Application of Arabidopsis thaliana AtNAC002 gene

Abstract

The present invention provides the application of Arabidopsis AtNAC002 gene. The Arabidopsis AtNAC002 gene can improve the copper toxicity resistance of Arabidopsis. Under the same copper toxicity concentration, the root length and fresh weight of the mutant strain that does not express the AtNAC002 gene decreased by 31.46% and 54% respectively. It can reduce the copper content in the Arabidopsis root system by reducing the copper absorption of the Arabidopsis root system under copper toxicity conditions.

Description

Technical Field

[0001] The invention relates to the technical fields of biotechnology and plant genetic engineering, and in particular to the application of Arabidopsis thaliana AtNAC002 gene. Background Art

[0002] Copper (Cu) is an essential trace nutrient for plant growth and development. It has a dual nature for plant growth and is essential at the optimal level. When the copper concentration in plant tissues is too high, the plant will show obvious symptoms of poisoning, inhibiting the normal growth and development of the plant. Excessive copper will not only reduce the yield and quality of crops, but may also endanger human health through the food chain. Therefore, controlling the accumulation and dynamic balance of copper in crops is very important for the normal growth and development of plants and human health. Especially in industry and coal mining, copper has become a more serious pollutant in the environment.

[0003] Copper is discharged into the ecosystem in large quantities through various agronomic activities due to the use of fungicides, algaecides and bactericides in agricultural production. The toxicity of copper is not only related to the increase in industrial activities, but also to metal processing, the use of sludge, electroplating, metal mining, dyes, fertilizers, fungicides and pesticides. Fungicides containing copper are widely used to protect against biodegradation caused by fungi or insects. Due to the increase in the use of copper in different agricultural and industrial activities, copper is mined in large quantities around the world. With the current rapid development of industry, different potentially toxic elements are released into the environment in large quantities, causing a lot of pollution to the environment. Therefore, with the impact of human activities on the ecosystem, the pollution of the environment by toxic elements such as heavy metals is becoming more and more common and serious. Summary of the invention

[0004] The inventors discovered during their research on the Arabidopsis thaliana AtNAC002 gene that the gene can improve the resistance of Arabidopsis thaliana to copper toxicity. Therefore, in order to solve the problems existing in the background technology, the present invention provides an application of the Arabidopsis thaliana AtNAC002 gene.

[0005] The technical solution of the present invention to solve the above technical problems is as follows:

[0006] In a first aspect, the present invention provides an application of the Arabidopsis thaliana AtNAC002 gene in improving the resistance of Arabidopsis thaliana to copper toxicity. The ID number of the gene in NCBI is At1g01720.

[0007] According to the above scheme, the nucleotide sequence of the Arabidopsis thaliana AtNAC002 gene is shown in SEQ ID NO:2.

[0008] In a second aspect, the present invention provides the use of Arabidopsis thaliana AtNAC002 transcription factor in improving the copper toxicity resistance of Arabidopsis thaliana, wherein the Arabidopsis thaliana AtNAC002 transcription factor has an amino acid sequence shown in Ⅰ) or Ⅱ):

[0009] I) The amino acid sequence shown in SEQ ID NO: 1; or

[0010] II) A protein having the same function obtained by substituting, deleting and / or adding one or more amino acids on the basis of the amino acid sequence described in I).

[0011] In a third aspect, the present invention provides an application of a recombinant vector in improving the copper toxicity resistance of Arabidopsis thaliana, and the recombinant vector contains the above-mentioned Arabidopsis thaliana AtNAC002 gene.

[0012] According to the above scheme, the Arabidopsis thaliana AtNAC002 gene is up-regulated by copper toxicity induction in the root system.

[0013] According to the above scheme, the Arabidopsis thaliana AtNAC002 gene improves the copper toxicity resistance of Arabidopsis thaliana by reducing the copper absorption of the Arabidopsis thaliana root system.

[0014] According to the above scheme, the Arabidopsis thaliana AtNAC002 transcription factor improves the copper toxicity resistance of Arabidopsis thaliana by reducing the copper absorption of the Arabidopsis thaliana root system.

[0015] According to the above scheme, the recombinant vector improves the copper toxicity resistance of Arabidopsis thaliana by reducing the copper absorption of the Arabidopsis thaliana root system.

[0016] According to the above scheme, the Arabidopsis thaliana AtNAC002 gene reduces the copper content in the Arabidopsis thaliana root system.

[0017] According to the above scheme, the Arabidopsis thaliana AtNAC002 transcription factor reduces the copper content in the Arabidopsis thaliana root system.

[0018] According to the above scheme, the recombinant vector reduces the copper content in the Arabidopsis thaliana root system.

[0019] The beneficial effect of the present invention is that: by comparing wild-type Arabidopsis thaliana and mutant strains in which a fragment is inserted into the AtNAC002 gene by T-DNA technology to make the AtNAC002 gene not expressed, it is found that the mutant strains are very sensitive to copper toxicity. Under the same copper toxicity concentration, the root length and fresh weight of the mutant strains decreased by 31.46% and 54% respectively, and under copper toxicity conditions, the expression level of the AtNAC002 gene in the roots of wild-type Arabidopsis thaliana was about 6 times that under non-copper toxicity conditions. It shows that the Arabidopsis thaliana AtNAC002 gene and its encoded AtNAC002 transcription factor have the effect of improving the copper toxicity resistance of Arabidopsis thaliana, and it can reduce the copper content in the Arabidopsis thaliana root system by reducing the copper absorption of the Arabidopsis thaliana root system under copper toxicity conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1Biomass of wild type and AtNAC002 gene mutant (nac002) in Example 1 of the present invention under different heavy metal treatments, Figure 1 where (a) in Figure 1 is the relative primary root length, and

[0021] Figure 2 Transcription of AtNAC002 gene in roots and above-ground parts of wild type Arabidopsis thaliana under copper toxicity treatment in Example 2 of the present invention;

[0022] Figure 3 Gene structure characteristics of AtNAC002 gene in Example 3 of the present invention, where Figure 3 in (a) is the schematic diagram of T-DNA insertion site, Figure 3 in (b) is the result of semi-quantitative verification of T-DNA insertion site, Figure 3 c is the phylogenetic tree analysis of AtNAC002 family;

[0023] Figure 4 Subcellular localization of AtNAC002 gene provided in Example 4 of the present invention;

[0024] Figure 5 Transcription activation activity results of AtNAC002 gene provided in Example 5 of the present invention, where Figure 5 in (a) is the schematic diagram of CDS sequences of different segments, Figure 5 in (b) is the schematic diagram of transcription activation activity results;

[0025] Figure 6 Statistical results of phenotypes and biomass (root length and fresh weight) of wild type and AtNAC002 mutant (nac002) under normal and copper toxicity conditions in Example 6 of the present invention, where Figure 6 in (a) are the phenotypes under normal and copper toxicity conditions, Figure 6 in (b) is the root length under normal and copper toxicity conditions, Figure 6 in (c) is the fresh weight under normal and copper toxicity conditions;

[0026] Figure 7 Copper concentration measurement results of different parts of wild type and AtNAC002 mutant (nac002) seedlings under normal and copper toxicity conditions in Example 6 of the present invention, where Figure 7 in (a) is the above-ground part, Figure 7 in (b) are the root system results;

[0027] Figure 8 Results of reactive oxygen species measurement in above-ground parts of wild type and AtNAC002 mutant (nac002) under normal and copper toxicity conditions provided in Example 6 of the present invention, whereFigure 8 In (a), (b), (c), and (d), they are the activities of SOD, CAT, POD, and MDA, respectively;

[0028] Figure 9 This is the result of the determination of reactive oxygen species in the roots of the wild type and the AtNAC002 mutant (nac002) provided in Example 6 of the present invention under normal and copper toxicity conditions, where Figure 9 in (a), (b), (c), and (d), they are the activities of SOD, CAT, POD, and MDA, respectively. Detailed implementation manners

[0029] The principles and features of the present invention will be described below in conjunction with the accompanying drawings and specific embodiments. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0030] It should be understood that the terms described in the present invention are only for describing specific embodiments and are not intended to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0031] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. Reagents not specifically described in detail in this application are all conventional reagents and can be obtained commercially; methods not specifically described in detail are all conventional experimental methods and can be learned from the prior art.

[0032] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the description of the present invention, which are obvious to those skilled in the art. Other implementation manners obtained from the description of the present invention are obvious to those skilled in the art. The description and examples of this application are only exemplary.

[0033] Regarding the terms "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, that is, they are meant to include but not be limited to.

[0034] In this application, the term "gene" refers to a nucleic acid fragment that expresses a specific protein or functional RNA molecule, and this nucleic acid fragment may include regulatory sequences (5' non-coding region) before the coding sequence and regulatory sequences (3' non-coding region) after it.

[0035] In the present application, the term "expression vector" refers to a vector that, on the basis of the basic backbone of a cloning vector (empty backbone), adds expression elements (such as promoters, RBSs, terminators, etc.) to enable the expression of a target gene.

[0036] In the present application, the term "homologous arm sequence" refers to a homologous sequence that functions to ligate the amplified target gene fragment to a linearized plasmid. During the ligation process, due to the presence of the homologous sequence, homologous recombination will occur between the target sequence and the plasmid DNA, inserting the target sequence fragment into the plasmid.

[0037] In the present application, the term "infusion ligation" refers to an enzyme-free ligation technique in the construction of expression vectors, mainly derived from the discovery of infusion enzymes. Infusion enzymes can recognize any 16 bases at the 5'-3' ends of linearized DNA fragments, forming sticky ends. After the target plasmid is linearized by restriction enzyme digestion or PCR, it can also be recognized by infusion enzymes. As long as the sticky ends formed by the vector and the gene are complementary, the construction of the vector can be completed through the annealing process.

[0038] In the present application, the term "nucleic acid molecule" refers to a polymer of RNA or DNA, which is single-stranded or double-stranded and optionally contains synthetic, non-natural or modified nucleobases. Nucleic acid molecules in the form of DNA polymers can be composed of one or more segments of cDNA, genomic DNA or synthetic DNA.

[0039] The embodiment provides a copper detoxification gene AtNAC002, which is located on the first chromosome of Arabidopsis thaliana, and its ID number in NCBI is: At1g01720. The amino acid sequence of the gene is shown in SEQ ID NO.1, and the coding nucleotide sequence (CDS sequence) of the gene is shown in SEQ ID NO.2.

[0040] The name and gene ID of the Arabidopsis AtNAC002 gene provided by the present invention both originate from the Arabidopsis thaliana sequencing database (www.arabidopsis.org), specifically from the wild-type Arabidopsis thaliana Col-0.

[0041] To determine the transcriptional activation activity of the AtNAC002 transcription factor, the AtNAC002 protein was divided into two fragments: a conserved N-terminal and an active C-terminal. The C-terminal sequence is shown in SEQ ID NO:3, with a length of 452 bp, and the N-terminal sequence is shown in SEQ ID NO:4, with a length of 418 bp.

[0042] In one or more embodiments, the nucleotide sequences of the amplification primers for the Arabidopsis thaliana AtNAC002 gene (SEQ ID NO: 2) are shown in SEQ ID NO: 10 and SEQ ID NO: 11.

[0043] In one or more embodiments, the nucleotide sequences of the amplification primers for the C-terminal sequence (SEQ ID NO: 3) of the Arabidopsis thaliana AtNAC002 gene are shown in SEQ ID NO: 12 and SEQ ID NO: 13.

[0044] In one or more embodiments, the nucleotide sequences of the amplification primers for the N-terminal sequence (SEQ ID NO: 4) of the Arabidopsis thaliana AtNAC002 gene are shown in SEQ ID NO: 14 and SEQ ID NO: 15.

[0045] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention.

[0046] The molecular biology experiments in the following examples include plasmid construction, restriction digestion, ligation, preparation of competent cells, transformation, medium preparation, etc. These are mainly carried out with reference to "Molecular Cloning: A Laboratory Manual" (Third Edition), edited by J. Sambrook et al., published by Science Press. Specific experimental conditions can be determined through simple experiments when necessary. The PCR amplification experiment is carried out according to the reaction conditions provided by the plasmid or DNA template supplier or the kit instructions, and can be adjusted through simple experiments when necessary.

[0047] Plants, genes and vectors: Arabidopsis thaliana Col-0 seeds, Arabidopsis thaliana mutant seeds (T-DNA insertion of the AtNAC002 gene, purchased from AraShare, product ID: 72973), PBI121 vector.

[0048] Media and buffers:

[0049] YPDA medium (1L): 20 g of tryptone, 10 g of yeast extract, 20 g of glucose, 30 mg of adenine sulfate, adjust the pH value to 5.8 with hydrochloric acid, add 1.2 - 1.5% agar powder for solid medium, sterilize at 115 °C;

[0050] SD / -TRP medium (1L): 6.7 g of yeast nitrogen base without amino acids, 0.64 g of supplement without tryptophan, 20 g of glucose, 15 g of agar powder, adjust the pH value to 5.8 with NaOH, sterilize at 115 °C;

[0051] SD / -TRP-HIS Medium (1L): 6.7 g of amino acid-free yeast nitrogen source, 0.64 g of supplement components lacking tryptophan and histidine, 20 g of glucose, 15 g of agar powder, adjust the pH value to 5.8 with NaOH, sterilize at 115 °C by high temperature;

[0052] Z-Buffer (pH 7.0, 1L): 21.5 g of Na2HPO4·12H2O, 6.22 g of NaH2PO4·2H2O, 0.75 g of KCl, 0.246 g of MgSO4·7H2O;

[0053] Yeast Chromogenic Solution (10 mL): 10 ml of Z-Buffer, 27 μL of β-mercaptoethanol, 167 μL of X-Gal stock solution;

[0054] 50% PEG 4000 (w / v, 100 mL): Dissolve 50 g of PEG 4000 in water, make up the volume to 100 mL, sterilize with a 0.22 μmol / L filter.

[0055] 1 mol / L LiAc (pH 7.5, 100 mL): Dissolve 10.2 g of C2H2LiO2·2H2O in water, make up the volume to 100 mL, sterilize with a 0.22 μmol / L filter.

[0056] ssDNA (5 mg / mL, 50 mL): Dissolve 250 mg of salmon sperm DNA in 50 mL of TE buffer.

[0057] X-Gal Stock Solution (20 mg / mL): Dissolve 0.5 g of 5-bromo-4-chloro-3-indolyl-β-D-galactoside in 25 mL of dimethylformamide, store in the dark at -20 °C.

[0058] Example 1 Specificity of Arabidopsis AtNAC002 Gene in Response to Copper Toxicity

[0059] Measure the growth of wild-type Arabidopsis Col-0 (WT) and AtNAC002 mutants (nac002) under different metal toxicities (manganese toxicity: 1 mmol / L MnCl2; zinc toxicity: 200 μmol / / L ZnSO4; iron toxicity: 300 μmol / L EDTA-Fe; arsenic toxicity: 20 mmol / L H3AsO3; copper toxicity: 30 μmol / L CuSO4) respectively. The specific operation is as follows:

[0060] A) Seed disinfection: Select Arabidopsis Col-0 seeds and mutant seeds of uniform size. First, disinfect them with 75% alcohol for 1 min, and then sterilize them with 1% NaClO for 20 min. In a laminar flow hood, wash them 6 - 8 times with autoclaved ultrapure water (>18.25 MΩ). Place the washed seeds in the dark in a 4°C refrigerator for 3 days.

[0061] B) Preparation of culture medium: Weigh a certain proportion of MS (Murashige and Skoog) at 2.215 g / L into an Erlenmeyer flask. According to different treatment conditions, add the corresponding metal salts to the appropriate concentrations. Add 1% (g / L) sucrose, dissolve it with ultrapure water and make up the volume, adjust the pH of the culture medium to 5.7 - 5.9, and add 1% (g / L) agar. Sterilize the prepared culture medium.

[0062] C) Sowing: Sterilize the workbench for 20 - 30 min. Write the seed number, treatment concentration, culture time, etc. on the culture dish. Pour the culture medium evenly into the culture dish and wait for it to solidify. After the culture medium solidifies, use a blue pipette tip to evenly sow the seeds on the culture medium at a certain spacing. Seal the culture dish with 3M breathable tape.

[0063] D) Cultivation: Place the culture dishes at a certain angle in a light incubator (temperature 22°C, light cycle 16 h (light) / 8 h (dark), light intensity 15000 lx, humidity 60% - 75%). During the growth period, regularly change the positions between the dishes to ensure uniform light.

[0064] Taking the root length and fresh weight of Arabidopsis as indicators, measure the root length and fresh weight of Arabidopsis on the 13th day. The results are as Figure 1 shown. Under copper toxicity conditions, the root length of the AtNAC002 gene mutant of Arabidopsis is 67.7% of that of the wild type, and the fresh weight is 50.7% of that of the wild type. This shows that in the case of the non-expression of the AtNAC002 gene, Arabidopsis shows sensitivity to copper toxicity and its growth is inhibited, proving that the AtNAC002 gene has a role in relieving copper toxicity in Arabidopsis. However, there is no significant difference between the AtNAC002 gene mutant material and the wild type under other heavy metal toxicity conditions, and it only shows sensitivity under copper toxicity conditions, indicating that the sensitivity of the AtNAC002 gene mutant to copper toxicity is specific.

[0065] Example 2 Spatial and temporal expression of the Arabidopsis AtNAC002 gene in response to copper toxicity

[0066] a) Arabidopsis thaliana copper toxicity culture conditions: After sterilized seeds were sown in normal medium and grown for 10 days, they were transferred to solid medium containing 30 μmol / L CuSO4 and treated at different time points (0, 3 h, 6 h, 12 h, 24 h, 3 d, 7 d). Samples were taken from the shoot and root, immediately put into liquid nitrogen, and stored in an -80 °C refrigerator.

[0067] b) Quantitative analysis process: Arabidopsis thaliana samples stored in an -80 °C refrigerator were used to extract total RNA from wild-type Arabidopsis thaliana using the EastepR SuperTotal RNA Extraction Kit (Promega, Madison, WI, United States). The total RNA (500 ng) was reverse transcribed into cDNA using ReverTra Ace qPCR RT Master Mix (TOYOBO, Osaka, Japan). According to the manufacturer's instructions, qRT-PCR was performed on a QuantStudio6Flex System (Applied Biosystems) using HieffqPCR SYBR Green Master Mix (Yeasen). The primers used in qRT-PCR were carefully checked, and the amplification efficiency was between 90% and 110%. By normalizing the relative expression levels.

[0068] Using UBC9 as the internal reference gene, the transcriptional level of gene AtNAC002 after copper toxicity treatment for different times was detected. The results are as Figure 2 shown. Taking the expression level of the shoot at 0 h of copper toxicity treatment as 1, the expression level of gene AtNAC002 in the root did not change significantly in the first 3 days, but increased significantly on the seventh day, about 6 times that of the control. This result indicates that gene AtNAC002 is up-regulated by copper toxicity induction in the root system.

[0069] Example 3 Structural characteristics of the Arabidopsis thaliana AtNAC002 gene

[0070] (1) Identification process of the T-DNA insertion site of gene AtNAC002

[0071] Download the T-DNA primers LP, RP, and BP provided by the website http: / / signal.salk.edu / tdnaprimers.2.html. Extract the DNA of the mutant using the TPS method and perform PCR amplification using the three-primer method. By comparing with the marker, determine whether it is a homozygous material according to the fragment length, and judge whether it is a homozygous material according to the amplified fragment length and the number of fragment bands.

[0072] (2) Design of PCR amplification primers

[0073] In this embodiment, a total of three primers were designed. According to the T-DNA insertion serial number SALK_057618 provided by the website http: / / signal.salk.edu / , the LP and RP primer sequences provided by the website were consulted. The nucleotide sequences are shown in SEQ ID NO: 5 (LP) and SEQ ID NO: 6 (RP). BP is a fragment on the T-DNA insertion sequence. According to the rhizosphere serial number category, LBb1.3 was selected as the BP primer for this gene. The corresponding nucleotide sequence of BP is shown in SEQ ID NO: 7 (BP).

[0074] (3) PCR reaction system and procedure

[0075] PCR reaction system: 0.2 μL of primer LP, 0.2 μL of primer RP, 0.2 μL of primer BP, 1 μL of cDNA, 5 μL of 2×Hieff PCR Master Mix (With Dye), 3.4 μL of ddH2O.

[0076] PCR amplification conditions: 5 min at 84 °C, 30 s at 94 °C, 30 s at 55 °C, 1 min / kb at 72 °C, 35 cycles, 10 min at 72 °C.

[0077] (4) Phylogenetic tree analysis of the AtNAC family

[0078] Construct a phylogenetic tree of the AtNAC family members in Arabidopsis thaliana and analyze the phylogeny among the AtNAC family members in Arabidopsis thaliana. Using MEGA7.0.21 software, select protein sequence alignment. After protein alignment with default parameters, select phylogenetic tree analysis. Construct a gene phylogenetic tree by the Neighbor-joining method (NJ). Check the parameter Bootstrap for 1000 repetitions. Select the Poisson distribution model for the substitution model, and keep other parameters as default values to construct the phylogenetic tree.

[0079] Figure 3Gene structure analysis: The AtNAC002 (AT1G01720) gene contains three exons and two introns. The full-length genome is 1827 bp and encodes 290 amino acids in total. The atnac002 (SALK_057618) mutant has a T-DNA insertion, and the insertion site is in the third exon near the 3' UTR region. With a 1-kb amplification range, it was identified by three primers (LP, RP, and BP). PCR confirmed the T-DNA insertion in the atnac002 mutant, and the identification result showed that this mutant is a homozygote. AtNAC002 was not detected at the mRNA level either, indicating that this mutant has changes at the transcriptional level. Previous studies reported that 151 NAC members were found in rice and there are a total of 105 NAC genes in Arabidopsis thaliana. We constructed a phylogenetic tree analysis using the 105 Arabidopsis thaliana NAC genes reported in the literature. Referring to the analysis of the rice NAC family, we divided the Arabidopsis thaliana NAC family genes into two major families, A and B, and 17 small subfamilies. The A family is divided into 4 small subfamilies, with a total of 29 genes; the B family is divided into 13 small subfamilies, with a total of 76 genes. The NAC002 we studied belongs to the B5 subfamily. This small subfamily includes a total of four genes, among which AtNAC002 and AtNAC032 have both been reported to be related to drought and salt stress before.

[0080] Example 4 Subcellular localization of the AtNAC002 gene

[0081] (1) The method for constructing and identifying the subcellular localization vector of the AtNAC002 gene is as follows:

[0082] Using the Arabidopsis thaliana root CDS as a template, the CDS fragment of NAC002 containing restriction enzyme sites (EcoRⅠ and XmaⅠ) was amplified. The amplification primers are shown in SEQ ID NO:8 and SEQ ID NO:9. After inserting it behind the 35S promoter of the PBI121 vector, the 35S:NAC002 vector was constructed. The CDS fragment of the AtNAC002 gene was amplified by blue enzyme from Tsingke Company, and the correct fragment was ligated to the expression vector pBI121-35S-NOS through restriction enzyme digestion.

[0083] (3) Transform Agrobacterium tumefaciens GV3101 with the AtNAC002 gene expression vector;

[0084] The detailed method is as follows: 25 μL of Agrobacterium competent cells (sterile operation), after slowly thawing on ice, add 1 μL of plasmid (about 200 ng), mix by flicking the bottom of the tube with your finger, immediately insert it into ice, quickly transfer the competent cell-plasmid mixture to an electroporation cuvette with a 200 μL pipette tip for electroporation, add 500 μL of LB liquid medium, incubate at 28 °C with shaking at 200 rpm for 2 h; spread the incubated bacterial solution on an LB plate containing the corresponding antibiotics (gentamicin Gen and kanamycin Kana), incubate at 28 °C in an inverted position for 2 days, pick monoclonal colonies for colony PCR and plasmid digestion identification.

[0085] (4) Transformation of tobacco using the recombinant expression vector

[0086] Pick the Agrobacterium clone transformed with the expression plasmid into 1 ml of LB containing the corresponding antibiotics, culture at 28 °C with shaking at 250 rpm for 24 h, add 100 μl of 0.5 M MES and 2 μl of 100 mM AS to 5 ml of LB containing the corresponding antibiotics, then inoculate 50 μl of the Agrobacterium bacterial solution, culture at 28 °C with shaking at 250 rpm until OD600 = 1.0 (about 12 - 18 h), centrifuge at 4000 rpm at room temperature for 10 min to collect the cells, resuspend with 10 mM MgCl2 to OD600 = 1.0, add 100 mM AS at a ratio of 2 μl per ml of the bacterial solution, and let stand for more than 3 h. Take Nicotiana benthamiana plants that are in the vigorous growth stage, suck the bacterial solution into a syringe, remove the needle, press against the front of the leaf with your finger, and infiltrate the bacterial solution from the back of the leaf. Place it under normal conditions and samples can be taken after 24 - 48 h.

[0087] Figure 4 To determine the subcellular localization of the gene AtNAC002, the principle of protein subcellular localization was observed using the tobacco transient expression system. The target gene was fused with the coding region of the GFP reporter gene, and the vector containing the exogenous target gene was introduced into tobacco leaves by Agrobacterium for expression. Fluorescence in the leaf epidermal cells was observed by a laser confocal microscope. For subcellular localization, in the tobacco leaf epidermal cells, the GFP signal fused with the gene AtNAC002 was detected in the nucleus and overlapped with the nuclear marker.

[0088] Example 5 Transcriptional activation activity of the AtNAC002 gene

[0089] (1) Yeast vector construction and ligation

[0090] The yeast empty vector used was the pGBKT7 empty vector. Yeast vector construction was carried out using the seamless cloning method (infusion ligation). According to the sequence information at both ends after vector linearization, the full-length target genes AtNAC002, AtNAC002-C, and AtNAC002-N were designed. Using the correctly sequenced CDS plasmid as a template, 15bp homologous arm sequences for ligation with the vector (nucleotide sequences shown in SEQ ID NO: 16 and SEQ ID NO: 17 respectively) were added to the front ends of three pairs of primers for the target genes AtNAC002, AtNAC002-C, and AtNAC002-N (nucleotide sequences shown in SEQ ID NO: 10 - SEQ ID NO: 15 respectively) to form the second primer pairs (nucleotide sequences shown in SEQ ID NO: 18 - SEQ ID NO: 23 respectively) for fragment amplification. Then, the amplified target fragments and the linearized vector backbone were subjected to infusion ligation, followed by Escherichia coli transformation.

[0091]

[0092]

[0093] (2) Preparation and transformation of yeast competent cells

[0094] The yeast strain AH109 was streaked on YPDA solid medium and cultured at 30 °C for 2 days to activate the yeast strain. Single colonies were selected and inoculated into 5 mL of YPDA liquid medium, and cultured with shaking at 30 °C until the OD 600 reached 1.6 - 1.8 (about 16 h); then transferred at a ratio of 1:10 to a new YPDA liquid medium and cultured with shaking at 30 °C until the OD 600 reached 0.8 - 1.0 (about 3 - 5 h); at room temperature, centrifuged at 3000 r / min for 5 min, discarded the supernatant, and washed the cells with 1 / 2 volume of sterile ddH2O; at room temperature, centrifuged at 3000 r / min for 5 min, discarded the supernatant, added an appropriate amount of sterile ddH2O to resuspend the cells. The ssDNA was boiled in boiling water for 5 - 10 min and immediately placed on ice for cooling and standby. The following solutions were added successively to a 1.5 mL sterile EP tube: 60 μL of yeast competent cells, 240 μL of 50% PEG 4000, 36 μL of 1 M LiAc, 10 μL of ssDNA (5 mg / mL), and 5 μL each of plasmid DNA (0.1 - 5 μg). Vortexed for 1 min to mix the transformation system evenly; incubated at 30 °C for 30 min; heat-shocked at 42 °C for 25 min, placed on ice for 5 min; centrifuged at 7200 r / min for 1 min, discarded the supernatant, added 200 μL of ddH2O to resuspend the cells, and spread them on SD / -TRP solid medium; cultured at 30 °C for 2 - 3 days until single colonies grew.

[0095] (3) Yeast clone gradient dilution spotting

[0096] Inoculate the monoclonal colonies obtained in the previous step into 2 mL of YPDA liquid medium and incubate overnight at 30 °C; centrifuge at 7200 r / min for 1 min, discard the supernatant, resuspend the cells with sterile ddH2O, and repeat 2 times; add 1 mL of sterile ddH2O to a new 1.5 mL sterile EP tube, add an appropriate amount of yeast solution, and adjust its OD 600 to about 0.8; gradient dilute the adjusted yeast solution 10-fold, 100-fold, and 1000-fold respectively; take 10 μL of the yeast solution under each concentration condition and spot it on SD / -TRP and SD / -TRP-HIS auxotrophic solid media, and culture it in the dark in a 30 °C incubator for 2 - 3 days.

[0097] (4) β-galactosidase activity detection

[0098] Take 2 pieces of filter paper and soak them with 2 mL of Z-Buffer containing X-Gal, and suck off the excess liquid; take out one piece of filter paper and cover it on the SD / -TRP-HIS auxotrophic solid medium with yeast transformants, carefully drive away the bubbles with a glass spreader, and make the filter paper contact the yeast colonies as much as possible, and mark the order of the clones on the filter paper; gently take out the filter paper and quickly freeze it in liquid nitrogen for 15 s; take out the filter paper, thaw it at room temperature, and then quickly freeze the filter paper in liquid nitrogen for 15 s again and thaw it at room temperature. Place the side of the filter paper with the bacterial solution facing up and closely attach it to another piece of filter paper previously soaked with Z-Buffer containing X-Gal. Place the filter paper in a petri dish and put it in a 30 °C incubator, observe the color development situation, and those that can develop color within 8 h are considered to have transcriptional activation activity.

[0099] Figure 5 According to the structural characteristics of transcription factors, we detected whether AtNAC002 has transcriptional activation activity and dissected the key regions of transcriptional activation. The AtNAC002 protein was divided into two fragments, a conserved N-terminal and an active C-terminal. The full-length AtNAC002 and the 2 fragments were respectively ligated to the pGBKT7 vector (containing the DNA-BD domain); the binding domain of GAL4 alone (pGBKT7) was used as a negative control, and then the vectors were introduced into yeast cells and grown on different media. All transformed yeast cells, including the negative control, grew on the SD / -Trp single-deficient medium. In contrast, when cultured on the SD / -Trp / -His double-deficient medium, only the yeast cells containing the full-length and C-terminal sequences could survive and convert the colorless X-Gal into blue by activating the reporter gene ( Figure 5 b). These results indicate that the gene AtNAC002 has transcriptional activation activity, and the C-terminal plays a very important role in the process of transcriptional activation.

[0100] Phenotypic Results of AtNAC002 Gene Responding to Copper Toxicity in Example 6

[0101] (1) Arabidopsis thaliana Agar Culture

[0102] Select Arabidopsis thaliana seeds of uniform size (wild type and AtNAC002 mutant (nac002)), sterilize them with 75% sterilizing alcohol for 1 min first, then sterilize them with 1% NaClO (w / v) for 10 min, and finally wash them 3 - 6 times with sterile water in a laminar flow hood and purify them at 4°C in the dark for 2 - 3 d. Then sow them in 1 / 2 MS medium with or without 30 μmol / L CuSO4 and grow them in a light incubator for about 13 d.

[0103] Measure the root length and fresh weight of Arabidopsis thaliana. The results are as Figure 6 shown. Under normal culture conditions, there is no significant difference in the root length and fresh weight between the wild type and the AtNAC002 mutant. Under copper toxicity conditions, the root length and fresh weight of the AtNAC002 mutant (nac002) are reduced by 31.46% and 54% respectively compared with the wild type, which is significantly lower than that of the wild type.

[0104] (2) Copper Content Determination

[0105] The samples for measuring copper content are the roots and shoots of Arabidopsis thaliana cultured for 13 d in vitro. Store the materials in kraft paper bags and blanch them in an oven at 105°C for 30 min, then dry them at 65°C until the samples reach a constant weight, and weigh the dry weight of the samples with a ten-thousandth balance. Weigh 0.1000 - 0.1500 g of shoot samples and 0.0150 - 0.0450 g of root samples into test tubes, add 5 mL of HNO3 / HClO4 mixed solution (v:v = 4:1), and then place them in a fume hood to soak overnight. After pre-digesting at 60°C for 1 h, raise the temperature to 1260°C until white smoke appears and the digestion solution becomes colorless, transparent or slightly yellow, then the digestion is completed. After cooling to room temperature, dilute the digestion solution to 10 mL in a volumetric flask with deionized water, shake well, filter, and conduct a reagent blank test at the same time. Use atomic absorption spectrometry (AAS) to measure the copper content in the digestion solution.

[0106] Measure the copper content in the roots and shoots. The results are as Figure 7 shown. Under normal conditions, there is no significant difference in the copper content between the shoots and roots; under copper toxicity conditions, there is no significant difference in the copper content in the shoots, while the copper content in the mutant roots is increased by 47% compared with the wild type, which is significantly higher than that of the wild type. The mutation of gene AtNAC002 will increase the absorption of copper by the roots of Arabidopsis thaliana, thus inhibiting the growth of plants.

[0107] (3) Determination of Antioxidant Enzyme Activity

[0108] Determination of Superoxide Dismutase (SOD) Enzyme Activity

[0109] The enzyme activity is determined based on the inhibition of the reduction of nitro - blue tetrazolium (NBT) by superoxide dismutase under light. In the presence of methionine and riboflavin, NBT undergoes a photoreduction reaction upon illumination to form blue formazan, which has a maximum light absorption at 560 nm. SOD can scavenge O2 - , thereby inhibiting the reduction of NBT to blue formazan. Therefore, after the photoreduction reaction, the darker the blue color of the reaction solution, the lower the SOD enzyme activity, and vice versa. The enzyme activity can be represented by the inhibition intensity of superoxide dismutase on the reduction of nitro - blue tetrazolium (NBT) under light.

[0110] Determination of Peroxidase (POD) Enzyme Activity

[0111] Under the catalysis of peroxidase, hydrogen peroxide oxidizes guaiacol into a tea - brown product. This product has a maximum light absorption at 470 nm. Therefore, the activity of peroxidase can be determined by measuring the change in absorbance at 470 nm.

[0112] Determination of Catalase (CAT) Activity

[0113] It has a strong absorption at 240 nm. Catalase can decompose hydrogen peroxide, causing the absorbance (A240) of the reaction solution to decrease with the prolongation of the reaction time. The activity of catalase can be measured according to the change rate of the measured absorbance.

[0114] Determination of Malondialdehyde (MDA)

[0115] MDA is a commonly used index of membrane lipid peroxidation. Under acidic and high - temperature conditions, it can react with thiobarbituric acid (TBA) to form a red - brown trimethyl complex, which has a maximum absorption wavelength of 532 nm and a minimum absorption peak at 600 nm.

[0116] The contents of the above - mentioned enzymes in the above - ground part and roots were measured respectively, and the results are as Figure 8 and 9 shown. Under normal and copper - toxic conditions, the contents of SOD, CAT, and POD in the roots of the mutant decreased, the SOD activity in the roots of the mutant increased, and only the POD activity in the above - ground part was significantly higher than that of the wild type, and there were no significant differences in other antioxidant enzymes. The results indicate that under copper - toxic conditions, the mutation of gene AtNAC002 reduces the activities of key antioxidant - related enzymes in the roots, thereby increasing the accumulation of ROS in the roots of Arabidopsis thaliana and further increasing the sensitivity to copper toxicity.

[0117] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. Application of Arabidopsis thaliana AtNAC002 gene in improving copper toxicity resistance of Arabidopsis thaliana, characterized in that The ID number of the gene in NCBI is At1g01720.

2. The application according to claim 1, characterized in that The Arabidopsis thaliana AtNAC002 The nucleotide sequence of the gene is shown in SEQ ID NO:

2.

3. Application of Arabidopsis thaliana AtNAC002 transcription factor in improving copper toxicity resistance of Arabidopsis thaliana, characterized in that The amino acid sequence of the Arabidopsis thaliana AtNAC002 transcription factor is shown in SEQ ID NO:

1.

4. Application of a recombinant vector in improving copper toxicity resistance of Arabidopsis thaliana, characterized in that The recombinant vector contains the Arabidopsis thaliana described in claim 1 or 2 AtNAC002 gene.

5. The application according to claim 1 or 2, characterized in that The Arabidopsis thaliana AtNAC002 gene is up-regulated by copper toxicity induction in the root system.

6. The application according to claim 4, characterized in that The Arabidopsis thaliana AtNAC002 gene is up-regulated by copper toxicity induction in the root system.

7. The application according to claim 1 or 2, characterized in that The Arabidopsis thaliana AtNAC002 gene improves the resistance of Arabidopsis thaliana to copper toxicity by reducing the absorption of copper by the roots of Arabidopsis thaliana.

8. The application according to claim 3, characterized in that The Arabidopsis thaliana AtNAC002 transcription factor improves the resistance of Arabidopsis thaliana to copper toxicity by reducing the absorption of copper by the roots of Arabidopsis thaliana.

9. The application according to claim 1 or 2, characterized in that The Arabidopsis thaliana AtNAC002 gene reduces the copper content in the roots of Arabidopsis thaliana.

10. The application according to claim 3, characterized in that The Arabidopsis thaliana AtNAC002 transcription factor reduces the copper content in the roots of Arabidopsis thaliana.

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