Application of maize ZmTIP2-1 gene in plant stress breeding

Abstract

The application discloses application of a corn ZmTIP2-1 gene in plant stress resistance breeding and belongs to the technical field of genetic engineering. The ZmTIP2-1 gene encodes an amino acid sequence as shown in SEQ ID NO. 2. The application can improve the salt resistance and drought resistance of plants by improving the expression of the ZmTIP2-1 gene, and the gene can be applied in plant stress resistance breeding.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to maize. ZmTIP2-1 Gene sequences and their application in plant stress resistance breeding. Background Technology

[0002] Under the dual pressures of global climate change and population growth, agricultural production faces unprecedented challenges. Salt stress and drought stress are major abiotic stressors affecting crop yield and quality. Salt stress, usually caused by soil salinization, leads to problems such as osmotic pressure imbalance in plant cells, ion toxicity, and oxidative stress, severely impacting plant growth, development, and metabolic activities. It is estimated that approximately 20% of irrigated land globally is threatened by salinization, and this proportion is even higher in some regions. Drought stress, caused by insufficient or uneven rainfall, results in water scarcity, preventing plants from obtaining enough water for photosynthesis and transpiration, ultimately leading to reduced yields or even crop failure. Drought stress is considered one of the most destructive environmental factors for agriculture in the coming decades.

[0003] While traditional agricultural measures such as irrigation management and soil improvement can alleviate these problems to some extent, they cannot fundamentally solve them. Therefore, genetic improvement breeding has become an important way to improve crop stress resistance. By selecting and breeding varieties with stronger stress resistance, crop adaptability and yield stability can be significantly improved without relying on external resources. Modern genetic improvement technology combines traditional breeding methods with molecular biology techniques, enabling more efficient screening and introduction of target genes and accelerating the breeding process of superior varieties.

[0004] In recent years, with the development of genomics and functional genomics, scientists have identified and cloned many genes related to salt and drought resistance. These genes mainly involve multiple aspects such as signal transduction, osmotic regulation, and antioxidant defense, enhancing plant tolerance by regulating their response mechanisms to stress. For example, some genes encode proteins that can regulate ion balance in plants and prevent excessive salt accumulation; other genes participate in the synthesis of osmotic regulators such as proline, helping plants maintain intracellular water balance; and still others activate antioxidant enzyme systems to scavenge reactive oxygen species and mitigate oxidative damage.

[0005] In summary, facing increasingly severe salt and drought stress, cloning and utilizing stress-resistance genes is a key strategy for improving crop adaptability and productivity. By deeply studying the functions and regulatory networks of these genes, combined with advanced genetic improvement techniques, we can expect to breed higher-quality stress-resistant varieties, making a significant contribution to ensuring global food security. However, the genetic backgrounds of different plants vary greatly. Further discovering new genes with stress-resistance functions and proposing new methods to improve plant stress resistance by regulating the expression of key genes remains crucial for the genetic improvement and breeding of crops. Summary of the Invention

[0006] To solve the above technical problems, the present invention provides a corn ( Zea mays ) ZmTIP2-1 Genes and their application in plant stress resistance breeding.

[0007] This invention utilizes cloning of corn ZmTIP2-1 Genes were constructed and compared ZmTIP2-1 Germination rate, seedling length, single plant weight, and single plant root weight of the overexpression lines and wild-type lines were compared. The results showed that the overexpression lines had advantages in all of the above indicators under stress conditions. ZmTIP2-1 The gene encodes a nucleotide sequence with the amino acid sequence shown in SEQ ID NO.2. This gene holds promise for application in plant genetic engineering breeding, providing a theoretical basis for improving plant stress resistance and creating stress-resistant germplasm. Considering the degeneracy of codons, modifications to the bases of the above nucleotide sequence without altering the amino acid sequence also fall within the scope of protection of this invention.

[0008] On the one hand, the present invention provides a protein that regulates the ability of plants to cope with abiotic stress, characterized in that the amino acid sequence of the protein is shown in SEQ ID NO.2.

[0009] Furthermore, the present invention provides a gene that regulates the ability of plants to cope with abiotic stress, characterized in that the gene encodes the amino acid sequence shown in SEQ ID NO.2.

[0010] Preferably, the gene sequence comprises a nucleotide sequence as shown in SEQ ID NO.1, or a nucleotide sequence that is completely inversely complementary to the sequence shown in SEQ ID NO.1.

[0011] On the other hand, the present invention provides an application of the aforementioned protein or gene in regulating the ability of plants to cope with abiotic stress.

[0012] Furthermore, the regulation of plant stress response is achieved by overexpressing the aforementioned proteins and / or genes to enhance the plant's tolerance to salt stress and / or drought stress.

[0013] Furthermore, the regulation of plant stress response is achieved by overexpressing the aforementioned proteins and / or genes to increase germination rate, seedling length, and / or individual plant weight under salt and / or drought stress.

[0014] Furthermore, the plant in question is a grass belonging to the Poaceae family.

[0015] Furthermore, the plant in question is a species of the genus *Zea*.

[0016] Furthermore, the plant in question is corn.

[0017] On the other hand, the present invention provides the application of the aforementioned protein or gene in the cultivation of plant varieties with high tolerance to abiotic stress.

[0018] Furthermore, the cultivation of plant varieties with high tolerance to abiotic stress is achieved by overexpressing the aforementioned proteins and / or genes to obtain varieties with high tolerance to salt stress and / or drought stress.

[0019] Furthermore, the cultivation of plant varieties with high tolerance to abiotic stress involves overexpressing the aforementioned proteins and / or genes to cultivate varieties with high germination rates, seedling lengths, and / or individual plant weights under salt and / or drought stress.

[0020] Furthermore, the plant in question is a grass belonging to the Poaceae family.

[0021] Furthermore, the plant in question is a species of the genus *Zea*.

[0022] Furthermore, the plant in question is corn.

[0023] Compared with the prior art, the present invention has the following advantages:

[0024] 1) This invention provides a novel method for cultivating stress-resistant plant varieties, namely, through overexpression ZmTIP2-1 Genes or their encoded proteins are used to obtain new varieties of stress-resistant plants.

[0025] 2) The method provided by this invention can simultaneously improve the plant's resistance to salt stress and drought stress. Attached Figure Description

[0026] The beneficial effects of the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0027] Figure 1 As shown ZmTIP2-1 TIP2-1 expression levels in overexpressing maize and control samples.

[0028] Figure 2 The figure shows the effects of salt and drought stress treatments. ZmTIP2-1 Phenotypic analysis of seedlings of relevant strains on day 7.

[0029] Figure 3 The figures shown are the control and treatment under salt and drought stress. ZmTIP2-1 Differences in related indicators of overexpression lines were analyzed, with Figure A representing germination rate, Figure B representing seedling length, Figure C representing single seedling weight, and Figure D representing single root weight. Detailed Implementation

[0030] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0032] Corn in the example ZmTIP2-1 The gene nucleotide sequence is shown in SEQ ID NO.1:

[0033] ATGGTGAAGCTCGCCTTCGGAAGCGTCGGCGACTCCTTCAGCGCCACCTCCATCAAGGCCTACGTGGCCGAGTTCATCGCCACCCTCCTCTTCGTCTTCGCCGGCGTCGGTTCCGCCATCGCCTACGGGCAACTGACGAATGGCGGCGCGCTGGACCCGGCGGGCCTGGTGGCGATCGCGATCGCGCACGCGCTGGCGCTGTTCGTGGGCGTGTCCGTCGCGGCGAACATCTCGGGCGGCCACCTGAACCCGGCCGTGACGTTCGGGCTGGCCGTGGGCGGCCACATCACCATCCTGACGGGCGTCTTCTACTGGGTGGCCCAGCTGCTGGGCGCCACCGTGGCGTGCCTGCTCCTCGGGTTCGTCACCCACGGCAAGGCCATCCCGACGCACGCCGTCGCGGGCATCAGCGAGCTGGAAGGCGTCGTGTTCGAGGTCGTCATCACCTTCGCGCTCGTCTACACCGTGTACGCCACCGCCGCCGACCCCAAGAAGGGCTCGCTCGGCACCATCGCGCCCATCGCCATCGGCTTCATCGTCGGCGCCAACATCCTCGCCGCGGGGCCCTTCAGCGGCGGCTCCATGAACCCCGCCCGCTCCTTCGGCCCCGCCGTCGCCGCGGGCGACTTCGCCGGAAACTGGGTCTACTGGGTCGGCCCGCTCGTCGGCGGCGGCCTCGCTGGCCTCGTCTACGGCGACGTCTTCATTGGCGGCTCCTACCAGCAGGTCGCGGACCAGGACTACGCCTAA

[0034] The amino acid sequence of maize ZmTIP2-1 protein is shown in SEQ ID NO.2:

[0035] MVKLAFGSVGDSFSATSIKAYVAEFIATLLFVFAGVGSAIAYGQLTNGGALDPAGLVAIAIAHALALFVGVSVAANISGGHLNPAVTFGLAVGGHITILTGVFYWVAQLLGATVACLLLGFVTH GKAIPTHAVAGISELEGVVFEVVITFALVYTVYATAADPKKGSLGTIAPIAIGFIVGANILAAGPFSGGSMNPARSFGPAVAAGDFAGNWVYWVGPLVGGGLAGLVYGDVFIGGSYQQVADQDYA

[0036] Example

[0037] Example 1: Establishment of ZmTIP2-1 transgenic maize plants

[0038] The CDS sequence of ZmTIP2-1 was cloned and constructed into the overexpression vector WMV013. This vector was transformed into Agrobacterium EHA105, which infected the immature embryos of the maize inbred line KN5585. The infected embryos were then transferred to a selection medium containing a selection marker (herbicide) and cultured for 2-4 weeks to induce callus tissue. Subsequently, the embryos were transferred to a regeneration medium to induce shoot and root differentiation, resulting in normal, complete seedlings. Positive seedlings were identified by PCR using ZmTIP2-1 specific primers. These positive seedlings were then transferred to the field for self-pollination to obtain T1 generation transgenic seeds. Multiple generations of self-pollination were then conducted to obtain homozygous ZmTIP2-1 overexpression lines.

[0039] The obtained transgenic seeds were identified, and the ZmTIP2-1 transgenic lines ZmTIP2-1_OE2, ZmTIP2-1_OE8, and ZmTIP2-1_OE9 were selected for qRT-RCR detection, followed by subsequent experiments and analysis.

[0040] 1. Use a plant RNA extraction kit (Magen) to extract total RNA from 4-day-old maize seedlings. Take 1 μg of high-quality RNA (OD260 / OD280: 1.8-2.0; OD260 / OD230 ≈ 2.0) and perform reverse transcription (Novizan reverse transcription kit) to obtain first-strand cDNA.

[0041] 2. Using the cDNA from step 1 above as a template, the expression of the ZmTIP2-1 gene was detected using primer pairs ZmTIP2-1-qF (GTTCGTGGGCGTGTC) and ZmTIP2-1-qR (CGTCAGGATGGTGATGTG). The expression of the maize housekeeping gene Actin-1 was detected using primer pairs ZmACT1-F (GCTACGAGATGCCTGATGGTC) and ZmACT1-R (CCCCCACTGAGGACAACG) as internal controls. The quantitative PCR reagent was ChamQ Blue Universal SYBR Qpcr Master Mix (Vazyme), and the quantitative PCR instrument was StepOne Software v2.3 (Applied Biosystems). The reaction mixture consisted of: 10.0 μL 2×PCR buffer, 0.4 μL qF primer, 0.4 μL qR primer, 2.0 μL cDNA template, and 7.2 μL sterile water, for a total reaction volume of 20.0 μL. The reaction program was: 95℃ for 30 sec; 95℃ for 5 sec, 60℃ for 30 sec, for 40 cycles.

[0042] The results are as follows Figure 1 As shown, the expression level of ZmTIP1-1 gene was significantly increased in the ZmTIPs overexpression lines.

[0043] Example 2: Phenotypic analysis of overexpression plants

[0044] Germination experiments were conducted on seeds of the homozygous ZmTIP2-1 overexpressing transgenic line and the control KN5585. Control (H2O) and salt stress (100 mM NaCl solution) treatments were included. The control was placed in a germination box using a sand bed (60% saturated moisture content), with 50 seeds per replicate. Watering was required from day 3 to day 7. Salt stress treatment was also conducted in a germination box using a sand bed (60% NaCl solution content, sand mixed with the treatment solution in the correct proportion), with 50 seeds per replicate, covered with plastic wrap. Drought stress (20% saturated moisture content) was also conducted in a germination box, with 50 seeds per replicate. Seedling growth phenotypic differences were continuously observed and recorded.

[0045] Phenotypic results and differential analysis of seedlings after 7 days of stress treatment are shown in [the table below]. Figures 2-3The results showed that under salt stress, the germination rate, seedling length, and seedling weight of the ZmTIP2-1 overexpressing material were significantly higher than those of the control; under drought stress, the germination rate, seedling length, seedling weight, and root weight per plant of the ZmTIP2-1 overexpressing material were also significantly higher than those of the control. In summary, these results indicate that ZmTIP2-1 positively regulates maize seedling tolerance to abiotic stress.

[0046] Based on the disclosure and teachings of the foregoing specification, those skilled in the art can make appropriate changes and modifications to the above embodiments. Therefore, the present invention is not limited to the specific embodiments disclosed and described above, and some modifications and changes to the present invention should also fall within the protection scope of the claims of the present invention. Furthermore, although some specific terms are used in this specification, these terms are only for convenience of explanation and do not constitute any limitation on the present invention.

Claims

1. Use of a protein that regulates the stress tolerance of a plant in regulating the ability of a plant to cope with stress, characterized in that, The amino acid sequence of the protein is shown as SEQ ID NO. 2, the ability of the plant to resist stress is the ability of the plant to resist salt stress and drought stress by overexpressing the protein, the ability of the plant to resist salt stress refers to the increase of the germination rate, seedling length and seedling weight of the plant under the condition of salt stress, and the ability of the plant to resist drought stress refers to the increase of the germination rate, seedling length, seedling weight and root weight of the plant under the condition of drought stress, and the plant is corn.

2. Use of a gene that regulates the stress resistance of a plant in regulating the ability of a plant to cope with stress, characterized in that, The gene encodes a protein with an amino acid sequence shown as SEQ ID NO. 2, the ability of the plant to resist stress is the ability of the plant to resist salt stress and drought stress by overexpressing the gene, the ability of the plant to resist salt stress refers to the increase of the germination rate, seedling length and seedling weight of the plant under the condition of salt stress, and the ability of the plant to resist drought stress refers to the increase of the germination rate, seedling length, seedling weight and root weight of the plant under the condition of drought stress, and the plant is corn.

3. Use of a protein that regulates stress tolerance of a plant in breeding a plant variety having high stress tolerance, characterized in that, The amino acid sequence of the protein is shown as SEQ ID NO. 2, the plant variety with high stress resistance is obtained by overexpressing the protein, and the plant variety has high resistance to salt stress and drought stress, the high resistance to salt stress refers to the increase of the germination rate, seedling length and seedling weight of the plant under the condition of salt stress, and the high resistance to drought stress refers to the increase of the germination rate, seedling length, seedling weight and root weight of the plant under the condition of drought stress, and the plant is corn.

4. Use of a gene regulating stress tolerance of a plant in breeding a plant variety with high stress tolerance, characterized in that, The gene encodes a protein with an amino acid sequence shown as SEQ ID NO. 2, the plant variety with high stress resistance is obtained by overexpressing the gene, and the plant variety has high resistance to salt stress and drought stress, the high resistance to salt stress refers to the increase of the germination rate, seedling length and seedling weight of the plant under the condition of salt stress, and the high resistance to drought stress refers to the increase of the germination rate, seedling length, seedling weight and root weight of the plant under the condition of drought stress, and the plant is corn.

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