WO2022213453A1 - 一种调控植物抗铝性的铝离子受体alr1基因或蛋白的应用 - Google Patents

一种调控植物抗铝性的铝离子受体alr1基因或蛋白的应用 Download PDF

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WO2022213453A1
WO2022213453A1 PCT/CN2021/094884 CN2021094884W WO2022213453A1 WO 2022213453 A1 WO2022213453 A1 WO 2022213453A1 CN 2021094884 W CN2021094884 W CN 2021094884W WO 2022213453 A1 WO2022213453 A1 WO 2022213453A1
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alr1
gene
aluminum
seq
protein
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PCT/CN2021/094884
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郑绍建
丁忠杰
徐晨
李桂新
颜晶莹
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浙江大学
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    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
    • C12N15/8205—Agrobacterium mediated transformation
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
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    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the invention belongs to the technical field of plant genetic engineering, in particular to the application of an aluminum ion receptor ALR1 gene or protein for regulating the aluminum resistance of plants.
  • Soil acidification and the consequent aluminum toxicity are widespread problems in agricultural production worldwide.
  • the intensification of soil acidification has greatly affected the yield of crops, posing a threat to sustainable agricultural production.
  • the decrease of soil pH leads to the leaching of base ions, and on the other hand, also causes some insoluble mineral aluminum (the most abundant metal element in the crust, with an average content of about 8%) to dissociate into ionic state and enter.
  • insoluble mineral aluminum the most abundant metal element in the crust, with an average content of about 8%
  • the micromolar level can significantly inhibit the growth of plant roots, which in turn affects the absorption of nutrients and water by the roots, and ultimately leads to the reduction of crop yields, or even extinction. Therefore, aluminum toxicity is recognized as a major limiting factor for acid soils affecting crop production.
  • the physiological mechanisms of plant resistance to aluminum can be divided into aluminum external repulsion represented by changes in the properties of organic acids and cell wall components, and aluminum internal tolerance mechanisms represented by the storage of aluminum in vacuoles for compartmentalization. These two mechanisms are currently recognized The main physiological mechanism of plant anti-aluminum toxicity. With the rapid development of molecular biology, a large number of genes and proteins involved in plant anti-aluminum signal transduction pathways have been identified. However, little is known about how plants sense aluminum ions. Identifying genes involved in the process of sensing aluminum ions and deeply analyzing their functions are of great significance for further clarifying the molecular mechanism of plant aluminum resistance, providing reference for breeding, and improving plant aluminum resistance and crop yield. The aluminum ion receptor gene and its function that regulate the aluminum resistance of plants have not yet been discovered, which hinders the cultivation process of aluminum-resistant plants to a certain extent.
  • the purpose of the present invention is to provide an application of the aluminum ion receptor ALR1 gene or protein for regulating the aluminum resistance of plants.
  • the primer of the invention can improve the aluminum resistance of plants, promote the growth of plant roots, and then promote the absorption of nutrients and water by plants, and improve the yield of crops.
  • the present invention provides a primer for regulating the ALR1 gene of plant aluminum resistance, the primer comprises an upstream primer whose nucleotide sequence is shown in SEQ ID NO.1 and a nucleotide sequence which is shown in SEQ ID NO.2 downstream primers.
  • the present invention also provides a vector for overexpressing the Arabidopsis aluminum ion receptor ALR1 gene prepared based on the primers described in the above technical solution.
  • ALR1 gene the nucleotide sequence of the ALR1 gene is shown in SEQ ID NO.3.
  • the present invention also provides the application of an Arabidopsis thaliana aluminum ion receptor ALR1 gene or protein or the above primer in the regulation of plant aluminum resistance, the nucleotide sequence of the ALR1 gene is shown in SEQ ID NO.
  • the amino acid sequence of ALR1 protein is shown in SEQ ID NO.4.
  • the present invention also provides the application of overexpressing the Arabidopsis thaliana aluminum ion receptor ALR1 gene or protein or the vector described in the above technical solution in improving the aluminum resistance of plants.
  • the nucleotide sequence of the ALR1 gene is SEQ ID NO.3
  • the amino acid sequence of the ALR1 protein is shown in SEQ ID NO.4.
  • the present invention also provides the application of an Arabidopsis thaliana aluminum ion receptor ALR1 gene or protein or the above primer in regulating the root elongation of aluminum-stressed plants.
  • the nucleotide sequence of the ALR1 gene is represented by SEQ ID NO.3.
  • the amino acid sequence of the ALR1 protein is shown in SEQ ID NO.4.
  • the present invention also provides the application of overexpressing the Arabidopsis aluminum ion receptor ALR1 gene or protein or the vector described in the above technical solution in improving the root elongation of aluminum-stressed plants, the nucleotide sequence of the ALR1 gene is SEQ ID As shown in NO.3, the amino acid sequence of the ALR1 protein is shown as SEQ ID NO.4.
  • the root system is the main root.
  • the present invention also provides the application of an Arabidopsis thaliana aluminum ion receptor ALR1 gene or protein or the primer described in the above technical solution in regulating the root aluminum content of aluminum-stressed plants.
  • the nucleotide sequence of the ALR1 gene is SEQ ID NO. .3, the amino acid sequence of the ALR1 protein is shown in SEQ ID NO.4.
  • the present invention also provides the application of overexpressing the Arabidopsis thaliana aluminum ion receptor ALR1 gene or protein or the vector described in the above technical solution in reducing the root aluminum content of plants under aluminum stress.
  • the nucleotide sequence of the ALR1 gene is SEQ ID NO. .3, the amino acid sequence of the ALR1 protein is shown in SEQ ID NO.4.
  • the plant comprises Arabidopsis thaliana.
  • the present invention provides a primer for regulating the ALR1 gene of plant aluminum resistance.
  • the invention clones the aluminum ion receptor gene ALR1 from the model plant Arabidopsis thaliana by using specific amplification primers, and constructs two mutant lines by knocking out and overexpressing the ALR1 gene, and the knockout lines obviously reduce the aluminum resistance.
  • the overexpression lines showed a significant increase in Al resistance.
  • the experimental results showed that under Al stress, the taproot elongation of transgenic Arabidopsis thaliana overexpressed was significantly higher than that of the wild-type control, and the root aluminum content was significantly decreased, while the taproot elongation of the ALR1 gene knockout mutant line was significantly lower than that of the wild-type control.
  • the root aluminum content was significantly higher than that in the wild type, and the main root elongation of the mutant functional recovery line was similar to that of the wild type.
  • the application of the invention is of great significance for improving the aluminum resistance of plants, promoting the growth of plant roots, thereby promoting the absorption of nutrients and water by plants, and increasing the yield of crops.
  • the plant seeds obtained by using the primers, genes, proteins or applications of the present invention are used as aluminum-resistant plants, which can greatly simplify industrial production operations, open up channels for genetic breeding of aluminum-resistant plants, and provide new production ideas for genetic breeding of aluminum-resistant plants.
  • the screening work in the traditional breeding process, and the use of aluminum-resistant plants reduces the chemical and artificial input of soil improvement, etc., and greatly reduces the production cost.
  • Fig. 1 is the schematic diagram of binary vector 35s-pCAMBIA1301 provided by the present invention.
  • Fig. 2 is the schematic diagram of the transgenic vector pOEALR1 provided by the present invention.
  • Fig. 3 is a graph showing the comparison of ALR1 gene expression between wild-type and ALR1 overexpression transgenic lines provided by the present invention
  • Figure 4 is a comparison diagram of the aluminum resistance of wild-type, ALR1 knockout mutants, functionally restored lines and overexpressed transgenic lines provided by the present invention
  • Figure 5 is a comparison diagram of the relative elongation of the main root of the wild type, ALR1 knockout mutant, functional recovery line and overexpression transgenic line provided by the present invention
  • Figure 6 is a graph showing the comparison of aluminum content of wild-type, ALR1 knockout mutants and overexpression transgenic lines provided by the present invention.
  • the present invention provides a primer for regulating the ALR1 gene of plant aluminum resistance, the primer comprising an upstream primer and a nucleotide sequence shown in SEQ ID NO.1 (5'-CGGATCCATGCGTGTTCATCGTTTTTGT-3') Downstream primer as shown in SEQ ID NO. 2 (5'-CGTCGACCTAGACATCATCAAGCCAAGAG-3').
  • the primers are designed according to the Arabidopsis aluminum ion receptor ALR1 gene, and enzyme cleavage sites are designed at both ends to facilitate the preparation of subsequent vectors.
  • the nucleotide sequence of the gene of the present invention is as shown in SEQ ID NO.3(ATGCGTGTTCATCGTTTTTGTGTGATCGTCATCTTCCTCACAGAGTTACTATGTTTCTTCTATTCCTCGGAATCTCAGACCACCTCCCAGGTGCCATCCACATGACCTCGAAGCCTTACGTGACTTCATAGCACATCTCGAACCAAAACCAGATGGTTGGATCAATTCTTCTTCTTCTACAGACTGCTGCAACTGGACCGGAATCACCTGCAATTCAAACAACACCGGAAGAGTTATTAGATTGGAGCTTGGGAACAAAAAAAGCTGTCGGGGAAGTTGTCTGAATCTCTCTCTCGGGAAGCTAGATGAGATTAGGGTTCTTAATCTCTCTCGAAACTTCATCAAAGATTCGATCCCTCTTTCGATTTTCAACTTGAAGAATCTACAAACTCTTGATTTGAGCTCTCTAATGATCTCTCTAATGATCTCTCTAATGATCTCTCTAATGATCTCTCTAATGATCTCTCTAATGATCTCT
  • the primers of the present invention can be used for artificial cloning of ALR1.
  • the source of the gene ALR1 there is no special limitation on the source of the gene ALR1, and the artificial gene synthesis method or amplification method well-known in the art can be used.
  • the acquisition of the gene is preferably carried out by the method of cloning, preferably using the Arabidopsis root cDNA as a template, and using the primers described in the above technical scheme to carry out PCR amplification to obtain a DNA containing enzyme cleavage sites at both ends.
  • the reaction procedure of the PCR amplification is preferably as follows: pre-denaturation at 94°C for 2 minutes; denaturation at 98°C for 10 seconds, annealing at 57°C for 30 seconds, extension at 68°C for 3 minutes, 30 cycles; final extension at 68°C for 5 seconds minute.
  • the plants preferably include all types of plants, such as Arabidopsis thaliana.
  • the present invention uses the model plant Arabidopsis thaliana as the material to carry out the experiment, and the amplified gene is recorded as AtALR1 in the embodiment.
  • the present invention also provides a vector for overexpressing the Arabidopsis aluminum ion receptor ALR1 gene prepared based on the primers described in the above technical solution.
  • ALR1 gene the nucleotide sequence of the gene is shown in SEQ ID NO.3.
  • the vector for overexpressing the Arabidopsis thaliana aluminum ion receptor ALR1 gene of the present invention can realize the overexpression of the Arabidopsis thaliana aluminum ion receptor ALR1 gene.
  • the present invention does not specifically limit the construction method of the vector for overexpressing the Arabidopsis aluminum ion receptor ALR1 gene, and a conventional vector construction method well known to those skilled in the art can be used, such as the enzyme cleavage ligation method.
  • the present invention preferably firstly uses the upstream primer whose nucleotide sequence is shown in SEQ ID NO.1 and the downstream primer whose nucleotide sequence is shown in SEQ ID NO.2 to perform amplification to obtain two
  • the ALR1 coding region sequence containing the restriction site at the end was then connected to the pMD19T vector, and then the gene ALR1 was excised from the pMD19T vector by double restriction digestion with BamHI and SalI, and connected to the binary vector pCAMBIA1301 containing the promoter CaMV35S ( 35s-pCAMBIA1301), the constructed vector was named pOEALR1 (Fig. 2, the schematic diagram of the transgenic vector pOEALR1).
  • the vector for overexpressing the Arabidopsis thaliana aluminum ion receptor ALR1 gene of the present invention is preferably transferred into plants by Agrobacterium-mediated method to achieve the overexpression of the gene.
  • the 35s-pCAMBIA1301 is a plant constitutive overexpression vector (Fig. 1, the schematic diagram of the binary vector 35s-pCAMBIA1301), which is a binary vector pCAMBIA1301 containing the promoter CaMV35S.
  • the construction method of the 35s-pCAMBIA1301 is preferably to insert a cauliflower mosaic virus constitutive promoter CaMV35S into the multiple cloning site of pCAMBIA1301 by using the enzyme cleavage sites SacI and Kpn1.
  • the present invention also provides an Arabidopsis thaliana aluminum ion receptor ALR1 gene or protein or the application of the primer described in the above technical solution in the regulation of plant aluminum resistance, and the nucleotide sequence of the ALR1 gene is as shown in SEQ ID NO.3 As shown, the amino acid sequence of the ALR1 protein is as shown in SEQ ID NO. TDLVGTLGYIPPEYGQASVATYKGDVYSFGVVLLELLTDKRPVDMCKPKGCRDLISWVVKMKHESRASEVFDPLIYSKENDKEMFRVLEIACLCLSENPKQRPTTQQLVSWLDDV).
  • the ALR1 is located in the coding region 1-3027 of the full-length cDNA of ALR1, and the length of the nucleotide sequence is 3027 bp; the protein is a sequence consisting of 1008 amino acids.
  • the evaluation method of the plant's aluminum resistance is preferably evaluated by detecting the elongation of the main root of the plant by aluminum treatment.
  • the plants preferably include all types of plants, such as Arabidopsis thaliana.
  • the present invention uses the model plant Arabidopsis thaliana as the material to conduct experiments.
  • the present invention also provides the application of overexpressing the Arabidopsis thaliana aluminum ion receptor ALR1 gene or protein or the vector described in the above technical solution in improving the aluminum resistance of plants.
  • the nucleotide sequence of the ALR1 gene is SEQ ID NO.3
  • the amino acid sequence of the ALR1 protein is shown in SEQ ID NO.4.
  • the overexpression method preferably includes the following steps: cloning the gene ALR1 into a plant constitutive overexpression vector to obtain a recombinant expression vector; mediated by Agrobacterium, transforming the recombinant expression vector into a in plants.
  • the plant constitutive overexpression vector is preferably 35s-pCAMBIA1301, and the construction method of 35s-pCAMBIA1301 is preferably as described above.
  • the present invention has no particular limitation on the transformation method, and conventional operation methods well known to those skilled in the art can be adopted.
  • the present invention preferably performs the operations of culturing, screening and harvesting transgenic seeds.
  • the present invention does not specifically limit the methods of culturing, screening and harvesting, and conventional operation methods well known to those skilled in the art can be used.
  • the plants preferably include all types of plants, such as Arabidopsis thaliana.
  • the present invention uses the model plant Arabidopsis thaliana as the material to conduct experiments.
  • the present invention also provides the application of an Arabidopsis thaliana aluminum ion receptor ALR1 gene or protein or the above primer in regulating the root elongation of aluminum-stressed plants.
  • the nucleotide sequence of the ALR1 gene is represented by SEQ ID NO.3.
  • the amino acid sequence of the ALR1 protein is shown in SEQ ID NO.4.
  • the regulation preferably includes promoting the elongation of plant roots through overexpression of ALR1 gene or inhibiting the elongation of plant roots through knockout or silencing of ALR1 gene.
  • the method of gene knockout or silencing is not particularly limited in the present invention, and a gene knockout or silencing method well known in the art can be used.
  • the plants preferably include all types of plants, such as Arabidopsis thaliana.
  • the present invention uses the model plant Arabidopsis thaliana as the material to conduct experiments.
  • the present invention also provides the application of overexpressing the Arabidopsis thaliana aluminum ion receptor ALR1 gene or protein in improving the root elongation of aluminum-stressed plants.
  • the nucleotide sequence of the ALR1 gene is shown in SEQ ID NO.
  • the amino acid sequence of the ALR1 protein is shown in SEQ ID NO.4.
  • the plants preferably include all types of plants, such as Arabidopsis thaliana.
  • the present invention uses the model plant Arabidopsis thaliana as the material to conduct experiments.
  • the root system is preferably a main root.
  • overexpression of the ALR1 gene increased the root elongation of Arabidopsis thaliana under the condition of aluminum stress, and the relative elongation of the Arabidopsis taproot was significantly higher than that of the wild type and the mutant Arabidopsis thaliana that knocked out the ALR1 gene The relative elongation of the taproot of the material.
  • the overexpression method is preferably the same as the above-mentioned overexpression method of the Arabidopsis thaliana aluminum ion receptor ALR1 gene or protein or the application of the vector in improving the aluminum resistance of plants.
  • the method for detecting the elongation of the main root preferably includes sterilizing the seeds of the plant with 75% alcohol by mass, then washing them with sterilized water for 3 to 5 times, and sowing the seeds on demand in 1/2MS solid culture On the base plate, the plate was placed in a refrigerator at 4°C for 2 to 3 days, and then the plate was placed in a light incubator (light 16h/dark 8h) for 7 to 10 days.
  • the present invention also provides the application of an Arabidopsis thaliana aluminum ion receptor ALR1 gene or protein or the primer described in the above technical solution in regulating the root aluminum content of aluminum-stressed plants.
  • the nucleotide sequence of the ALR1 gene is SEQ ID NO. .3, the amino acid sequence of the ALR1 protein is shown in SEQ ID NO.4.
  • the regulation preferably includes reducing the aluminum content of plant roots by overexpression of ALR1 gene or increasing the aluminum content of plant roots by knockout or silencing of ALR1 gene.
  • the plants preferably include all types of plants, such as Arabidopsis thaliana.
  • the present invention uses the model plant Arabidopsis thaliana as the material to conduct experiments.
  • the present invention also provides the application of overexpressing the Arabidopsis thaliana aluminum ion receptor ALR1 gene or protein or the vector described in the above technical solution in reducing the root aluminum content of plants under aluminum stress.
  • the nucleotide sequence of the ALR1 gene is SEQ ID NO. .3, the amino acid sequence of the ALR1 protein is shown in SEQ ID NO.4.
  • the plants preferably include all types of plants, such as Arabidopsis thaliana.
  • the present invention uses the model plant Arabidopsis thaliana as the material to conduct experiments.
  • overexpression of the ALR1 gene reduces the aluminum content of Arabidopsis roots under aluminum stress conditions, and the aluminum content of Arabidopsis roots is significantly lower than that of the wild-type and ALR1 gene knockout Arabidopsis mutant materials aluminum content.
  • the overexpression method is preferably the same as the above-mentioned overexpression method of the Arabidopsis thaliana aluminum ion receptor ALR1 gene or protein or the application of the vector in improving the aluminum resistance of plants.
  • the method for detecting the content of aluminum in the root system preferably includes treating the seedlings with 0.5 mM calcium chloride plus 50 ⁇ M aluminum chloride solution for 24 hours. The roots of the seedlings were rinsed 3 times with ultrapure water to remove the aluminum solution on the surface of the roots, and the ultrapure water was blotted with filter paper. The main roots of the seedlings were cut off with a clean blade, and the roots of the same lines were combined and weighed.
  • the roots were digested and lysed with a mixture of nitric acid and perchloric acid (4:1 by volume).
  • the completely lysed samples were filtered through filter paper and collected in clean tubes for testing.
  • the aluminum content in the extract was determined by ICP-AES (inductively coupled plasma-atomic emission spectrometry).
  • the surface-sterilized Arabidopsis thaliana seeds were planted in 1/2MS solid medium, vernalized for 2-3 days in the dark at 4°C, and then moved to light for 6 days. After culturing for 6 days, the roots of the seedlings were collected for RNA extraction.
  • the cassette was reverse transcribed to synthesize cDNA as a template for subsequent gene cloning.
  • Upstream primer 5'-ATGCGTGTTCATCGTTTTTGT-3' (SEQ ID NO.5);
  • Downstream primer 5'-CTAGACATCATCAAGCCAAGAG-3' (SEQ ID NO. 6).
  • PCR amplification was performed using KOD FX enzyme from TOYOBO company.
  • the reaction program of PCR amplification was: pre-denaturation: 94°C, 2 minutes; denaturation: 98°C, 10 seconds; annealing at 57°C, 30 seconds; extension at 68°C, 3 minutes ( 30 cycles); final extension: 5 min at 68°C.
  • the reaction system of PCR amplification is as follows:
  • the PCR amplification product was sent to sequencing to obtain the CDS sequence of AtALR1 (SEQ ID NO.3).
  • a cauliflower mosaic virus constitutive promoter CaMV35S was inserted into the multi-cloning site through the digestion sites SacI and KpnI, so that the promoter CaMV35S was successfully connected to the pCAMBIA1301 vector,
  • the vector 35s-pCAMBIA1301 (Fig. 1), which can be used to construct constitutively overexpressed transgenic material, was obtained through transformation.
  • Example 2 Using primers ALR1-F: 5'-CGGATCCATGCGTGTTCATCGTTTTTGT-3' (SEQ ID NO. 1) and ALR1-R: 5'-CGTCGACCTAGACATCATCAAGCCAAGAG-3' (SEQ ID NO. 2), the cDNA sequences obtained in Example 1 above were used As a template, referring to the PCR amplification reaction procedure in the above Example 1, amplify the coding region sequence of the Arabidopsis aluminum ion receptor gene AtALR1 containing restriction sites at both ends.
  • the Arabidopsis aluminum ion receptor gene AtALR1 coding region sequence was linked to pMD19T according to the instructions of the pMD19T vector produced by Takara Company, and then the Arabidopsis aluminum ion receptor gene was ligated with BamHI and SalI double digestion and ligation method.
  • the AtALR1 coding region sequence was excised from the pMD19T vector and then connected to the promoter CaMV35S on the constitutive overexpression vector 35s-pCAMBIA1301 to obtain the transgenic vector pOEALR1 (binary transgenic vector pOEALR1 plasmid) that promotes the Arabidopsis gene AtALR1 from the promoter CaMV35S. (figure 2).
  • Example 2 0.5 ⁇ g of the binary transgenic vector pOEALR1 plasmid prepared in Example 2 was transferred into Agrobacterium tumefaciens strain GV3101 competent cells, followed by ice bath for 5 minutes, liquid nitrogen for 5 minutes, 37°C water bath for 5 minutes and ice bath for 5 minutes, and then added.
  • Antibiotic-free LB culture was based on activation in a shaker at 28°C for 1 h to obtain Agrobacterium strains containing binary plasmid vectors.
  • the Agrobacterium containing the binary plasmid vector was cultured in LB medium containing 50 mg/L kanamycin (Kan) and 50 mg/L rifampicin (Rif) at 28°C with shaking overnight to OD 600 absorbance value. 1.0, the cells were collected by centrifugation at 4000 rpm for 15 min, and resuspended in 1/2 MS medium containing 50 g/L sucrose. And select the wild-type (Col-0) Arabidopsis thaliana that has been bolted and partially completed flowering as the transgenic material, subtract the mature pods, retain the flowers and buds, and use the vacuum transformation method to infuse the above-mentioned Arabidopsis thaliana aerial part.
  • Kan kanamycin
  • Rif rifampicin
  • transgenic generation T1 generation seeds were harvested.
  • ARR1 ox 1 Homozygous transgenic T2 generation material was obtained after T1 generation seeds were screened for another generation on 1/2MS medium containing 50 mg/L hygromycin.
  • the wild-type and overexpressed transgenic plants were sampled to extract RNA from young whole plantlets. After reverse transcription, fluorescence real-time quantitative PCR was performed using TOYOBO's SYBR Green Realtime PCR Master Mix, and the Actin2 gene was used as an internal reference.
  • the detection system and primers used are as follows:
  • the primers used in the real-time quantitative PCR reaction were:
  • qALR1-F 5'-AGCGAGGTTTTCGATCCGTT-3' (SEQ ID NO. 7)
  • qALR1-R 5'-CTGTTGAGTCGTTGGCCTCT-3' (SEQ ID NO. 8)
  • qActin2-F 5'-GGTAACATTGTGCTCAGTGGTGG-3' (SEQ ID NO. 9)
  • qActin2-R 5'-AACGACCTTAATCTTCATGCTGC-3' (SEQ ID NO. 10).
  • Pre-denaturation 95°C, 1 minute; PCR cycles: 95°C, 15 seconds; 60°C, 15 seconds; 72°C, 45 seconds (40 cycles).
  • the reaction system of real-time quantitative PCR is as follows:
  • Figure 3 is a comparison chart of ALR1 gene expression between wild-type and ALR1 overexpressed transgenic lines
  • the expression of ALR1 gene in non-transgenic lines was 1.00 ⁇ 0.36, while the overexpression transgenic plants
  • the expression level of ALR1 gene was 8.15 ⁇ 0.97.
  • the ALR1 gene was expressed 15-20 times higher in the overexpressed transgenic plants.
  • the relative elongation of Arabidopsis taproots was measured by Al treatment to evaluate the Al resistance of plants.
  • the wild type and the Arabidopsis ALR1 knockout mutant material (alr1) purchased from the Arabidopsis Biological Resource Center (ABRC) were re-transformed into the alr1 mutant.
  • the functional recovery material and the seeds of the overexpressed transgenic material obtained in Example 3 of the present invention were surface-sterilized with 75% alcohol by mass, and then washed with sterilized water for 3 to 5 times.
  • Figure 4 a comparison chart of the aluminum resistance of wild type, ALR1 knockout mutants and overexpression transgenic lines; wherein the scale bars are all 1 cm in length
  • Figure 5 wild type, ALR1 knockout mutants and Comparison of the relative elongation of the main root of the overexpressed transgenic lines
  • ALR1 ox 1 the transgenic overexpressed Arabidopsis thaliana
  • alr1 knockout mutant material
  • Table 1 the transgenic overexpressed Arabidopsis thaliana
  • the root length of the materials was not significantly different from that of the wild-type control, while the relative elongation of the taproot of the transgenic (ALR1 ox 1) Arabidopsis thaliana was significantly increased under Al stress compared with the wild-type control, while the ALR1 gene
  • the relative elongation of the main root of the knockout mutant lines was significantly lower than that of the wild type.
  • the wild type grown for 7 days, the Arabidopsis ALR1 knockout mutant material (alr1) purchased from the Arabidopsis Biological Resource Center (ABRC), and the overexpressed transgenic material obtained in Example 3 of the present invention were used.
  • the seedlings were treated with 0.5 mM calcium chloride plus 50 ⁇ M aluminum chloride (Al) solution for 24 h.
  • the roots of the seedlings were rinsed 3 times with ultrapure water to remove the aluminum solution on the surface of the roots, and the ultrapure water was blotted with filter paper.
  • the seedling taproots were cut off with a clean blade, and the roots of the same lines were combined and weighed.
  • the roots were digested and lysed with a mixture of nitric acid and perchloric acid (4:1 by volume).
  • the completely lysed samples were filtered through filter paper and collected in clean tubes for testing.
  • the aluminum content in the extract was determined by ICP-AES (inductively coupled plasma-atomic emission spectrometry).
  • Figure 6 is a comparison chart of root aluminum content of wild-type, ALR1 knockout mutants and overexpression transgenic lines provided by the present invention
  • Table 2 under aluminum stress, transgene overexpression (ALR1 ox 1 ) of Arabidopsis thaliana was significantly lower than that of the wild-type control, while the root aluminum content of the ALR1 knockout mutant line was significantly higher than that of the wild-type.
  • ALR1 ox 1 Arabidopsis thaliana is significantly higher than that of the wild-type control, while the aluminum resistance of the ALR1 knockout mutant line is significantly lower than that of the wild type, indicating that the gene ALR1 is indeed Involved in the regulation of plant aluminum resistance.

Abstract

提供一种调控植物抗铝性的铝离子受体ALR1基因或蛋白的应用。利用核苷酸序列如SEQ ID NO.1所示的上游引物和核苷酸序列如SEQ ID NO.2所示的下游引物扩增获得核苷酸序列如SEQ ID NO.3所示的ALR1基因。在拟南芥中过表达该基因时，可以提升植物抗铝性，促进植物根系的生长。

Description

一种调控植物抗铝性的铝离子受体ALR1基因或蛋白的应用
本申请要求于2021年04月06日提交中国专利局、申请号为202110367334.2、发明名称为“一种调控植物抗铝性的铝离子受体ALR1基因或蛋白的应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明属于植物基因工程技术领域，具体涉及一种调控植物抗铝性的铝离子受体ALR1基因或蛋白的应用。
背景技术
土壤酸化以及随之而来的铝毒害是世界范围内农业生产中普遍存在的问题。土壤酸化的加剧，使得农作物的产量受到很大的影响，对农业可持续生产构成威胁。土壤pH的下降，一方面导致了盐基离子淋失，另一方面也使得部分不溶性的矿物态铝(地壳中含量最丰富的金属元素，平均含量在8％左右)解离为离子态而进入土壤溶液中，而大量研究表明，微摩尔级水平就可以显著抑制植物根系的生长，进而影响根系对养分和水分的吸收，并最终导致农作物的减产，甚至绝产。因此，铝毒被公认为酸性土壤影响作物生产的主要限制因素。尽管传统上人们可以通过土壤改良的方法(如施用石灰等改良剂)降低土壤中的铝活度，但这种举措一方面需要大量人力、物力及财力的投入，另一方面对心底层土壤的改造仍旧十分困难。而通过遗传改良来获得抗铝毒能力较强的植物品种，是持续、高效解决酸性土壤铝毒害的有效途径。
植物抗铝的生理机制可分为以有机酸和细胞壁组分特性改变为代表的铝外部排斥和将铝储存在液泡内进行区室化为代表的铝内部忍耐机制，这两大机制是目前公认的植物抗铝毒最主要的生理机制。随着分子生物学的快速发展，参与植物抗铝信号转导途径的基因和蛋白也大量被鉴定出来。然而，目前人们对于植物如何感受铝离子却仍一无所知。鉴定参与感受铝离子过程的基因，并深入剖析其功能，对进一步明确植物抗铝的分子 机制，为育种提供参考，以及提高植物抗铝性和作物产量具有重要意义。调节植物抗铝性的铝离子受体基因及其功能尚未被发现，在一定程度上阻碍了抗铝性植物的培育进程。
发明内容
本发明的目的在于提供一种调控植物抗铝性的铝离子受体ALR1基因或蛋白的应用。本发明所述引物在具体应用时可以提升植物抗铝性，促进植物根系的生长，进而促进植物对养分和水分的吸收，提高农作物的产量。
本发明提供了一种调控植物抗铝性的ALR1基因的引物，所述引物包括核苷酸序列如SEQ ID NO.1所示的上游引物和核苷酸序列如SEQ ID NO.2所示的下游引物。
本发明还提供了一种基于上述技术方案所述引物制备得到的过表达拟南芥铝离子受体ALR1基因的载体，所述载体以35s-pCAMBIA1301作为骨架载体，还包括拟南芥铝离子受体ALR1基因，所述ALR1基因的核苷酸序列SEQ ID NO.3所示。
本发明还提供了一种拟南芥铝离子受体ALR1基因或蛋白或上述引物在植物抗铝性调控中的应用，所述ALR1基因的核苷酸序列SEQ ID NO.3所示，所述ALR1蛋白的氨基酸序列如SEQ ID NO.4所示。
本发明还提供了过表达拟南芥铝离子受体ALR1基因或蛋白或上述技术方案所述载体在提高植物的抗铝性中的应用，所述ALR1基因的核苷酸序列SEQ ID NO.3所示，所述ALR1蛋白的氨基酸序列如SEQ ID NO.4所示。
本发明还提供了一种拟南芥铝离子受体ALR1基因或蛋白或上述引物在调控铝胁迫植物的根系伸长量中的应用，所述ALR1基因的核苷酸序列SEQ ID NO.3所示，所述ALR1蛋白的氨基酸序列如SEQ ID NO.4所示。
本发明还提供了过表达拟南芥铝离子受体ALR1基因或蛋白或上述技术方案所述载体在提高铝胁迫植物的根系伸长量中的应用，所述ALR1基因的核苷酸序列SEQ ID NO.3所示，所述ALR1蛋白的氨基酸序列如SEQ ID NO.4所示。
优选的是，所述根系为主根。
本发明还提供了一种拟南芥铝离子受体ALR1基因或蛋白或上述技术方案所述引物在调控铝胁迫植物的根系铝含量中的应用，所述ALR1基因的核苷酸序列SEQ ID NO.3所示，所述ALR1蛋白的氨基酸序列如SEQ ID NO.4所示。
本发明还提供了过表达拟南芥铝离子受体ALR1基因或蛋白或上述技术方案所述载体在降低铝胁迫植物的根系铝含量中的应用，所述ALR1基因的核苷酸序列SEQ ID NO.3所示，所述ALR1蛋白的氨基酸序列如SEQ ID NO.4所示。
优选的，所述植物包括拟南芥。
本发明提供了一种调控植物抗铝性的ALR1基因的引物。本发明利用特定扩增引物从模式植物拟南芥中克隆了铝离子受体基因ALR1，并通过敲除和过表达ALR1基因构建了两种突变株系，敲除株系抗铝性明显降低，而过表达株系则表现出抗铝性显著提高。试验结果表明，在铝胁迫下，转基因过表达拟南芥比野生型对照的主根伸长量明显提高，根系铝含量明显降低，而ALR1基因的敲除突变株系的主根伸长量则明显低于野生型，根系铝含量明显高于野生型，突变功能回复株系的主根伸长量与野生型相近，提高ALR1的表达有利于植物抗铝性的提高。本发明所述应用对提升植物抗铝性，促进植物根系的生长，进而促进植物对养分和水分的吸收，提高农作物的产量具有重要意义。利用本发明引物、基因、蛋白或应用获得的植物种子作为抗铝植物使用，能够大大简化工业生产操作，开拓遗传育种抗铝植物的渠道，为遗传育种抗铝植物提供新的生产思路，减少了传统育种过程中的筛选工作，同时抗铝植物的使用减少了土壤改良等的药剂和人工投入，大大降低生产成本。
说明书附图
图1为本发明提供的双元载体35s-pCAMBIA1301的示意图；
图2为本发明提供的转基因载体pOEALR1的示意图；
图3为本发明提供的野生型和ALR1过表达转基因株系ALR1基因表达量对比图；
图4为本发明提供的野生型、ALR1敲除突变体、功能回复株系和过表达转基因株系的抗铝性的对比图；
图5为本发明提供的野生型、ALR1敲除突变体、功能回复株系和过表达转基因株系的主根相对伸长量的对比图；
图6为本发明提供的野生型、ALR1敲除突变体和过表达转基因株系的铝含量对比图。
具体实施方式
本发明提供了一种调控植物抗铝性的ALR1基因的引物，所述引物包括核苷酸序列如SEQ ID NO.1(5'-CGGATCCATGCGTGTTCATCGTTTTTGT-3')所示的上游引物和核苷酸序列如SEQ ID NO.2(5'-CGTCGACCTAGACATCATCAAGCCAAGAG-3')所示的下游引物。在本发明中，所述引物是根据拟南芥铝离子受体ALR1基因进行设计的，两端设计有酶切位点，方便后续载体的制备，本发明所述基因的核苷酸序列如SEQ ID NO.3(ATGCGTGTTCATCGTTTTTGTGTGATCGTCATCTTCCTCACAGAGTTACTATGTTTCTTCTATTCCTCGGAATCTCAGACCACCTCCAGGTGCCATCCACATGACCTCGAAGCCTTACGTGACTTCATAGCACATCTCGAACCAAAACCAGATGGTTGGATCAATTCTTCTTCTTCTACAGACTGCTGCAACTGGACCGGAATCACCTGCAATTCAAACAACACCGGAAGAGTTATTAGATTGGAGCTTGGGAACAAAAAGCTGTCGGGGAAGTTGTCTGAATCTCTCGGGAAGCTAGATGAGATTAGGGTTCTTAATCTCTCTCGAAACTTCATCAAAGATTCGATCCCTCTTTCGATTTTCAACTTGAAGAATCTACAAACTCTTGATTTGAGCTCTAATGATCTCTCCGGCGGAATCCCAACAAGTATAAATCTCCCAGCTCTGCAAAGTTTTGATCTTTCTTCAAATAAATTCAATGGGTCGCTTCCGTCTCATATCTGCCATAACTCTACTCAAATTAGGGTTGTGAAACTTGCGGTGAACTACTTCGCCGGAAACTTCACTTCCGGGTTTGGGAAATGTGTCTTGCTTGAGCATCTCTGTCTTGGTATGAACGATCTTACTGGTAACATCCCTGAGGATTTGTTTCATCTCAAAAGATTGAATCTTTTAGGGATTCAAGAGAATCGTCTCTCT GGTTCGTTGAGTCGTGAGATTAGGAATCTCTCAAGTCTTGTTCGTCTTGATGTTTCTTGGAATTTGTTTTCCGGTGAAATCCCTGATGTGTTCGACGAATTGCCTCAGTTAAAGTTTTTCTTAGGTCAGACCAATGGATTCATTGGAGGAATACCTAAATCGTTGGCGAATTCACCGAGTTTGAATCTGCTTAACTTGAGGAACAATTCTTTATCGGGTCGTTTGATGTTGAATTGTACGGCGATGATTGCTTTGAACTCTCTTGATTTAGGTACCAATAGATTCAATGGGAGGTTACCTGAGAATCTACCGGATTGCAAGCGGTTAAAGAACGTTAACCTCGCGAGGAACACCTTCCATGGACAAGTACCAGAGAGTTTCAAGAACTTCGAGAGCTTATCTTACTTCTCGTTATCGAATTCGAGTTTGGCTAATATCTCTTCAGCGCTTGGGATACTTCAGCATTGCAAGAACTTGACGACTTTGGTTCTTACATTGAATTTCCATGGAGAGGCTTTACCCGATGATTCAAGTCTTCATTTCGAGAAGCTTAAGGTGCTTGTAGTGGCGAATTGTAGGCTTACTGGTTCGATGCCGAGGTGGTTAAGCTCGAGTAATGAACTTCAGTTGTTGGATCTTTCTTGGAACCGTTTAACCGGCGCTATCCCGAGCTGGATTGGTGACTTCAAGGCTCTGTTCTACTTGGATTTATCTAACAACTCGTTTACAGGAGAGATCCCTAAGAGCTTAACTAAGTTAGAGAGTCTCACTAGCCGTAATATCTCAGTCAATGAGCCATCTCCTGATTTCCCGTTCTTTATGAAAAGAAACGAGAGCGCGAGAGCGTTGCAATACAATCAGATTTTCGGGTTCCCGCCAACGATTGAGCTTGGTCATAACAATCTCTCTGGACCTATTTGGGAGGAGTTTGGTAATCTGAAGAAGCTTCATGTGTTTGATTTGAAATGGAATGCATTATCTGGATCAATACCTAGCTCGCTTTCTGGTATGACGAGCTTGGAAGCTCTTGATCTCTCTAATAACCGTCTTTCGGGTTCGATCCCGGTTTCTCTGCAACAGCTCTCGTTTCTGTCGAAGTTCAGTGTTGCTTATAACAATCTCTCGGGAGTAATACCTTCCGGTGGTCAGTTTCAGACGTTTCCAAACTCGAGCTTTGAGAGTAACCATCTCTGCGGGGAACACAGATTCCCCTGTTCTGAAGGTACTGAGAGTGCATTGATCAAACGGTCAAGAAGAAGCAGAGGAGGTGACATTGGAATGGCGATTGGGATAGCGTTTGGTTCGGTTTTTCTTTTGACTCTTCTCTCGTTGATTGTGTTGCGTGCTCGTAGACGGTCAG GAGAAGTTGATCCGGAGATAGAAGAATCCGAGAGCATGAATCGTAAAGAACTCGGAGAGATTGGATCTAAGCTTGTGGTTTTGTTTCAGAGCAATGATAAAGAGCTCTCTTATGATGACCTTTTGGACTCAACAAATAGTTTTGATCAAGCTAACATCATTGGCTGTGGCGGGTTTGGTATGGTTTACAAAGCAACGTTACCAGACGGTAAGAAAGTTGCGATCAAGAAGTTATCCGGTGATTGCGGTCAAATCGAAAGAGAATTCGAAGCAGAAGTTGAAACACTCTCAAGAGCACAGCATCCAAATCTTGTTCTTCTCCGAGGATTCTGTTTCTACAAAAACGACCGGCTTTTAATCTACTCGTATATGGAAAACGGAAGCTTAGACTATTGGCTACACGAGCGTAACGACGGTCCAGCGTTGTTGAAGTGGAAAACACGTCTTAGAATCGCTCAAGGTGCTGCAAAAGGGTTACTTTACTTGCATGAAGGGTGTGATCCTCATATCTTACACCGCGATATTAAATCGAGTAATATTCTTCTCGACGAGAATTTCAACTCTCATTTAGCGGATTTCGGACTCGCAAGGCTGATGAGTCCTTACGAGACGCATGTAAGTACTGATTTGGTTGGAACTTTAGGTTACATTCCTCCGGAATACGGGCAAGCTTCGGTTGCTACTTACAAAGGCGATGTGTATAGTTTCGGAGTTGTGCTTCTCGAGCTTTTAACCGATAAAAGACCGGTGGATATGTGTAAACCGAAAGGGTGTAGGGATCTGATCTCGTGGGTCGTCAAGATGAAGCATGAGAGTCGAGCAAGCGAGGTTTTCGATCCGTTAATATACAGTAAAGAGAATGATAAAGAGATGTTTCGGGTTCTCGAGATTGCTTGTTTATGTTTAAGCGAAAACCCGAAACAGAGGCCAACGACTCAACAGTTAGTCTCTTGGCTTGATGATGTCTAG)所示。本发明所述引物能够用于ALR1的人工克隆。本发明对所述基因ALR1的来源没有特殊限制，采用本领域所熟知的基因人工合成方法或扩增方法即可。如，在本发明中，所述基因的获取优选采用克隆的方法进行，优选以拟南芥根系cDNA为模板，采用上述技术方案所述引物进行PCR扩增，得到两端包含酶切位点的基因ALR1。在本发明中，所述PCR扩增的反应程序优选如下：94℃预变性2分钟；98℃变性10秒，57℃退火30秒，68℃延伸3分钟，30个循环；68℃终延伸5分钟。
在本发明中，所述植物优选包括所有类型的植物，如拟南芥。为了举 例说明所述ALR1基因的调控方式，本发明以模式植物拟南芥为材料进行实验，扩增得到的基因在实施例中记为AtALR1。
本发明还提供了一种基于上述技术方案所述引物制备得到的过表达拟南芥铝离子受体ALR1基因的载体，所述载体以35s-pCAMBIA1301作为骨架载体，还包括拟南芥铝离子受体ALR1基因，所述基因的核苷酸序列如SEQ ID NO.3所示。本发明所述过表达拟南芥铝离子受体ALR1基因的载体能够实现拟南芥铝离子受体ALR1基因的过表达。本发明对所述过表达拟南芥铝离子受体ALR1基因的载体的构建方法没有特殊限定，采用本领域技术人员熟知的常规载体构建方法即可，如酶切连接法。在本发明具体实施例中，本发明优选先利用核苷酸序列如SEQ ID NO.1所示的上游引物和核苷酸序列如SEQ ID NO.2所示的下游引物进行扩增，得到两端包含酶切位点的ALR1编码区序列，然后将此序列连接到pMD19T载体，再利用BamHI和SalI双酶切将基因ALR1从pMD19T载体切下，连接到含启动子CaMV35S的双元载体pCAMBIA1301(35s-pCAMBIA1301)上，构建完成的载体被命名为pOEALR1(图2，转基因载体pOEALR1的示意图)。本发明所述过表达拟南芥铝离子受体ALR1基因的载体优选利用农杆菌介导法转入植物中实现基因的过表达。在本发明中，所述35s-pCAMBIA1301为植物组成型过表达载体(图1，双元载体35s-pCAMBIA1301的示意图)，为含有启动子CaMV35S的双元载体pCAMBIA1301。在本发明中，所述35s-pCAMBIA1301的构建方法优选为利用酶切位点SacI和Kpn1，在pCAMBIA1301的多克隆位点插入一个花椰菜花叶病毒组成型启动子CaMV35S。
本发明还提供了一种拟南芥铝离子受体ALR1基因或蛋白或上述技术方案所述引物在植物抗铝性调控中的应用，所述ALR1基因的核苷酸序列如SEQ ID NO.3所示，所述ALR1蛋白的氨基酸序列如SEQ ID NO.4(MRVHRFCVIVIFLTELLCFFYSSESQTTSRCHPHDLEALRDFIAHLEPKPDGWINSSSSTDCCNWTGITCNSNNTGRVIRLELGNKKLSGKLSESLGKLDEIRVLNLSRNFIKDSIPLSIFNLKNLQTLDLSSNDLSGGIPTSINLPALQSFDLSSNKFNGSLPSHICHNSTQIRVVKLAVNYFAGNFTSGFGKCVL LEHLCLGMNDLTGNIPEDLFHLKRLNLLGIQENRLSGSLSREIRNLSSLVRLDVSWNLFSGEIPDVFDELPQLKFFLGQTNGFIGGIPKSLANSPSLNLLNLRNNSLSGRLMLNCTAMIALNSLDLGTNRFNGRLPENLPDCKRLKNVNLARNTFHGQVPESFKNFESLSYFSLSNSSLANISSALGILQHCKNLTTLVLTLNFHGEALPDDSSLHFEKLKVLVVANCRLTGSMPRWLSSSNELQLLDLSWNRLTGAIPSWIGDFKALFYLDLSNNSFTGEIPKSLTKLESLTSRNISVNEPSPDFPFFMKRNESARALQYNQIFGFPPTIELGHNNLSGPIWEEFGNLKKLHVFDLKWNALSGSIPSSLSGMTSLEALDLSNNRLSGSIPVSLQQLSFLSKFSVAYNNLSGVIPSGGQFQTFPNSSFESNHLCGEHRFPCSEGTESALIKRSRRSRGGDIGMAIGIAFGSVFLLTLLSLIVLRARRRSGEVDPEIEESESMNRKELGEIGSKLVVLFQSNDKELSYDDLLDSTNSFDQANIIGCGGFGMVYKATLPDGKKVAIKKLSGDCGQIEREFEAEVETLSRAQHPNLVLLRGFCFYKNDRLLIYSYMENGSLDYWLHERNDGPALLKWKTRLRIAQGAAKGLLYLHEGCDPHILHRDIKSSNILLDENFNSHLADFGLARLMSPYETHVSTDLVGTLGYIPPEYGQASVATYKGDVYSFGVVLLELLTDKRPVDMCKPKGCRDLISWVVKMKHESRASEVFDPLIYSKENDKEMFRVLEIACLCLSENPKQRPTTQQLVSWLDDV)所示。在本发明中，所述ALR1位于ALR1全长cDNA的1～3027编码区，核苷酸序列长度为3027bp；所述蛋白为1008个氨基酸组成的序列。在本发明中，所述植物抗铝性的评价方法优选用铝处理检测植物主根的伸长量来评价。在本发明中，所述植物优选包括所有类型的植物，如拟南芥。为了举例说明所述ALR1基因的调控方式，本发明以模式植物拟南芥为材料进行实验。
本发明还提供了过表达拟南芥铝离子受体ALR1基因或蛋白或上述技术方案所述载体在提高植物的抗铝性中的应用，所述ALR1基因的核苷酸序列SEQ ID NO.3所示，所述ALR1蛋白的氨基酸序列如SEQ ID NO.4所示。在本发明中，所述过表达的方法，具体优选包括以下步骤：将基因ALR1克隆至植物组成型过表达载体中，得到重组表达载体；以农杆菌介导，将所述重组表达载体转化入植物中。在本发明中，所述植物组成型过表达载体优选为35s-pCAMBIA1301，35s-pCAMBIA1301的构建方法优选 如上文所述。本发明对所述转化的方法没有特殊限定，采用本领域技术人员熟知的常规操作方法即可。转化后，本发明优选进行培养，筛选和收获转基因种子的操作。本发明对所述培养、筛选和收获的方法没有特殊限定，采用本领域技术人员熟知的常规操作方法即可。在本发明中，所述植物优选包括所有类型的植物，如拟南芥。为了举例说明所述ALR1基因的过表达方法，本发明以模式植物拟南芥为材料进行实验。
本发明还提供了一种拟南芥铝离子受体ALR1基因或蛋白或上述引物在调控铝胁迫植物的根系伸长量中的应用，所述ALR1基因的核苷酸序列SEQ ID NO.3所示，所述ALR1蛋白的氨基酸序列如SEQ ID NO.4所示。在本发明中，所述调控优选包括通过ALR1基因过表达促进植物根系的伸长量或通过ALR1基因的敲除或沉默抑制植物根系的伸长量。本发明对所述基因敲除或沉默的方法没有特殊限制，采用本领域所熟知的基因敲除或沉默方法即可。在本发明中，所述植物优选包括所有类型的植物，如拟南芥。为了举例说明所述ALR1基因的调控方式，本发明以模式植物拟南芥为材料进行实验。
本发明还提供了过表达拟南芥铝离子受体ALR1基因或蛋白在提高铝胁迫植物的根系伸长量中的应用，所述ALR1基因的核苷酸序列SEQ ID NO.3所示，所述ALR1蛋白的氨基酸序列如SEQ ID NO.4所示。在本发明中，所述植物优选包括所有类型的植物，如拟南芥。为了举例说明所述ALR1基因的过表达方式，本发明以模式植物拟南芥为材料进行实验。在本发明实施例中，过表达ALR1基因提高了铝胁迫条件下拟南芥根系的伸长量，拟南芥根系的相对伸长量显著高于野生型和敲除ALR1基因的拟南芥突变材料的根系相对伸长量。在本发明中，所述根系优选为主根。在本发明实施例中，过表达ALR1基因提高了铝胁迫条件下拟南芥根系的伸长量，拟南芥主根的相对伸长量显著高于野生型和敲除ALR1基因的拟南芥突变材料的主根相对伸长量。
在本发明中，所述过表达的方法优选与上述过表达拟南芥铝离子受体ALR1基因或蛋白或载体在提高植物的抗铝性中的应用的过表达方法相 同。在本发明中，所述主根伸长量的检测方法优选包括将植物的种子用质量浓度75％酒精表面消毒后，再用灭菌水清洗3～5遍，将种子点播在1/2MS固体培养基平板上，然后将平板置于4℃冰箱2～3天，再将平板放置于光照培养箱(光照16h/黑暗8h)中生长7～10天。用尺子测量每种材料幼苗主根的长度，计算相应材料对照条件下(不进行铝处理)的相对伸长量，即对照条件下的根长/对照条件下的平均根长*100，然后计算相应材料铝处理下相对伸长量，即铝处理下的根长/对照条件下的平均根长*100。
本发明还提供了一种拟南芥铝离子受体ALR1基因或蛋白或上述技术方案所述引物在调控铝胁迫植物的根系铝含量中的应用，所述ALR1基因的核苷酸序列SEQ ID NO.3所示，所述ALR1蛋白的氨基酸序列如SEQ ID NO.4所示。在本发明中，所述调控优选包括通过ALR1基因过表达降低植物根系的铝含量或通过ALR1基因的敲除或沉默提高植物根系的铝含量。在本发明中，所述植物优选包括所有类型的植物，如拟南芥。为了举例说明所述ALR1基因的调控方式，本发明以模式植物拟南芥为材料进行实验。
本发明还提供了过表达拟南芥铝离子受体ALR1基因或蛋白或上述技术方案所述载体在降低铝胁迫植物的根系铝含量中的应用，所述ALR1基因的核苷酸序列SEQ ID NO.3所示，所述ALR1蛋白的氨基酸序列如SEQ ID NO.4所示。在本发明中，所述植物优选包括所有类型的植物，如拟南芥。为了举例说明所述ALR1基因的过表达方式，本发明以模式植物拟南芥为材料进行实验。在本发明实施例中，过表达ALR1基因降低了铝胁迫条件下拟南芥根系的铝含量，拟南芥根系的铝含量显著低于于野生型和敲除ALR1基因的拟南芥突变材料的铝含量。
在本发明中，所述过表达的方法优选与上述过表达拟南芥铝离子受体ALR1基因或蛋白或载体在提高植物的抗铝性中的应用的过表达方法相同。在本发明中，所述根系铝含量的检测方法优选包括将小苗用0.5mM氯化钙加50μM氯化铝溶液处理24h。用超纯水将小苗根冲洗3次，去除根表面的铝溶液，并用滤纸吸干超纯水。用干净刀片切下小苗主根，相 同株系的根进行合并并称重。用硝酸和高氯酸(体积比4:1)的混合液对根进行消煮裂解。完全裂解后的样品经滤纸过滤后收集于干净的管子中待测。提取物中的铝含量用ICP-AES(inductively coupled plasma-atomic emission spectrometry)测定。
下面结合具体实施例对本发明所述的一种调控植物抗铝性的铝离子受体ALR1基因或蛋白的应用做进一步详细的介绍，本发明的技术方案包括但不限于以下实施例。
实施例1
ALR1基因的克隆
将表面消毒后的拟南芥种子种于1/2MS固体培养基，黑暗4℃条件下春化2～3天后移到光照条件下培养6天后，收集小苗的根用于RNA的提取，采用试剂盒进行逆转录合成cDNA作为后续基因克隆的模板。
根据目前已公开的拟南芥全基因组测序结果，分别设计上游引物和下游引物：
上游引物：5'-ATGCGTGTTCATCGTTTTTGT-3'(SEQ ID NO.5)；
下游引物：5'-CTAGACATCATCAAGCCAAGAG-3'(SEQ ID NO.6)。
使用TOYOBO公司KOD FX酶进行PCR扩增，PCR扩增的反应程序为：预变性：94℃，2分钟；变性：98℃，10秒；退火57℃，30秒；延伸68℃，3分钟(30个循环)；终延伸：68℃，5分钟。PCR扩增的反应体系为：
Figure PCTCN2021094884-appb-000001
将PCR扩增产物送至测序，得到AtALR1的CDS序列(SEQ ID NO.3)。
实施例2
组成型过表达转基因载体的构建
利用DNA片段双酶切和连接的方法，通过酶切位点SacI和KpnI，在多克隆位点中正向插入一个花椰菜花叶病毒组成型启动子CaMV35S，使得启动子CaMV35S成功连接到pCAMBIA1301载体上，改造获得可用于构建组成型过表达转基因材料的载体35s-pCAMBIA1301(图1)。
使用引物ALR1-F:5'-CGGATCCATGCGTGTTCATCGTTTTTGT-3'(SEQ ID NO.1)和ALR1-R：5'-CGTCGACCTAGACATCATCAAGCCAAGAG-3'(SEQ ID NO.2)，以上述实施例1中获得的cDNA序列作为模板，参照上述实施例1中PCR扩增反应程序，扩增获得两端包含酶切位点的拟南芥铝离子受体基因AtALR1编码区序列。按照Takara公司生产的pMD19T载体使用说明将拟南芥铝离子受体基因AtALR1编码区序列连接到pMD19T上，然后再利用BamHI和SalI双酶切和连接的方法，将拟南芥铝离子受体基因AtALR1编码区序列从pMD19T载体切下后连接到组成型过表达载体35s-pCAMBIA1301上的启动子CaMV35S后，得到由启动子CaMV35S启动拟南芥基因AtALR1的转基因载体pOEALR1(双元转基因载体pOEALR1质粒)(图2)。
实施例3
拟南芥的转化
将0.5μg实施例2制备的双元转基因载体pOEALR1质粒转入农杆菌(Agrobacterium tumefaciens)株系GV3101感受态细胞中，依次冰浴5min，液氮5min，37℃水浴5min和冰浴5min后，加入无抗LB培养基于28℃摇床中活化1h，得含有双元质粒载体的农杆菌菌株。用制备的含有双元质粒载体的GV3101菌株来转化拟南芥，具体步骤如下：
将含有双元质粒载体的农杆菌在含有50mg/L的卡那霉素(Kan)和50mg/L的利福平(Rif)的LB培养基中，28℃振荡过夜培养至OD 600吸光值为1.0，在4000rpm条件下离心15min收集菌体，并用含有50g/L蔗糖的1/2MS培养基重悬。并选取已经抽薹并部分完成开花的野生型(Col-0)拟南芥作为转基因材料，减去已成熟的荚果，保留花和花苞，采用抽真空转化法，将拟南芥地上部分浸染到上述制备的菌液中，真空抽 取5min后，在黑暗、23℃条件下培养24h后，在含有50mg/L潮霉素的1/2MS培养基上筛选1周后获得抗性苗，移栽土壤培养后收获转基因一代(T1代)种子。T1代种子通过在含有50mg/L潮霉素的1/2MS培养基上再筛选一代后得到纯合的转基因T2代材料(ALR1 ox 1)。
实施例4
目的基因表达的分子检测
野生型和过表达转基因植株的幼嫩全株小苗取样提取RNA，经逆转录，采用TOYOBO公司SYBR Green Realtime PCR Master Mix进行荧光实时定量PCR检测，以Actin2基因作为内参；检测体系和所用引物如下：
所用荧光实时定量PCR反应引物为：
qALR1-F：5'-AGCGAGGTTTTCGATCCGTT-3'(SEQ ID NO.7)
qALR1-R：5'-CTGTTGAGTCGTTGGCCTCT-3'(SEQ ID NO.8)
qActin2-F:5'-GGTAACATTGTGCTCAGTGGTGG-3'(SEQ ID NO.9)
qActin2-R:5'-AACGACCTTAATCTTCATGCTGC-3'(SEQ ID NO.10)。
荧光实时定量PCR的反应程序如下：
预变性：95℃，1分钟；PCR循环：95℃，15秒；60℃，15秒；72℃，45秒(40个循环)。
荧光实时定量PCR的反应体系为：
Figure PCTCN2021094884-appb-000002
检测后发现，如图3所示(图3为野生型和ALR1过表达转基因株系ALR1基因表达量对比图)，未转基因株系中ALR1基因的表达量为1.00±0.36，而过表达转基因植株中ALR1基因的表达量为8.15±0.97。相比未转基因株系，ALR1基因在过表达转基因植株中有着15～20倍的高表达。
实施例5
种子抗铝性的检测
用铝处理检测拟南芥主根的相对伸长量来评价植物的抗铝性。分别将野生型，以及从拟南芥生物资源中心(ABRC)购买获得的拟南芥ALR1基因的敲除突变体材料(alr1)，将ALR1基因重新转入到alr1突变体中构建的突变体的功能回复材料，和本发明实施例3所得的过表达转基因材料的种子用质量浓度75％酒精表面消毒后，再用灭菌水清洗3～5遍，将种子点播在含有(铝处理)或不含有1mM氯化铝(不进行铝处理，即对照)的1/2MS固体培养基平板上，然后将平板置于4℃冰箱2～3天，再将平板放置于光照培养箱(光照16h/黑暗8h)中生长7～10天。用尺子测量每种材料幼苗主根的长度，计算相应材料对照条件下(不进行铝处理)的相对伸长量，即对照条件下的根长/对照条件下的平均根长*100，然后计算相应材料铝处理下相对伸长量，即铝处理下的根长/对照条件下的平均根长*100。
如图4(野生型、ALR1敲除突变体和过表达转基因株系的抗铝性的对比图；其中标尺长度均为1cm)、图5(本发明提供的野生型、ALR1敲除突变体和过表达转基因株系的主根相对伸长量的对比图)和表1所示，在无铝胁迫条件下，转基因过表达拟南芥(ALR1 ox 1)、敲除突变材料(alr1)及功能回复材料(Com1和Com2)与野生型对照的根长无显著差别，而在铝胁迫下，转基因过表达(ALR1 ox 1)拟南芥比野生型对照主根的相对伸长量显提高，而ALR1基因的敲除突变株系的主根相对伸长量则明显低于野生型。
表1主根相对伸长量(％)
Figure PCTCN2021094884-appb-000003
Figure PCTCN2021094884-appb-000004
实施例6
根系铝含量检测
将生长7天的野生型，以及从拟南芥生物资源中心(ABRC)购买获得的拟南芥ALR1基因的敲除突变体材料(alr1)，和本发明实施例3所得的过表达转基因材料的小苗用0.5mM氯化钙加50μM氯化铝(Al)溶液处理24h。用超纯水将小苗根冲洗3次，去除根表面的铝溶液，并用滤纸吸干超纯水。用干净刀片切下小苗主根，相同株系的根进行合并并称重。用硝酸和高氯酸(体积比4:1)的混合液对根进行消煮裂解。完全裂解后的样品经滤纸过滤后收集于干净的管子中待测。提取物中的铝含量用ICP-AES(inductively coupled plasma-atomic emission spectrometry)测定。
如图6(图6为本发明提供的野生型、ALR1敲除突变体和过表达转基因株系的根系铝含量对比图)和表2所示，在铝胁迫下，转基因过表达(ALR1 ox 1)拟南芥比野生型对照根系铝含量明显降低，而ALR1基因的敲除突变株系的根系铝含量则明显高于野生型。
表2根系铝含量(μg/g)
编号 WT ALR1 ALR1ox1
1 403.2573 789.3843 350.5266
2 356.3158 587.0902 400.3358
3 340.1515 478.5032 280.5766
4 360.6925 428.1437 320.1371
5 405.4054 399.7126 312.8168
6 467.3469 617.551 250.6913
7 452.0629 440 330.581
8 338.4137 610.2334 236.6282
9 420.6221 580.6346 258.4818
由此可见，转基因过表达(ALR1 ox 1)拟南芥比野生型对照的抗铝性明显提高，而ALR1基因的敲除突变株系的抗铝性则明显低于野生型，说明基因ALR1确实参与了植物抗铝性的调控。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (10)

  1. 一种调控植物抗铝性的ALR1基因的引物，其特征在于，所述引物包括核苷酸序列如SEQ ID NO.1所示的上游引物和核苷酸序列如SEQ ID NO.2所示的下游引物。
  2. 一种基于权利要求1所述引物制备得到的过表达拟南芥铝离子受体ALR1基因的载体，所述载体以35s-pCAMBIA1301作为骨架载体，还包括拟南芥铝离子受体ALR1基因，所述ALR1基因的核苷酸序列SEQ ID NO.3所示。
  3. 一种拟南芥铝离子受体ALR1基因或蛋白或权利要求1所述引物在植物抗铝性调控中的应用，所述ALR1基因的核苷酸序列SEQ ID NO.3所示，所述ALR1蛋白的氨基酸序列如SEQ ID NO.4所示。
  4. 过表达拟南芥铝离子受体ALR1基因或蛋白或权利要求2所述载体在提高植物的抗铝性中的应用，所述ALR1基因的核苷酸序列SEQ ID NO.3所示，所述ALR1蛋白的氨基酸序列如SEQ ID NO.4所示。
  5. 一种拟南芥铝离子受体ALR1基因或蛋白或权利要求1所述引物在调控铝胁迫植物的根系伸长量中的应用，所述ALR1基因的核苷酸序列SEQ ID NO.3所示，所述ALR1蛋白的氨基酸序列如SEQ ID NO.4所示。
  6. 过表达拟南芥铝离子受体ALR1基因或蛋白或权利要求2所述载体在提高铝胁迫植物的根系伸长量中的应用，所述ALR1基因的核苷酸序列SEQ ID NO.3所示，所述ALR1蛋白的氨基酸序列如SEQ ID NO.4所示。
  7. 根据权利要求6所述的应用，其特征在于，所述根系为主根。
  8. 一种拟南芥铝离子受体ALR1基因或蛋白或权利要求1所述引物在调控铝胁迫植物的根系铝含量中的应用，所述ALR1基因的核苷酸序列SEQ ID NO.3所示，所述ALR1蛋白的氨基酸序列如SEQ ID NO.4所示。
  9. 过表达拟南芥铝离子受体ALR1基因或蛋白或权利要求2所述载体在降低铝胁迫植物的根系铝含量中的应用，所述ALR1基因的核苷酸序列SEQ ID NO.3所示，所述ALR1蛋白的氨基酸序列如SEQ ID NO.4所示。
  10. 根据权利要求3～9任一项所述的应用，其特征在于，所述植物包括拟南芥。
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