CN116144675B - SlCNR8 gene and application, protein, detection method and acquisition method thereof - Google Patents

Abstract

The invention provides SlCNR gene and application, protein, detection method and acquisition method thereof, belonging to the technical field of genetic engineering. The nucleotide sequence of SlCNR gene is shown as SEQ ID No. 1. The invention discovers that the gene SlCNR in the mongolian willow responds to the stress of metal copper for the first time. Compared with a wild poplar, slCNR transgenic poplar can improve the heavy metal Cu resistance of plants under the stress of heavy metal copper, and can be used for repairing heavy metal copper pollution of soil.

Description

SlCNR8 gene and application, protein, detection method and acquisition method thereof

Technical Field

The invention relates to the field of genetic engineering, in particular to SlCNR gene and application, protein, detection method and acquisition method thereof.

Background

Copper in soil can be absorbed and accumulated by plants, the copper absorbed by the plants can promote plant growth at low concentration, high concentration can cause damage to plant bodies, copper can collapse a thylakoid membrane system, increase of photosynthetic pigments is inhibited, and photosynthesis is damaged. Copper is used as an essential component of intracellular enzymes and a donor or an acceptor in an electron transfer chain, copper stress can reduce the activity of various enzymes in plants, excessive copper can induce the intracellular production of a large amount of active oxygen and generate membranous peroxidation with cytoplasmic membranes and organelle membranes to damage cell membranes, organelle membranes and nuclear membranes, and prevent root systems from absorbing nutrient substances.

Copper in the environment can be enriched in crops and enter the human body through a food chain, copper is an important element for assisting endocrine activities of the human body, but the intake content exceeds the normal range accepted by the human body, so that the damage to the central system, liver, gall and intestines and stomach of the human body can be caused.

The method reduces copper pollution of soil, improves and repairs degraded soil, and improves soil production quality to be the problem to be solved at present. The soil pollution treatment mainly comprises physical repair, chemical repair and biological repair. Physical repair includes physical separation and the like, chemical repair includes in-situ passivation, redox, leaching techniques and the like, and biological repair includes microbial repair, plant enrichment and the like. The traditional physical and chemical methods have the defects of high cost, limited application scale, easiness in forming secondary pollution and the like. The plant restoration restores heavy metal polluted soil through the actions of plant extraction, root filtration and the like, has low cost and no secondary pollution, can effectively protect surface soil, reduces water and soil loss, and can be widely applied to ecological restoration and reconstruction of heavy metal copper polluted soil and mining areas. Copper ion tolerant plants are planted on the copper ion polluted soil, so that the method has important ecological benefits.

Disclosure of Invention

In order to solve the problems, the invention provides SlCNR gene and application, protein, detection method and acquisition method thereof, and the invention discovers that the gene SlCNR in the mongolian willow can respond to the stress of metallic copper for the first time, compared with a wild poplar, the growth condition of the transgenic poplar is less affected under the stress of heavy metal copper ions in the poplar transformed into the SlCNR gene, and the transgenic poplar is planted in copper contaminated soil to protect surface soil and reduce water and soil loss.

In order to achieve the above object, the present invention provides the following technical solutions:

The invention provides SlCNR genes, and the nucleotide sequence of the SlCNR genes is shown as SEQ ID No. 1.

The invention also provides the protein coded by the SlCNR gene in the technical scheme, and the amino acid sequence of the protein is shown as SEQ ID No. 2.

The invention also provides application of SlCNR gene in improving copper resistance of plants.

The invention also provides application of SlCNR gene in regulating copper resistance of plants.

The invention also provides application of SlCNR gene in breeding plants with copper resistance improvement.

The invention also provides application of SlCNR gene in repairing copper-polluted soil.

Preferably, the plant comprises poplar.

The invention also provides a method for detecting the copper stress tolerance of the plant, which is used for detecting whether the plant contains SlCNR genes according to the technical scheme, and when the plant contains SlCNR genes, the plant is subjected to the copper stress tolerance.

Preferably, the nucleotide sequence of the upstream primer used for detecting SlCNR gene is shown as SEQ ID No.3, and the nucleotide sequence of the downstream primer is shown as SEQ ID No. 4.

The invention also provides a method for obtaining the transgenic poplar, which comprises the following steps:

1) Connecting SlCNR genes in the technical scheme into a plant expression vector to obtain a target gene vector;

2) Transforming the target gene vector obtained in the step 1) into escherichia coli, and extracting plasmids to obtain a recombinant vector;

3) Transforming the recombinant vector obtained in the step 2) into agrobacterium to obtain transformed agrobacterium;

4) And 3) infecting poplar with the transformed agrobacterium obtained in the step 3) by a leaf disc method to obtain transgenic poplar.

The beneficial effects are that:

Experiments provided by the invention prove that SlCNR genes are over-expressed in poplar, and compared with wild poplar, the obtained transgenic poplar has the advantages that plant height, root length, fresh weight and dry weight are increased, and resistance to metallic copper is obviously enhanced, so that SlCNR genes have important application value in the aspect of genetic improvement of metallic copper resistance.

In the invention, compared with a wild poplar, the transgenic poplar plant grows better after being subjected to copper sulfate stress treatment, and the transgenic poplar plant can be seen to be less damaged after being dyed by NBT. Therefore, the SlCNR gene modified poplar has high copper stress resistance and has wide application prospect in improving natural environment in copper-polluted soil.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments will be briefly described below.

FIG. 1 is a SlCNR gene clone electrophoresis pattern;

FIG. 2 is a molecular characterization of transgenic poplar;

FIG. 3 shows SlCNR gene subcellular localization results;

FIG. 4 is a phenotype of a wild type poplar and a transgenic poplar after heavy metal copper treatment;

FIG. 5 is a plant height, root length, weight of a wild poplar and transgenic poplar after heavy metal copper treatment;

FIG. 6 shows the results of NBT staining of wild type poplar and transgenic poplar after heavy metal copper treatment.

Detailed Description

The invention provides a SlCNR gene, the nucleotide sequence of the SlCNR gene is shown as SEQ ID No.1, and the nucleotide sequence is as follows:

ATGGCTGACAACAACAACCAGAGCTTCGACAGCAACAATCATGATGGCGAGGAAGCGAGCCCTCTTTTGAACAAGAACCTTGAAGAACAAAACGACAAGAAACCCACCAAGGTTTCTCCTGATGAAAAGACGGCCACCGCCTCCCCGGGATCTGCTTTGCCTGAATATGGGTGGACCGTTAATGGGCTGCCGTTGAGTCATGGGAGCGTGGTGGGTGAACCTATGGGTCGGACTCAGTGGGACTCCAGCCTTTTTGCTTGCCTTGGTCGCAACGATGAATTCTGTAGCAGCGATCTTGAAGTCTGTCTTCTTGGAAGCACTGTCCCTTGCATGCTGTACGGAAGCAACGCGGAGAGACTTGGATCTACTCCTGGAACATTTGCAACCCACTGCTTGCCATACTGTGGTCTGTACCTGATCGGCAATTCATTTTTCGGTGGGAATTGCATCGCACCATGGTTTTCATATCCTAGCCGCACAGCTATCCGTCGGAAGTTTAACCTAGAGGGTAGCTGTGAGGCACTTGATAGGTCATGTGGCTGCTGTGGGAGCTTTGTGGAAGATGATCTGCAACGTGAGCAATGCGAGTCAGCATGTGATTTTGCAACTCATGTTTTCTGCCACCCATGTGCCCTTTGCCAGGAAGGTCGTGAGATCCGCCGTAGGGTGCCCCATCCTGGGTTTAATGCTCAACCGATCTTGGTTATGATCCCACCAGGGGAGCAATCCATGGGCCGTGGAGCCTGA.

The invention also provides a protein coded by SlCNR genes in the technical scheme, the amino acid sequence of the protein is shown as SEQ ID No.2, and the protein is specifically as follows:

MADNNNQSFDSNNHDGEEASPLLNKNLEEQNDKKPTKVSPDEKTATASPGSALPEYGWTVNGLPLSHGSVVGEPMGRTQWDSSLFACLGRNDEFCSSDLEVCLLGSTVPCMLYGSNAERLGSTPGTFATHCLPYCGLYLIGNSFFGGNCIAPWFSYPSRTAIRRKFNLEGSCEALDRSCGCCGSFVEDDLQREQCESACDFATHVFCHPCALCQEGREIRRRVPHPGFNAQPILVMIPPGEQSMGRGA.

the invention also provides application of SlCNR gene in improving copper resistance of plants.

The invention also provides application of SlCNR gene in regulating copper resistance of plants.

The invention also provides application of SlCNR gene in breeding plants with copper resistance improvement.

The invention also provides application of SlCNR gene in repairing copper-polluted soil.

In the present invention, the plant preferably comprises a poplar, which is preferably an 84K poplar.

The invention also provides a method for detecting copper stress tolerance of plants, which is used for detecting whether the plants contain SlCNR genes as claimed in claim 1, and when the plants contain SlCNR genes, the plants are subjected to copper stress.

In the invention, the nucleotide sequence of an upstream primer used for detecting SlCNR genes is shown as SEQ ID No.3, and the nucleotide sequence of a downstream primer is shown as SEQ ID No.4, and the specific steps are as follows:

SEQ ID No.3：ATGGCTGACAACAACAACCAGAG；

SEQ ID No.4：TCAGGCTCCACGGCCCATG。

The invention also provides a method for obtaining the transgenic poplar, which comprises the following steps:

1) Connecting SlCNR genes in the technical scheme into a plant expression vector to obtain a target gene vector;

2) Transforming the target gene vector obtained in the step 1) into escherichia coli, and extracting plasmids to obtain a recombinant vector;

3) Transforming the recombinant vector obtained in the step 2) into agrobacterium to obtain transformed agrobacterium;

4) And 3) infecting poplar with the transformed agrobacterium obtained in the step 3) by a leaf disc method to obtain transgenic poplar.

The SlCNR gene of the technical scheme is connected into a plant expression vector to obtain a target gene vector. In the present invention, the plant expression vector preferably includes a plant expression vector 1300. In the present invention, the SlCNR gene and the plant expression vector are preferably connected by a T4 ligase after cleavage, and the system and reaction conditions for the cleavage and the T4 ligase are not particularly limited, and those skilled in the art may operate according to conventional procedures.

The invention converts the obtained target gene vector into escherichia coli, extracts plasmids and obtains a recombinant vector. In the present invention, the E.coli preferably includes E.coli DH5a. The method for transforming the target gene vector into escherichia coli and extracting plasmids is not particularly limited, and can be performed by a person skilled in the art according to conventional operation.

The recombinant vector obtained is transformed into agrobacterium to obtain transformed agrobacterium. In the present invention, the agrobacterium preferably includes agrobacterium EHA105. The method of transforming the recombinant vector into Agrobacterium according to the present invention is not particularly limited, and a person skilled in the art may use a conventional method.

The obtained transformed agrobacterium is used for infecting poplar by a leaf disc method to obtain transgenic poplar. The method for infecting poplar by the leaf disc method is not particularly limited, and conventional operation is adopted.

The present invention will be described in detail with reference to examples for further illustration of the invention, but they should not be construed as limiting the scope of the invention. The quantitative experiments in the invention are all provided with three repetitions if no special description exists, and the results are the average value of the three results.

Example 1

Genetic transformation method of poplar and molecular identification of transgenic poplar

Polymerase chain reaction (PCR amplification) of SlCNR gene and construction of plant expression vector

According to the transcriptome sequencing result of the mongolian willow, designing an upstream primer and a downstream primer by taking the full-length SlCNR gene as a template, and carrying out polymerase chain reaction of the SlCNR gene by taking cDNA of the mongolian willow as the template, wherein an amplification system is as follows: 10 XEx Taq Buffer 5. Mu.l, ex Taq enzyme 0.25. Mu.l, dNTP 1.25. Mu.l, 10. Mu. Mol/L upstream primer 1. Mu.l, 10. Mu. Mol/L downstream primer 1. Mu.l cDNA template 1. Mu.l, sterile water 40.5. Mu.l, total system 50. Mu.l, PCR amplification procedure of 3min pre-denaturation at 95 ℃,30 s annealing at 60.5 ℃, 45s extension at 72 ℃,30 cycles, 10min extension at 72℃full length, and storage at 4 ℃. The PCR amplified product is obtained through the steps, and is recovered through gel electrophoresis.

The upstream primer of SlCNR gene PCR amplification is:

5′-ATGGCTGACAACAACAACCAGAG-3′。

the SlCNR gene PCR amplified downstream primer is: 5'-TCAGGCTCCACGGCCCATG-3'.

The PCR amplification product was ligated with the plant expression vector 1300 using a double enzyme digestion method, and the T4 DNA ligase manufacturer was Takara, cat# 2011A.

The double enzyme digestion connection steps are as follows: the total amount of the double enzyme digestion reaction system is 50 mu l, wherein the SlCNR gene double enzyme digestion reaction system is 10 XFD Green Buffer5 mu l, scaI, xbaI endonuclease which is 1.2 mu l, 20 mu l of PCR amplified product and 23 mu l of sterile water respectively. Wherein the 1300 vector double enzyme digestion reaction system is 10 XFD Green Buffer5 mu l, scaI, xbaI which is 1.2 mu l, 1300 vector plasmid 20 mu l and sterile water 23 mu l respectively. The double enzyme digestion reaction condition is 37 ℃ for 1h. And detecting the target gene and the vector enzyme digestion product through gel electrophoresis, and recovering the correct target strip by using a gel recovery kit gel. The gel recovery kit was purchased from century biotechnology limited under the code CW2302.

The ligation system consisted of 1. Mu.l of T4 Buffer, 1. Mu.l of T4 DNA ligase, 3. Mu.l of gene gel recovery product and 5. Mu.l of enzyme-digested vector gel recovery product. The ligation conditions were 16℃overnight.

The method comprises the steps of transforming a vector with a target gene into escherichia coli competent DH5a by utilizing the principle of thermal expansion and cold contraction, uniformly coating the escherichia coli competent DH5a on an LB solid medium with Kana resistance, and after the incubator is inverted at 37 ℃ and grows overnight, selecting a monoclonal colony for PCR identification. The PCR reaction system is as follows: 2X HieffPCR MasterMix. Mu.l, 10. Mu. Mol/L of upstream primer 1. Mu.l, 10. Mu. Mol/L of downstream primer 1. Mu.l, 1. Mu.l of monoclonal bacteria solution as template and 7. Mu.l of sterile water. The PCR amplification procedure was 95℃pre-denatured for 3min,95℃denatured for 30s,60.5℃annealed for 30s,72℃extended for 45s,30 cycles, 72℃full length extended for 10min, and 4℃stored. Positive colonies with the target gene were screened by gel electrophoresis using 2000bp DNAMarker as a control, and cultured in 5ml of LB liquid medium and 5 μ lKana antibiotics overnight with shaking at 37 ℃ at 200rpm. Extracting plasmid from amplified positive bacteria liquid, and sequencing and detecting. The plasmid kit is purchased from Tiangen Biochemical technology Co., ltd, and the product number is DP103-03.

Sequencing the positive bacterial liquid in biological company, comparing the sequencing result with template, and maintaining the correct positive bacterial liquid for subsequent work

Agrobacterium-mediated genetic transformation of poplar

Transformation of Agrobacterium competence: the correctly sequenced recombinant vector was transformed into Agrobacterium competent EHA 105. Generally, 5. Mu.l of plasmid DNA is added to 100. Mu.l of Agrobacterium competence, stirred and mixed uniformly by a gun head, placed in ice for 5min, placed in liquid nitrogen for 5min, and placed in a water bath at 37 ℃ for 5min and ice for 5min. Mu.l of YEP liquid medium was added thereto, and the culture was continued at 28℃for 3 hours with shaking. About 150. Mu.l of the mixture was spread on a plate with Rif and Kana antibiotics, and cultured upside down in an incubator at 28℃for 2-3d. After single colony grows out, picking the single colony, carrying out shake culture for 1d at 28 ℃, and carrying out PCR identification on the obtained bacterial liquid, wherein a PCR reaction system is as follows: 2X HIEFF PCR MASTER Mix 10. Mu.l, 10. Mu. Mol/L upstream primer 1. Mu.l, 10. Mu. Mol/L downstream primer 1. Mu.l, template 1. Mu.l monoclonal bacteria solution, sterile water 7. Mu.l. The PCR amplification procedure was 95℃pre-denatured for 3min,95℃denatured for 30s,60.5℃annealed for 30s,72℃extended for 45s,30 cycles, 72℃full length extended for 10min, and 4℃stored. By using 2000bp DNAMarker as a control, a positive colony with a target gene is screened out through agarose gel electrophoresis, 200 μl of positive bacterial liquid is sucked and cultured in 5mlYEP liquid culture medium, 5 μl Kana and 5 μ lRif antibiotics for one day under shaking conditions of 28 ℃ and 200rpm. 200. Mu.l of the resulting bacterial liquid was pipetted into a new centrifuge tube and stored in a-80℃refrigerator.

Agrobacteria-mediated leaf disk method infection poplar experiments: firstly, carrying out activation culture on agrobacterium liquid, then carrying out amplification culture, and infecting the leaves when the bacterial liquid OD _600nm = 0.6-0.8. Selecting 2 nd to 3 rd leaves which are spread at the top end of 84K poplar with the seedling age of 3-4 weeks, carefully cutting the leaves by a tissue culture knife, removing leaf tips, the periphery of the leaves, leaf bases and edge parts of the leaves, cutting 3-4 wounds perpendicular to veins, moving the leaves into agrobacterium tumefaciens bacteria liquid to infect the leaves for 25min, putting the leaves into a differentiation medium for co-culture (inversion culture) after infection, and carrying out dark culture at 25 ℃ for 3d.

After co-culture, bacteria are washed: and (3) transferring the co-cultured leaves into a liquid differentiation medium, washing off surface floating bacteria, and washing for 3 to 5 minutes. Then, the leaves are subjected to dark culture in a resistance screening medium after being washed by sterile water added with the cephalosporin for about six times, 5 to 8 minutes each time.

Screening and culturing transgenic poplar: the medium was changed weekly thereafter and the medium formulation was adjusted according to the status of the shoots: screening culture for about 7d (second plate change): WPM+0.03mg/L6-BA+0.01 mg/LIBA+0.001mg/L TDZ; screening culture about 14d (third plate change): WPM+0.03mg/L6-BA+0.01mg/L IBA+0.0005mg/L TDZ; about 21d (fourth plate change): WPM+0.03mg/L6-BA+0.01mg/L IBA+0.0004mg/L TDZ; screening culture for 28d (fifth plate change): WPM+0.03mg/L6-BA+0.01mg/L IBA+0.0004mg/LTDZ; screening culture for about 35d (sixth plate change): WPM+0.03 mg/L6-BA+0.02 mg/L IBA+0.0002mg/LTDZ, and gradually discarding the yellow-blackened leaf discs, separating small buds from the leaf discs, and after the leaves differentiate and grow strong adventitious buds, separating buds and placing the buds in a rooting culture medium to induce rooting; about 42d (seventh flask change): MS+0.02mg/LNAA+0.05mg/L IBA; about 49d (eighth flask change): MS+0.02mg/LNAA+0.05mg/L IBA, the rooting process can also screen out some false positive plants which cannot root, and the rooted plants are cultivated in a plant incubator in a relay mode.

Identification of transgenic poplar

DNA level identification of transgenic poplar

The upstream primer identified by SlCNR gene PCR is:

5′-ATGGCTGACAACAACAACCAGAG-3′；

The downstream primers identified by SlCNR gene PCR are: 5'-TCAGGCTCCACGGCCCATG-3'.

The amplified target band size was 747bp.

Extraction of poplar in-vivo DNA by CTAB method: selecting transgenic poplar leaves which are strong in growth, and putting the transgenic poplar leaves into liquid nitrogen for freezing and grinding. To the ground sample, 500. Mu.l of CTAB extract was added, incubated in a 65℃water bath for 20min, and mixed carefully and upside down every 5min. Centrifuge at 12000rpm for 10min at room temperature, add 250. Mu.l chloroform and 250. Mu.l Tris-saturated phenol to the supernatant, mix well and centrifuge at 12000rpm for 5min at room temperature. Repeating once. The supernatant was pipetted into a new centrifuge tube and 800. Mu.l of absolute ethanol and 100. Mu.l of 3M NaAc were added to precipitate the DNA for 20min. Centrifuge at 12000rpm for 20min at room temperature. The supernatant was discarded, and the DNA was washed with 500. Mu.l of 75% ethanol, and the ethanol was removed by pipetting. The DNA was dissolved with 30-50. Mu.l of sterile water.

The DNA of the transgenic poplar and the wild poplar is extracted by a CTAB method, and PCR identification is carried out by taking the DNA of the transgenic poplar and the wild poplar as templates. The PCR reaction system is as follows: 2X HieffPCRMasterMix. Mu.l, 10. Mu. Mol/L of upstream primer 1. Mu.l, 10. Mu. Mol/L of downstream primer 1. Mu.l, 1. Mu.l of template DNA and 7. Mu.l of sterile water. The PCR amplification procedure was 95℃pre-denatured for 3min,95℃denatured for 30s,60.5℃annealed for 30s,72℃extended for 45s,30 cycles, 72℃full length extended for 10min, and 4℃stored. And (3) performing gel electrophoresis on the PCR reaction product, and screening out the transgenic poplar with the target gene by taking 2000bp DNAMarker as a control.

RNA level identification of transgenic poplar

Firstly, selecting transgenic poplar leaves which grow robustly, sampling in an ultra-clean workbench, loading the leaves into a 2ml centrifuge tube for removing RNase, then placing the centrifuge tube into liquid nitrogen for quick freezing, and grinding the poplar leaves into white powder by using an oscillation grinder. To the ground sample, 1ml of Trizol reagent was added to lyse the cells, and the mixture was allowed to stand at room temperature for 5min. 200. Mu.l of chloroform was added to the sample, and after shaking and mixing, the mixture was allowed to stand at room temperature for 2 to 3 minutes. Centrifuge at 12000rpm for 15min at 4℃and transfer supernatant to a new centrifuge tube and record the approximate volume of supernatant. Adding isopropanol with the same volume as the supernatant, standing at room temperature for 10min, and centrifuging at 12000rpm at 4deg.C for 10min. The supernatant was discarded, 500. Mu.l of 75% DEPC ethanol solution was added, and the precipitate was flicked up and mixed with the solution, and centrifuged at 12000rpm for 2min at 4 ℃. The supernatant was discarded, 500. Mu.l of 75% DEPC ethanol solution was added thereto, the precipitate was flicked up and mixed with the solution, and the mixture was centrifuged at 12000rpm for 2 minutes at 4℃to discard the supernatant. The mixture was allowed to air-stand at 12000rpm for 2min at 4℃and the ethanol was removed by pipetting. The DNA was dissolved with 30-50. Mu.l of sterile water.

PCR identification was performed using cDNA of transgenic poplar and wild poplar as a template. The PCR reaction system is as follows: 2X HIEFFPCR MASTER Mix 10. Mu.l, 10. Mu. Mol/L upstream primer 1. Mu.l, 10. Mu. Mol/L downstream primer 1. Mu.l, template cDNA 1. Mu.l, sterile water 7. Mu.l. The PCR amplification procedure was 95℃pre-denatured for 3min,95℃denatured for 30s,60.5℃annealed for 30s,72℃extended for 45s,30 cycles, 72℃full length extended for 10min, and 4℃stored. And (3) performing gel electrophoresis detection on the PCR reaction product, and screening out the transgenic poplar with the target gene by taking 2000bp DNAMarker as a control.

In order to determine subcellular localization of SlCNR gene, since 1300 expression vector contains GFP sequence, recombinant vector in transformed Agrobacterium is used to infect tobacco, and its expression position is observed by using laser confocal microscope, and protein encoded by SlCNR gene is expressed in cell nucleus and cytoplasm.

Example 2

Effect of SlCNR Gene overexpression on plant copper resistance

The wild poplar was subcultured with the transgenic poplar of example 1 in 1/2MS medium and rooting after 7 d.

Wild poplars of consistent growth (similar 1-2cm plant height, leaf number, number of fibrous roots and root length) were selected and transferred to 1/2MS medium supplemented with 40 and 80. Mu. Mol/L CuSO ₄ with the transgenic poplar of example 1, and each treatment was repeated 5 groups. Photographing is carried out on observation every day, poplar is cultivated for 15d after copper treatment, photographing is carried out after treatment, plant height, main root length and fresh weight are measured, then the roots and She Fenkai are dried for 48h at the temperature of 65 ℃, and dry weight is measured.

When no stress exists, the growth vigor of the wild poplar is consistent with that of the transgenic poplar, the plant height, root length, fresh weight and dry weight of the poplar after 80 mu mol/L CuSO ₄ stress treatment are obviously reduced, and the growth vigor of the transgenic poplar after stress treatment is stronger than that of the wild poplar, so that the aspects of plant height, root length, fresh weight and dry weight are reflected.

Example 3

Influence of SlCNR Gene overexpression on plant antioxidant Capacity

The wild poplar was subcultured with the transgenic poplar of example 1 in 1/2MS medium and the poplar was rooted after 7 days.

Wild poplars of consistent growth (plant height, leaf number, number of fibrous roots and root length were similar 1-2 cm) were selected and transferred to 1/2MS medium supplemented with 80. Mu. Mol/L copper sulfate with the transgenic poplar of example 1, and each treatment was repeated 5 groups. NBT staining was performed at the end of the treatment.

The plants were placed in a 50ml large tube, NBT dye solution was added, the leaves were immersed, after the leaves were observed to be dyed, the dye solution was poured off, carnot fixative solution was added, after 2h, fixative solution was poured off, and transparence was added, and after 1d, the photographs were observed.

Carnot fixative: 1ml glacial acetic acid is added into 3ml absolute ethanol and mixed

And (3) a transparent agent: 1ml of glycerol is added into 4ml of absolute ethanol and mixed

The NBT staining result shows that the wild poplar leaves are darker in staining after copper stress than the transgenic poplar leaves, and the result shows that the accumulation of superoxide anions of the transgenic poplar is smaller than that of the wild poplar, and the transgenic poplar is stronger in antioxidant capacity and resistance after copper stress.

Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.

Claims (8)

1. The SlCNR gene is characterized in that the nucleotide sequence of the SlCNR gene is shown as SEQ ID No. 1.

2. The protein encoded by SlCNR gene according to claim 1, wherein the amino acid sequence of said protein is shown in SEQ ID No. 2.

3. Use of SlCNR gene according to claim 1 to increase copper resistance in poplar.

4. Use of SlCNR gene according to claim 1 for regulating copper resistance of poplar.

5. The use of SlCNR gene according to claim 1 for breeding poplar with increased copper resistance.

6. A method for detecting copper stress tolerance of poplar, comprising detecting whether the poplar contains SlCNR gene according to claim 1, wherein the poplar is copper stress tolerant when the poplar contains SlCNR gene.

7. The method according to claim 6, wherein the nucleotide sequence of the upstream primer used for detecting SlCNR gene is shown in SEQ ID No.3, and the nucleotide sequence of the downstream primer is shown in SEQ ID No. 4.

8. A method for obtaining a transgenic poplar, comprising the steps of:

1) Connecting SlCNR genes described in claim 1 into a plant expression vector to obtain a target gene vector;

2) Transforming the target gene vector obtained in the step 1) into escherichia coli, and extracting plasmids to obtain a recombinant vector;

3) Transforming the recombinant vector obtained in the step 2) into agrobacterium to obtain transformed agrobacterium;

4) And 3) infecting poplar with the transformed agrobacterium obtained in the step 3) by a leaf disc method to obtain transgenic poplar.