Corn gene Zmhta1 and application thereof in improving salt tolerance of corn

Abstract

This invention belongs to the field of plant bioscience and technology, specifically relating to maize. ZmHTA1 Genes and their application in improving salt tolerance in maize. This invention clones a maize salt tolerance-related gene. ZmHTA1 For the first time, maize genes were obtained through gene knockout and transgenic technology. ZmHTA1 The study elucidated the salt tolerance regulatory function of this gene in loss-of-function and overexpression lines, revealing its positive regulatory role in enhancing salt tolerance in maize. This gene regulates the salt tolerance of maize varieties, laying the foundation for creating and obtaining new salt-tolerant maize materials and for breeding new salt-tolerant maize varieties.

Description

Technical Field

[0001] This invention belongs to the field of plant bioscience and technology, specifically relating to maize. ZmHTA1 Genes and their application in improving the salt tolerance of maize. Background Technology

[0002] In recent years, with rapid economic growth, human demand for animal fats and proteins has increased dramatically. Simultaneously, the booming ethanol fuel industry has led to a continuous rise in demand for corn. However, due to environmental pollution causing a continuous reduction in arable land, it is impossible to expand corn planting areas on a large scale. Therefore, increasing yield per unit area has become a practical way to increase corn production. Corn has a strong ability to survive in saline-alkali soil, and the scientific and rational utilization of saline-alkali land resources is an important way to solve the contradiction between population and land. Breeding salt-tolerant corn using genetic engineering technology has significant strategic importance and is crucial for improving the ecological environment and promoting sustainable socio-economic development.

[0003] ZmHTA1 These belong to the category of heat shock proteins (HSPS), an ancient and highly conserved class of molecular chaperones widely distributed in prokaryotes and eukaryotes. When organisms are subjected to stresses such as high temperature, drought, heavy metals, and high salt, HSPS can maintain cellular function by stabilizing cell structure, transporting proteins, and assisting in protein folding, thereby enhancing the organism's adaptability to stress. Based on amino acid sequence homology and molecular weight, heat shock proteins in plants do indeed play an important role under adverse conditions, especially small heat shock proteins (SHSPS). These proteins are present in low amounts under normal plant growth conditions, but are activated and expressed under biotic and abiotic stresses. Studies have found that these genes are expressed in large quantities when organisms are subjected to external stimuli or abiotic stresses, thereby enhancing the organism's adaptability to external conditions. Salt stress may lead to upregulation of the expression of these proteins to enhance their stability and function. Overall, salt stress can regulate the expression of plant heat shock proteins to protect cells from salt stress-induced damage.

[0004] Traditional breeding involves selecting superior varieties through hybridization and selection to gradually breed more salt-tolerant varieties. This method requires significant time, manpower, and resources, and is suitable for breeding some crops, such as wheat and corn in desert regions. Molecular breeding, on the other hand, uses molecular biology techniques to identify genes related to salt tolerance and introduce these genes into target crops to improve their salt tolerance. This method requires less time and resources and has more significant effects. In recent years, with the rapid development of genome editing technology, the process of crop genetic breeding has been greatly accelerated. This technology has been widely used in improving the yield, quality, and resistance of various crops such as rice, wheat, and corn. Combining gene editing technology, genetic engineering technology, and corn transgenic technology not only helps in the study of corn gene function but also lays the foundation for creating salt-tolerant germplasm and breeding salt-tolerant varieties. Summary of the Invention

[0005] Identifying key maize salt tolerance regulatory genes and creating and rationally utilizing salt-tolerant materials through genetic engineering are crucial for breeding salt-tolerant maize varieties. Based on this, the present invention provides maize... ZmHTA1 The invention utilizes gene editing technology to improve the salt tolerance of maize, taking B104 maize as an example, and knocks out maize using gene editing technology. ZmHTA1 The gene was used to obtain new transgenic maize materials with homozygous mutants that showed reduced resistance to salt stress. At the same time, overexpression plants were obtained by constructing an overexpression vector to overexpress the gene.

[0006] In one aspect, this invention discloses a salt-tolerant gene for maize, wherein the salt-tolerant gene is... ZmHTA1 Gene, ZmHTA1 The nucleotide sequence of the gene is shown in SEQ ID NO. 1. ZmHTA1 The amino acid sequence encoded by the gene is shown in SEQ ID NO. 2. ZmHTA1 The CDS sequence of the gene is shown in SEQ ID NO. 4.

[0007] In one aspect, the present invention discloses a salt tolerance gene. ZmHTA1 The application of genes, the ZmHTA1 Genes can regulate the salt tolerance of maize. ZmHTA1 The gene plays a positive regulatory role in maize's resistance to salt stress. Homozygous mutants have reduced resistance to salt stress, while overexpressed plants have enhanced resistance to salt stress.

[0008] In one aspect, the present invention discloses a method for identifying salt-tolerant maize varieties, the method comprising detecting the variety's... ZmHTA1 Gene expression, if the ZmHTA1 If the gene is expressed at a higher level than that of the wild-type variety, then the variety is a salt-tolerant variety.

[0009] In one aspect, the present invention discloses a salt tolerance gene. ZmHTA1 Application in the genetic production of salt-tolerant maize varieties, wherein the application is to improve... ZmHTA1 Gene expression in maize can improve the salt tolerance of maize varieties.

[0010] In one embodiment, a method for preparing a salt-tolerant maize variety includes the following steps: (1) Construction ZmHTA1 Gene overexpression vector pCAMBIA3301- ZmHTA1 ; (2) Overexpression vector pCAMBIA3301- ZmHTA1 The bacteria were transformed into Agrobacterium strain EHA105, which has a strong ability to infect maize embryos, using an electroporation method, for the next step of maize genetic transformation. (3) The above-mentioned Agrobacterium vector was transferred into maize by the genetic transformation method of Agrobacterium infection of maize embryos to obtain transgenic maize plants. The overexpressing plants were screened to obtain salt-tolerant varieties.

[0011] This invention is the first to be constructed in maize. ZmHTA1 The gene was knocked out using a gene mutation vector, resulting in homozygous mutant transgenic maize. Simultaneously, overexpression plants were obtained by constructing an overexpression vector to overexpress the gene, elucidating the regulatory role of this gene in maize salt tolerance. Compared to the non-transgenic wild type, the homozygous mutant showed decreased resistance to salt stress, while the overexpressing plants showed increased resistance to salt stress, indicating... ZmHTA1 It strongly positively regulates the salt tolerance of maize, providing a theoretical basis for the future breeding of new salt-tolerant maize varieties. Beneficial effects

[0012] The corn provided by this invention ZmHTA1 This gene is a high-quality resource for regulating salt tolerance. Utilizing this gene to obtain salt-tolerant maize materials has advantages such as strong resistance to salt stress. ZmHTA1 Gene expression is crucial for enhancing salt tolerance in maize. Rapid advancements in sequencing and transgenic technologies have made rapid screening of salt-tolerant genes in maize a reality. Genetic engineering techniques are used to validate these genes, serving as a technological reserve for salt-tolerant maize screening. Simultaneously, combining conventional breeding with marker-assisted selection, core salt-tolerant maize populations are screened to support the development of core germplasm resources. Traditional breeding methods suffer from drawbacks such as long breeding cycles and high labor costs, while genetic engineering breeding methods utilizing maize salt-tolerant regulatory genes offer advantages such as wider disease resistance, simpler operation, shorter cycle, economic efficiency, and suitability for cultivating broad-spectrum, durable, and highly disease-resistant varieties. Combining genetic engineering technology with... ZmHTA1 Genetically created salt-tolerant maize germplasm for maize ZmHTA1This laid the foundation for gene function research and the breeding of new salt-tolerant maize varieties. Attached Figure Description

[0013] Figure 1 This invention provides phenotypic observations of salt stress-treated homozygous mutants, wild-type materials, and overexpression materials. Figure 1 A represents maize from mutant, wild-type, and overexpression plants. ZmHTA1 Gene expression level detection showed that the expression level of this gene in the knockout plant was close to 0, while the expression level of this gene in the overexpression plant was significantly higher than that in the wild type, indicating that knockout plants and overexpression plants were successfully obtained. Figure 1 In Figure B, the growth of plants after salt stress treatment with 100 mM / L NaCl is shown. The results show that the growth rate of homozygous mutants is significantly slowed down after salt stress, the leaves are severely curled, and the plant height is severely limited. In contrast, the overexpressing plants have better growth and significantly enhanced resistance to salt stress. KO#1 is a knockout mutant transgenic plant; OE#1 is an overexpressing transgenic plant.

[0014] Figure 2 Wild type in the embodiments of the present invention ZmHTA1 homozygous mutant ( WHO # 1 and WHO # 2 Results of POD activity detection in OE#1 and OE#2 plants after salt stress treatment. Figure 2 The results are as follows: POD activity was measured on day 7; after 7 days of salt stress, ZmHTA1 Knockout maize exhibited significantly lower POD activity than wild-type maize under salt stress, while overexpression plants showed higher levels of POD activity, significantly higher than both wild-type and mutant plants. This indicates that... ZmHTA1 Knockout maize significantly reduced its resistance to salt stress. Detailed Implementation

[0015] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified, the equipment and reagents used in the embodiments and experimental examples are commercially available. Unless otherwise stated, all reagents used in this invention are analytical grade reagents. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0016] Example 1: Corn ZmHTA1 Acquisition of genes In the early stages of this invention, bioinformatics functional analysis of the maize genome was performed, revealing a gene suspected to be related to maize salt tolerance, which was named... ZmHTA1 Genes, the ones mentioned ZmHTA1The gene is a heat shock protein gene, and its nucleotide sequence is shown in SEQ ID NO. 1. ZmHTA1 The CDS sequence of the gene is shown in SEQ ID NO. 4. ZmHTA1 The amino acid sequence encoded by the gene is shown in SEQ ID NO. 2.

[0017] Example 2: Corn ZmHTA1 Gene function identification The main steps include: 1.1 Construction of CRISPR / CAS9 vectors and overexpression vectors Combining a corn database and an online website for CRISPR / P target design, we can design targets that can be targeted. ZmHTA The sgRNA was obtained. Primers were designed based on the target quantity and sequence, and PCR amplification was performed. After fragment recovery, the linearized vector pCXB053 was recombined with the target and related sequences through recombination ligation to obtain the desired vector. PCR verification and sequencing alignment yielded the desired result. ZmHTA1 CRISPR-20 gene knockout vector ZmHTA1 Simultaneously, using pCAMBIA3301 as the carrier framework, a system was constructed. ZmHTA1 Gene overexpression vectors.

[0018] 1.2. CRISPR- ZmHTA1 Agrobacterium transformation of vectors and overexpression vectors CRISPR-CRI carrying sgRNA target sequence ZmHTA1 The vector and the overexpression vector of the gene were transformed into Agrobacterium strain EHA105, which has a strong infectivity for maize, by electroporation. Positive strains were screened on LB medium containing kanamycin and rifampicin antibiotics at 28°C. The positive single colonies were shaken and stored in a -80°C freezer with 30% glycerol for the next step of maize genetic transformation.

[0019] 1.3. Corn ZmHTA1 Obtaining mutants and overexpression plants Using freshly peeled corn embryos approximately 1 mm in size as material, the peeled corn embryos were placed in 2 ml plastic centrifuge tubes containing 1.8 mL of suspension. Approximately 150 immature embryos were processed within 30 minutes. The suspension was then removed, leaving the corn embryos in the tubes. 1.0 ml of Agrobacterium suspension was added to the remaining embryos, and the tubes were incubated for 5 minutes. The embryos in the centrifuge tubes were then resuspended and transferred to a co-culture medium. Excess Agrobacterium suspension on the surface was removed using a pipette, and the embryos were co-cultured in the dark at 23°C for 3 days. After co-culture, the embryos were transferred to resting medium and incubated in the dark at 28°C for 6 days. They were then transferred to selection medium containing diammonium phosphate for two weeks of selection culture, followed by two weeks of selection culture on a new selection medium. Resistant callus tissue was transferred to differentiation medium and cultured at 25°C, 5000 lx, under light for 3 weeks. The differentiated seedlings were then transferred to rooting medium and cultured at 25°C, 5000 lx, under light until rooting occurred. The seedlings were then transferred to small pots for further growth. After a certain growth stage, they were transplanted into a greenhouse. Genomic DNA was extracted from leaves, and PCR amplification was performed on positive plants. The expression level of positive plants was then detected. Figure 1 (A) Primers for editing detection were designed, and DNA from positive plants was amplified. After sequencing, the editing status was analyzed, revealing that the transformation event M231538A012a resulted in homozygous editing at both target sites. ZmHTA1 Homozygous mutant plants (Table 1) were obtained, and overexpressing transgenic lines were obtained after positive plant identification. Seeds of the offspring were harvested 3-4 months later.

[0020] See Table 1: Transformed plants of generation T0 in the embodiments of the present invention. ZmHTA1 Results of homozygous mutation editing detection. The results showed that the transformation event M231538A012a involved a deletion of one base at sgRNA1 (SEQ ID NO. 3) and an insertion of an A base at sgRNA2 (SEQ ID NO. 5), resulting in the base insertion or deletion alterations described above, leading to changes in the encoded amino acid and causing… ZmHTA1 Changes in the function of genes.

[0021] Table 1 Mutation types of the T0 generation ZmHTA1 mutant

[0022] 1.4. ZmHTA1 Salt tolerance identification of homozygous mutants and overexpression transgenic lines The above obtained ZmHTA1 Using homozygous mutants and overexpressing transgenic lines as materials, salt stress was applied using 100 mM / L NaCl. Antioxidant enzyme-related physiological indicators were measured during seed germination and seedling stages, and the activity of POD in seed embryos and three-leaf stage seedlings was determined. Results showed that knockout… ZmHTA1After gene modification, maize (KO#1) became more sensitive to salt stress, its growth rate slowed significantly, its leaves curled severely, and its plant height was severely limited. Figure 1 (B), while overexpression ZmHTA1 The genetically modified plant (OE#1) grew better. Figure 1 (B) indicates ZmHTA1 Genes play a crucial regulatory role in the salt tolerance of maize. Secondly, ZmHTA1 There is a close relationship between genes and the antioxidant enzyme POD. After 7 days of salt stress, ZmHTA1 Knockout maize (KO#1, KO#2) showed significantly lower POD activity than WT maize under salt stress, while overexpression plants (OE#1, OE#2) exhibited higher levels of POD activity, significantly higher than wild-type and mutants. This indicates that... ZmHTA1 Knockout maize significantly reduced its resistance to salt stress. Figure 2 ).

[0023] In summary, this invention cloned a maize salt tolerance-related gene. ZmHTA1 For the first time, maize genes were obtained through gene knockout and transgenic technology. ZmHTA1 The loss-of-function and overexpression lines elucidated the salt tolerance regulatory function of this gene, revealing that this gene has a positive regulatory function in improving the salt tolerance of maize. ZmHTA1 Mutations in this gene significantly weaken the salt tolerance of maize, while overexpression of this gene enhances its salt tolerance. Therefore, by constructing an overexpression vector for this gene and using genetic engineering techniques to create and obtain new salt-tolerant maize materials, we can lay the foundation for breeding new salt-tolerant maize varieties.

[0024] The above description, in conjunction with specific embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, several simple deductions or substitutions can be made without departing from the concept of the present invention, and all such deductions or substitutions should be considered to fall within the scope of protection defined by the claims submitted herein.

Claims

1. A salt-tolerant gene for maize, characterized in that, The salt tolerance gene is ZmHTA1 Gene, ZmHTA1 The nucleotide sequence of the gene is shown in SEQ ID NO.

1. ZmHTA1 The amino acid sequence encoded by the gene is shown in SEQ ID NO.

2. ZmHTA1 The CDS sequence of the gene is shown in SEQ ID NO.

4.

2. A method for identifying salt-tolerant maize varieties, characterized in that, The method includes detecting varieties. ZmHTA1 Gene expression, if the ZmHTA1 If the gene is expressed at a higher level than in the wild-type variety, then the variety is a salt-tolerant variety. ZmHTA1 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

3. Salt tolerance gene ZmHTA1 Its application in the genetic production of salt-tolerant maize varieties is characterized by, The application is to improve ZmHTA1 The expression of the gene in maize improves the salt tolerance of maize varieties. ZmHTA1 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

4. A method for preparing salt-tolerant maize varieties, characterized in that, The method includes the following steps: (1) Construction ZmHTA1 Gene overexpression vector pCAMBIA3301- ZmHTA1, The ZmHTA1 The nucleotide sequence of the gene is shown in SEQ ID NO. 1; (2) Overexpression vector pCAMBIA3301- ZmHTA1 The bacteria were transformed into Agrobacterium strain EHA105, which has a strong ability to infect maize embryos, using an electroporation method, for the next step of maize genetic transformation. (3) The above-mentioned Agrobacterium vector was transferred into maize by the genetic transformation method of Agrobacterium infection of maize embryos to obtain transgenic maize plants. The overexpressing plants were screened to obtain salt-tolerant varieties.

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