Application of corn ZmTIP2-1 gene in plant stress resistance breeding

Abstract

The invention discloses application of a corn ZmTIP2-1 gene in plant stress resistance breeding, and belongs to the technical field of gene engineering. The ZmTIP2-1 gene is used for coding an amino acid sequence as shown in SEQ ID NO. 2. The salt resistance and drought resistance of the plant can be improved by improving the expression of the ZmTIP2-1 gene, so that the gene can be applied to stress-resistant breeding of the plant.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a maize ZmTIP2-1 gene sequence and an application thereof in plant stress resistance breeding. Background Art

[0002] Under the dual pressures of global climate change and population growth, agricultural production is facing unprecedented challenges. Salt stress and drought stress are the main abiotic stress factors that affect crop yield and quality. Salt stress is usually caused by soil salinization, which leads to problems such as osmotic pressure imbalance, ion toxicity and oxidative stress in plant cells, seriously affecting plant growth, development and metabolic activities. It is estimated that about 20% of irrigated land in the world is threatened by salinization, and this proportion is even higher in some areas. Drought stress is a lack of water caused by insufficient or uneven rainfall, which makes it impossible for plants to obtain enough water for photosynthesis and transpiration, ultimately leading to reduced yields or even crop failure. Drought stress is considered to be one of the most destructive environmental factors to agriculture in the next few decades.

[0003] In order to cope with these adversities, traditional agricultural measures such as irrigation management and soil improvement can alleviate the problem to a certain extent, but it is difficult to solve the problem fundamentally. Therefore, genetic improvement breeding has become an important way to improve the stress resistance of crops. By selecting and cultivating varieties with stronger stress resistance, the adaptability and yield stability of crops can be significantly improved without relying on external resources. Modern genetic improvement technology combines traditional breeding methods with molecular biological methods, which can more efficiently screen and introduce target genes and accelerate the selection and breeding process of excellent 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 are mainly involved in signal transduction, osmotic regulation, antioxidant defense and other aspects, and enhance the tolerance of plants by regulating their response mechanisms to adversity. For example, some genes encode proteins that can regulate the ion balance in plants and prevent excessive salt accumulation; other genes are involved in the synthesis of osmotic regulating substances such as proline, helping plants maintain water balance in cells; and some genes activate antioxidant enzyme systems to remove reactive oxygen free radicals and reduce oxidative damage.

[0005] In conclusion, in the face of increasingly severe salt and drought stress, cloning and utilizing stress-resistant genes is a key strategy to improve crop adaptability and productivity. By deeply studying the functions of these genes and their regulatory networks, combined with advanced genetic improvement technologies, we are expected to breed better-quality stress-resistant varieties and make important contributions to ensuring global food security. However, the genetic backgrounds of different plants vary greatly, and further discovering new genes with stress-resistant functions and proposing a new method to improve plant stress resistance by regulating the expression of key genes is still very important for genetic improvement and breeding of crops. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides a maize (Zea mays) ZmTIP2-1 gene and its application in plant stress resistance breeding.

[0007] The present invention clones the maize ZmTIP2-1 gene, constructs and compares the germination rate, seedling length, plant weight and root weight of the ZmTIP2-1 overexpression strain and the wild-type strain. The results show that the overexpression strain has advantages in the above indicators under adverse conditions. The ZmTIP2-1 gene encodes a nucleotide sequence of the amino acid sequence shown in SEQ ID NO.2. The gene is expected to be applied to genetic engineering breeding of plants, providing a theoretical basis for improving plant resistance to stress and creating stress-resistant germplasm. Considering the degeneracy of codons, the bases of the above nucleotide sequence are modified without changing the amino acid sequence, which also belongs to the protection scope of the present invention.

[0008] In one aspect, the present invention provides a protein for regulating the ability of a plant to cope with adverse 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 for regulating the ability of a plant to cope with adverse stress, characterized in that the gene encodes an amino acid sequence as shown in SEQ ID NO.2.

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

[0011] In another aspect, the present invention provides a use of the aforementioned protein or the aforementioned gene in regulating the ability of a plant to cope with adverse stress.

[0012] Furthermore, the regulation of the plant's ability to cope with adverse stress is to improve the plant's ability to tolerate salt stress and / or drought stress by overexpressing the aforementioned protein and / or gene.

[0013] Furthermore, the regulation of the plant's ability to cope with adverse stress is to increase the germination rate, seedling length and / or plant weight of the plant under salt stress and / or drought stress by overexpressing the aforementioned protein and / or gene.

[0014] Furthermore, the plant is a grass plant.

[0015] Furthermore, the plant is a plant of the genus Zea mays.

[0016] Furthermore, the plant is corn.

[0017] In another aspect, the present invention provides a use of the aforementioned protein or the aforementioned gene in breeding plant varieties with high resistance to adverse stress.

[0018] Furthermore, the plant variety cultivated with high tolerance to adverse stress is obtained by overexpressing the aforementioned protein and / or gene to obtain a variety with high tolerance to salt stress and / or drought stress.

[0019] Furthermore, the method of cultivating plant varieties with high resistance to adverse stress is to cultivate varieties with high germination rate, seedling length and / or single plant weight under salt stress and / or drought stress by overexpressing the aforementioned proteins and / or genes.

[0020] Furthermore, the plant is a grass plant.

[0021] Furthermore, the plant is a plant of the genus Zea mays.

[0022] Furthermore, the plant is corn.

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

[0024] 1) The present invention provides a novel method for breeding new stress-resistant plant varieties, namely, obtaining new stress-resistant plant varieties by overexpressing the ZmTIP2-1 gene or the protein encoded by it.

[0025] 2) The method provided by the present invention can simultaneously improve the plant's resistance to salt stress and drought stress. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The beneficial effects of the present invention are described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0027] Figure 1 Shown are the TIP2-1 expression levels in ZmTIP2-1 overexpressing maize and control samples.

[0028] Figure 2 Shown is the phenotypic analysis of 7-day seedlings of ZmTIP2-1 related lines under salt and drought stress treatments.

[0029] Figure 3 The figure shows the difference analysis of relevant indicators between the control and ZmTIP2-1 overexpression lines under salt and drought stress, where Figure A is the germination rate, Figure B is the seedling length, Figure C is the seedling size per plant, and Figure D is the root weight per plant. DETAILED DESCRIPTION

[0030] The technical solutions in the embodiments of the present invention are described clearly and completely below. 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 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 ordinary technicians in the technical field to which this application belongs.

[0032] The nucleotide sequence of the maize ZmTIP2-1 gene in the embodiment is shown in SEQ ID NO.1:

[0033] ATGGTGAAGCTCGCCTTCGGAAGCGTCGGCGACTCCTTCAGCGCCACCTCCATCAAGGCCTACGTGGCCGAGTTCATCGCCACCCTCCTCTTCGTCTTCGCCGGCGTCGGTTCCGCCATCGCCTACGGGCAACTGACGAATGGCGGCGCGCTGGACCCGGCGGGCCTGGTGGCGATCGCGATCGCGCACGCGCTGGCGCTGTTCGTGGGCGTGTCCGTCGCGGCGAACATCTCGGGCGGCCACCTGAACCCGGCCGTGACGTTCGGGCTGGCCGTGGGCGGCCACATCACCATCCTGACGGGCGTCTTCTACTGGGTGGCCCAGCTGCTGGGCGCCACCGTGGCGTGCCTGCTCCTCGGGTTCGTCACCCACGGCAAGGCCATCCCGACGCACGCCGTCGCGGGCATCAGCGAGCTGGAAGGCGTCGTGTTCGAGGTCGTCATCACCTTCGCGCTCGTCTACACCGTGTACGCCACCGCCGCCGACCCCAAGAAGGGCTCGCTCGGCACCATCGCGCCCATCGCCATCGGCTTCATCGTCGGCGCCAACATCCTCGCCGCGGGGCCCTTCAGCGGCGGCTCCATGAACCCCGCCCGCTCCTTCGGCCCCGCCGTCGCCGCGGGCGACTTCGCCGGAAACTGGGTCTACTGGGTCGGCCCGCTCGTCGGCGGCGGCCTCGCTGGCCTCGTCTACGGCGACGTCTTCATTGGCGGCTCCTACCAGCAGGTCGCGGACCAGGACTACGCCTAA

[0034] The amino acid sequence of the 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, constructed into the overexpression vector WMV013, transformed into Agrobacterium EHA105, infected the immature embryos of the maize inbred line KN5585, and the infected immature embryos were transferred to a selection medium containing a selection marker (herbicide), cultured for 2-4 weeks to induce callus tissue, and then transferred to a regeneration medium to induce the differentiation of buds and roots, and grow into normal complete seedlings. PCR detection with ZmTIP2-1 specific primers was used to identify positive seedlings. The positive seedlings were transferred to the field for growth, self-pollination was used to obtain T1 generation transgenic seeds, and multiple generations of self-pollination were used 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, and subsequent experiments and analyses were carried out.

[0040] 1. The total RNA of 4-day-old corn seedlings was extracted using a plant RNA extraction kit (Magen). 1 μg of high-quality RNA (OD260 / OD280: 1.8-2.0; OD260 / OD230≈2.0) was reverse transcribed (Novagen reverse transcription kit) to obtain the first-strand cDNA.

[0041] 2. Using the cDNA in step 1 as a template, the expression of the ZmTIP2-1 gene was detected by the primer pair ZmTIP2-1-qF (GTTCGTGGGCGTGTC) and ZmTIP2-1-qR (CGTCAGGATGGTGATGTG), and the expression of the maize Actin-1 gene was detected by the primer pair ZmACT1-F (GCTACGAGATGCCTGATGGTC) and ZmACT1-R (CCCCCACTGAGGACAACG) of the maize housekeeping gene Actin-1 as an internal reference. 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 system was: 2×PCR buffer 10.0μL, qF primer 0.4μL, qR primer 0.4μL, cDNA template 2.0μL, sterile water 7.2μL, total reaction volume 20.0μL. Reaction procedure: 95℃30sec; 95℃5sec, 60℃30sec, 40 cycles.

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

[0043] Example 2: Phenotypic analysis of overexpressing plants

[0044] Seeds of homozygous ZmTIP2-1 overexpressing transgenic lines and control KN5585 were subjected to germination experiments, and control (H2O) and salt stress (100 mM NaCl solution) treatments were set up. The control was placed in a germination box with a sand bed (water content of 60% of the saturated water content), and 50 seeds were repeated each time for experimental germination. Supplementary water was required from the 3rd to the 7th day; the salt stress treatment was placed in a germination box with a sand bed (NaCl solution with a saturated water content of 60%, and sand was mixed with the treatment solution in proportion), and 50 seeds were repeated each time for experimental germination, and covered with plastic wrap; drought stress (water content of 20% of the saturated water content) was carried out in a germination box, and 50 seeds were repeated each time, and the differences in seedling growth phenotypes were continuously observed and recorded.

[0045] See the phenotypic results and differential analysis of seedlings at 7 days of growth under stress treatment. Figures 2-3The results showed that under salt stress, the germination rate, seedling length and seedling weight of ZmTIP2-1 overexpressing materials were significantly higher than those of the control; under drought stress, the germination rate, seedling length, seedling weight and single plant root weight of ZmTIP2-1 overexpressing materials were significantly higher than those of the control. The above results indicate that ZmTIP2-1 positively regulates maize seedling tolerance to abiotic stress.

[0046] According to the disclosure and teaching of the above description, those skilled in the art to which the present invention belongs may also 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 scope of protection of the claims of the present invention. In addition, although some specific terms are used in this specification, these terms are only for the convenience of description and do not constitute any limitation to the present invention.

Claims

1. Use of a protein for regulating plant stress resistance in regulating the ability of plants to cope with adverse stress, characterized in that: The amino acid sequence of the protein is shown in SEQ ID NO.

2.

2. The use according to claim 1, characterized in that: The method for regulating the ability of plants to cope with adverse stress is to improve the ability of plants to tolerate salt stress and / or drought stress by overexpressing the protein according to claim 1.

3. Use of a gene for regulating plant stress resistance in regulating the ability of plants to cope with adverse stress, characterized in that: The gene encodes a protein with an amino acid sequence as shown in SEQ ID NO.

2.

4. The use according to claim 3, characterized in that: The method for regulating the ability of plants to cope with adverse stress is to improve the ability of plants to tolerate salt stress and / or drought stress by overexpressing the gene described in claim 3.

5. Use of a protein for regulating plant stress resistance in cultivating plant varieties with high stress resistance, characterized in that: The amino acid sequence of the protein is shown in SEQ ID NO.

2.

6. The use according to claim 5, characterized in that: The plant variety cultivated with high tolerance to adverse stress is a variety with high tolerance to salt stress and / or drought stress obtained by overexpressing the protein according to claim 5.

7. Use of a gene for regulating plant stress resistance in breeding plant varieties with high stress resistance, characterized in that: The gene encodes a protein with an amino acid sequence as shown in SEQ ID NO.

2.

8. The use according to claim 7, characterized in that: The plant variety cultivated with high tolerance to adverse stress is a variety with high tolerance to salt stress and / or drought stress obtained by overexpressing the gene described in claim 7.

9. The use according to any one of claims 1 to 8, characterized in that: The plant is a grass plant.

10. The use according to any one of claims 1 to 8, characterized in that: The plant is corn.

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