soybean, sorghum and millet.

[0022] The present invention also provides a method for improving plant yield and stress resistance, which is any one of the following:

[0023] (1) Improving the yield and stress resistance of a target plant by increasing the expression or activity of a maize ZmTST1 protein in the target plant;

[0024] (2) Improving the yield and stress resistance of the target plant by promoting the expression of the maize ZmTST1 gene in the target plant;

[0025] The nucleotide sequence of the maize ZmTST1 gene is shown in SEQ ID NO.1.

[0026] The implementation method of "promoting the expression of ZmTST1 gene in target plants" can be as follows (1)~

[0027] (3) Any of the following:

[0028] (1) Introducing the ZmTST1 gene into target plants;

[0029] (2) introduction of strong promoters and / or enhancers;

[0030] (3) Other common methods in this field.

[0031] The target plants of the present invention are preferably corn and Arabidopsis thaliana.

[0032] In some embodiments, the promoting the expression of the maize ZmTST1 gene in the target plant is specifically any one of the following:

[0033] (1) transferring the expression construct or vector of the maize ZmTST1 gene into the target plant;

[0034] (2) The expression construct or vector containing the maize ZmTST1 gene is transferred into the target plant.

[0035] The present invention also provides a method for producing food, comprising the following steps: planting crops and harvesting food from the crops; increasing the expression or activity of the corn ZmTST1 gene or the protein encoded by it in the crops;

[0036] The nucleotide sequence of the maize ZmTST1 gene is shown in SEQ ID NO.1.

[0037] In some embodiments, the use includes detecting the use of plants in breeding high-yield or stress-tolerant plants.

[0038] In the present invention, the application of ZmTST1 gene in plant high yield and stress resistance is that ZmTST1 gene is used as a molecular marker for plant high yield and stress resistance, that is, the yield level and stress resistance of the plant are explained by detecting the expression level of ZmTST1 gene in the plant; it can also be bred according to whether the ZmTST1 gene exists, such as increasing the yield and stress resistance of the plant by transferring the ZmTST1 gene or increasing the expression of the ZmTST1 gene, or obtaining new plant varieties by inhibiting, knocking out or silencing the ZmTST1 gene.

[0039] In the present invention, there is no particular limitation on the plants applicable to the present invention, as long as they are suitable for gene transformation operations, such as various crops, flower plants, or forestry plants, etc. The plants can be (but not limited to): dicotyledons, monocotyledons or gymnosperms.

[0040] As a preferred embodiment, the "plant" includes but is not limited to: corn, Arabidopsis, and any plant having the gene or a gene homologous thereto is applicable.

[0041] The "plant" mentioned in the present invention includes the whole plant, its parent and progeny plants and different parts of the plant, including seeds, fruits, buds, stems, leaves, roots (including tubers), flowers, tissues and organs, and our target gene or nucleic acid is present in these different parts. The "plant" mentioned here also includes plant cells, suspension cultures, callus, embryos, meristem regions, gametophytes, sporophytes, pollen and microspores, and similarly, each of the aforementioned objects contains the target gene / nucleic acid.

[0042] The present invention includes any plant cell, or any plant obtained or obtainable by the method therein, and all plant parts and propagules thereof. This patent also includes transfected cells, tissues, organs or whole plants obtained by any of the aforementioned methods. The only requirement is that the progeny exhibit the same genotypic or phenotypic characteristics, and the progeny obtained using the method of this patent have the same characteristics.

[0043] The invention also extends to the harvestable parts of the plants as described above, but not limited to seeds, leaves, fruits, flowers, stems, roots, rhizomes, tubers and bulbs. It also further relates to other derivatives of the plants after harvest, such as dry granules or powders, oils, fats and fatty acids, starch or proteins. The invention also relates to foods or food additives obtained from the relevant plants.

[0044] The present invention also provides a method for detecting high yield and stress resistance of plants, which detects the expression level of the ZmTST1 gene of the sample to be detected. If the expression level of the ZmTST1 gene in the sample to be detected is upregulated, it is judged to have high yield and high stress resistance.

[0045] Among them, the present invention can detect the expression level of ZmTST1 gene in the sample to be detected in a variety of ways, such as directly detecting the ZmTST1 gene itself, or detecting the product produced by the ZmTST1 gene, the product includes a direct product or an indirect product or a secondary product, etc. The product can be a gene, a protein, or a certain compound, etc.

[0046] For example, the ZmTST1 gene itself can be directly detected using a specific primer pair for the ZmTST1 gene, or using a probe or chip designed for the ZmTST1 gene.

[0047] Furthermore, the sample to be detected is detected by using a primer pair or a probe or a chip of the ZmTST1 gene.

[0048] The primer pair, probe or chip for the ZmTST1 gene provided by the present invention can be designed according to conventional methods. By detecting the gene, it is possible to effectively confirm whether the ZmTST1 gene is contained, so as to illustrate the stress resistance of the tested plant to a certain extent.

[0049] Furthermore, the nucleic acid sequence of the primer pair is shown in SEQ ID NO.5 and SEQ ID NO.6; the primer pair SEQID NO.5 and SEQ ID NO.6 are detection primers using cDNA as a template and have strong specificity, but the method for detecting the ZmTST1 gene itself is not limited to this, and any method that can be implemented in molecular biology is within the scope of protection of the present invention.

[0050] Similarly, the detection of the product produced by the ZmTST1 gene can also be carried out by various means, such as various ELISA detection kits.

[0051] Furthermore, in various embodiments, the sample to be tested includes tissue culture material suitable for sexual reproduction, asexual reproduction or regenerative cells.

[0052] These samples to be tested can be materials suitable for sexual reproduction, such as selected from pollen, ovary, ovule, embryo sac, etc.; materials suitable for asexual reproduction can be selected from cuttings, roots, stems, protoplasts, etc.; materials suitable for tissue culture of regenerable cells can be selected from leaves, pollen, embryos, cotyledons, hypocotyls, meristem cells, roots, root tips, anthers, flowers, seeds and stems, etc.

[0053] Specifically, the sample to be detected includes any one of the following materials: seeds, leaves, roots, stems, radicles and embryos.

[0054] In the method for detecting plant stress resistance, the plants to be detected can be various crops, flower plants, or forestry plants.

[0055] The plant may be, for example (but not limited to): dicotyledon, monocotyledon or gymnosperm. As a preferred embodiment, the "plant" includes but is not limited to: corn, Arabidopsis, and any plant having the gene or a homologous gene thereof is applicable.

[0056] In summary, compared with the prior art, the present invention achieves the following technical effects:

[0057] The present invention has been subjected to sequence analysis and functional verification, and it has been found that overexpression of the ZmTST1 gene can promote plant growth and increase plant yield. At the same time, the drought resistance and salt tolerance of the ZmTST1-expressing plant are significantly stronger than those of the wild type, proving that the gene ZmTST1 has a significant effect on high yield and stress resistance. Plants with high stress resistance can be selected by identifying the ZmTST1 gene, which can shorten the cultivation period of plants and provide a new method and idea for future plant breeding. The present invention provides a theoretical basis and technical means for the molecular mechanism of increasing plant yield and resisting drought and salt stress, and has a wide application prospect in production for increasing plant yield and enhancing plant stress resistance, and has great application value; using the gene for plant variety improvement can effectively increase plant yield and reduce the damage caused by drought and high salt to plants, thereby increasing yield and economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0059] Figure 1 This is the phylogenetic analysis of maize, rice and Arabidopsis TSTs in Example 2 of the present invention; A is a phylogenetic tree constructed using MEGA11.0; B is a multiple sequence alignment of maize, rice and Arabidopsis TSTs, and the 12 conserved transmembrane domains are marked with black lines.

[0060] Figure 2 A is the expression pattern of ZmTSTs in Example 3 of the present invention; A is the tissue-specific expression of ZmTST1, ZmTST2, ZmTST3 and ZmTST4 detected by qRT-PCR; B is the expression result of ZmTSTs in leaves under 200 mM NaCl; C is the expression result of ZmTSTs in leaves under 20% PEG6000.

[0061] Figure 3The growth phenotype, silique and seed trait results of WT and ZmTST1 transgenic plants in Example 4 of the present invention; A is the growth phenotype of Arabidopsis plants after 7 weeks of growth under normal conditions; B is the plant height of WT and ZmTST1 transgenic plants after seven weeks of growth in soil; C is the fresh weight of WT and ZmTST1 transgenic plants after seven weeks of growth in soil; D is the silique of WT and ZmTST1 transgenic plants after seven weeks of growth in soil; E is the seeds of WT and ZmTST1 transgenic plants; F is the seed area of ​​WT and ZmTST1 transgenic plants under normal conditions; G is the silique length of WT and ZmTST1 transgenic plants grown under normal conditions; H is the number of siliques per plant of WT and ZmTST1 transgenic plants after seven weeks of growth in soil; I is the thousand-grain weight of WT and ZmTST1 transgenic plants under normal conditions; J is the number of seeds per silique of WT and ZmTST1 transgenic plants after seven weeks of growth in soil.

[0062] Figure 4 is the germination rate of WT and ZmTST1 overexpressing plants in Example 5 of the present invention; A is the phenotype of WT, OE#4, OE#7 and OE#10 after germination for 7 days on 1 / 2MS containing 100mM NaCl or 250mM mannitol; B is the germination rate of WT, OE#4, OE#7 and OE#10 on 1 / 2MS; C is the germination rate of WT, OE#4, OE#7 and OE#10 on 1 / 2MS containing 100mM NaCl; D is the germination rate of WT, OE#4, OE#7 and OE#10 on 1 / 2MS containing 250mM mannitol; E is the cotyledon greening rate of WT, OE#4, OE#7 and OE#10.

[0063] Figure 5 The figures are the growth phenotypes of WT and ZmTST1 overexpressing plants on 1 / 2MS under normal or stress conditions in Example 5 of the present invention; A is the growth phenotypes of WT, OE#4, OE#7 and OE#10 after growing on 1 / 2MS medium containing 100 mM NaCl or 300 mM mannitol for one week; B is the root length of WT, OE#4, OE#7 and OE#10 seedlings; C is the fresh weight of WT, OE#4, OE#7 and OE#10 seedlings; D is the root length inhibition rate of WT, OE#4, OE#7 and OE#10; E is the fresh weight inhibition rate of WT, OE#4, OE#7 and OE#10.

[0064] Figure 6The figure is the growth phenotype of WT and ZmTST1 overexpressing plants grown on soil under normal or stress conditions in Example 6 of the present invention; A is the three-week-old WT, OE#4, OE#7 and OE#10 subjected to 200 mM NaCl or drought treatment for 2 weeks; B is the plant height of WT, OE#4, OE#7 and OE#10 after 200 mM NaCl or drought for two weeks; C is the growth inhibition rate of WT, OE#4, OE#7 and OE#10 after 200 mM NaCl or drought for two weeks; D is the survival rate of WT, OE#4, OE#7 and OE#10 after 200 mM NaCl or drought for two weeks; E is the leaf phenotype of one-month-old wild type, OE#4, OE#7 and OE#10 plants after natural drying for 6 hours; F is the leaf water loss rate.

[0065] Figure 7 The physiological parameters and expression levels of stress response genes of WT and ZmTST1 overexpression lines under NaCl or drought stress conditions in Example 7 of the present invention; A is the SOD activity of leaves of WT, OE#4, OE#7 and OE#10 plants treated with 200 mM NaCl or drought for one week, B is the POD activity, C is the MDA content, D is the proline content, E is the expression level of AtSOS1, F is the expression level of AtRD29A, G is the expression level of AtRAB18 and H is the expression level of AtP5CS1. DETAILED DESCRIPTION

[0066] In order to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention are clearly and completely described. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0067] Unless otherwise specified, the implementation of the present invention will use conventional botanical techniques, microorganisms, tissue culture, molecular biology, chemistry, biochemistry, DNA recombination and bioinformatics techniques that are obvious to those skilled in the art. These techniques are fully explained in the published literature. In addition, the methods of DNA extraction, construction of phylogenetic trees, gene editing methods, construction of gene editing vectors, and obtaining gene-edited plants used in the present invention can be achieved by using methods already disclosed in existing literature, except for the methods used in the following examples.

[0068] The gene sequence number (Sequence ID) of ZmTST1 gene in NCBI is NM_001158464.3. The length of the messenger RNA (mRNA) sequence of ZmTST1 gene is 2874bp, and the length of the coding sequence of ZmTST1 gene is 2244bp, including 747 amino acids. In order to explore the application of ZmTST1 gene in improving plant yield and drought and salt stress tolerance, the overexpression vector pCAMBIA3301-ZmTST1 of ZmTST1 was constructed, and the wild-type Arabidopsis thaliana (Clo-0, WT) was transformed by Agrobacterium inflorescence infection method to obtain overexpression plants. The analysis results showed that compared with the wild type, the growth rate of ZmTST1 overexpression plants was significantly accelerated throughout the growth and development stage. In addition, the seed size and 1000-grain weight of the overexpression line were significantly greater than those of the wild type. Under osmotic stress and salt stress conditions, the root length of ZmTST1 overexpression plants was longer, the biomass was increased, and its drought and salt tolerance were significantly enhanced. This indicates that the ZmTST1 gene positively regulates the tolerance of Arabidopsis to adverse stress. Therefore, it can be seen that the ZmTST1 gene plays an important role in regulating plant yield and adverse stress, and is of great significance for breeding high-yield corn varieties that can resist external stress conditions.

[0069] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials and reagents used are all commercially available unless otherwise specified.

[0070] (1) Biological materials: B73 corn seeds, Arabidopsis Col-0 seeds, overexpression vector pCAMBIA3301, Escherichia coli DH5α and Agrobacterium tumefaciens GV3101;

[0071] Primer synthesis and sequencing were completed by Qingke Biotechnology Co., Ltd.

[0072] (2) Experimental reagents:

[0073] RNA extraction kit, reverse transcription kit, and fluorescence quantification kit were purchased from Novozyme Biotechnology Co., Ltd.;

[0074] Common reagents such as Mannitol, NaCl, and PEG were purchased from Solebao Company;

[0075] Kanamycin was purchased from Solebro Biotech;

[0076] MS medium was purchased from Beijing Coolaibo Technology Co., Ltd.;

[0077] Various endonucleases were purchased from Mona Biotechnology Co., Ltd.;

[0078] One-step cloning enzyme was purchased from Novozyme Biotech Co., Ltd.;

[0079] Plasmid miniprep kit and gel recovery kit were purchased from Beijing Tiangen Biotechnology Co., Ltd.

[0080] (3) Experimental equipment:

[0081] The PCR instrument was purchased from Bio-rad;

[0082] The refrigerated centrifuge was purchased from Eppendorf;

[0083] The quantitative PCR instrument was purchased from Bio-rad;

[0084] The high-temperature and high-pressure sterilizer MLS-3750 was purchased from Sanyo Company of Japan;

[0085] The nucleic acid detector Nanodrop 2000C was purchased from Thermo Scientific;

[0086] The room temperature centrifuge was purchased from Thermo Scientific.

[0087] Example 1

[0088] 1. Planting and processing of plant materials

[0089] Seeds of corn variety B73 were sown on sterile filter paper, soaked in a petri dish with deionized water, and germinated in a growth chamber at 22°C, 16h light and 8h dark photoperiod for 5 days. Subsequently, the seedlings with stable growth were transferred to soil in plastic pots and grown in a greenhouse at 22°C, 16h light and 8h dark photoperiod. After four weeks of growth, soil-grown seedlings with strong growth and uniform size were selected, and the seedlings were treated with an aqueous solution containing 200mM NaCl for 0h, 1h, 3h, 6h, 9h, 12h and 24h, and then corn leaf samples were taken, quickly placed in liquid nitrogen, RNA was extracted, and cDNA was obtained after reverse transcription. Soil-grown seedlings with strong growth and uniform size were selected, and the seedlings were treated with an aqueous solution containing 20% ​​PEG for 0h, 1h, 3h, 6h, 9h, 12h and 24h, and then corn leaf samples were taken, quickly placed in liquid nitrogen, RNA was extracted, and cDNA was obtained after reverse transcription.

[0090] 2. RNA extraction and real-time quantitative RT-PCR (qRT-PCR)

[0091] Total RNA was isolated from each sample using TRIzol reagent (TIANGEN, Beijing, China). RNA content was detected by Nanodrop2000 nucleic acid analyzer, and quality was checked by gel electrophoresis. cDNA was synthesized using the reverse transcription kit DRR047A (TaKaRa, Dalian, China). qRT-PCR primers were designed using Primer3 software (http: / / frodo.wi.mit.edu / ), and primer specificity was verified by NCBI-primer blast (https: / / www.ncbi.nlm.nih.gov / tools / primer-blast / index.cgi), and ZmACTIN was used as an internal reference gene for qRT-PCR analysis. qRT-PCR experiments used the CFX96 real-time fluorescence quantitative PCR detection system (Bio-Rad, Hercules, CA, USA) and SYBR Premix ExTaq reagent TM Each sample was subjected to three biological replicates and three technical replicates using a PCR amplification kit (Takara). The amplification parameters were as follows: 95°C for 30 seconds, 95°C for 5 seconds, 60°C for 30 seconds, and 40 cycles.

[0092] 3. Construction of ZmTST1 overexpression vector

[0093] In order to further analyze the function of ZmTST1, this example constructed an overexpression vector pCAMBIA3301-ZmTST1 of ZmTST1 and obtained overexpression Arabidopsis plants, and the steps were as follows:

[0094] First, design primers with restriction endonuclease BamHI cleavage sites, the sequences are as follows:

[0095] 3301-ZmTST1-F: (SEQ ID NO.3)

[0096] 5'-ctctctctcaagcttggatccATGGGGGGCGCCGTGATG-3';

[0097] 3301-ZmTST1-R: (SEQ ID NO.4)

[0098] 5'-caagtcgactctagaggatccTCAACCTTCCCTGGCCTCC-3';

[0099] Second, using the cDNA sample as a template, PCR amplification is performed and the amplified product is purified and recovered;

[0100] Third, the pCAMBIA3301 vector was digested with BamHI and the digested product was purified;

[0101] Fourth, the PCR amplification product and the vector after restriction digestion were connected by homologous recombination to construct the pCAMBIA3301-ZmTST1 overexpression vector;

[0102] Fifth, the ligation product was transformed into Escherichia coli DH5α by heat shock transformation method, and Basta (8 mg / L) resistance screening was performed. The positive colonies were selected for PCR detection, and the colonies identified correctly by PCR were amplified and sent for sequencing. The plasmids were extracted from the bacterial solution with correct sequencing for future use;

[0103] Sixth, the extracted plasmid was transformed into Agrobacterium competent cells GV3101 and stored at -80°C for future use.

[0104] Seventh, wild-type Arabidopsis thaliana (Clo-0, WT) was transformed using the Agrobacterium inflorescence infection method, and the harvested seeds were screened on a 1 / 2MS medium containing Basta. Seeds of potential transgenic plants were harvested and screened again on a medium containing Basta until potential homozygous transgenic plants were obtained from the T3 generation.

[0105] Example 2 Maize ZmTSTs sequence alignment and phylogenetic tree analysis

[0106] The phylogenetic relationships of TST proteins were investigated based on the amino acid sequences of the TST subfamily in Arabidopsis, maize, and rice. Phylogenetic analysis of maize and Arabidopsis showed that all ZmTST proteins had the highest homology with AtTST2 of Arabidopsis. In maize and rice, ZmTST1, ZmTST2, ZmTST3, and ZmTST4 had the highest sequence identities with OsTST1 (88.4%), OsTST4 (90.9%), OsTST5 (72.8%), and OsTST6 (71.8%) of rice, respectively. Figure 1 As shown in A. Similar to AtTSTs and OsTsTs, all ZmTsTs have 12 conserved transmembrane domains, which is a common feature of the TST subfamily ( Figure 1 B).

[0107] Example 3 Expression pattern of maize ZmTSTs gene under adverse stress

[0108] Soil salinization is one of the key factors limiting crop yield. In order to understand the response pattern of ZmTSTs genes to osmotic stress and salt stress, this example analyzed the expression pattern of ZmTSTs genes in corn leaves under salt stress and osmotic stress by qPCR. The results showed that all ZmTST family genes were induced by salt or osmotic stress ( Figure 2). Four ZmTSTs showed up-regulated expression at at least two time points under salt treatment. ZmTST1, ZmTST2, and ZmTST3 showed up-regulated expression at six time points under osmotic stress treatment. The expression of ZmTST1 was higher than that of other ZmTSTs under both stress conditions.

[0109] Example 4 Overexpression of ZmTST1 can promote plant growth and increase seed yield

[0110] In order to further study the biological function of ZmTST1, this example introduced the recombinant pCAMBIA3301-ZmTST1 vector into wild-type Arabidopsis. Ten transgenic lines were generated, and the expression levels of ZmTST1 in these transgenic lines were detected using qRT-PCR. Three homozygous ZmTST1 lines (OE#4, OE#7, and OE#10) representing low, medium, and high transgenic expression levels were screened for further study. In order to understand the effect of ZmTST1 on the growth and development of Arabidopsis, this example compared the growth phenotypes of transgenic plants (OE#4, OE#7, and OE#10) with those of wild-type plants. The results are shown in Figure 2. Figure 3 As shown, 7 weeks after sowing, significant growth phenotype differences were observed between transgenic plants and wild-type plants ( Figure 3 In addition, the plant height and fresh weight of all transgenic Arabidopsis were higher than those of wild-type plants ( Figure 3 B and C).

[0111] In addition to promoting growth, expression of ZmTST1 also affected silique and seed development. ZmTST1 transgenic plants produced longer siliques and more seeds ( Figure 3 In addition, the transgenic plants had more siliques per plant, an increased thousand-seed weight, and the number of seeds per silique remained unchanged, resulting in an increase in seed yield per plant ( Figure 3 These results indicate that overexpression of ZmTST1 affects seed development and increases seed yield in transgenic plants.

[0112] Example 5 Overexpression of ZmTST1 can significantly improve the stress resistance of Arabidopsis seedlings

[0113] In order to study whether the expression of ZmTST1 can improve the stress tolerance of transgenic Arabidopsis thaliana, this example first compared the seed germination rate and cotyledon greening rate of wild-type and transgenic plants under salinity and osmotic stress.

[0114] The germination rate determination method is as follows: After the WT and OE#4, OE#7 and OE#10 seeds are disinfected, they are sown on 1 / 2MS medium, 1 / 2MS medium containing 100mM NaCl, and 1 / 2MS medium containing 250mM mannitol. The appearance of radicle is regarded as germination. The germination is observed every day for seven consecutive days and the germination rate is recorded.

[0115] The method for determining the cotyledon greening rate is as follows: After disinfecting the WT and OE#4, OE#7 and OE#10 seeds, sow them on 1 / 2MS medium, 1 / 2MS medium containing 100mM NaCl, and 1 / 2MS medium containing 250mM mannitol. If the seeds germinate and unfold green cotyledons, the cotyledons are considered green. Observe the cotyledon greening every day for seven consecutive days and record the cotyledon greening rate.

[0116] The root length detection method is as follows: all seedlings on 1 / 2MS medium, 1 / 2MS medium containing 100mM NaCl, and 1 / 2MS medium containing 300mM mannitol are taken out, their root lengths are measured with a ruler, and then the root lengths of different strains and under different treatment conditions are calculated.

[0117] The biomass detection method is as follows: all seedlings on 1 / 2MS medium, 1 / 2MS medium containing 100mM NaCl, and 1 / 2MS medium containing 300mM mannitol were taken out, their biomass was measured with an analytical balance, and then the biomass of different strains and different treatment conditions was calculated.

[0118] The growth inhibition rate detection method is as follows: by measuring the root length and biomass, the root length inhibition rate and fresh weight inhibition rate of WT and OE#4, OE#7 and OE#10 after treatment with 100 mM NaCl and 300 mM mannitol were calculated.

[0119] Under normal conditions, there was no significant difference in seed germination rate and cotyledon greening rate between transgenic plants and wild type plants after 3 days ( Figure 4 A, B and E). However, under NaCl and mannitol stress conditions, the seed germination rate and cotyledon greening rate of transgenic plants were higher than those of the wild type ( Figure 4 Moreover, the seed germination rate and cotyledon greening rate were consistent with the expression level of ZmTST1. The seed germination rate and cotyledon greening rate of OE#10 were higher than those of transgenic lines OE#4 and OE#7, which was due to the highest expression level of ZmTST1 in OE#10 plants.

[0120] The resistance of plants overexpressing ZmTST1 to salt and osmotic stress during the early developmental stage of seedlings was then investigated. Seeds of the wild type and three transgenic lines OE#4, OE#7, and OE#10 were sown on 1 / 2MS medium supplemented with 0, 100 mM NaCl, or 300 mM mannitol. In the presence of 100 mM NaCl or 300 mM mannitol, the growth of both WT and transgenic seedlings was impaired, while the growth of transgenic seedlings was less affected ( Figure 5 Under control and stress treatment conditions, seedlings expressing ZmTST1 had longer roots, higher biomass, and lower growth inhibition than wild-type plants ( Figure 5 These results indicate that the expression of ZmTST1 increases the salt and osmotic tolerance of Arabidopsis seedlings.

[0121] Example 6 Overexpression of ZmTST1 can significantly improve the stress resistance of Arabidopsis seedlings

[0122] To further investigate the effects of salt and drought on the growth of WT and ZmTST1-overexpressing adult plants, three-week-old seedlings grown in greenhouse soil were treated with 200 mM NaCl or drought for two weeks.

[0123] Under normal conditions, plants expressing ZmTST1 grow taller than wild-type plants ( Figure 6 However, under NaCl or drought stress, the growth of wild-type and ZmTST1-overexpressing plants was significantly inhibited, while the growth inhibition of transgenic plants was less severe ( Figure 6 In addition, the water loss rate of detached leaves of transgenic plants was lower than that of wild-type plants ( Figure 6 E and F). These results indicate that ZmTST1 expression increases drought and salt tolerance in adult Arabidopsis plants.

[0124] Example 7 ZmTST1 expression significantly improves oxidative stress tolerance and the expression of stress-related genes

[0125] To further understand how ZmTST1 improves the tolerance of transgenic plants to salt and drought stress, this example studied the antioxidant enzyme activities, proline and MDA contents between wild-type and ZmTST1 overexpressing plants.

[0126] Kit assay: Superoxide dismutase (SOD) activity detection kit (BC5165), Peroxidase (POD) activity detection kit (BC0090), Malondialdehyde (MDA) content detection kit (BC0025), Proline (Pro) content detection kit (BC0290).

[0127] Under normal conditions, there were no significant differences between wild-type and transgenic plants. However, under salt and drought conditions, SOD and POD activities and proline content in transgenic plants increased significantly, but MDA content decreased significantly ( Figure 7 These findings suggest that ZmTST1-overexpressing plants improve salt and drought tolerance by regulating ROS scavenging and ion homeostasis.

[0128] In order to further study the correlation between ZmTST1 and abiotic stress, the expression levels of salt- and drought-related genes were also detected in this example. The results showed that under normal conditions, there was no significant difference in the expression levels of AtSOS1, AtRD29A, AtRAB18, and AtP5CS1. However, under salt and drought stress conditions, the expression levels of AtSOS1, AtRD29A, AtRAB18, and AtP5CS1 were significantly upregulated in both WT and ZmTST1 overexpressing plants, and the expression levels in transgenic plants were significantly higher than those in wild-type plants ( Figure 7 Therefore, overexpression of ZmTST1 upregulated the expression of stress-responsive genes, altered antioxidant enzymes and ion homeostasis, and thus conferred stress resistance to transgenic plants.

[0129] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

[0130]

[0131]

Claims

1. Application of any one of (a) to (e) in improving plant yield and stress resistance: (a) Maize ZmTST1 gene; (b) a recombinant vector comprising the maize ZmTST1 gene; (c) an expression cassette comprising the maize ZmTST1 gene; (d) a cell comprising the maize ZmTST1 gene; (e) a recombinant bacterium comprising the corn ZmTST1 gene; The nucleotide sequence of the maize ZmTST1 gene is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that: The stress resistance is tolerance to salt stress and drought stress.

3. The use according to claim 1, characterized in that: The plant is any one of corn, Arabidopsis, cucumber, tomato, wheat, oat, rice, corn, poplar, lawn grass, alfalfa, peanut, soybean, sorghum and millet.

4. A method for cultivating high-yield and stress-resistant plants, characterized in that: The method comprises: cloning the corn ZmTST1 gene described in claim 1 into an expression vector, introducing the expression vector into plant cells, screening the plant cells carrying the expression vector, and cultivating the plant cells into plants to obtain high-yield and stress-resistant plants.

5. The method according to claim 4, characterized in that The plant cells are callus cells or embryonic cells.

6. The method according to claim 4, characterized in that The expression vector is a monocotyledonous plant expression vector.

7. The method according to claim 4, characterized in that The plant is any one of corn, Arabidopsis, cucumber, tomato, wheat, oat, rice, corn, poplar, lawn grass, alfalfa, peanut, soybean, sorghum and millet.

8. A plant breeding method, characterized in that: Any of the following: (1) Improving the yield and stress resistance of the target plant by increasing the content or activity of maize ZmTST1 protein in the target plant; (2) Improving the yield and stress resistance of the target plant by promoting the expression of the maize ZmTST1 gene in the target plant; The nucleotide sequence of the maize ZmTST1 gene is shown in SEQ ID NO.

1.

9. The method according to claim 8, characterized in that The method of promoting the expression of the maize ZmTST1 gene in the target plant is specifically any one of the following: (1) transferring the expression construct or vector of the maize ZmTST1 gene into the target plant; (2) Transform the expression construct or vector containing the maize ZmTST1 gene into the target plant.

10. A method for producing food, characterized in that: The method comprises the following steps: planting crops and harvesting the food of the crops; increasing the expression or activity of the corn ZmTST1 gene or the protein encoded by it in the crops; The nucleotide sequence of the maize ZmTST1 gene is shown in SEQ ID NO.1.

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