Plant oxalate transport candidate gene and application thereof in relieving aluminum toxicity

Abstract

The application provides a plant oxalic acid transport candidate gene and application thereof in relieving aluminum toxicity, wherein the plant oxalic acid transport candidate gene is an oxalic acid transporter candidate gene HbOT1 and HbOT2 with a SNARE_assoc domain in rubber trees and a homologous gene AtOT with a SNARE_assoc domain in Arabidopsis thaliana, the DNA sequences of the genes are shown as SEQ ID NO. 1-3, the HbOT1, HbOT2 and the homologous gene AtOT can change the oxalic acid metabolism rule of plants, have certain tolerance to high-concentration aluminum stress, and can be applied to oxalic acid transport in roots and relieving aluminum toxicity.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a candidate gene for oxalic acid transport in plants and its application in detoxifying aluminum toxicity. Background Technology

[0002] Aluminum (Al) is the most abundant metallic element in the Earth's crust. When the soil is neutral or slightly acidic, aluminum generally exists in the form of stable silicate minerals and oxides. When the soil pH is less than 5, aluminum is released from the solid phase into the soil solution and adsorbed onto the cation exchange sites on the soil surface as exchangeable aluminum, forming free Al. 3+ Aluminum toxicity increases the activity of aluminum in the soil, leading to aluminum toxicity and adversely affecting plant growth and soil microbial activity. Currently, due to the increasing frequency of global acid rain and the excessive use of chemical fertilizers, soil acidification is severe, and soil aluminum toxicity has become a significant factor affecting global crop yields. Therefore, research on plant aluminum toxicity and its detoxification mechanisms is of great practical significance and is currently a hot topic in plant stress biology research.

[0003] To adapt to high-alumina environments, plants have developed two physiological mechanisms for detoxifying aluminum toxicity: external rejection and internal tolerance. In the external rejection mechanism, the secretion of aluminum ligands is considered the most important pathway for aluminum detoxification in plants. These ligands mainly include organic acids and phosphates. Phosphates primarily function within the plant cell wall, while most of the aluminum ligands secreted outside the plant are organic acids. Once secreted, these organic acids form stable complexes with aluminum, preventing aluminum from entering the symptom and thus achieving detoxification. In the internal tolerance mechanism, the formation of low-toxicity organoaluminum complexes by the complexation of organic acid anions and phenolic substances with aluminum is also a prerequisite for plants to tolerate high concentrations of aluminum within the plant. Therefore, aluminum-induced organic acid synthesis and secretion play a crucial role in plant aluminum tolerance mechanisms.

[0004] The most important organic acids involved in aluminum release in plants are citric acid, malic acid, and oxalic acid. The first malic acid transporter gene, TaALMT1, was identified in wheat, which induces the secretion of malic acid from the root tip to release aluminum. Citric acid transporter genes have also been identified in plants such as Arabidopsis thaliana, barley, and sorghum, which induce the secretion of citric acid from the plant roots to chelate aluminum. 3+ The mechanisms involved in aluminum release have been demonstrated. However, apart from Lv et al. (2021) who screened an aluminum-induced oxalate secretion-related regulatory gene, MsDHN1, through transcriptome analysis of alfalfa under aluminum stress, no other oxalate transporter genes in plants have been identified or reported to date. Summary of the Invention

[0005] Therefore, this invention proposes a candidate gene for oxalic acid transport in plants and its application in detoxifying aluminum toxicity.

[0006] The technical solution of this invention is implemented as follows:

[0007] A candidate gene for oxalic acid transport in plants, wherein the candidate gene is HbOT1 and HbOT2, which are oxalic acid transporter candidate genes with SNARE_assoc domains in rubber trees, and AtOT, a homologous gene with SNARE_assoc domains in Arabidopsis thaliana.

[0008] To further illustrate, the DNA sequences of the oxalate transporter candidate genes HbOT1 and HbOT2 are as shown in SEQ ID NO.1-2; the DNA sequence of the homologous gene AtOT is as shown in SEQ ID NO.3.

[0009] Further explanation: the SNARE-assoc domain sequences of the HbOT1 and HbOT2 genes are shown in SEQ ID NO.4-5; the SNARE-assoc domain sequence of the homologous gene AtOT is shown in SEQ ID NO.6.

[0010] A protein encoded by a candidate gene for oxalate transport, comprising HbOT1 and HbOT2 proteins, wherein HbOT1 and HbOT2 proteins have 4 and 5 transmembrane domains, respectively.

[0011] To further illustrate, the amino acid sequence of the HbOT1 protein is shown in SEQ ID NO.7; and the amino acid sequence of the HbOT2 protein is shown in SEQ ID NO.8.

[0012] Further explanation includes the protein encoded by the Arabidopsis homolog AtOT, whose amino acid sequence is shown in SEQ ID NO. 9.

[0013] Application of an oxalic acid transporter candidate gene, specifically the application of the oxalic acid transporter candidate genes HbOT1 and HbOT2 in detoxification of aluminum toxicity.

[0014] Application of a candidate gene for oxalic acid transport, specifically the application of the Arabidopsis thaliana homolog AtOT in detoxification of aluminum toxicity.

[0015] Application of a candidate gene for rubber oxalic acid transporter, HbOT1, HbOT2, and its homolog AtOT, in root oxalic acid transport.

[0016] Compared with existing technologies, the beneficial effects of this invention are as follows: This invention identifies and clones candidate genes HbOT1 and HbOT2 for oxalate transporters from rubber trees through sequence alignment and homologous cloning; conserved domain prediction analysis reveals that HbOT1 and HbOT2 are both stable hydrophobic proteins with transmembrane structures and possess SNARE_assoc domains, belonging to the SNARE_assoc subfamily of the SNARE superfamily; HbOT1 and HbOT2 show the highest homology with AtOT, a member of the Arabidopsis thaliana SNARE_assoc Golgi protein family, and both HbOT1 and HbOT2 are upregulated in response to aluminum stress; yeast strains transgenic with HbOT1, HbOT2, and AtOT genes exhibit anti-oxalate activity, altering the oxalate metabolism patterns of yeast cells; and they show a certain tolerance to high concentrations of aluminum stress. These genes can serve as candidate genes related to the transport of oxalate and detoxification of aluminum toxicity in rubber tree roots, representing the first report of oxalate transporters in plants, and providing a theoretical reference for in-depth research on the molecular mechanisms of oxalate secretion to detoxify aluminum toxicity in rubber trees and other plants, as well as for aluminum-tolerant genetic engineering breeding. Attached Figure Description

[0017] Figure 1 This is a schematic diagram showing the amplification results of HbOT1, HbOT2, and Arabidopsis thaliana AtOT genes in an embodiment of the present invention; wherein, a represents the amplification results of HbOT1 and HbOT2 genes; and b represents the amplification results of the ATOT gene.

[0018] Figure 2 This is a schematic diagram of the HbOT1 and HbOT2 protein structure prediction and analysis according to an embodiment of the present invention; wherein, A is the prediction of conserved domains of HbOT1 and HbOT2; B is the prediction of secondary structure of HbOT1 and HbOT2; and C is the construction of tertiary structure models of HbOT1 and HbOT2.

[0019] Figure 3 Phylogenetic tree clustering analysis of HbOT1, HbOT2 and Arabidopsis thaliana AtOT proteins from rubber trees and other SNARE family members from other plants, according to embodiments of the present invention.

[0020] Figure 4 This is a schematic diagram showing the subcellular localization results of HbOT1 and HbOT2, candidate genes for oxalic acid transport in rubber trees, according to an embodiment of the present invention.

[0021] Figure 5 This is a schematic diagram showing the tissue expression analysis results of HbOT1 and HbOT2, candidate genes for oxalic acid transport in rubber trees, according to an embodiment of the present invention.

[0022] Figure 6 This is a schematic diagram showing the expression analysis results of HbOT1 and HbOT2 genes in the roots of rubber trees under different concentrations of aluminum stress according to an embodiment of the present invention.

[0023] Figure 7 This is a schematic diagram showing the expression analysis results of HbOT1 and HbOT2 genes in the roots of rubber trees under different aluminum stress treatment times according to an embodiment of the present invention.

[0024] Figure 8 This is a schematic diagram illustrating the identification of the oxalic acid resistance activity of rubber tree oxalate transport candidate genes HbOT1, HbOT2, and AtOT in a yeast system according to an embodiment of the present invention.

[0025] Figure 9 This is a graph showing the changes in oxalic acid content in the culture medium and cells of different yeast cells under 2 mmol / L oxalic acid stress according to an embodiment of the present invention; where A represents the oxalic acid content in the cells and B represents the oxalic acid content in the culture medium.

[0026] Figure 10 This is a schematic diagram illustrating the aluminum tolerance identification of HbOT1, HbOT2, and AtOT yeasts according to an embodiment of the present invention.

[0027] Figure 11 This is a comparative analysis diagram of the promoter-cis-acting elements of HbALOT1, HbALOT2 and Arabidopsis homolog AtOT in this invention. Detailed Implementation

[0028] To better understand the technical content of this invention, specific embodiments are provided below to further illustrate the invention.

[0029] Unless otherwise specified, the experimental methods used in the embodiments of this invention are all conventional methods.

[0030] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available.

[0031] This invention uses tissue-cultured 'Reyan 7-33-97' rubber tree seedlings with uniform growth and two tufts of leaves as plant material, and conducts hydroponic cultivation in a light incubator using Hoagland nutrient solution. The incubator conditions are set as follows: 28℃ for 16 hours of light, 25℃ for 8 hours of darkness, with a light intensity of 200 μmol·m⁻². -2 ·s -1 Before aluminum stress treatment, rubber seedlings were cultured in Hoagland nutrient solution (containing 40 μmol / L AlCl3, pH = 5.5) for 5 days to prevent potential shock responses to aluminum stress. After recovery, the tissue-cultured rubber seedlings were treated with 0 (CK), 50, 100, and 200 mmol / L AlCl3, respectively. During aluminum stress treatment (including the control), the pH of the Hoagland nutrient solution was adjusted to 4.2 with 1 mmol / L HCl or ammonia, and the nutrient solution was changed every 2 days. Roots, stems, leaves, and bark of rubber trees under normal conditions were collected for tissue expression analysis; different concentrations of AlCl3 were also collected. 3+Roots of rubber tree tissue culture seedlings treated for 5 days were used for gene expression analysis under different concentrations of aluminum stress; 200 mmol / L Al was collected. 3+ Root systems of rubber tree tissue culture seedlings treated for 0, 6, 12, 24, 48, and 120 hours were used for gene expression analysis under aluminum stress at different time points. Samples were flash-frozen in liquid nitrogen and then stored at -80°C for later use.

[0032] Example 1 - Extraction of total RNA and synthesis of cDNA

[0033] Total RNA was extracted from different tissues and treated samples of *Rhizopus rubra* using the TIANGEN Plant Total RNA Extraction Kit (Tiangen Biotech Co., Ltd., Beijing). RNA concentration and purity were detected using a Thermo Fisher NanoDrop2000 micro-volume nucleic acid and protein analyzer. RNA extracted from the roots of *Rhizopus rubra* tissue culture seedlings was reverse transcribed into cDNA using the TaKaRa Reverse Transcription Kit (Baori Biotechnology Co., Ltd., Beijing). RNA integrity and cDNA concentration and purity were detected by 1% agarose gel electrophoresis.

[0034] Example 2 - Cloning of the full-length HbOT1 and HbOT2 genes in rubber trees

[0035] This invention uses the identified oxalic acid transporter FpOAR protein (GeneBank: BAJ10704.1) from the brown rot fungus *Fomitopsis palustris* as the query sequence. BLASTP alignment was performed in the rubber tree genome database, identifying two unidentified rubber tree proteins, XP_021645179.1 and XP_021655511.1, and obtaining the corresponding candidate genes XM_021789487.1 (HbOT1) and XM_021799819.1 (HbOT2). Simultaneously, a highly homologous Unigene sequence, NM_117026.5 (AT4G09580), was obtained from the *Arabidopsis thaliana* genome database.

[0036] To obtain the full-length HbOT1, HbOT2, and AtOT genes, cloning primers were designed based on sequences from the NCBI database (Table 1). Using *Rubberia rubra* tissue culture seedlings ("Reyan 7-33-97") and *Arabidopsis thaliana* root cDNA as templates, the full-length sequences of HbOT1 and HbOT2 were amplified using PCR. The PCR products were recovered using an OMEGA gel extraction kit (Feiyang Biotechnology Co., Ltd., Guangzhou) and followed the instructions. After ligation into the pMD-18T vector, the cells were transformed into *E. coli* DH5α competent cells and cultured overnight on LB agar plates containing ampicillin. Positive clones were then picked and cultured in LB liquid medium containing ampicillin for 24 hours. After bacterial culture PCR detection, the positive clones were sequenced. Bacterial cultures with accurate sequencing results were stored at -80°C for later use.

[0037] Table 1 Primers used in the experiment

[0038]

[0039]

[0040] The full-length cDNA of the target sequence was obtained by PCR cloning. After sequencing verification and alignment, the cDNA sequences were temporarily named HbOT1 (ID_NCBI: LOC110638805), HbOT2 (ID_NCBI: LOC110646400), and AtOT (ID_NCBI: AT4G09580), respectively. The amplification results of the HbOT1, HbOT2, and ATOT genes (M.Marker DL2000) are shown below. Figure 1 As shown, the HbOT1 gene is 1163 bp long, with a CDS region of 792 bp, encoding 263 amino acid residues; the HbOT2 gene is 1647 bp long, with a CDS region of 840 bp, encoding 279 amino acid residues; and the AtOT gene is 1374 bp long, with a CDS region of 864 bp, encoding 287 amino acid residues.

[0041] Example 3 - Bioinformatics Analysis of HbOT1, HbOT2 and AtOT in Rubber Trees

[0042] Bioinformatics analysis of the HbOT1 and HbOT2 genes was performed using online tools (Table 2). DNAMAN 7 software was used for multiple sequence alignment and homology similarity analysis, and MEGA 7 software was used for phylogenetic analysis. All protein sequences used in the phylogenetic analysis were retrieved from the NCBI database.

[0043] Table 2 Online tools for bioinformatics

[0044]

[0045]

[0046] (1) Physicochemical properties analysis of HbOT1, HbOT2 and AtOT proteins

[0047] The physicochemical properties of the protein were analyzed using the ProtParam online tool. The molecular formula of HbOT1 is C1. 1322 H 2059 N 335 O 352 S 11 The relative molecular weight is 28630.58, the theoretical isoelectric point is 9.61, the instability index is 33.70, and the overall average hydrophilicity is 0.473; the molecular formula of HbOT2 is C 1473 H 2309 N 359 O 376 S 12 The relative molecular weight is 31448.44, the theoretical isoelectric point is 9.71, the instability index is 37.40, and the overall average hydrophilicity is 0.411; the molecular formula of AtOT is C2. 1497 H 2343 N 375 O 387 S 11 It has a relative molecular weight of 32139.01 Da, a theoretical isoelectric point (pI) of 9.74, an instability index (II) of 46.11, and a total average hydrophilicity (GRAVY) of 0.407.

[0048] Using the online website TMPRED to predict transmembrane domains, the results showed that there were four transmembrane helices at positions 46-68, 78-100, 159-181, and 201-220 of the HbOT1 amino acid sequence; five transmembrane helices at positions 45-67, 103-125, 135-157, 218-240, and 255-272 of the HbOT2 amino acid sequence; and five transmembrane helices at positions 53-75, 111-133, 143-165, 226-248, and 263-280 of the AtOT amino acid sequence. It is speculated that all three are transmembrane proteins. Signal peptide prediction using the online website SignalIP showed that HbOT1 had a probability of functioning as a signal peptide of 0.0376, HbOT2 had a probability of functioning as a signal peptide of 0.0003, and AtOT had a probability of functioning as a signal peptide of 0.0001, all less than the threshold of 0.5000. Amino acid hydrophilicity / hydrophobicity analysis using the online website ProtScale showed that the hydrophobic portions of HbOT1, HbOT2, and AtOT were all larger than their hydrophilic portions.

[0049] (2) Predictive analysis of HbOT1, HbOT2 and AtOT protein structures

[0050] Protein conserved domain analysis was performed using the NCBI CDD database and the online SMART website. The results showed that the HbOT1 protein contains a SNARE_assoc domain located at positions 62–184 of the N-terminus, with an E-value of 4.88e-20; the HbOT2 protein contains a SNARE_assoc domain located at positions 85–278 of the N-terminus, with an E-value of 1.51e-17; and the AtOT protein contains a SNARE_assoc domain located at positions 104–284 of the N-terminus, with an E-value of 4.86e-16 (e.g., ...). Figure 2 (As shown in A). The secondary structure of the proteins was predicted using the online website NPS@SOPMA. The results showed that in the amino acid sequence encoding the HbOT1 protein, 126, 41, 19, and 77 amino acid residues were involved in the formation of the α-helix, the extended chain, the β-turn, and the random coil, respectively, accounting for 47.91%, 15.59%, 7.22%, and 29.28% of the secondary structure. In the amino acid sequence encoding the HbOT2 protein, the residues involved in the formation of the α-helix, the extended chain, the β-turn, and the random coil were 77.77%, accounting for 47.91%, 15.59%, 7.22%, and 29.28% of the secondary structure, respectively. The number of amino acid residues involved in α-helix, 32, 13, and 78 random coils, respectively, accounts for 55.91%, 11.47%, 4.66%, and 27.96% of the secondary structure. In the amino acid sequence encoding the AtOT protein, the number of amino acid residues involved in the formation of the α-helix, outer extension chain, β-turn, and random coils are 165, 32, 8, and 82, respectively, accounting for 57.49%, 11.15%, 2.79%, and 28.57% of the secondary structure (e.g., ...). Figure 2 (As shown in B). Tertiary structure models of HbOT1, HbOT2, and AtOT proteins were constructed using the online website I-TASSER, as shown in Figure B. Figure 2 As shown in C, it is basically consistent with the prediction results of the secondary structure.

[0051] (3) Subcellular localization prediction analysis of HbOT1 and HbOT2 proteins

[0052] Using the online website PSORT to predict the subcellular localization of the genes, the results showed that HbOT1 was likely located on the plasma membrane and vacuoles, with confidence scores of 6 and 5, respectively; HbOT2 was likely located on the plasma membrane and vacuoles, with confidence scores of 10 and 2, respectively. Agrobacterium GV3101, successfully transformed with HbOT1, HbOT2, and empty vectors, was used to infect Tobacco Benzovia leaves. Epidermal cells of tobacco leaves containing the 35S::HbOT1-GFP and 35S::HbOT2-GFP fusion proteins showed fluorescence only on the plasma membrane, while epidermal cells injected with the 35S::1300-GFP fusion protein showed fluorescence throughout the cell. This indicates that HbOT1 and HbOT2 are located on the plasma membrane (e.g., cellular localization). Figure 4).

[0053] (4) Multiple sequence alignment of HbOT1 and HbOT2 and phylogenetic tree analysis

[0054] The amino acid sequences of HbOT1 and HbOT2 were searched using NCBIBLASTP and found to be similar to those of Arabidopsis thaliana (AtOT, NP_175116.2), cassava (Manihot esculenta, XP_021601880.1, XP_021621868.1), castor bean (Ricinus communis, XP_015579430.1), jatropha curcas (Jatropha curcas, XP_012065770.1), plum (Prunus mume, XP_008237264.1), peach (Prunus persica, XP_007221446.1), American black poplar (Populus deltoides, KAH8513031.1), sweet cherry (Prunus avium, XP_021834470.1), and hairy poplar (Populus). The SNARE proteins of 10 plant species, including *Trichocarpa* (XP_002325990.2, XP_006376293.1) and *Populus alba* (XP_034924621.1, XP_034897016.1), showed high homology.

[0055] Among them, HbOT1 showed the highest homology (90.87%) with cassava SNARE protein XP_021621868.1, while HbOT2 showed the highest homology (96.42%). Based on the finding that rubber tree HbOT1 and HbOT2 proteins are located in the plasma membrane, other membrane-localized plant SNARE protein subfamilies that have been biologically identified were added for comparative analysis. A phylogenetic tree was constructed using MEGA 7 software and the neighbor-joining method to further explore the evolutionary relationship between HbOT1 and HbOT2 and other plant SNARE proteins.

[0056] The results showed that HbOT1 and HbOT2 belong to the SNARE_assoc subfamily. A common characteristic of proteins in this subfamily is the presence of a SNARE_assoc domain. Evolutionarily, HbOT1 and HbOT2 are most closely related to the cassava SNARE proteins XP_021621868.1 and XP_021601880.1, respectively, suggesting their functional similarity (e.g., ...). Figure 3 (As shown).

[0057] Example 4 - Expression analysis of HbOT1 and HbOT2 genes in rubber trees

[0058] Based on the ORF region sequences of the HbOT1 and HbOT2 genes, quantitative real-time PCR primers were designed (Table 1). The rubber tree HbUBC4 gene, which is most stably expressed under aluminum stress, was selected as the internal control gene. Quantitative PCR was performed using the SYBR Green method on a CFX96 TOUCH real-time PCR instrument (Bio-Rad Laboratories, USA). The quantitative PCR reaction system was 20 μL, including 10 μL of SYBR Premix Ex Taq™ (TaKaRa), 0.4 μL each of forward and reverse primers (10 mol / L), 1 μL of rubber tree root cDNA, and 8.2 μL of ddH2O. The reaction program consisted of three steps: the first step was 95℃ for 3 min, then 95℃ for 10 s; the second step was 60℃ for 20 s; and the third step was 72℃ for 30 s, for a total of 43 cycles. Through 2... -ΔΔCT The experimental results were analyzed using the method.

[0059] Tissue-specific expression of HbOT1 and HbOT2 genes was analyzed using qRT-PCR, and the results are as follows: Figure 5 As shown, the results indicate that HbOT1 and HbOT2 are expressed in the roots, stems, leaves, bark, and latex of rubber trees. HbOT1 showed relatively high expression levels in roots, stem tips, bark, and latex, but the lowest relative expression level in leaves, significantly lower than in roots, stem tips, bark, and latex. There were no significant differences between any of the roots, stem tips, bark, and latex. HbOT2 showed the highest relative expression level in latex, significantly higher than in roots, stem tips, leaves, and bark, and the lowest relative expression level in bark. (Different lowercase letters on the bars indicate significant differences between data points (P < 0.05), the same applies below.)

[0060] Example 5 - Identification of oxalic acid resistance and aluminum tolerance functions of HbOT1 and HbOT2 genes in rubber trees

[0061] (1) Analysis of the expression of HbOT1 and HbOT2 genes in roots in response to aluminum stress

[0062] Rubber seedlings were subjected to aluminum stress treatment at different concentrations. It was found that after 120 hours of aluminum stress treatment, both HbOT1 and HbOT2 levels were below 50 mmol / L Al. 3+ 100 mmol / L Al 3+ and 200 mmol / L Al 3+ Compared to CK, HbOT1 expression was significantly upregulated under the treatment, with HbOT1 expression levels being 1.78, 1.64, and 2.40 times higher than CK, respectively; while HbOT2 expression was significantly upregulated under 50 mmol / L Al. 3+ 100 mmol / L Al3+ and 200 mmol / L Al 3+ The gene expression levels under the treatments were 15.34, 2.91, and 4.89 times that of CK, respectively (e.g., Figure 6 (As shown).

[0063] For 200 mmol / L Al 3+ Further investigation into the expression of HbOT1 and HbOT2 genes after different treatment times revealed that HbOT1 gene expression was significantly upregulated at 6h, 12h, and 24h, with expression levels 346.87, 99.59, and 695.32 times higher than that of the control group (CK), respectively. However, no significant changes were observed at 48h and 120h. HbOT2 gene expression was also significantly upregulated at 6h, 12h, 24h, 48h, and 120h, with expression levels 9.83, 10.66, 11.38, 5.41, and 4.50 times higher than that of the CK, respectively. Figure 7 ).

[0064] (2) Identification of the antioxalate activity of HbOT1 and HbOT2 genes in yeast system

[0065] The oxalic acid resistance and aluminum tolerance of the rubber tree HbOT1 and HbOT2 genes and the Arabidopsis thaliana homolog AtOT were identified using the yeast mutant strain AD1-8 (kindly provided by Professor Tang Kexuan of Shanghai Jiao Tong University and Professor Richard Cannon of the University of Otago) and the pDR196 vector (Zhuangmeng International Biotechnology Co., Ltd., Beijing).

[0066] SalⅠ (F-terminus) and EcoRI (R-terminus) were selected as restriction enzyme sites, and recombinant expression vectors pDR196-HbOT1, pDR196-HbOT2, pDR196-AtOT, and pDR196-FpOAR (positive control) were constructed using homologous recombination. After sequencing confirmed to be correct, the recombinant expression vectors and the empty pDR196 vector (negative control) were transformed into the yeast mutant strain AD1-8 using lithium acetate conversion.

[0067] Successfully transformed yeast cells were cultured in SD(-Ura) liquid culture medium at 30°C and 180 rpm until OD. 600 The value is 0.5. Dilute with distilled water to a final concentration of 10. -1 10 -2 10 -3 10 -4 10 -5SD(-Ura) solid medium was prepared, and after adjusting the pH to the same level, 0, 2, 4, 8, and 10 mmol / L oxalic acid were added respectively. Yeast cells with different concentration gradients were spotted on the medium containing oxalic acid and cultured in a 30°C incubator for 4 days. After culture, the growth status of yeast cells under different oxalic acid concentrations was recorded (Watanabe et al., 2010). The same culture method was used to record the growth of yeast cells in medium containing 2.4, 2.5, 2.6, 2.7, and 2.8 mmol / L Al. 3+ The growth status of yeast cells under certain environmental conditions.

[0068] Absorb OD separately 600 100 μL of each yeast cell culture (1.0) was added to 50 mL of SD(-Ura) liquid containing 2 mmol / L oxalic acid. The cultures were incubated at 30℃ and 180 rpm for 0, 1, 3, 5, 7, 9, 11, and 13 days. The yeast cells were separated from the culture medium by centrifugation at 1000 x g for 10 min and collected separately (the yeast cells were washed twice with an equal volume of ddH2O). The oxalic acid content in the cells and culture medium was determined. The pH of the yeast cell culture at different incubation times was measured using a METTLER TOLEDO FE plus pH meter. The yeast cells were freeze-dried using a SCIENTZ-30YG / A freeze dryer, and the dry weight of the cells was measured. The oxalic acid content in the cells and culture medium was determined according to the instructions of the Boxbio Oxalic Acid (OA) Content Assay Kit (Boxbio Biotechnology Co., Ltd., Beijing). The pH of the yeast cell culture, dry weight of the cells, and oxalic acid content are the average of three biological replicates ± standard error.

[0069] Results analysis:

[0070] The constructed yeast recombinant expression vector was transformed into the yeast mutant strain AD1-8. FpOAR-transformed yeast cells served as a positive control, and pDR196 empty vector-transformed yeast cells served as a negative control. The yeast system was used to verify the oxalic acid resistance activity of the HbOT1 and HbOT2 genes in rubber trees. The results showed that after culturing yeast cells at different concentration gradients of 0, 2, 4, 8, and 10 mmol / L oxalic acid on SD(-Ura) solid medium for 4 days, when the oxalic acid concentration was not higher than 2 mmol / L, the growth of the yeast cells was not significantly different due to the inherent resistance of the AD1-8 yeast mutant strain to low concentrations (≤2 mmol / L) oxalic acid stress. When the oxalic acid concentration reached 4-8 mmol / L, the growth of the yeast cells increased significantly. -3 10 -4 10 -5At a certain concentration, no colonies formed in the negative control, while the colonies formed in the positive control and recombinant yeast changed from white, smooth, round colonies to white, wavy, dotted colonies; when the oxalic acid concentration reached 10 mmol / L, at 10 -1 At a certain concentration, the positive control formed three tiny white dot-like colonies, the recombinant yeast formed irregular white colonies, and the negative control showed no colony formation. However, at 10... -2 10 -3 10 -4 10 -5 No colonies formed in any of the yeast cells at the specified concentrations. Figure 8 ).

[0071] It is evident that the HbOT1, HbOT2, and AtOT genes have a positive regulatory effect on yeast cell oxalate resistance in terms of colony phenotype, and their oxalate resistance activity is stronger than that of the positive control FpOAR.

[0072] In this invention, different yeast cells were cultured in SD(-Ura) liquid medium containing 2 mmol / L oxalic acid for 13 days. The pH value of each yeast culture was continuously observed every 48 hours, showing a decreasing trend over time. At the same culture period, there were no significant differences in pH value and trend among recombinant yeast cells and between recombinant yeast cells and positive control cells. The pH value of the negative control cell was significantly higher than that of the recombinant yeast cells and positive control cells. At the end of the culture, the pH value of each yeast culture reached the critical value. The pH value of the recombinant yeast cells and positive control cells remained at 2.3-2.5, while the pH value of the negative control cell was maintained at around 2.8. The results are shown in Table 3.

[0073] The dry weight of each yeast cell increased with culture time. At the same culture time, the dry weight of the negative control was significantly lower than that of the recombinant yeast and the positive control, and the dry weight remained at about 0.2g at the end of culture. There was little difference in the dry weight and trend among the recombinant yeasts. There was little difference in the dry weight between the recombinant yeast and the positive control from 0 to 7 days. From 7 to 13 days, the dry weight of the positive control was significantly smaller than that of the recombinant yeast, remaining at 0.225-0.25g, while the dry weight of the recombinant yeast reached 0.325g at the end of culture. The results are shown in Table 4.

[0074] Table 3. Changes in pH of bacterial cultures of different yeast cells under 2 mmol / L oxalate stress.

[0075]

[0076]

[0077] Note: Different uppercase letters indicate significant differences between rows, and different lowercase letters indicate significant differences between columns (P < 0.05), the same applies below.

[0078] Table 4. Changes in cell dry weight (g) of different yeast cells under 2 mmol / L oxalic acid stress.

[0079]

[0080] The results of the determination of oxalic acid content in yeast showed that ( Figure 9 A) The oxalic acid environment caused a significant increase in the oxalic acid content in yeast cells within a short period of time (≤1 day), from 3.5-5 mmol / L to about 10 mmol / L. At this time, there was no significant difference in the oxalic acid content among the yeast cells. From 1 to 13 days, the oxalic acid content in the recombinant yeast and the positive control yeast showed a decreasing trend with the progress of culture time, reaching about 3-4.5 mmol / L at the end of culture. However, the oxalic acid content in the negative control remained at a high level with the progress of culture time, and its oxalic acid content at the end of culture was significantly higher than that of the recombinant yeast and the positive control.

[0081] To verify whether the transformed yeast would transfer yeast to other parts of the culture medium, the oxalic acid content in the culture medium was also measured. Figure 9 B). It can be seen that the oxalic acid content in the negative control medium was not stable with culture time, but from day 7 until the end of culture, the oxalic acid content in the medium was significantly lower than that in the recombinant yeast and positive control. With the progression of culture time, the oxalic acid content in both the recombinant yeast and positive control media generally showed an upward trend; the oxalic acid content in the positive control medium reached the critical value on day 7, and then fluctuated around 8.5 mmol / L until the end of culture; while the oxalic acid content in the recombinant yeast medium showed a gradual increase, reaching 14.17 mmol / L and 12.04 mmol / L in the media for HbOT1 and HbOT2 yeast respectively at the end of culture. This indicates that recombinant HbOT1 and HbOT2 yeast have the ability to transport oxalic acid into the culture medium.

[0082] The above experimental results indicate that oxalic acid stress affects the normal growth of yeast cells and causes disordered oxalic acid metabolism in the negative control. Transgenic HbOT1 and HbOT2 genes significantly enhance the adaptability of yeast cells to oxalic acid, change the oxalic acid metabolism pattern of yeast cells in an oxalic acid environment, and may be involved in the efflux of oxalic acid from cells.

[0083] (3) Identification of aluminum tolerance of HbOT1 and HbOT2 genes in yeast system

[0084] To further verify the roles of HbOT1 and HbOT2 in yeast aluminum tolerance, yeast cells were cultured for 4 days on aluminum-stressed medium with different concentration gradients. The results showed that yeast cells transformed with HbOT1 and HbOT2 exhibited certain aluminum tolerance, and this tolerance was concentration-dependent. 3+When the concentration reached 2.4 mmol / L, the colony size of each yeast cell was significantly smaller than under normal conditions, with no significant difference between them. When Al 3+ At concentrations of 2.5-2.6 mmol / L, the yeast cells at 10 -1 No significant difference in growth was observed at concentrations of 10; -4 10 -5 No colonies formed at any concentration; when Al 3+ When the concentration reaches 2.7 mmol / L, at 10 -1 At the specified concentration, FpOAR, HbOT1, and pDR196-AtOT yeasts formed white, smooth, round colonies, while HbOT2 yeast formed white, dotted colonies. The control yeast showed no colony formation. At 10... -2 10 -3 At the specified concentration, FpOAR and HbOT2 yeasts formed white, pinpoint-shaped colonies, while HbOT1 yeast and the control group showed no colony formation; at 10 -4 10 -5 No colonies formed in any of the yeast cells at the specified concentrations. When Al... 3+ When the concentration reached 2.8 mmol / L, HbOT1, HbOT2, and pDR196-AtOT yeast were transformed at 10... -1 At certain concentrations, white, pinhead-shaped colonies form; at other concentrations, no colonies form (e.g.) Figure 10 These results indicate that the HbOT1, HbOT2, and AtOT genes can increase the tolerance limit of yeast cells to aluminum stress and have a positive regulatory effect on the tolerance of yeast cells to high concentrations (≥2.7 mmol / L) of aluminum stress. This demonstrates the role of the HbOT1 and HbOT2 genes in enhancing plant aluminum tolerance.

[0085] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of a candidate gene for oxalate transport, characterized in that: The application is the use of oxalate transporter candidate genes in the detoxification of aluminum toxicity in rubber trees or Arabidopsis thaliana. The oxalic acid transporter candidate gene is an oxalic acid transporter candidate gene from rubber trees. HbOT1 , HbOT2; The oxalate transporter candidate gene HbOT1 and HbOT2 The DNA sequence is shown in SEQ ID NO.1-2.

2. The application of a candidate gene for oxalate transport, characterized in that: The application is the use of oxalate transporter candidate genes in the detoxification of aluminum toxicity in Arabidopsis thaliana. The oxalate transporter candidate gene is a gene from Arabidopsis thaliana. AtOT; The gene AtOT The DNA sequence is shown in SEQ ID NO.

3.

3. Candidate genes for rubber oxalic acid transporter HbOT1 , HbOT2 Application in the translocation of oxalic acid in the roots of rubber trees or Arabidopsis thaliana; The oxalic acid transporter candidate gene is an oxalic acid transporter candidate gene from rubber trees. HbOT1 , HbOT2; The oxalate transporter candidate gene HbOT1 and HbOT2 The DNA sequence is shown in SEQ ID NO.1-2.

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