Application of OsATL12 gene in regulation and control of absorption and transportation of rice to alkyl quaternary ammonium salt compound

Abstract

The invention provides an application of an OsATL12 gene in regulation and control of absorption and transportation of rice to alkyl quaternary ammonium salt compounds, and the gene plays a positive regulation and control role in the process. The nucleotide sequence of the OsATL12 gene is as shown in SEQ ID NO. 1, and the amino acid sequence of the protein coded by the OsATL12 gene is as shown in SEQ ID NO. 2. The OsATL12 gene in the rice is knocked out through a gene editing technology, so that the function of the OsATL12 gene is inactivated, the absorption amount of the alkyl quaternary ammonium salt compound at the overground part of the rice can be reduced, and the accumulation of the alkyl quaternary ammonium salt compound in the rice body is reduced. Important molecular biological resources can be provided for cultivating alkyl quaternary ammonium salt compound low-accumulation plant varieties, and environment and food safety problems caused by alkyl quaternary ammonium salt compounds can be solved.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology. OsATL12 Application of genes in regulating the absorption and transport of alkyl quaternary ammonium compounds in rice. Background Technology

[0002] Alkyl quaternary ammonium compounds (QACs) have the general formula (R4N) + )X - (X) - QACs (chemically active organic compounds) are a class of major cationic surfactants widely used in household and industrial products such as disinfectants, preservatives, fabric softeners, and catalysts. As a high-yield chemical recognized by the OECD, global annual production exceeds 500,000 tons. During the COVID-19 pandemic, QACs saw a dramatic increase in usage due to their key active ingredients in many anti-coronavirus disinfectants, leading to a large influx of QACs into wastewater treatment systems. However, they are often not completely removed during wastewater treatment and ultimately enter the environment with effluent and reused sludge. Studies have found that QACs are toxic to microalgae even at low concentrations, inhibiting their growth and nutrient absorption; similar toxicity has been found in terrestrial plants. Furthermore, QACs may contribute to the development of antibiotic resistance, and these potential risks have increasingly drawn attention to their environmental and health impacts. In 2021, the state of California listed QACs as a priority monitoring chemical, with particular focus on exposure to children. Due to the positively charged nitrogen groups in their molecules, QACs readily adsorb into negatively charged environmental media such as sludge, sediment, and soil. The application of sludge, the use of quaternary ammonium herbicides, and irrigation with reclaimed water are all significant sources of QACs contamination in soil. QACs have been widely detected in farmland where sludge has been applied for a long period, and even in soils of different land use types. More concerningly, QACs have also been detected in crops, sometimes at concentrations exceeding food safety thresholds, suggesting that QACs may enter the food chain via the soil-crop pathway.

[0003] Rice is the staple food for more than half of the world's population, especially in Asia. Research on the accumulation and metabolism of pollutants in rice is directly related to food safety and health risk assessment for the vast majority of people, and its public health significance is extremely important. Furthermore, QACs (chemically active organic pollutants) are stable and difficult to degrade under anaerobic conditions, and rice is often exposed to flooding, raising concerns about the potential absorption and transport risks of QACs. Existing studies have shown that aquaporins and lysine-histidine transporters are involved in the absorption and transport of non-ionic organic pollutants (such as neonicotinoid pesticides and polybrominated diphenyl ethers) in rice. However, research on the absorption and transport mechanisms of ionic organic pollutants in rice is severely lacking.

[0004] Therefore, elucidating the molecular mechanism of QACs absorption and translocation in rice and regulating QACs absorption and translocation in crops is of great significance for ensuring safe crop production. Summary of the Invention

[0005] To address the above problems, the purpose of this invention is to provide... OsATL12 The application of genes in regulating the absorption and transport of alkyl quaternary ammonium compounds in rice: To achieve the above objectives, the technical solution adopted in this invention is as follows: OsATL12 The nucleotide sequence of the gene is shown in SEQ ID NO.1. OsATL12 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.2.

[0006] Preferably, knock out the OsATL12 Genes, obtained osatl12 mutant plants, the osatl12 The mutant plants have reduced ability to transport alkyl quaternary ammonium compounds.

[0007] Preferably, the knockout of the OsATL12 The gene is the result of gene editing technology. OsATL12 The gene was knocked out, making the OsATL12 Gene function is inactivated.

[0008] Preferably, reducing the OsATL12 The expression level of genes reduces the ability of rice to transport alkyl quaternary ammonium compounds.

[0009] Preferably, the alkyl quaternary ammonium salt compound is dodecyl dimethyl benzyl ammonium chloride.

[0010] Preferably, after treatment with the dodecyl dimethyl benzyl ammonium chloride for 72 hours, the roots and aboveground parts of the rice... OsATL12 Gene expression levels increase.

[0011] Preferably, after treatment with the dodecyl dimethyl benzyl ammonium chloride, the osatl12 The concentration of dodecyl dimethyl benzyl ammonium chloride in the aboveground parts of the mutant plants was lower than that in the wild-type plants.

[0012] Preferably, the plant is rice, and the rice variety is Zhonghua 11.

[0013] A rice breeding method, based on OsATL12 Gene, the method includes: According to the above OsATL12 Designing sgRNA targets based on specific exon sequences of genes; Construct a knockout vector; The knockout vector was transformed into rice embryogenic callus using Agrobacterium-mediated transformation. After resistance screening, plant differentiation and regeneration, T0 generation transgenic plants were obtained. The process involves self-pollinating identified, effectively edited T0 generation transgenic plants to obtain T1 generation seeds. Genomic DNA is extracted from the T1 generation plants, amplified by PCR, and sequenced to obtain homozygous T1 plants. osatl12 The mutant plants were self-crossed with the T1 generation homozygous mutants, and the T2 generation seeds were harvested for subsequent experiments.

[0014] The construction of the knockout vector includes: Using a DNA fragment containing a tRNA-sgRNA architecture as a template, PCR amplification was performed to obtain the amplified product. The amplified product was subjected to gel electrophoresis and gel extraction for recovery to obtain the recovered product; The recovered product was ligated with the enzyme-digested vector pYL-HU-U3-CCDB-tRNA to obtain the ligation product; The ligation product was transferred into competent E. coli cells to obtain the transformed product. The transformed product was coated onto a kanamycin-resistant plate for screening to obtain transformants; For the transformants, single colonies were picked from the plate and subjected to colony PCR identification to screen out positive clones; The positive clones were sequenced and verified to obtain the knockout vector.

[0015] Compared with the prior art, the beneficial effects of the present invention are: Addressing the problem of dodecyl dimethylbenzylammonium chloride (DBAC) pollution in plants, the present invention screens a gene from rice that regulates the absorption and transport of DBAC in plants. OsATL12 Using gene editing technology to modify genes OsATL12 Knockout, making the gene OsATL12 The gene function is inactivated, resulting in osatl12 mutant plants, osatl12 The mutant plants exhibited reduced DBAC translocation, decreased DBAC concentration in the aboveground parts, and reduced gene... OsATL12 The discovery provides technical support for reducing the absorption and translocation of DBACs in plants, and can provide important molecular biological resources for breeding plant varieties with low accumulation of QACs, which can help solve the environmental and food safety problems caused by QACs. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0017] Figure 1 For rice treated with DBAC for different durations, the genes OsATL12 The relative expression level; Figure 2 The image shows the recombinant plasmid pYES2-OsATL12. Figure 3 A comparison of intracellular DBAC content after DBAC treatment of yeast expression vectors pYES2-OsATL12 and pYES2, respectively. Figure 4 A map of the recombinant vector; Figure 5 for osatl12 mutant plants ( osatl12-1, osatl12-2 Identification diagram; Figure 6 Wild-type plants (WT) and osatl12 mutant plants ( osatl12-1, osatl12-2 Comparison of DBAC absorption in roots and aboveground parts. Detailed Implementation

[0018] The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the invention. Modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and substance of the invention are all within the scope of protection of the present invention. Unless otherwise specified, the experimental materials, reagents, instruments, etc., used in the embodiments of the present invention are all commercially available; unless specifically specified, all technical means in the embodiments of the present invention are conventional means well known to those skilled in the art.

[0019] Example 1: Screening of key genes regulating DBAC uptake in plants This embodiment provides a method for amplifying genes. OsATL12 The primer pairs are as follows: F: 5'-AATGGTGTCAATGCCCTCTC-3'; R: 5'-TCGCTGCTATGGTTATGAAGA-3'.

[0020] Using rice cDNA as a template, real-time quantitative PCR was performed using primer pairs to determine gene expression levels and obtain the DBAC-sensitive gene OsATL12. The nucleotide sequence of this gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by this gene is shown in SEQ ID NO.2.

[0021] Alkyl quaternary ammonium compounds (QACs) have the general formula (R4N).+ X - (X) - Halogen ions (halogen ions) are a major class of cationic surfactants, widely used in household and industrial products such as disinfectants, preservatives, fabric softeners, and catalysts.

[0022] This embodiment uses alkyl quaternary ammonium salt compounds (QACs) as an example, specifically dodecyldimethylbenzylammonium chloride (DBAC).

[0023] This embodiment provides a method for screening key genes that regulate the uptake and transport of DBAC in plants, including the following steps: Wild-type Zhonghua 11 rice seedlings with a seedling age of 14 days were transplanted into an environment containing 3 μmol / L... -1 Samples were taken after 12 h and 72 h of exposure to 1 / 2 KB of DBAC nutrient solution. A control group without DBAC was set up in the experiment.

[0024] RNA was extracted from the sample and used for gene amplification as described above. OsATL12 The gene expression level was determined by real-time quantitative PCR using primer pairs. The forward primer was F: 5'-AATGGTGTCAATGCCCTCTC-3', and the reverse primer was R: 5'-TCGCTGCTATGGTTATGAAGA-3'.

[0025] like Figure 1 As shown, based on the experimental results, compared with the control group (CK), the gene count in the rice roots of the treatment group (DBAC) was significantly lower after 12 h of treatment. OsATL12 Expression levels were induced to downregulate, but were induced to upregulate 1.53-fold after 72 h treatment; genes in the aboveground parts of rice OsATL12 Expression levels were induced to rise in the treatment group (DBAC) at different treatment times, specifically after 12 hours and 72 hours, with an upregulation of 2.05-2.54 times compared to the control group (CK). Given the gene... OsATL12 OsATL12 protein is continuously induced to express in the aerial parts, suggesting that it plays an important role in the uptake and transport of DBAC.

[0026] The above results suggest that the OsATL12 protein may be involved in the absorption and transport of DBACs, providing a potential strategy for regulating the absorption and transport of QACs in crops.

[0027] Example 2: Yeast System Validation To further identify candidate genes that regulate DBAC uptake and transport, the genes OsATL12The coding region sequence was cloned into the yeast expression vector pYES2 and transformed into yeast strain W303-1A. The specific steps included: According to genes OsATL12 To design primers, use the following primer pairs. OsATL12 Gene cloning was performed using the forward primer F: 5'-cttggtaccgagctcggatccATGGAAACCAACACTCCTCCAA-3' and the reverse primer R: 5'-gcggccgttactagtggatccTCAGAAGCTCTGCACAATCTGTTT-3'. The target fragment was obtained by PCR amplification. Single-restriction digestion was performed at the BamHI site to obtain the pYES2 vector linearized with restriction endonuclease.

[0028] The target fragment was recombined with the enzyme-digested yeast expression vector pYES2. Single colonies were screened and sequenced to obtain the recombinant plasmid pYES2-OsATL12. The map of the recombinant plasmid pYES2-OsATL12 is shown below. Figure 2 As shown.

[0029] The recombinant plasmid pYES2-OsATL12 was transformed into yeast strain W303-1A. The mixed bacterial solution was evenly spread on SD-Ura medium and incubated in a 30 °C incubator for 2-3 days. Single colonies were picked from the plate.

[0030] The obtained single colonies were added to SD-Ura liquid medium containing 2% glucose and incubated overnight at 30 °C and 200 rpm in a shaker. After incubation, the OD of the bacterial culture was measured. 600 Set the value to 1, then centrifuge to collect the bacterial cells.

[0031] The collected bacterial cells were added to a 6 μmol / L solution. -1 DBAC was placed in 20 mL of SD-Ura liquid medium containing 2% galactose and incubated in a shaker at 30 °C and 200 rpm for 3 h. After centrifugation, the supernatant was discarded and the mixture was resuspended in sterile water. This step was repeated three times.

[0032] Finally, the bacterial cells were collected, freeze-dried, and the DBAC content in the yeast cells was measured. An empty vector pYES2 control group was set up in the experiment.

[0033] like Figure 3 As shown, the experimental results indicate that, compared with cells containing the empty vector pYES2, the expression of... OsATL12 The DBAC content in yeast cells was 88.40% higher than that in the control, indicating that the gene... OsATL12 It participates in regulating the absorption and transport of DBAC in yeast cells.

[0034] Example 3: Validation of Gene Editing Materials To further verify the genes OsATL12 The function utilizes CRISPR / Cas9 gene editing technology to construct rice using Zhonghua 11 as the background. OsATL12 Mutant. Specific sgRNA target primers were designed and synthesized based on the target gene sequence. The forward primer was F: 5'-cagtGGTCTCatgcaagcagcgatctgcttctacagtttcag-3', and the reverse primer was R: 5'-cagtGGTCTCaaaaccgtcaagacctacccggacatg-3'. Two target sites were set for single-gene editing. Subsequently, using a template containing a tRNA-sgRNA structure, a target fragment of approximately 203 bp was amplified by PCR. This fragment was separated by agarose gel electrophoresis and purified by gel extraction. The target fragment was then ligated to the restriction endonuclease (BsaI)-linearized vector pYL-HU-U3-CCDB-tRNA using T4 ligase to construct a recombinant vector. The map of the recombinant vector is shown below. Figure 4 As shown; after the ligation product was transformed into competent E. coli cells, transformants were screened on kanamycin-resistant plates, and colony PCR was performed using vector-specific primers (expected band approximately 1040 bp); PCR-positive clones were sequenced to verify the sgRNA sequence, and recombinant plasmids were extracted. These plasmids were then transformed into rice callus tissue via Agrobacterium-mediated transformation. After resistance selection and plant regeneration, T0 generation transgenic seedlings were obtained. T0 generation transgenic plants were self-pollinated to obtain T1 generation seeds. Genomic DNA was extracted from T1 generation plants, and two stable genetic sequences were obtained through PCR amplification and sequencing. osatl12 The mutant plants were named respectively. osatl12-1 , osatl12-2 The T1 generation homozygous mutants were self-crossed, and the T2 generation seeds were harvested for subsequent functional studies. Using forward primer F: 5'-CTTCCATCCTGCCTCTCGTG-3' and reverse primer R: 5'-CAGAACGCGTACAGGCTCAT-3', homozygous primers were identified. osatl12 mutant plants ( osatl12-1 , osatl12-2 The identification results are as follows: Figure 5 As shown.

[0035] To further elucidate genes OsATL12 Functions in plants, comparing 30-day-old wild-type plants (WT) and osatl12 mutant plants ( osatl12-1 , osatl12-2 ) Transfer into a solution containing 3 μmol / L -1Rice samples were collected after 48 hours of exposure to 1 / 2 KB of DBAC in nutrient solution. The roots were then immersed in 10 mM CaCl2 solution for 10 minutes to remove DBAC adsorbed on the root surface. The samples were then separated into roots and aboveground parts, freeze-dried, weighed, and the DBAC concentration in different parts of different rice genotypes was determined. Figure 6 As shown, the results indicate that osatl12 mutant plants ( osatl12-1 , osatl12-2 The DBAC concentration in the roots was not different from that in the wild-type plants (WT). osatl12 mutant plants ( osatl12-1 , osatl12-2 The DBAC concentration in the aboveground parts was 30.10% to 30.64% lower than that in wild-type plants (WT).

[0036] In summary, OsATL12 Genes play a regulatory role in the absorption and transport of DBAC in plants.

[0037] This invention addresses the problem of DBAC pollution by screening a gene from rice that regulates the uptake of DBAC by plants. OsATL12 Using gene editing technology to modify genes OsATL12 Knockout inactivates its function, reducing the plant's transport of DBAC and its genes. OsATL12 The discovery provides technical support for reducing the absorption and translocation of DBACs in plants, and can provide important molecular biological resources for breeding plant varieties with low accumulation of QACs, which can help solve the environmental and food safety problems caused by QACs.

[0038] It is understood that the above embodiments only illustrate preferred embodiments of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can freely combine the above technical features without departing from the concept of the present invention, and can also make several modifications and improvements, all of which fall within the protection scope of the present invention. Therefore, all equivalent transformations and modifications made with respect to the scope of the claims of the present invention should fall within the scope of the claims of the present invention.

Claims

1. OsATL12 The application of genes in regulating the absorption and transport of alkyl quaternary ammonium compounds in rice is characterized by, OsATL12 The nucleotide sequence of the gene is shown in SEQ ID NO.

1. OsATL12 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.

2.

2. The application according to claim 1, characterized in that, Knock out the OsATL12 Genes, obtained osatl12 mutant plants, the osatl12 The mutant plants have reduced ability to transport alkyl quaternary ammonium compounds.

3. The application according to claim 2, characterized in that, The knockout OsATL12 The gene is the result of gene editing technology. OsATL12 The gene was knocked out, making the OsATL12 Gene function is inactivated.

4. The application according to claim 1, characterized in that, Reduce the OsATL12 The expression level of genes reduces the ability of rice to transport alkyl quaternary ammonium compounds.

5. The application according to any one of claims 1-4, characterized in that, The alkyl quaternary ammonium salt compound is dodecyl dimethyl benzyl ammonium chloride.

6. The application according to claim 5, characterized in that, After treatment with the dodecyl dimethyl benzyl ammonium chloride for 72 hours, the roots and aboveground parts of rice... OsATL12 Gene expression levels increase.

7. The application according to claim 5, characterized in that, After treatment with the dodecyl dimethyl benzyl ammonium chloride, the osatl12 The concentration of dodecyl dimethyl benzyl ammonium chloride in the aboveground parts of the mutant plants was lower than that in the wild-type plants.

8. The application according to any one of claims 1-4, characterized in that, The plant is rice, and the rice variety is Zhonghua 11.

9. A method for breeding rice, characterized in that, Based on the application described in claim 1 OsATL12 Gene, the method includes: According to the above OsATL12 Designing sgRNA targets based on specific exon sequences of genes; Construct a knockout vector; The knockout vector was transformed into rice embryogenic callus using Agrobacterium-mediated transformation. After resistance screening, plant differentiation and regeneration, T0 generation transgenic plants were obtained. The process involves self-pollinating identified, effectively edited T0 generation transgenic plants to obtain T1 generation seeds. Genomic DNA from the T1 generation plants is then extracted, amplified by PCR, and sequenced to obtain homozygous T1 plants. osatl12 The mutant plants were self-crossed with the T1 generation homozygous mutants, and the T2 generation seeds were harvested for subsequent experiments.

10. The method according to claim 9, characterized in that, The construction of the knockout vector includes: Using a DNA fragment containing a tRNA-sgRNA architecture as a template, PCR amplification was performed to obtain the amplified product. The amplified product was subjected to gel electrophoresis and gel extraction for recovery to obtain the recovered product; The recovered product was ligated with the enzyme-digested vector pYL-HU-U3-CCDB-tRNA to obtain the ligation product; The ligation product was transferred into competent E. coli cells to obtain the transformed product. The transformed product was coated onto a kanamycin-resistant plate for screening to obtain transformants; For the transformants, single colonies were picked from the plate and subjected to colony PCR identification to screen out positive clones; The positive clones were sequenced and verified to obtain the knockout vector.

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