CN108866081B - Gene for improving cadmium resistance and cadmium content and application thereof - Google Patents

Abstract

The invention relates to the technical field of genetic engineering, in particular to a gene for improving cadmium resistance and cadmium content and application thereof; the invention constructs cDNA library of cadmium hyper-accumulation ore mountain type sedum alfredii hance, screens out the cDNA librarySaCTPThe gene is verified to improve the cadmium resistance of the yeast by a yeast dot plate experiment; will be provided withSaCTPThe gene is heterologously expressed in the arabidopsis thaliana, and the cadmium resistance and the cadmium ion content of the transgenic arabidopsis thaliana are improved; obtained by the inventionSaCTPThe gene provides gene resources for cultivating the novel plant germplasm with high cadmium resistance and high cadmium content, and can be applied to the field of plant bioremediation of heavy metal contaminated soil.

Description

Gene for improving cadmium resistance and cadmium content and application thereof

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a gene for improving cadmium resistance and cadmium content and application thereof.

Background

China is a big agricultural country, soil is the basis of agriculture, and the soil is taken as an important natural environment element to passively bear most pollutants including heavy metals; the cultivated land area polluted by heavy metal in China reaches 2000 hectares, the grain polluted by cadmium only reaches 1200 million tons every year, and the direct economic loss caused by the cadmium only exceeds 200 million yuan. The difficult degradation and easy enrichment of heavy metal elements and the transferability of a food chain bring serious threats to ecological environment and food safety, are 'invisible killers' for human life health and safety, in recent years, the heavy metal such as cadmium rice and the like frequently pollutes food, is firstly painful and is not easy to treat cadmium-polluted soil. The functional research of cadmium super-accumulation type plant resistance genes is developed, the molecular mechanism of cadmium tolerance of plants is clarified, and the important significance is achieved in cultivating cadmium resistance transgenic new germplasm and treating and repairing heavy metal contaminated soil in China.

The super-accumulation type sedum alfredii hance is a zinc/cadmium (Cd/Zn) super-accumulation type plant grown in China indigenous soil, and can accumulate 9,000 mu g.g on the overground part^-1Cd and 29,000 μ g.g^-1Zn does not present any toxic symptoms, and is extremely researchedA value; the sedum alfredii transports high-concentration cadmium ions to the overground part through a high-efficiency root-stem transport mechanism, and finally stores the cadmium ions in parenchyma cells of mesophyll, medulla parts of stems and cortex tissues.

In order to analyze the high resistance and the super accumulation capacity of the super accumulation type sedum alfredii hance, researchers develop researches on genes participating in processes of ion transport, chelation, oxidative damage repair and the like; however, it is still unclear whether there is a specific cadmium ion transport, detoxification, response-related protein in the hyperaccumulating sedum alfredii. The existing research shows that the super-accumulation type sedum alfredii hanceSaNramp6The gene can improve the cadmium ion accumulation amount of plants, but the excessive cadmium ion accumulation reduces the cadmium resistance of transgenic plants. Whether a gene exists in sedum alfredii hance or not can improve the cadmium resistance of transgenic plants and the cadmium content and needs to be researched urgently.

Disclosure of Invention

The invention discovers a gene with the function of improving cadmium resistance and cadmium content from the sedum alfredii cDNA library for the first time by constructing the sedum alfredii cDNA library and screening cadmium resistance genes by means of cadmium sensitive yeast, and names the gene as the gene with the function of improving cadmium resistance and cadmium content Sedum alfrediiCd tolerance protein (SaCTP) A reaction of the above-mentionedSaCTP geneTransferring cadmium sensitive yeast mutant strain (ycf1) In the culture medium containing cadmium, the growth condition of the transgenic yeast is obviously superior to that of a control group through yeast spotting experiments. Will be described inSaCTPThe gene is expressed heterologously in Arabidopsis thalianaSaCTPThe gene can obviously improve the cadmium resistance and the cadmium ion absorption capacity of the transgenic arabidopsis thaliana.

The invention aims to provide a gene for improving cadmium resistance and cadmium content.

Another object of the present invention is to provide transgenic Arabidopsis plants containing the above-mentioned genes related to the improvement of cadmium resistance and cadmium content.

The invention also provides the application of the gene for improving cadmium resistance and cadmium uptake in improving cadmium resistance and cadmium content of plants.

In order to achieve the above object, the present invention disclosesA gene for increasing the resistance and content of Cd is disclosedSaCTPThe geneSaCTPThe nucleotide sequence of the cDNA is shown in SEQ.ID.No. 1.

The invention also provides a protein coded by the gene for improving cadmium resistance and cadmium content, and the amino acid sequence of the protein is shown in SEQ.ID.No. 2.

The invention also provides an arabidopsis transgenic plant containing the gene for improving cadmium resistance and cadmium content.

The invention also provides application of the gene for improving cadmium resistance and cadmium content in improving cadmium resistance and cadmium content of plants.

Compared with the prior art, the invention has the beneficial effects that:

(1) the invention provides a plant coding gene capable of improving cadmium resistance and cadmium contentSaCTPThe gene is not reported and researched at present, and is derived from cadmium stress screening after yeast heterologous expression of a rhodiola sachalinensis cDNA library; by constructionSaCTPThe yeast expression vector can verify that the gene can improve the cadmium resistance of the yeast by using a yeast dot-plate experiment.

(2) The invention is toSaCTPThe gene is expressed heterologously in arabidopsis thaliana, and the gene is found to be capable of remarkably improving the cadmium resistance and the cadmium ion content of transgenic arabidopsis thaliana seedlings and seedlings, compared with the common arabidopsis thaliana, the biomass of the seedlings after cadmium stress is improved by 35-50%, the root length of the seedlings is improved by 75-90%, and the cadmium ion content of the seedlings is improved by 20-60%.

(3) According to the inventionSaCTPThe gene provides gene resources for cultivating new plant germplasm with high cadmium resistance and high cadmium content, can be applied to soil monitoring and treatment in cadmium-polluted areas, and plays an important role in researching plant bioremediation technology, improving heavy metal pollution conditions of soil and the like.

Drawings

FIG. 1 shows the hybrid plasmid electro-stimulation transformation of cDNA library of cadmium hyper-accumulator mountain type Sedum alfredii Hance into yeast mutantycf1Then containing 15 mu M.L^-1 CdCl₂The growth of colonies on SD-U medium of (1);

FIG. 2 shows the present inventionSaCTPPCR amplification electrophoretogram of yeast colony of the gene;

FIG. 3 is T-easy-SaCTPPCR amplification electropherograms in E.coli;

FIG. 4 is a comparison of the phenotypes of transgenic Arabidopsis seeds of T3 generation and wild type Arabidopsis seeds after ten days of cultivation on 1/2MS solid medium; wherein FIG. 4A shows a control group containing no cadmium, and FIG. 4B shows a control group containing 100 uM CdCl₂The stress group of (1);

FIG. 5 is a comparison of root length and average fresh weight of transgenic Arabidopsis seeds T3 generation and wild type Arabidopsis seeds after ten days of cultivation on 1/2MS solid medium; wherein, FIG. 5A is a comparison graph of root length after ten days of cultivation, and FIG. 5B is a comparison graph of average fresh weight of 5 seedlings;

FIG. 6 shows the post-transplantation wild type Arabidopsis plants and overexpressionSaCTPA phenotype comparison map of the transgenic Arabidopsis plants of (1); wherein FIG. 6A shows the absence of CdCl₂Stress treated arabidopsis plants as controls; FIG. 6B is 0.5mM CdCl₂Stress arabidopsis plants at 2 weeks;

FIG. 7 is a graph comparing the amount of metal accumulation in transgenic Arabidopsis plants and wild type Arabidopsis plants.

Detailed Description

The present invention will be further explained with reference to the accompanying drawings, which are provided for illustrative purposes only and are not intended to limit the scope of the present invention; the reagents and biomaterials, if not specifically indicated, are commercially available.

Experimental materials and reagents:

(1) strain and carrier: coli/yeast shuttle vector pYES2.0G, E.coli/yeast shuttle expression vector pYES2-DEST, and Agrobacterium EHA105 were all purchased from Invitrogen. Coli DH5 α was purchased from Kong as a century Biotechnology Ltd. The T-easy vector was purchased from Promega corporation. Cadmium sensitive Yeast mutant Strain (ycf1) Purchased from Euroscarf.

(2) Enzyme and kit: total RNA Purification Kit Total RNA extraction Kit for plant tissues was purchased from Norgen. SMART^TMcDNA library constructsKit construction was purchased from Clontech. DNA gel recovery kits and plasmid DNA miniprep kits were purchased from Axygen Biotech. Restriction enzymesfi I Purchased from New England Biolabs.

(3) Reaction substrate: SMART ™ IV Oligonucleotide, CDS III/3 'PCR Primer and 5' PCR Primer were purchased from Clontech.

Example 1:

construction of cDNA library of cadmium hyper-accumulator mine type sedum alfredii hance

(1) Selecting selected cadmium hyper-accumulator ore type southeast rhodiola rosea (collected from an old lead-zinc ore area in Quzhou city of Zhejiang province and transplanted to the laboratory) water culture seedling as material, and performing 400 mu M.L^-1Cadmium chloride (CdCl)₂) After the stress treatment, using a plant tissue Total RNA extraction Kit (Total RNA Purification Kit) to extract the Total RNA of the sedum alfredii leaves under the cadmium stress, and filtering through an oligo (dT) -fiber column to obtain purified mRNA;

(2) obtaining of full-Length cDNA

Synthesis of cDNA Strand Using SMART^TMcompleting a cDNA library construction kit;

first strand cDNA Synthesis: SMART IV Oligonucleotide (10. mu.M) (SEQ ID No. 3: 5'-AAGCAGTGGTATCAACGCAGAGTGGCCATTACGGCCGGG-3') and CDS III/3' PCR Primer (10. mu.M) using the purified mRNA obtained in the above step (1) as a template, the sequences of which are shown in SEQ ID No. 4: 5' -ATTCTAGAGGCCGAGGCGGCCGACATG-d (T)₃₀N_-1N-3’(N = A, G, C, or T; N_-1 = A, G, or C) as primers, and loading and synthesizing a first cDNA chain according to kit instructions;

second strand cDNA Synthesis: a first strand of cDNA is taken as a template, CDS III/3 'PCR Primer (shown as SEQ.ID.No. 4) and 5' PCR Primer (shown as SEQ.ID.No. 5: 5'-AAGCAGTGGTATCAACGCAGAGT-3') are taken as primers, Buffer solution (Buffer), dNTP Mix, Polymerase Mix and double distilled water are sequentially added according to the kit specification, and the full-length cDNA is synthesized on a PCR instrument.

PCR reaction procedure: 95 ℃→ 1 min; (95 ℃→ 15 sec; 68 ℃→ 6 min). times.20 cycles.

(3) Construction of cDNA library

By usingsfi IEnzyme-cutting the Escherichia coli/yeast shuttle vector pYES2.0G and the full-length cDNA obtained in the step (2) by using enzyme, observing the enzyme-cut product through agarose gel electrophoresis, and recovering a band more than 500bp by using a DNA gel recovery kit;

connecting the recovered product of Escherichia coli/yeast shuttle vector pYES2.0G enzyme digestion with the full-length cDNA enzyme digestion product with the length of more than 500bp at 16 ℃ overnight; the ligation products were transferred into E.coli DH 5. alpha. by electric shock transformation, the electric shock transformed products were smeared on LB medium containing ampicillin resistance overnight at 37 ℃ for inversion culture, and the single colonies randomly selected were tested with primers T7 (SEQ. ID. No. 6: 5'-TAATACGACTCACTATAGGG-3') and pYES2-R (SEQ. ID. No. 7: 5'-TCG GTT AGA GCG GAT GTG-3') for PCR identification.

Example 2:

screening and identification of cadmium-resistant transformants

(1) Extracting mixed plasmid of cDNA library of cadmium hyper-accumulator mountain type southeast rhodiola root obtained in example 1, and electrically exciting and transforming cadmium sensitive yeast mutant (ycf1）：

Extracting mixed plasmid of cDNA library by using plasmid DNA miniprep extraction kit, and electrically exciting and transforming the extracted mixed plasmid intoycf1In the strain, the voltage for electric excitation transformation is 1.5V; the conversion product was plated to contain 15. mu.M.L^-1 CdCl₂On SG-U plate; the yeast colonies were evenly sized and grown well as can be seen in FIG. 1 after 3 days of inverted culture at 28 ℃.

(2) TA cloning and sequencing was performed to obtain insert sequence information:

the yeast colonies obtained in step (1) of example 2 were individually identified by PCR, separated by agarose gel electrophoresis and observedSaCTPThe target fragment band of the gene, and primers used in PCR were T7 (shown in SEQ. ID. No. 6) and pYES2-R (shown in SEQ. ID. No. 7). As shown in FIG. 2, M is a DNA molecular weight standard; 1 represents detection with T7 as primerSaCTPPCR product of Yeast colonies of Gene, 2 represents detection with pYES2-R as primerSaCTPPCR products of yeast colonies of the gene; recovery Using DNA gel recovery kitSaCTPPCR bands of the colonies of the transgenic yeast transformants. The PCR-recovered product was ligated with T-easy vector by TA cloning technique, transformed into E.coli DH 5. alpha. by heat shock method, and plated on ampicillin-resistant LB medium and cultured overnight by inversion at 37 ℃. Picking single clone for T-easy-SaCTPThe results of PCR detection in E.coli are shown in FIG. 3, where M is the DNA molecular weight standard, and 1-3 represent M13F/M13R as primers for detection of T-easy-SaCTPPCR products in E.coli.

And (3) sending the bacterial liquid to Shanghai biological engineering Co., Ltd for sequencing, and obtaining the sequence SEQ.ID.No.1 in the sequence table through sequence splicing. Obtained by sequencingSaCTPThe Open Reading Frame (ORF) of the gene, and the amino acid sequence of the gene coding protein SEQ.ID.No. 2. The PCR and sequencing primers are T-easy vector universal primers M13-F (shown as SEQ ID No. 8: 5'-TGTAAAACGACGGCCAGT-3') and M13-R (shown as SEQ ID No. 9: 5'-CAGGAAACAGCTATGACC-3').

Example 3

Yeast spotting plate experiment verificationSaCTPGene cadmium resistance

Obtained by sequencingSaCTPPrimers NG3-F1 (shown as SEQ ID No. 10: atggcttccggcacgttctt) and NG3-R1 (shown as SEQ ID No. 11: tcaagcgacttgaattgtatc) are respectively designed from the Open Reading Frame (ORF) sequence of the gene, and are cloned from a cDNA librarySaCTPA gene coding sequence;

through Gateway technology willSaCTPThe gene is inserted into an escherichia coli/yeast shuttle expression vector pYES2-DEST to obtain the geneSaCTPExpression vector pYES2- SaCTP。

pYES2- SaCTPThe yeast expression vector plasmid and the pYES2-DEST empty vector plasmid are respectively electrically stimulated to be transformed into the cadmium sensitive yeast mutantycf1In the strain, the obtained yeast transformant and the empty vector were streaked on an SD-U plate for 3 days.

Selecting single colony, performing shake culture until OD600 is 1.0, sequentially diluting by 10 times, and respectively diluting in a medium containing 15 μ M.L^-1And 30. mu.M.L^-1 CdCl₂And carrying out a plate spotting experiment on the SG-U flat plate to verify the cadmium resistance function. As a result, it was found that the colony size and number of yeast were substantially uniform on the medium without adding cadmium, indicating that the medium contained the empty expression vector andSaCTPthe growth vigor of the yeast of the gene expression vector is basically consistent, and the growth conditions are also more consistent; at 15. mu.M.L^-1And 30. mu.M.L^-1Growth was inhibited to some extent by both yeasts on cadmium treatment, but transformants were expressed in the unloaded yeast at a specific ratioSaCTPThe suppression of the yeast transformants of the genes was severe. The results show that, under the cadmium treatment conditions,SaCTPthe gene can obviously improve the cadmium sensitive yeast mutantycf1The resistance to cadmium of (1) was confirmedSaCTPThe heterologous expression of the gene can play a role in improving cadmium resistance and cadmium ion content.

Example 4:

transgenic arabidopsis seedling cadmium stress experiment verificationSaCTPGene heterologous expression for improving arabidopsis cadmium resistance

Designing a forward primer (the primer sequence is shown as SEQ ID No. 12: caccatggcttccggcacgttctt) and a reverse primer (the primer sequence is shown as SEQ ID No. 13: tcaagcgacttgaattgtatc), and carrying out PCR amplification to obtain a cDNA fragmentSaCTPThe whole coding sequence of the gene. Will utilize Gateway methodSaCTPThe gene is inserted into a plant expression vector pH2GW7.0 to obtain a gene containingSaCTPExpression vector pH2GW7.0-SaCTP。

pH2GW7.0-SaCTPTransferring agrobacterium to EHA105 by electric excitation method to obtain agrobacterium engineering cell line; adopting an inflorescence infection method to transform arabidopsis thaliana, horizontally laying the infected arabidopsis thaliana into a light-proof plastic basin, spraying a small amount of water, covering a preservative film to prevent moisture from evaporating, covering newspaper on the preservative film to keep a dark environment, culturing for 16-24 h, removing the preservative film, taking out the arabidopsis thaliana, and continuously culturing in an arabidopsis thaliana chamber; collecting seeds after the arabidopsis thaliana is mature, drying and storing at 4 ℃ for later use.

Sterilizing dried T0 generation seeds with 75% ethanol on a clean bench, and uniformly spreading on a container containing hygromycin (Hyg) 20 mg.L ^-11/2MS solid medium; dark at 4 deg.CCulturing for 2 days, then transferring the culture dish into an arabidopsis thaliana chamber for culturing for about one week; normally growing Arabidopsis plants with Hyg resistance are then transferred to nutrient soil and grown in Arabidopsis rooms. After PCR and RT-qPCR detection, selecting positive plants with high expression quantity as overexpressionSaCTPThe transgenic Arabidopsis plant lines of (1) were grown generation by generation until T3 generation homozygote seeds were harvested and stored at-4 ℃ for subsequent biological function analysis.

Overexpression to be obtainedSaCTPTransgenic Arabidopsis thaliana T3 generation seeds (OE 1, OE2 and OE 3) and wild type Arabidopsis thaliana seeds (WT) were sown with sterile toothpick containing 100 uM CdCl ₂1/2MS solid medium, while overexpressing 1/2MS solid medium grown in the absence of cadmiumSaCTPThe transgenic Arabidopsis thaliana T3 generation seeds (OE 1, OE2 and OE 3) and wild type Arabidopsis thaliana seeds (WT) were used as controls, and observations were made after 10 days, as shown in FIG. 4, wild type Arabidopsis thaliana grown in a cadmium-free 1/2MS solid medium and overexpressionSaCTPThe transgenic arabidopsis thaliana grows normally without obvious difference, and contains 100 uM CdCl₂The growth of wild type Arabidopsis plants on 1/2MS solid medium was significantly inhibited.

As shown in FIG. 5A, the root length of Arabidopsis plants is about 4.5 cm under the unstressed state, and the root length of wild Arabidopsis plants is about 0.8 cm under the heavy metal cadmium stress, so that the wild Arabidopsis plants are over-expressedSaCTPThe root length of the transgenic Arabidopsis thaliana is obviously longer than that of the wild Arabidopsis thaliana, and the statistical average value of the root lengths of three lines of OE-1, OE-2 and OE-3 is 1.47 cm, 1.55 cm and 1.40 cm. As shown in FIG. 5B, overexpression was also shown by measurement of biomassSaCTPThe average fresh weight of 5 seedlings of the three lines after the transgenic arabidopsis thaliana is subjected to cadmium stress is 17.87 mg, 18.72 mg and 16.04 mg, while the wild type is 12.23 mg, which is obviously higher than that of the wild type line. Seedling stress experiments show that the cadmium stress tolerance of the seedlings of the transgenic arabidopsis is higher than that of wild type seedlings,SaCTPthe cadmium resistance of the transgenic arabidopsis can be obviously improved through overexpression.

Wild type Arabidopsis WT cultured as described aboveAnd overexpressionSaCTPThe transgenic arabidopsis strains OE1, OE2 and OE3 are transplanted for about 30 days, wild type plants and transgenic arabidopsis plants with consistent growth states are selected, and the wild type plants and the transgenic arabidopsis plants are started to be used by plants containing 0.5mM CdCl₂The aqueous solution of (A) was drenched into Arabidopsis thaliana, and after the soil was dried, drenched with the same solution for 2 weeks without CdCl₂Stress treated arabidopsis thaliana served as a control. As shown in fig. 6, observation of the phenotypic differences of the wild type and transgenic arabidopsis plants during 2 weeks of cadmium stress revealed that both wild type and transgenic arabidopsis plants can grow after being subjected to cadmium stress, and the initial phenotypic difference of the stress is less obvious; however, as time goes on, the color of the leaf of the wild arabidopsis begins to become dark and gradually becomes purple, while the periphery of the outer leaf of the transgenic arabidopsis appears weak yellowing, and the leaf purple is not obvious. Therefore, it is phenotypically assumed that wild type Arabidopsis thaliana has poor tolerance to cadmium.

Separating and obtaining the overground parts of wild type and transgenic arabidopsis plants with different treatment times, and cleaning for three times by using double distilled water; all samples were dried in a105 ℃ oven for 30 min and then 80 ℃ until the samples were thoroughly dried to constant weight. The high content cadmium element is determined by flame atomic absorption photometry, and the low content cadmium element is determined by graphite furnace atomic absorption photometry. And calculating the cadmium content in the plant tissue through the on-machine concentration, the sample weighing and the constant volume. As a result, as shown in FIG. 7, the cadmium content was not substantially detected in wild type and transgenic Arabidopsis thaliana when not subjected to cadmium treatment; after 2 w of 0.5mM Cd treatment, the cadmium content of the wild type Arabidopsis line is 50.17 mug/g FW, the cadmium content of the three transgenic lines is 82.56 mug/g FW, 71.96 mug/g FW and 60.33 mug/g FW respectively, and the result shows that the over-expression is realizedSaCTPAfter the gene is obtained, the content of cadmium in transgenic arabidopsis thaliana can be increased, and a relatively normal growth state can be maintained.

SaCTPThe gene coding sequence is shown in SEQ.ID.No. 1:

ATGGCTTCCGGCACGTTCTTCTATGCAGAGTGCGATGATCGTTGCACTCTTGTATCAAAATCGCCTTGTAAAGAGTCTTGCTTTATGTGCGAGAAGCCGCTTGGATTCAGCGCTGATATATTCATGTACATGGGGAATACACCGTTTTGTAGCACGGAGTGTAGACAAGAGCAGATTGAAATGGACGATGCGGAGGAGAGGAGGAAGAGGAAGAAATCCGCGCTGAGGGCTAAGGCGGAGACAGCAAGATCGACGGCCGGGGGTAAATCGGTCAGGACGGATACAATTCAAGTCGCTTGA。

SaCTPthe amino acid sequence is shown as SEQ ID No. 2:

MASGTFFYAECDDRCTLVSKSPCKESCFMCEKPLGFSADIFMYMGNTPFCSTECRQEQIEMDDAEERRKRKKSALRAKAETARSTAGGKSVRTDTIQVA。

sequence listing

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ttcatgtaca tggggaatac accgttttgt agcacggagt gtagacaaga gcagattgaa 180

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<213> Artificial Sequence (Artificial Sequence)

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Claims (3)

1. The gene for improving the cadmium resistance and the cadmium content is characterized in that the gene for improving the cadmium resistance and the cadmium content is obtained by separating from a leaf cDNA library of sedum alfredii hance, and the nucleotide sequence of the gene is shown as SEQ ID No: 1 is shown.

2. The protein encoded by the gene for improving cadmium resistance and cadmium content of claim 1, wherein the amino acid sequence of the protein is SEQ ID No: 2, respectively.

3. The use of the gene of claim 1 for increasing the cadmium resistance and cadmium content of Arabidopsis thaliana.

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