Tartary buckwheat sourced vacuolar amino acid transporter gene as well as expression vector and application thereof

Abstract

The invention discloses a tartary buckwheat sourced vacuole amino acid transporter gene as well as an expression vector and application thereof. The method comprises the following steps: separating a vacuolar amino acid transporter FtYPQ1 gene from tartary buckwheat; the polynucleotide sequence of the FtYPQ1 gene is as shown in SEQ ID NO.1, and the amino acid sequence of the encoded protein of the FtYPQ1 gene is as shown in SEQ ID NO.2. The FtYPQ1 gene is genetically transformed into tartary buckwheat to obtain transgenic tartary buckwheat hairy roots, and the growth vigor of the transgenic tartary buckwheat hairy roots is better than that of a control group under excessive zinc treatment; the FtYPQ1 gene is genetically transformed into arabidopsis thaliana to obtain transgenic arabidopsis thaliana, under excessive zinc treatment, compared with wild arabidopsis thaliana, the accumulation tolerance details of the transgenic arabidopsis thaliana are more than those of a control group, and it is proved that the FtYPQ1 gene has the function of regulating and controlling the zinc stress resistance or zinc accumulation of plants, so that the zinc stress resistance or zinc accumulation of the plants can be regulated and controlled. The method has an application prospect in cultivation or breeding of high-zinc plant varieties with strong zinc stress tolerance.

Description

Technical Field

[0001] The invention relates to a vacuolar amino acid transporter gene of plant origin, in particular to a vacuolar amino acid transporter gene separated from tartary buckwheat and an expression vector thereof, and an application thereof in regulating plant tolerance to zinc stress or regulating zinc accumulation in plants, belonging to the field of vacuolar amino acid transporter genes and their applications. Background Art

[0002] Zinc plays many vital roles in life. 2+ It plays a central role in determining the structure and catalytic function of nearly 10% of the proteins in the proteome of most eukaryotic organisms. Plants are the ultimate source of zinc in the human diet, and a variety of approaches have been used to increase the bioavailable zinc in dietary systems. Nutritional programs using supplements have proven to be particularly effective in the short term, and the addition of zinc oxide to flour has also been shown to be a cost-effective strategy. Biofortification, which increases the micronutrient content and availability of the edible part of a crop during its cultivation, is a more sustainable approach.

[0003] Tartary buckwheat is a dicotyledonous plant of the genus Fagopyrum in the family Polygonaceae. It is a cultivated species of buckwheat that is rich in mineral nutrients and trace elements, and has the characteristics of strong environmental adaptability, short growth period, and tolerance to cold and barren conditions. Compared with the main food crops wheat and rice, tartary buckwheat contains relatively high zinc, but little is known about the genetic background and key regulatory genes of zinc content in tartary buckwheat. Summary of the invention

[0004] One of the purposes of the present invention is to provide a vacuolar amino acid transporter gene isolated from tartary buckwheat and its encoded protein; The second object of the present invention is to provide an expression cassette containing the vacuolar amino acid transporter gene, a recombinant expression vector or a recombinant host cell containing the recombinant expression vector; The third purpose of the present invention is to use the vacuolar amino acid transporter gene isolated from tartary buckwheat and its encoded protein, an expression cassette containing the vacuolar amino acid transporter gene, a recombinant expression vector or a recombinant host cell containing the recombinant expression vector, etc. to regulate plant tolerance to zinc stress or regulate zinc accumulation in plants.

[0005] In order to achieve the above object, the main technical solutions adopted by the present invention include: One aspect of the present invention provides a vacuolar amino acid transporter isolated from tartary buckwheat FtYPQ1 Gene.

[0006] The vacuolar amino acid transporter described in the present invention FtYPQ1The nucleotide sequence of the gene is the nucleotide sequence shown in (a) or (b): (a) the polynucleotide sequence shown in SEQ ID NO.1; (b) A polynucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.2.

[0007] Another aspect of the present invention provides a protein FtYPQ1 encoded by a vacuolar amino acid transporter gene isolated from Tartary Buckwheat.

[0008] The amino acid sequence of the vacuolar amino acid transporter FtYPQ1 described in the present invention is shown in (a) or (b): (a) the amino acids shown in SEQ ID NO.2; (b) A protein variant derived from the amino acid shown in SEQ ID NO. 2 by substitution, deletion or / and insertion of one or more amino acid residues, which still has the function or activity of transporting heavy metal ions.

[0009] The protein variants of the present invention may be produced by genetic polymorphism or artificial manipulation, and these manipulation methods are generally known in the art. For example, amino acid sequence variants or fragments of the vacuolar amino acid transporter FtYPQ1 can be prepared by mutation of DNA, where methods for mutagenesis or alteration of polynucleotides are known in the art. Among them, conservative substitutions are to replace one amino acid residue with another amino acid having similar properties.

[0010] The present invention also provides a vacuolar amino acid transporter containing the FtYPQ1 The invention relates to a recombinant plant expression vector of a gene and a host cell containing the recombinant plant expression vector.

[0011] The chimeric gene or expression cassette obtained by chimerizing or connecting the gene shown in SEQ ID NO.1 of the present invention with other genes belongs to the protection scope of the present invention; the recombinant expression vector containing the chimeric gene or expression cassette also belongs to the protection scope of the present invention.

[0012] Another aspect of the present invention is to apply the vacuolar amino acid transporter gene isolated from tartary buckwheat and its encoded protein, an expression cassette containing the vacuolar amino acid transporter gene, a recombinant expression vector or a recombinant host cell containing the recombinant expression vector to regulating plant tolerance to zinc stress or regulating zinc accumulation in plants.

[0013] In a preferred embodiment of the present invention, the regulation of plant tolerance to zinc stress is to improve the tolerance of plants under zinc stress treatment or to promote plant growth.

[0014] In a preferred embodiment of the present invention, the regulation of zinc accumulation in plants is to increase the zinc content in plants.

[0015] For reference, the present invention provides an embodiment, that is, by overexpressing the coding gene of the vacuolar amino acid transporter, the expression amount or activity of the vacuolar amino acid transporter is increased, thereby improving the tolerance of the plant under zinc stress treatment or increasing the zinc content in the plant.

[0016] A preferred embodiment of the present invention is a method for improving the tolerance of plants under zinc stress or increasing the zinc content in plants, comprising: overexpressing a vacuolar amino acid transporter in a plant FtYPQ1 Gene, vacuolar amino acid transporter FtYPQ1 The expression of the gene is increased or the function or activity of the vacuolar amino acid transporter FtYPQ1 is enhanced; for example, the vacuolar amino acid transporter from bitter buckwheat is increased. FtYPQ1 The gene is connected to the expression control element to obtain a recombinant plant expression vector for expressing the gene in plants; the recombinant plant expression vector is transformed into plants to make the vacuolar amino acid transporter FtYPQ1 The gene is overexpressed in plants, and the resulting transgenic plants have enhanced tolerance to zinc stress or increased zinc content in the body.

[0017] As a reference, the present invention provides a vacuolar amino acid transporter FtYPQ1 Gene plant recombinant expression vector, comprising: vacuolar amino acid transporter derived from bitter buckwheat FtYPQ1 The gene is connected to the expression control element to obtain a recombinant plant expression vector; the recombinant plant expression vector may be composed of a 5' non-coding region, the nucleotides shown in SEQ ID NO.1 and a 3' non-coding region; wherein the 5' non-coding region may include a promoter sequence, an enhancer sequence and / or a translation enhancement sequence; the promoter may be a constitutive promoter, an inducible promoter, a tissue or organ specific promoter; the 3' non-coding region may include a terminator sequence, an mRNA cleavage sequence, etc. Suitable terminator sequences may be taken from the Ti-plasmid of Agrobacterium tumefaciens, such as the octopine synthase and nopaline synthase terminator regions.

[0018] The recombinant plant expression vector may also contain a selective marker gene for selecting transformed cells, for selecting transformed cells or tissues. The marker gene includes genes encoding antibiotic resistance and genes conferring resistance to herbicidal compounds. In addition, the marker gene also includes phenotypic markers, such as β-galactosidase and fluorescent protein.

[0019] The transformation protocol and the protocol for introducing the polynucleotide or polypeptide into the plant may vary depending on the type of plant or plant cell used for transformation. Suitable methods for introducing the polynucleotide into plant cells include: microinjection, electroporation, Agrobacterium-mediated transformation, direct gene transfer, and high-speed ballistic bombardment. In a specific embodiment, various transient transformation methods can be used to introduce the vacuolar amino acid transporter of Tartary buckwheat into the plant cell. FtYPQ1 Genes are provided to plants. Transformed cells can be regenerated into stably transformed plants using conventional methods (McCormick et al. Plant Cell Reports. 1986. 5:81-84).

[0020] The present invention also provides a method for cultivating a plant variety with high zinc content or strong tolerance to zinc stress, comprising: (1) constructing a vacuolar amino acid transporter FtYPQ1 gene or its chimeric gene; (2) transforming the constructed recombinant plant expression vector into plant tissues or plant cells; (3) FtYPQ1 The gene is overexpressed in plant tissues or cells.

[0021] In a preferred embodiment of the present invention, the plant is buckwheat or Arabidopsis thaliana.

[0022] The present invention cloned the vacuolar amino acid transporter from bitter buckwheat FtYPQ1 gene and transformed it into a zinc-sensitive yeast strain pYES2- FtYPQ1 Functional analysis of the recombinant plasmid revealed that the vacuolar amino acid transporter FtYPQ1 has the ability to transport zinc, which can increase the accumulation of zinc in plants. FtYPQ1 Genetic transformation of buckwheat explants to obtain overexpression FtYPQ1 Compared with the control group, the transgenic buckwheat hairy roots overexpressed FtYPQ1 The hairy roots of Tartary buckwheat are more tolerant to zinc stress; the present invention is overexpressed in Arabidopsis FtYPQ1 Gene discovery, overexpression FtYPQ1 The zinc content in Arabidopsis thaliana with the gene was higher than that in wild-type Arabidopsis thaliana; FtYPQ1 The gene can regulate the tolerance of plants to zinc stress or regulate the accumulation of zinc in plants. The invention enriches the research on the function of vacuolar amino acid transporter family proteins and provides guidance for the molecular breeding of high-zinc buckwheat and other crops in the future.

[0023] Terminology Definitions Related to the Present Invention Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0024] The term "polynucleotide" or "nucleotide" means deoxyribonucleotides, deoxyribonucleosides, ribonucleosides or ribonucleotides and polymers thereof in single-stranded or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides, which have binding properties similar to reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides. Unless specifically limited otherwise, the term also means oligonucleotide analogs, which include PNA (peptide nucleic acid), DNA analogs (phosphorothioates, phosphoramidates, etc.) used in antisense technology. Unless otherwise specified, a specific nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (including, but not limited to, degenerate codon substitutions) and complementary sequences as well as explicitly specified sequences. Specifically, degenerate codon substitutions can be achieved by generating a sequence in which the 3rd position of one or more selected (or all) codons is substituted with mixed bases and / or deoxyinosine residues.

[0025] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acid residues. That is, descriptions directed to polypeptides apply equally to describing peptides and describing proteins, and vice versa. The terms apply to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues are non-naturally encoded amino acids. As used herein, the terms encompass amino acid chains of any length, including full-length proteins (i.e., antigens), in which the amino acid residues are linked via covalent peptide bonds.

[0026] The "stringent hybridization conditions" described herein refer to conditions of low ionic strength and high temperature as known in the art. Generally, under stringent conditions, the detectable degree of hybridization of the probe to its target sequence is higher than the detectable degree of hybridization to other sequences (e.g., at least 2 times above background). Stringent hybridization conditions are sequence-dependent and will be different under different environmental conditions, with longer sequences specifically hybridizing at higher temperatures. By controlling the stringency of hybridization or washing conditions, target sequences that are 100% complementary to the probe can be identified. For detailed guidance on nucleic acid hybridization, reference can be made to the relevant literature (Tijssen, Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays. 1993). More specifically, the stringent conditions are usually selected to be lower than the thermal melting point (T) of the specific sequence at a specified ionic strength pH. m ) about 5-10℃. T m The temperature (under specified ionic strength, pH, and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (because the target sequence is present in excess, the temperature at T mStringent conditions may be those in which the salt concentration is less than about 1.0 M sodium ion concentration, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is at least about 30°C for short probes (including but not limited to 10 to 50 nucleotides) and at least about 60°C for long probes (including but not limited to greater than 50 nucleotides). Stringent conditions may also be achieved by adding destabilizing agents such as formamide. For selective or specific hybridization, a positive signal may be at least two times the background hybridization, optionally 10 times the background hybridization. Exemplary stringent hybridization conditions may be as follows: 50% formamide, 5×SSC and 1% SDS, incubated at 42°C; or 5×SSC, 1% SDS, incubated at 65°C, washed in 0.2×SSC and washed in 0.1% SDS at 65°C. The washing may be performed for 5, 15, 30, 60, 120 minutes or longer.

[0027] The term "recombinant host cell strain" or "host cell" means a cell comprising a polynucleotide of the present invention, regardless of the method used for insertion to produce a recombinant host cell, such as direct uptake, transduction, f-mating, or other methods known in the art. The exogenous polynucleotide may be maintained as a non-integrating vector such as a plasmid or may be integrated into the host genome. The host cell may be a prokaryotic cell or a eukaryotic cell, and the host cell may also be a monocotyledonous or dicotyledonous plant cell.

[0028] The term "operably linked" refers to a functional connection between two or more elements. The operably linked elements may be contiguous or non-contiguous.

[0029] The term "recombinant plant expression vector" means one or more DNA vectors used to achieve plant transformation; these vectors are often referred to as binary vectors in the art. Binary vectors, together with vectors with helper plasmids, are most commonly used for Agrobacterium-mediated transformation. Binary vectors typically include: cis-acting sequences required for T-DNA transfer, selection markers engineered to be expressed in plant cells, heterologous DNA sequences to be transcribed, etc.

[0030] The term "transformation" refers to a process by which a heterologous DNA sequence is introduced into a host cell or organism.

[0031] The term "expression" refers to the transcription and / or translation of an endogenous gene or a transgene in a plant cell. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 for FtYPQ1 Figure 2 shows the cloning results of the gene CDS.

[0033] Figure 2 for FtYPQ1 Genes for zinc transport activity in yeast; among them, Figure 2 -a is the growth condition of yeast in culture medium; Figure 2 -b is the growth curve of yeast.

[0034] Figure 3 For hairy roots containing the empty vector pCAMBIA 1307 and overexpression FtYPQ1 Gene hairy roots FtYPQ1 The relative expression levels of genes were detected by qRT-PCR.

[0035] Figure 4 Wild-type hairy roots and overexpression FtYPQ1 Determination of zinc tolerance of genetic hairy roots; Figure 4 -a represents wild type hairy roots and overexpression FtYPQ1 Phenotype of genetic hairy roots under the treatment of excess ZnCl2; Figure 4 -b represents wild type hairy roots and overexpression FtYPQ1 Fresh weight statistics of genetic hairy roots under the treatment of excess ZnCl2.

[0036] Figure 5 For overexpression FtYPQ1 The positive identification results of Arabidopsis thaliana of the gene; N is the negative control, the material is wild-type Arabidopsis thaliana; P is the positive control, the material is based on FtYPQ1 pCAMBIA 1307- FtYPQ1 Recombinant plasmid.

[0037] Figure 6 Wild-type Arabidopsis and overexpressing FtYPQ1 Phenotypic diagram of the Arabidopsis thaliana gene.

[0038] Figure 7 For wild-type Arabidopsis and overexpression FtYPQ1 Zn 2+ Content statistics chart. DETAILED DESCRIPTION

[0039] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, it should be understood that the embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications or replacements all fall within the protection scope of the present invention.

[0040] Example FtYPQ1 CDS cloning of genes Four-week-old buckwheat seedlings were selected, 100 mg of leaves were taken, and after being fully ground with liquid nitrogen, total RNA was extracted using the Trizol method. This RNA was used as a template and reverse transcribed using the HiScript® III 1st Strand cDNA Synthesis Kit (+gDNAwiper) (Nanjing Novozyme Biotechnology Co., Ltd.) to obtain the bitter buckwheat cDNA.

[0041] Using the cDNA of buckwheat 'Pinku' as template, primers FtYPQ1- F / R was cloned by PCR amplification using Phanta high-fidelity enzyme (Phanta® Max Master Mix, Vazyme, Beijing) FtYPQ1 The nucleotide sequence of the CDS of the gene ( Figure 1 ).

[0042] The PCR program was 95°C for 3 min; 95°C for 30 s, 57°C for 60 s, and 72°C for 90 s for 32 cycles. The PCR purified products were sequenced, analyzed, and spliced ​​to obtain FtYPQ1 The full-length sequence of the gene's CDS.

[0043] FtYPQ1

[0044] The amino acid sequence of the vacuolar amino acid transporter FtYPQ1 is shown in SEQ ID NO.2: MNPMLFNSKQSNSIVEYCYKENKPCIAWIQTYFSDCLCNFRDRISFGFGFISVLAWIVAEVPQIITNFQTKSSHGLSFLFLFTWVIGDVFNVAGCLLEPATLPTQYYTALLYTVSTLVLVFQSLYYDHFYKWWKCRQKKARTTQQVEDEKTPLKPSKQEDEQGRGGIPIPSAAEPQAQTARKEFYYMSARSLVGSNTPPFR SYLRIAKSGPSAMAIGGDHHFSSEDENENDSTVSAVAPVKTQPRSIPRSVGRGAFVAASLNLPAQSNAIAAVYQGFTGRRFLQESDVDYSAFGQWLGWTMAAIY MGGRIPQIVLNIKRGSVEGLNPLMFIFALIGNVTYVASILVRSTEWEKLKANLPWLLDAVVCVALDLFILLQYVYYKYLRKKDFDGDDDKKDYYVEVHPKAFVA (SEQ ID NO. 2).

[0045] Primers FtYPQ1- The nucleotide sequence of F / R is as follows: FtYPQ1- F:ATGAATCCAATGCTCTTCAACTCC (SEQ ID NO.3); FtYPQ1- R: TTAAGCTACAAAGGCTTTTGGATG (SEQ ID NO. 4).

[0046] Test Example 1 FtYPQ1 Gene function analysis in yeast 1 Test methods In order to verify whether the vacuolar amino acid transporter FtYPQ1 has the ability to transport zinc, a yeast expression vector was constructed. Plasmid pYES2 was cleaved with restriction endonucleases EcoR I and Xho I linearization; FtYPQ1 The PCR purified product of the gene was used as a template, and pYES2- FtYPQ1 -F / R are primers, PCR amplification FtYPQ1 The full-length sequence of the gene; FtYPQ1The full-length sequence of the gene was recovered and ligated. After transformation, the amplified FtYPQ1 The CDS fragment of the gene was inserted into the yeast expression vector pYES2 EcoRI and XhoI Between the two restriction sites, a positive E. coli single clone was selected for PCR amplification. After the amplified product was confirmed by 1% agarose gel electrophoresis, the target colony was streaked for overnight culture. The next day, the plasmid was extracted using the Plasmid Mini Kit I kit (OMEGA). After sequencing and identification, the recombinant plasmid, plasmid pYES2, and positive control pYES2- OsIRT1 The recombinant plasmid (OsIRT1 is a ferrous ion transporter with Zn transport capacity reported in rice) was introduced into the yeast using a yeast transformation kit (Frozen-EZ Yeast Transformation Ⅱ TM, ZYMO research, T2001). Δzrc1 Zinc-sensitive yeast strains ( BY4741 ; MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 YMR243c::kanMX4 ) and cultured in SD / -Ura solid medium (purchased from Beijing Coolaibo Company) at 30°C for 72 hours.

[0047] Primer pYES2- FtYPQ1 The nucleotide sequence of -F / R is as follows: pYES2- FtYPQ1 -F:gccgccagtgtgctggaattcATGAATCCAATGCTCTTCAACTCC (SEQ IDNO.5); pYES2- FtYPQ1 -R:ccctctagatgcatgctcgagTTAAGCTACAAAGGCTTTTGGATG (SEQ ID NO. 6).

[0048] Prepare yeast seed solution and culture the transformed yeast monoclonal strain in SD / -Ura liquid medium overnight (approximately 12-16 h) until the logarithmic growth phase (OD 600 =0.8-1.0), culture conditions are 30℃, 200 rpm shaker. Take 1 mL of bacterial solution and transfer it to 5 mL of untreated SD / -Ura liquid medium, and culture it at 30℃, 200 rpm to the logarithmic growth stage. Use a centrifuge precooled to 4℃ to centrifuge the bacterial solution at 4000 rpm for 5 min to enrich the bacteria, then pour out the supernatant, repeat 3 times, use sterile water to resuspend the bacteria, and then centrifuge to precipitate the bacteria. During this period, use a 1 mL pipette to repeatedly blow the bacterial block at the bottom of the centrifuge tube to completely suspend the bacteria.

[0049] The prepared OD 600 = 1.0 was diluted 4 times by 10 times (10 0 , 10 -1 , 10 -2 , 10 -3 and 10 -4 ), take 5 μL of the bacterial solution with different dilution multiples and titrate it on the galactose / glucose SD / -Ura plate containing ZnCl2 (0 μM, 200 μM, 400 μM), and culture it upside down in a 30℃ incubator away from light for 3-5 days, and observe and record it regularly.

[0050] Yeast cells in the logarithmic growth phase were transferred to 30 mL SD / -Ura liquid medium (galactose as carbon source) and the initial OD was adjusted to 600 =0.05, set the treatment group and control group (add the same volume of sterile water), each treatment contained 3 biological replicates, the culture conditions were 30℃, 200 rpm shaking, and the OD of the bacterial solution at different time points was measured by UV / visible spectrophotometer 600 .

[0051] 2 Test results The test results are as follows Figure 2 As shown, it contains empty vector pYES2, pYES2- FtYPQ1 and pYES2- OsIRT1 The recombinant plasmid yeast was added with glucose and different concentrations of Zn 2+ The growth state of yeast culture medium was consistent with that of yeast containing empty vector pYES2; compared with yeast containing pYES2- FtYPQ1 and pYES2- OsIRT1 The recombinant plasmid yeast was grown in the presence of galactose and 200 μM and 400 μM Zn 2+ The growth rate was significantly weaker in the medium ( Figure 2 -a), indicating transformation of pYES2- FtYPQ1 and pYES2- OsIRT1 Recombinant yeast plasmid with Zn 2+ transport capacity, high concentrations of zinc in the body affect yeast growth. 2+ Processing medium and low, containing pYES2- FtYPQ1 and pYES2- OsIRT1 The growth curve of yeast containing the recombinant plasmid was significantly lower than that of yeast containing the empty vector pYES2 ( Figure 2 -b).

[0052] Test Example 2 FtYPQ1 Experiment on Gene Regulation of Tartary Buckwheat's Tolerance to Zinc Stress 1 OverexpressionFtYPQ1 Construction of Tartary Buckwheat Hairy Roots with Gene by FtYPQ1 The PCR purified product of the gene was used as a template, 1307- FtYPQ1 -F / R are primers, PCR amplification FtYPQ1 After recovery and ligation, the full-length sequence of the gene FtYPQ1 The full-length sequence of the gene was inserted into the downstream of the CaMV35S promoter of the pCAMBIA 1307 vector in the forward direction, and the overexpression vector pCAMBIA 1307- FtYPQ1 .

[0053] Primer 1307- FtYPQ1 The nucleotide sequence of -F / R is as follows: 1307- FtYPQ1 -F:gtatctagaactagtggatccATGAATCCAATGCTCTTCAACTCCA (SEQ IDNO.7); 1307- FtYPQ1- R:gtcgacggtatcgataagcttAGCTACAAAGGCTTTTGGATGAACT (SEQ ID NO. 8).

[0054] The sequencing confirmed the correct pCAMBIA 1307- FtYPQ1 The recombinant plasmid and the pCAMBIA 1307 empty vector plasmid were transformed into Agrobacterium A4 and GV3101 competent cells respectively by heat shock method. FtYPQ1 Recombinant plasmid positive bacteria, containing pCAMBIA 1307 empty vector positive bacteria. FtYPQ1 The colonies of A4 rhizogenes containing the recombinant plasmid and A4 rhizogenes containing the pCAMBIA 1307 empty vector were picked into liquid culture medium and shaken at 200 rpm at 28°C until the OD 600 =0.8, and the leaves and stems of bitter buckwheat variety No. 1 were infected with bacterial solution and placed on MS solid medium containing 25 mg / L hygromycin (Hyg) resistance. After culturing for 1-2 weeks, those that could grow hairy roots were initially identified as candidate positive hairy roots. FtYPQ1 -qPCR-F / R for qRT-PCR detection FtYPQ1 The expression level of the gene. FtACTINThe gene was used as an internal reference gene, and qRT-PCR was performed using primers FtACTIN-qPCR-F / R. Real-time fluorescence quantitative PCR (qRT-PCR) was used to detect the expression of 5-mercaptoethanol in the BAI7500 real-time fluorescence quantitative PCR instrument. FtYPQ1 The expression level of the gene. Use RQ (relative expression) = 2 -ΔΔCT The algorithm calculates FtYPQ1 The relative expression level of the gene was set as 1 in the hairy roots containing the empty vector pCAMBIA 1307, and the experiment was repeated three times.

[0055] Primers FtYPQ1 The nucleotide sequences of -qPCR-F / R and FtACTIN-qPCR-F / R are as follows: FtYPQ1 -qPCR-F: CCATGTATAGCCTGGATTCAAACC (SEQ ID NO.9); FtYPQ1 -qPCR-R: ATGATCTGAGGGACTTCAGCGAC (SEQ ID NO. 10).

[0056] FtACTIN-qPCR-F: ATGGCAGAATCTGAGGACATTCAG (SEQ ID NO. 11); FtACTIN-qPCR-R: GAAACACTTTCTGTGGACAATTGATG (SEQ ID NO. 12).

[0057] The qRT-PCR test results are as follows Figure 3 As shown, overexpression FtYPQ1 Hairy roots FtYPQ1 The gene expression level was higher than that in hairy roots transformed with pCAMBIA-1300 empty vector.

[0058] 2 Overexpression FtYPQ1 Determination of the tolerance of hairy roots to zinc stress The wild-type hairy roots (CK) and overexpressing FtYPQ1 The same amount of three positive strains of the gene's hairy roots (OE1, OE2, and OE3) were placed on MS solid medium with 1 mM ZnCl2 and cultured in the dark for 2 weeks. Three biological replicates were set up to observe the growth of the hairy roots and weigh the fresh weight of the hairy roots.

[0059] The results of the test are as follows Figure 4 As shown, under zinc stress, overexpression FtYPQ1 The growth of hairy roots of the gene was stronger than that of wild-type hairy roots (Figure 4 -a), overexpression FtYPQ1 The fresh weight of hairy roots of the gene was higher than that of wild-type hairy roots ( Figure 4 -b), indicating overexpression FtYPQ1 Genes enhance zinc tolerance in buckwheat.

[0060] Test Example 3 FtYPQ1 Experiment on Gene Regulation of Zinc Content in Arabidopsis 1 Overexpression FtYPQ1 Construction of Arabidopsis thaliana The plasmid containing pCAMBIA 1307- FtYPQ1 The GV3101 Agrobacterium liquid of the recombinant plasmid was used to infect Arabidopsis thaliana by the floral dipping method to obtain transgenic Arabidopsis thaliana seeds. The infected Arabidopsis thaliana seeds were disinfected with 10% sodium hypochlorite for 8 minutes, washed 4-5 times with sterile water, and then placed on MS solid medium containing hygromycin (Hyg) resistance and cultured for 1-2 weeks. Arabidopsis thaliana plants that can grow true leaves are initially identified as candidate positive plants. Arabidopsis thaliana DNA was extracted for PCR verification, and pCAMBIA 1307 vector primers 1307-F / R were used to detect whether the gene was successfully transformed. The test results are as follows Figure 5 As shown, overexpression FtYPQ1 The Arabidopsis thaliana gene has been constructed.

[0061] The nucleotide sequence of primer 1307-F / R is as follows: 1307-F:GAGAGCTTGGGCGACCTCA (SEQ ID NO. 13); 1307-R: GAGAGAGACTGGTGATTTTTGC (SEQ ID NO. 14).

[0062] 2 Overexpression FtYPQ1 Determination of zinc content in Arabidopsis thaliana Overexpression FtYPQ1 The Arabidopsis thaliana with the gene (OE#1, OE#2, OE#3) and wild-type Arabidopsis thaliana (Col-0) were sown in MS solid medium containing 0.2 mM ZnCl2 and cultured for 3 weeks. Three biological replicates were set up to observe the growth of Arabidopsis thaliana.

[0063] ICP-MS analysis of Zn content in different Arabidopsis thaliana was performed. Arabidopsis thaliana was dried at 80 °C for 5 days, crushed and filtered through a 40-mesh sieve. 0.2000 g (accurate to 0.0001 g) of sample was accurately weighed and placed in a 15 ml digestion tube, and 1 ml of distilled water and 4 ml of HNO3-H2O2 mixed solution were added. After standing for a while, the digestion tube was placed in an inner barrel and digested using an ultra-microwave digestion system (EXPEC 790S, China). After digestion, the sample was transferred to a 50 ml plastic volumetric flask and the concentration of Zn element was determined by an inductively coupled plasma mass spectrometer (SUPEC 7000, Puyu Technology, Hangzhou, China). Three biological replicates were set.

[0064] The growth of Arabidopsis Figure 6 As shown, overexpression FtYPQ1 The growth and root length of Arabidopsis thaliana with the gene were better than those of wild-type Arabidopsis thaliana; Zn 2+ The content of Figure 7 As shown, overexpression FtYPQ1 The zinc content of the three strains of Arabidopsis thaliana with the gene was higher than that of wild-type Arabidopsis thaliana, confirming that FtYPQ1 Genes that can transport Zn 2+ , increase zinc accumulation and improve plants' tolerance to zinc.

Claims

1. Vacuolar amino acid transporter isolated from Tartary buckwheat QUR A gene characterized by Its polynucleotide sequence is shown as (a) or (b): (a) the polynucleotide sequence shown in SEQ ID NO.1; (b) A polynucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.

2.

2. The vacuolar amino acid transporter isolated from tartary buckwheat according to claim 1 QUR The encoded protein of the gene is characterized in that Its amino acid sequence is shown in SEQ ID NO.

2.

3. Containing the vacuolar amino acid transporter according to claim 1 QUR Gene expression cassette or recombinant expression vector.

4. A recombinant host cell containing the expression cassette or recombinant expression vector according to claim 3.

5. The vacuolar amino acid transporter according to claim 1 QUR Use of the gene, the coding protein according to claim 2, the expression cassette or recombinant expression vector according to claim 3, or the recombinant host cell according to claim 4 in regulating plant tolerance to zinc stress or regulating zinc accumulation in plants.

6. The use according to claim 5, characterized in that: The regulating plant tolerance to zinc stress is to improve the tolerance of the plant under zinc stress treatment or promote plant growth; the regulating zinc accumulation in the plant is to increase the zinc content in the plant.

7. The use according to claim 5, characterized in that: include: (1) Constructing a vacuolar amino acid transporter containing the QUR (1) transforming the constructed recombinant plant expression vector into plant tissues or plant cells; (2) transforming the constructed recombinant plant expression vector into plant tissues or plant cells; (3) transforming the vacuolar amino acid transporter into the QUR The gene is overexpressed in plant tissues or cells.

8. The use according to claim 5, characterized in that: The plant is tartary buckwheat or Arabidopsis thaliana.

9. A method for cultivating plant varieties with high zinc content or strong tolerance to zinc stress, characterized in that: include: (1) Construction of vacuolar amino acid transporters QUR gene or its chimeric gene; (2) transforming the constructed recombinant plant expression vector into plant tissues or plant cells; (3) QUR The gene is overexpressed in plant tissues or cells.

10. The method according to claim 9, characterized in that The plant is tartary buckwheat or Arabidopsis thaliana.

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