Application and method of osgasr1 gene in regulating aluminum toxicity stress tolerance of rice

Abstract

The application provides application and methods of OsGASR1 genes in regulating aluminum toxicity stress tolerance of rice, and belongs to the technical field of plant genetic engineering. By adjusting the expression amount of the OsGASR1 gene in the rice genome, the aluminum toxicity stress tolerance of the rice can be changed; overexpression of the OsGASR1 gene in the target rice germplasm genome can improve the aluminum toxicity stress tolerance of the rice, and the application not only provides new theoretical evidence for the research on the aluminum toxicity tolerance of the rice, but also provides a new target gene or new material for cultivating new varieties of high-quality aluminum toxicity tolerance rice (crops).

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to the application and method of the OsGASR1 gene in regulating the tolerance of rice to aluminum toxicity stress. Background Technology

[0002] With the continuous advancement of breeding and cultivation techniques, rice yields have steadily increased. However, various biotic and abiotic stresses in the soil remain the main factors affecting the planting area and quality of rice in my country. Among these, toxic metal ion stress has a significant impact on rice, with aluminum toxicity being a major threat in acidic soils. The mechanism of rice's tolerance to aluminum toxicity is highly complex. Although some genes regulating aluminum detoxification in rice have been cloned, the molecular mechanisms of rice's tolerance to aluminum toxicity still need further exploration. Therefore, developing and cultivating crops resistant to acidic aluminum-toxic soils has become a focus for researchers, which is of paramount importance for improving land utilization and crop quality and yield in areas with acidic aluminum-toxic soils.

[0003] Roots are vital organs for plants to absorb water and nutrients from the surrounding soil. When plants grow in an aluminum-containing environment, the root system is the first to be affected. Aluminum mainly accumulates in the epidermis and cortical tissue of the roots, with micromolar concentrations of Al... 3+ It can inhibit root growth. When plants are exposed to aluminum for extended periods, the roots are damaged, limiting their access to water and minerals, leading to reduced root volume, inhibited root elongation, a significant decrease in the number of lateral roots, and a reduction in biomass. For example, aluminum toxicity affects the division and elongation of citrus root tip cells, resulting in thickening of the root cell walls. After aluminum binds to the cell wall, it reduces cell wall extensibility, leading to a significant slowdown in soybean root growth. Studies have shown that aluminum toxicity reduces root growth by inhibiting auxin transport, thereby causing changes in cytosolic calcium levels. 2+ This leads to an imbalance. Furthermore, aluminum toxicity can ultimately limit root growth by altering the stability of microtubules and microfilaments.

[0004] Members of the GAST gene family are mostly expressed in the most vigorous growth areas of plant tissues or organs, and have a variety of biological functions. For example, in Arabidopsis, AtGASA4 accelerates seed germination by promoting GA signaling and inhibiting redox activity, while AtGASA5 inhibits stem elongation and flowering time. However, in tomato, GAST1 and in petunia, GIP2 promote stem elongation by promoting cell elongation. AtGASA4 and AtGASA14 proteins can interact with the cell membrane localization receptor-like kinase protein VH1 / BRL2, participating in leaf vein development. Furthermore, in rice, OsGASR1 and OsGASR2 are involved in cell division and play different roles in panicle differentiation. In addition, GAST is also involved in many plant hormone responses, including gibberellin (GA), abscisic acid (ABA), auxin (IAA), brassinosteroids (BR), salicylic acid (SA), cytokinin (CTK), jasmonic acid (JA), and more. Most members of the GAST gene family are upregulated by GA, such as tomato GAST1, Arabidopsis thaliana AtGASA4 / 6 / 7 / 8 / 13, rice OsGASR1 / 2 / 7, maize ZmGSL1, and soybean GsGASA1. However, the expression of some GAST genes is downregulated by GA, such as Arabidopsis thaliana AtGASA1, AtGASA5, AtGASA9, and AtGASA11. ABA induces the expression of AtGASA2 / 3 / 5 / 14 in Arabidopsis thaliana and inhibits the expression of AtGASA7 and AtGASA9. GAST1, FsGASA4, and GsGASA1 are antagonistically regulated by GA and ABA. The expression of rice OsGASR7 is suppressed by GA and induced by BR. Furthermore, AtGASA4 and AtGASA6 are simultaneously upregulated by IAA, BR, CTK, and GA, and downregulated by ABA, JA, and salicylic acid (SA). These results suggest that the GASA gene family may be involved in a broad plant hormone signal transduction network. In the rice GAST gene family, only four genes were functionally investigated. OsGASR1 and OsGASR2 were isolated by EST analysis and showed high expression in the apical meristems of shoots and roots, indicating their roles in cell division and proliferation. OsGASR1 has been reported to be involved in rice tolerance to salt and reactive oxygen species and has a negative effect on plant responses to GA and the expression of α-amylase genes. OsGASR1 is antagonistically regulated by GA and BR and is thought to be involved in the regulation between the GA and BR signaling pathways.

[0005] Rice (Oryza sativa), as one of the staple foods for most of the world's population, has a yield directly related to the normal and orderly development of human society. Aluminum (Al), as the most abundant metallic element in the Earth's crust, is naturally deposited in bauxite as insoluble oxides or aluminosilicates in neutral or slightly acidic soils. It has low activity, is not easily absorbed by plants, and is non-toxic. However, when the soil pH is below 5.5, the low-activity aluminum dissolves and becomes activated, releasing into the soil as soluble aluminum ions. This leads to excessive absorption of aluminum by plant roots, causing toxicity to plants. With increasingly severe industrial development, the area of ​​acidic soil caused by acid rain is becoming more extensive. Therefore, researching different strategies to enhance the tolerance of rice plants to aluminum toxicity is of paramount importance. Summary of the Invention

[0006] The purpose of this invention is to provide the application and method of the OsGASR1 gene in regulating aluminum toxicity stress tolerance in rice. Overexpression of the OsGASR1 gene can improve the aluminum toxicity stress tolerance of rice. This invention not only provides new theoretical evidence for the study of aluminum toxicity tolerance in rice, but also provides a new target gene or new material for breeding high-quality aluminum-tolerant rice (crop) varieties.

[0007] This invention provides the application of the OsGASR1 gene in regulating the tolerance of rice to aluminum toxicity stress, and the CDS nucleotide sequence of the OsGASR1 gene is shown in SEQ ID NO.1.

[0008] The present invention also provides the application of overexpression of the OsGASR1 gene in improving the tolerance of rice to aluminum toxicity stress, wherein the CDS nucleotide sequence of the OsGASR1 gene is shown in SEQ ID NO.1.

[0009] This invention also provides the application of overexpression of the OsGASR1 gene in increasing root elongation in rice under aluminum toxicity stress, enhancing the accumulation of reactive oxygen species in rice root tips under aluminum toxicity stress, and / or upregulating genes related to cell wall modification and oxidative stress under aluminum toxicity stress. The CDS nucleotide sequence of the OsGASR1 gene is shown in SEQ ID NO.1. This invention, using the maize Ubiquitin1 promoter to drive the OsGASR1 gene, found that the roots of the overexpressing lines treated with aluminum toxicity showed better growth than the control group, with a significantly greater relative root elongation than the wild type.

[0010] Preferably, the strong promoter used for the overexpression of the OsGASR1 gene includes the maize Ubiquitin1 strong promoter, the nucleotide sequence of which is shown in SEQ ID NO.4.

[0011] The present invention also provides an expression cassette comprising a maize Ubiquitin1 strong promoter and an OsGASR1 gene; the CDS nucleotide sequence of the OsGASR1 gene is shown in SEQ ID NO.1, and the nucleotide sequence of the maize Ubiquitin1 strong promoter is shown in SEQ ID NO.4.

[0012] The present invention also provides an expression vector, which includes the expression cassette described in the above technical solution.

[0013] Preferably, the base vector for constructing the expression vector includes the pCAMBIA1300 vector.

[0014] The present invention also provides an engineered bacterium, wherein the engineered bacterium includes the expression cassette or the expression vector described in the above technical solution.

[0015] The present invention also provides a method for improving the tolerance of rice to aluminum toxicity stress, comprising the following steps: overexpressing the OsGASR1 gene in the genome of a target rice germplasm, wherein the CDS nucleotide sequence of the OsGASR1 gene is shown in SEQ ID NO.1.

[0016] Preferably, the expression of the OsGASR1 gene is driven by the maize Ubiquitin1 strong promoter, the nucleotide sequence of which is shown in SEQ ID NO.4.

[0017] This invention provides the application of the OsGASR1 gene in regulating rice's tolerance to aluminum toxicity stress. By regulating the expression level of the OsGASR1 gene in the rice genome, the rice's tolerance to aluminum toxicity stress can be altered. Overexpression of the OsGASR1 gene in the genome of target rice germplasm can improve rice's tolerance to aluminum toxicity stress. This invention not only provides new theoretical evidence for the study of rice's aluminum toxicity tolerance but also provides new target genes or new materials for breeding high-quality aluminum-tolerant rice (crop) varieties. This invention uses homologous recombination technology to construct the Ubiquitin1 strong promoter and the OsGASR1 gene into the pCAMBIA1300 vector. Rice callus tissue was transformed via Agrobacterium-mediated transformation, and two plants with different expression levels were screened. Further phenotypic experiments were conducted, observing the root growth of aluminum-treated and untreated plants. The results showed that, compared with the wild type, the overexpressing plants had longer roots and significantly higher relative root elongation when treated with aluminum toxicity. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 Phylogenetic tree analysis of OsGASR1 homologous genes in rice and Arabidopsis thaliana provided for this invention;

[0020] Figure 2 The diagram shows the relative expression levels of the OsGASR1 gene in different tissues and roots of rice, as provided by this invention.

[0021] Figure 3 The graph shows the relative expression levels under different metal stress treatments, different aluminum toxicity concentrations, and different durations of aluminum toxicity treatment, as provided by this invention.

[0022] Figure 4 The diagram shows the overexpression pattern of the OsGASR1 gene and the results of screening and identifying the relative expression levels of plants overexpressing OsGASR1, which are provided for this invention.

[0023] Figure 5 The diagram shows the subcellular localization of OsGASR1 in rice protoplasts provided by this invention.

[0024] Figure 6 The results of tissue localization of OsGASR1 in rice roots provided by this invention are shown in the figure.

[0025] Figure 7 Phenotypic diagrams of wild-type plants and OsGASR1-overexpressing plants under normal and aluminum toxicity stress, as well as root length and root-to-root length results after treatment, are provided for the present invention.

[0026] Figure 8 The diagram shows the results of ROS accumulation in the roots of wild-type plants and plants overexpressing OsGASR1 under normal and aluminum toxicity stress conditions provided by this invention.

[0027] Figure 9 The figure shows the effect of OsGASR1 overexpression on the expression of genes related to oxidative stress and cell wall modification, as provided by this invention. Detailed Implementation

[0028] This invention provides the application of the OsGASR1 gene in regulating aluminum toxicity stress tolerance in rice. The CDS nucleotide sequence of the OsGASR1 gene is shown in SEQ ID NO.1: ATGAAGCTCAACACCACCACCACCCTGGCTCTCCTCCTGCTCCTGCTCTTGGCCTCCTCTTCCCTCCAAGTTTCCATGGCTGGATCAGATTTCTGCGACGGCAAGTGCAAGGTGAGGTGCTCGAAGGCGAGCAGGCACGACGACTGCCTCAAGTACTGCGGCGTGTGCTGCGCCTCCTGCAACTGCGTGCCGTCGGGGACGGCCGGCAACAAGGACGAGTGCCCCTGCTACCGCGACATGACCACCGGCCATGGCGCTCGCAAGAGGCCCAAGTGCCCATGA. In the present invention, the genomic nucleotide sequence of the OsGASR1 gene is such as SEQ ID Shown in NO.2: ATGAAGCTCAACACCACCACCCTGGCTCTCCTCCTGCTCCTGCTCTTGGCCTCCTCTCCTCCAAGTTTCCATGGCTGGATCAGGTCAGTTTTCACAGGCATTTTTTTTAGCAGAATAATCTGCACAAGTACACTGTTTTTCTTTCTTGGTGTTAACATGGTGAGCTGAAACAATGAATGTTTTGG TAGATTTCTGCGACGGCAAGTGCAAGGTGAGGTGCTCGAAGGCGAGCAGGCACGACGACTGCCTCAAGTACTGCGGCGTGTGCTGCGCCTCCTGCAACTGCGTGCCGTCGGGGACGGCCGGCAACAAGGACGAGTGCCCCTGCTACCGCGACATGACCACCGGCCATGGCGCTCGCAAGAGGCCCAAGTGCCCATGA. In this invention, the amino acid sequence of the OsGASR1 protein is shown in SEQ ID NO.3: MKLNTTTTLALLLLLLLASSSLQVSMAGSDFCDGKCKVRCSKASRHDDCLK YCGVCCASCNCVPSGTAGNKDECPCYRDMTTGHGARKRPKCP.The OsGASR1 gene is located in the nucleus and endoplasmic reticulum of cells. The full-length CDS of OsGASR1 is 282 bp, containing two exons and one intron. It is expressed in both the roots and stems of rice, with higher expression levels in the stems than in the roots. Under aluminum toxicity treatment, the expression level of OsGASR1 in the roots significantly increases, while the expression level in the stems remains unaffected. Furthermore, in rice roots, expression is mainly concentrated 1–2 cm from the root tip and is highly induced by aluminum toxicity. OsGASR1 expression is primarily induced by Al, with lower induction by Cd. Its expression level increases with increasing aluminum toxicity concentration. Further time-series expression analysis shows that the expression level of OsGASR1 is rapidly upregulated in the presence of aluminum toxicity. Modulating the expression level of the OsGASR1 gene in the rice genome can alter the rice's tolerance to aluminum toxicity stress; overexpression of the OsGASR1 gene in the genome of target rice germplasm can improve rice's tolerance to aluminum toxicity stress. This invention provides new theoretical evidence for the study of aluminum toxicity tolerance in rice, and also provides new target genes or new materials for the breeding of high-quality aluminum-tolerant rice (crop) varieties.

[0029]

[0030] This invention also provides the application of overexpression of the OsGASR1 gene in increasing root elongation in rice under aluminum toxicity stress, enhancing the accumulation of reactive oxygen species in rice root tips under aluminum toxicity stress, and / or upregulating genes related to cell wall modification and oxidative stress under aluminum toxicity stress. The CDS nucleotide sequence of the OsGASR1 gene is shown in SEQ ID NO.1. In this invention, the strong promoter used for overexpression of the OsGASR1 gene preferably includes the maize Ubiquitin1 strong promoter, the nucleotide sequence of which is shown in SEQ ID NO.4. In this invention, the genes related to cell wall modification and oxidative stress preferably include OsPME1, OscytME2, OsGRP2, OsPRP, PRX115, OsIAA16, OsABCG11, and Os09g0330000.

[0031] The present invention also provides an expression cassette comprising a maize Ubiquitin1 strong promoter and an OsGASR1 gene; the CDS nucleotide sequence of the OsGASR1 gene is shown in SEQ ID NO.1, and the nucleotide sequence of the maize Ubiquitin1 strong promoter is shown in SEQ ID NO.4.

[0032] The present invention also provides an expression vector, which includes the expression cassette described in the above technical solution.

[0033] In this invention, the base vector for constructing the expression vector includes the pCAMBIA1300 vector.

[0034] The present invention also provides an engineered bacterium, wherein the engineered bacterium includes the expression cassette or the expression vector described in the above technical solution.

[0035] In this invention, homologous recombination technology is preferably used to construct the strong promoter of Ubiquitin1 and the CDS of the OsGASR1 gene into the pCAMBIA1300 vector to obtain the pCAMBIA1300-ProUbi:OsGASR1 plasmid. This plasmid is then transformed into rice callus tissue via Agrobacterium-mediated transformation to construct rice gene overexpression plants.

[0036] This invention also provides a method for improving the tolerance of rice to aluminum toxicity stress, comprising the following steps: overexpressing the OsGASR1 gene in the genome of a target rice germplasm, wherein the CDS nucleotide sequence of the OsGASR1 gene is shown in SEQ ID NO.1. In this invention, the expression of the OsGASR1 gene is preferably driven by the maize Ubiquitin1 strong promoter, wherein the nucleotide sequence of the maize Ubiquitin1 strong promoter is shown in SEQ ID NO.4. After overexpressing the OsGASR1 gene in the genome of the target rice germplasm, phenotypic experiments were conducted on the plants. After aluminum toxicity treatment, it was found that under aluminum toxicity treatment conditions, compared with the wild type, the overexpressing plants were more tolerant to aluminum toxicity stress, and their root growth was significantly longer than that of the wild type, indicating that OsGASR1 is a gene involved in rice aluminum toxicity tolerance. This invention, through observation of traits, confirms that OsGASR1 participates in the aluminum toxicity stress response process of rice.

[0037] To further illustrate the present invention, the application and method of the OsGASR1 gene provided by the present invention in regulating rice aluminum toxicity stress tolerance are described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0038] Example 1

[0039] 1. Plant materials and growing conditions

[0040] The materials used in this embodiment are wild-type Nipponbare rice and two OsGASR1 overexpressing plants (the construction method is detailed in Part 3 below).

[0041] Rice seeds were soaked in distilled water and then placed in an incubator at 30℃ for 2 days to germinate. After germination, the seeds were placed on a net floating with a 0.5 mM CaCl2 solution. After 3 days of growth, seedlings with good growth and uniform root length were selected and placed in 0.5 mM CaCl2 solutions (pH 4.5) containing and without aluminum toxicity, respectively, and cultured in an incubator (14 h light / 10 h dark) for 24 h for physiological phenotypic analysis.

[0042] 2. Obtaining the complete CDS sequence of the OsGASR1 gene and constructing its phylogenetic tree.

[0043] To obtain the CDS sequence of OsGASR1, total RNA was extracted from rice roots using a Trizol kit (Life Technologies) following the product instructions. The first strand of cDNA was synthesized using 1 μg of total RNA using a Hiscript IIQ RT SuperMix Kit (Vazyme) following the product instructions. The obtained cDNA was used as a template to obtain the full-length CDS sequence of OsGASR1 via PCR amplification.

[0044] Phylogenetic trees of the GAS family proteins in rice and Arabidopsis thaliana were constructed primarily by downloading the full-length amino acid sequences of this family from TAIR (https: / / www.arabidopsis.org / ) and NCBI (www.ncbi.nlm.nih.gov). The phylogenetic trees were then constructed using MEGAX software via the nearest neighbor method. The results are as follows: Figure 1 As shown.

[0045] Depend on Figure 1 Phylogenetic analysis revealed that OsGASR1 has the highest homology with AtGASA10 (At5g59845) in Arabidopsis thaliana. Among the OsGASRs in the rice family, Os03g0607200 and OsGASR9 (Os07g0592000) are the closest in homology to OsGASR1. Phylogenetic analysis suggests that OsGASR1 may have similar functions to these members.

[0046] 3. RNA extraction and gene expression level analysis

[0047] To analyze the expression patterns of OsGASR1 in different tissues and the expression levels in different parts of the root, roots, stems, and root samples (0–1 cm and 1–2 cm from the root tip) were taken from wild-type plants at the heading stage and immediately placed in liquid nitrogen. Total RNA was extracted from rice roots using the Trizol kit (Life Technologies) according to the product instructions. The first strand of cDNA was synthesized using the Hiscript IIQ RT SuperMix Kit (Vazyme) according to the product instructions at a rate of 1 μg of total RNA. ChanQ was used... TM RT-qPCR experiments were performed using the SYBR Color qPCR Master Mix (Vazyme) kit and the StepOnePlus Real-Time PCR system (Analytikjena).

[0048] The primer sequences used for OsGASR1 gene expression were (SEQ ID NO.5: 5'-ATGAAGCTCAACACCACCAC-3' and SEQ ID NO.6: 5'-CAGTACTTGAGGCAGTCGTC-3'); the internal control was histone H3, with primer sequences (SEQ ID NO.7: 5'-GGTCAACTTGTTGATTCCCCTCT-3' and SEQ ID NO.8: 5'-AACCGCAAAATCCAAAGAACG-3'). The results are as follows... Figure 2 As shown.

[0049] Depend on Figure 2 It can be seen that, in the absence of aluminum, the expression level of the OsGASR1 gene in the root is low, and the expression level in the stem is about 7 times that in the root. However, in the presence of aluminum, the expression level in the root is upregulated by nearly 10 times, while the expression level in the stem is basically unaffected. Figure 2 The relative expression levels of A and OsGASR1 in rice roots and stems were analyzed. Spatial expression analysis showed that OsGASR1 was expressed at distances of 0–1 cm and 1–2 cm from the root tip, and its expression levels in both regions were upregulated by aluminum. Figure 2 (Relative expression levels of B, OsGASR1 in different segments of the root).

[0050] To analyze the response of OsGASR1 expression levels to different metal elements, wild-type rice seedlings aged 5 days were first treated with aluminum, cadmium, lanthanum, and zinc for 6 hours, with no metal element treatment serving as a control. Next, seedlings were treated with 0 μM, 10 μM, 30 μM, 50 μM, and 100 μM Al for 6 hours. Finally, wild-type seedlings were treated with 50 μM Al at 0 h, 2 h, 4 h, 8 h, 12 h, and 24 h. RNA was extracted from these samples and analyzed by RT-qPCR. Each experiment was performed in triplicate. The results are shown below. Figure 3 As shown, A represents the relative expression level of OsGASR1 in response to different metal elements; B represents the dose-dependent relative expression level of OsGASR1 in rice roots in response to aluminum; and C represents the time-dependent relative expression level of OsGASR1 in rice roots.

[0051] Depend on Figure 3 It can be seen that the expression of OsGASR1 is mainly induced by Al, less so by Cd, and not induced by La and Zn. Figure 3 (A) Concentration gradient expression analysis showed that the expression level of OsGASR1 increased with increasing aluminum toxicity concentration. Figure 3 (B in the text). Time-series expression analysis showed that the expression level of OsGASR1 was rapidly upregulated by aluminum, reaching a maximum of 13 times that under normal conditions at 8 hours after aluminum treatment. Figure 3 (C in the middle).

[0052] 4. Construction of OsGASR1 overexpression plants

[0053] The full-length genome of the OsGASR1 gene (SEQ ID NO.2) is 387 bp, containing 3 exons and 2 introns, and its CDS (SEQ ID NO.1) is 283 bp in length.

[0054] OsGASR1 overexpression plants were constructed by driving OsGASR1 expression using a strong promoter of maize Ubiquitin1 (SEQ ID NO.4). First, specific primer 1 (SEQ ID NO.9: 5'-TACGAATTCGAGCTCGGTACCGTCGTGCCCCTCTCTAGAGATAAT-3' and SEQ ID NO.10: 5'-GTGTTGAGCTTCATCTGCAGAAGTAACACCAAACAACAG-3') was designed to amplify the promoter sequence of Ubiquitin1 from the maize genome. Then, specific primer 2 (SEQ ID NO.11: 5'-CTGCAGATGAAGCTCAACACCACCACCA-3' and SEQ ID NO.12: 5'-CAGGTCGACTCTAGAGGATCCTTGCACAGTTGTGTGGGGG-3') was designed to amplify the genomic CDS sequence of OsGASR1. The ProUbi:OsGASR1 sequence was constructed into the pCAMBIA1300 vector using homologous recombination to obtain the pCAMBIA1300-ProUbi:OsGASR1 vector, which was then transformed into DH5α competent cells. After extracting plasmids from positive clones, they were sent to the company for sequencing. The plasmid pCAMBIA1300-ProUbi:OsGASR1 with the correct results was selected, and wild-type Nipponbare rice was constructed using Agrobacterium-mediated transformation to induce overexpression. RNA was extracted from both wild-type and overexpression plants and reverse-engineered into cDNA. Quantitative primers for the OsGASR1 gene were designed (SEQ ID NO.5: 5'-ATGAAGCTCAACACCACCAC-3' and SEQ ID NO.6: 5'-CAGTACTTGAGGCAGTCGTC-3'). Histone H3 was selected as the internal control, with primer sequences of (SEQ ID NO.7: 5'-GGTCAACTTGTTGATTCCCCTCT-3' and SEQ ID NO.8: 5'-AACCGCAAAATCCAAAGAACG-3'). The relative expression level of OsGASR1 was verified, and two independent overexpression plants with different expression levels were screened. The results are as follows: Figure 4 As shown.

[0055] 5. Subcellular localization analysis of OsGASR1

[0056] To analyze the subcellular localization of OsGASR1, an OsGASR1-GFP fusion expression vector was constructed.

[0057] The CDS sequence of OsGASR1 (with the stop codon removed) was amplified by PCR using OsGASR1-specific primers (SEQ ID NO.13: 5'-CCCGGAATTCTATGAAGCTCAACACCACCAC-3' and SEQ ID NO.14: 5'-CCGGGGTACCTCATGGGCACTTGGGCCTCT-3'). The amplified fragment was then cloned before the GFP coding region of the pUC18-GFP vector (purchased from Takara, with the GFP sequence added at the multiple cloning site) to generate the OsGASR1-GFP recombinant plasmid.

[0058] The experimental group used the OsGASR1-GFP recombinant plasmid, the nuclear marker plasmid Ghd7, and the endoplasmic reticulum marker plasmid HDEL, while the control group used the empty GFP vector and the nuclear marker plasmid Ghd7. These three co-transformed plasmids were used to transform protoplasts in rice protoplast cells via PEG-mediated transformation. After incubation at room temperature, subcellular localization imaging was performed using a laser confocal scanning microscope (TCS SP8; Leica). The results are as follows: Figure 5 As shown.

[0059] Depend on Figure 5 a~ Figure 5 As shown in d, the empty GFP vector (green fluorescence) is localized in the cytoplasm and nucleus, while the nuclear marker protein Ghd7 (red fluorescence) is localized only in the nucleus. However, due to... Figure 5 e in Figure 5 As shown in Figure 1, OsGASR1-GFP can co-localize with both nuclear Ghd7 and endoplasmic reticulum HDEL markers. These results indicate that OsGASR1 is localized in both the nucleus and endoplasmic reticulum.

[0060] 6. Tissue localization analysis of OsGASR1 in rice roots

[0061] To analyze the tissue localization of OsGASR1 in roots, a pOsGASR1:OsGASR1-GFP fusion expression vector was constructed.

[0062] Use pOsGASR1-specific primers (primers designed based on the CDS sequence of the OsGASR1 gene).

[0063] The promoter sequence of OsGASR1 was amplified by PCR using primers (SEQ ID NO.15: 5'-ACGACGGCCAGTGCCAAGCTTTTCTCGCCGTGTGCCATC-3' and SEQ ID NO.16: 5'-GGTGTTGAGCTTCATTGTGCTTGTGAATGCTGGTCC-3'). The CDS sequence of OsGASR1 was amplified by PCR using OsGASR1-CDS-specific primers (SEQ ID NO.17: 5'-GCACAATGAAGCTCAACACCACCACCA-3' and SEQ ID NO.18: 5'-GCCCTTGCTCACCATGGATCCTGGGCACTTGGGCCTCTT-3'). The pOsGASR1:OsGASR1 sequence was constructed into the pCAMBIA1300-GFP vector via homologous recombination to obtain the pCAMBIA1300-ProUbi:OsGASR1-GFP vector, which was then transformed into DH5α competent cells. After extracting plasmids from positive clones, they were sent to the company for sequencing. The plasmid pCAMBIA1300-ProUbi:OsGASR1-GFP with the correct results was selected, and wild-type Nipponbare rice was infected with Agrobacterium tumefaciens-mediated genetic transformation to construct tissue-localized plants.

[0064] like Figure 6 As shown, GFP antibody immunostaining experiments revealed that in transgenic lines, fluorescence signals were observed in all cells except the epidermis at a distance of 10 mm from the rice root tip. Figure 6 E- Figure 6 The H in the root was the strongest signal, while no fluorescence signal was observed in the wild-type root. Figure 6 A- Figure 6 The D in the figure indicates that the antibody is specific. These results show that OsGASR1 is expressed in all cells outside the root epidermis, with the highest expression in the stele region.

[0065] 7. Phenotypic analysis of OsGASR1 overexpression lines under aluminum stress

[0066] To further investigate whether overexpression of OsGASR1 in rice can improve the rice's tolerance to aluminum, this invention conducted aluminum stress treatment experiments using wild-type Nipponbare (WT) and OsGASR1 overexpression lines (OE-1, OE-2). The results are as follows... Figure 7 As shown in Figure A, under normal conditions without aluminum, the root elongation of OsGASR1 overexpressing lines was not significantly different from that of wild-type rice. However, under conditions with 30 μM AlCl3 present (pH 4.5), the roots of both overexpressing lines were longer than those of the wild-type. Figure 7 (B in the original text). This invention further analyzed the relative elongation of roots in WT, OE-1, and OE-2 after 24 hours of aluminum treatment. The results showed that the relative elongation of roots in the overexpression line was significantly greater than that in the wild type (B in the original text). Figure 7 C in Figure 7 (D in the text). These results indicate that overexpression of OsGASR1 can significantly enhance rice's tolerance to aluminum toxicity.

[0067] 8. ROS accumulation in the roots of OsGASR1 overexpression lines under aluminum stress

[0068] Aluminum (Al) treatment can induce plants to produce large amounts of highly reactive and unstable reactive oxygen species (ROS). To further analyze ROS accumulation in the roots of OsGASR1 overexpression lines, wild-type and two independent OsGASR1 overexpression lines of rice seedlings were treated with 0 μM or 50 μM aluminum chloride (AlCl3) for 24 h. Subsequently, the roots were immersed in 10 μM of the reactive oxygen species (ROS) fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) and incubated in the dark at 37°C for 30 min, followed by rinsing three times with deionized water. The results showed that under normal conditions, the fluorescence signal intensity of H2DCFDA in the root tips of the OsGASR1 overexpression lines was significantly lower than that of the wild type, indicating reduced ROS production. After treatment with 50 μM AlCl3 for 24 h, the root tips of the OsGASR1 overexpression lines showed a significantly enhanced fluorescence signal compared to the wild type. Figure 8 This indicates that OsGASR1 overexpression enhances the accumulation of reactive oxygen species in rice root tips.

[0069] 9. OsGASR1 overexpression affects the expression profiles of genes related to oxidative stress and cell wall modification.

[0070] To identify the genes regulated by OsGASR1 under aluminum stress, this invention analyzed the root transcriptomes of wild-type (WT) and OsGASR1-overexpressing lines under normal and aluminum-treated conditions using RNA-seq technology. Transcriptome data revealed that OsGASR1 overexpression regulates the expression of many genes related to aluminum toxicity stress. Real-time quantitative PCR validation results showed that OsPME1 (Os03g0309400) (SEQ ID NO.19: CCTCCAAGATCAACGCCAAG and SEQ ID NO.20: GGCTTGGTTTCCGGGATCAT), OscytME2 (Os05g0186300) (SEQ ID NO.21: CATCCATCGTCTCGCAGGA and SEQ ID NO.22: AGCACTCCTCCACGTTGT), OsGRP2 (Os10g0450900) (SEQ ID NO.23: CTACCCAGCTCACTGAAGTA and SEQ ID NO.24: AACCTAGGAGGGTTCTTGCA), OsPRP (Os05g0226900) (SEQ ID NO.25: TAGCGAAGAGGAGGAGCTT and SEQ ID NO.24: AGCACGAAGAGGAGGAGCTT and SEQ ID NO.25: AGCACGAAGAGGAGGAGCTT and SEQ ID NO.25: AGCACGAAGAGGAGGAGCTT and SEQ ID NO.24: AGCACGAAGAGGAGGAGCTT and SEQ ID NO.25: AGCACGAAGAGGAGGAGCTT and SEQ ID NO.24: AGCACGAAGAGGAGGAGCTT and SEQ ID NO.25: AGCACGAAGAGGAGGAGGAGCTT and SEQ ID NO.24: AGCACGAAGAGGAG ... NO.26: CCGCCGCTTCATCAACTAAA), PRX115 (Os07g0677600) (SEQ ID NO.27: GGACTTCTACAGCGAGACGT and SEQ ID NO.28: AGCAGAACGGAGCCATCAC), OsIAA16 (Os05g0186900) (SEQ ID NO.29: GCCCTCAAAGTTTCCTGCTT and SEQ ID NO.30: CTTGAACCCATCGTTTCCCT), OsABCG11 (Os05g0120200) (SEQ ID NO.31: TTCCACTACCTGTCGCTCGT and SEQ ID NO.32: CATCGAGCACCTTGAGCTTG) and Os09g0330000 (SEQ ID NO.33: TTCGTCACCTTCACCGACG and SEQ ID NO.34: AGAAGGACATCTTGGGGACG). Under aluminum treatment conditions, the expression levels of these genes in the overexpression lines were significantly higher than those in the wild type. Figure 9 ).

[0071] From the above examples, it can be concluded that OsGASR1 is a protein located in the cell nucleus and endoplasmic reticulum. Overexpression of OsGASR1 in rice can significantly upregulate genes related to cell wall modification and oxidative stress, thereby enabling overexpressing lines to participate in the tolerance of rice to aluminum toxicity stress.

[0072] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Overexpression OsGASR1 The application of genes in improving rice tolerance to aluminum toxicity stress, the aforementioned OsGASR1 The CDS nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. Overexpression OsGASR1 The application of genes in increasing root elongation in rice under aluminum toxicity stress, enhancing the accumulation of reactive oxygen species in rice root tips under aluminum toxicity stress, and / or upregulating genes related to cell wall modification and oxidative stress under aluminum toxicity stress. OsGASR1 The CDS nucleotide sequence of the gene is shown in SEQ ID NO.1; the gene related to cell wall modification and oxidative stress is... OsPME1 , OscytME2 , OsGRP2 , OsPRP , PRX115 , OsIAA16 , OsABCG11 and Os09g0330000 .

3. The application according to claim 1 or 2, characterized in that, The overexpression OsGASR1 Strong promoters used in genes include maize Ubiquitin1 Strong promoter, the corn Ubiquitin1 The nucleotide sequence of the strong promoter is shown in SEQ ID NO.

4.

4. A method for improving the tolerance of rice to aluminum toxicity stress, characterized in that, Includes the following steps: Overexpression in the target rice germplasm genome OsGASR1 Genes, the ones mentioned OsGASR1 The CDS nucleotide sequence of the gene is shown in SEQ ID NO.

1.

5. The method according to claim 4, characterized in that, Use corn Ubiquitin1 Strong starter driver OsGASR1 Gene expression, the corn Ubiquitin1 The nucleotide sequence of the strong promoter is shown in SEQ ID NO.4.

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