CN117050153A - Application of camphor tree CcNRAMP2 in promoting cadmium absorption and accumulation - Google Patents
Application of camphor tree CcNRAMP2 in promoting cadmium absorption and accumulation Download PDFInfo
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Abstract
The invention belongs to the technical field of genetic engineering, and particularly discloses application of camphor tree CcNRAMP2 in promoting cadmium absorption and accumulation, wherein the nucleotide sequence of camphor tree CcNRAMP2 is shown as SEQ ID NO.1, and the amino acid sequence is shown as SEQ ID NO. 2. The application provided by the invention not only can provide important candidate genes for the development of cadmium pollution repair of woody plants, but also can provide scientific basis for cultivating new germplasm of cadmium-enriched forest by utilizing modern biotechnology, and has higher application value and research value.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to application of camphor tree CcNRAMP2 in promoting cadmium absorption and accumulation.
Background
Cadmium (Cd) is the primary inorganic pollutant of the soil in our country. Cd accumulation in plants can inhibit their growth and even die. Cd in plants is also absorbed by the human body through the food chain to affect human health. Some rapidly growing woody plants, such as poplar, willow, camphor tree and acacia, have high biomass, high tolerance to heavy metals, show great potential for Cd uptake in Cd contaminated soil, and are of increasing interest in Cd contaminated soil remediation and importance. Therefore, the fast-growing woody plants have great potential value in the field of phytoremediation.
Along with the increasing serious environmental pollution problem, the search of functional genes capable of resisting Cd stress and reducing the Cd content in soil has become a matter related to the life and health of people, and has important scientific significance and application value for developing new germplasm of Cd-enriched forests.
Disclosure of Invention
The invention aims to provide application of camphor tree CcNRAMP2 in promoting cadmium absorption and accumulation so as to solve the technical problems.
One of the objects of the present invention is to provide: the application of camphor tree CcNRAMP2 in promoting cadmium absorption and accumulation is that the nucleotide sequence of camphor tree CcNRAMP2 is shown as SEQ ID NO.1, and the amino acid sequence is shown as SEQ ID NO. 2.
The second object of the present invention is to provide: recombinant vectors containing camphor tree CcNRAMP2, wherein the recombinant vectors are microorganism expression vectors pYES2-CcNRAMP2 and/or plant expression vectors pCAMBIA3300-CcNRAMP2.
Preferably, the engineering bacteria are yeast containing pYES2-CcNRAMP2 recombinant vector and/or agrobacterium containing pCAMBIA3300-CcNRAMP2.
The third object of the present invention is to provide: the application of camphor tree CcNRAMP2 in promoting the absorption and accumulation of yeast or camphor tree leaf cadmium is provided, the nucleotide sequence of camphor tree CcNRAMP2 is shown as SEQ ID NO.1, and the amino acid sequence is shown as SEQ ID NO. 2.
The principle and the beneficial effects of the invention are as follows:
1. the CcNRAMP2 gene disclosed by the invention can be used for remarkably promoting the absorption and accumulation of cadmium by microbial saccharomycetes and plant camphor tree leaves by over-expressing natural resistance related macrophage proteins (Natural resistance associated macrophage protein, NRAMP) through a genetic transformation technology, and remarkably improving the tolerance of the microbial saccharomycetes, agrobacterium and plant camphor tree leaves to Cd. The application provided by the invention not only can provide important candidate genes for the development of Cd pollution repair of woody plants, but also can provide scientific basis for cultivating novel germplasm of Cd enriched forest by using modern biotechnology, and has higher application value and research value.
2. The CcNRAMP2 cloned from camphor trees has Cd transfer activity, and the application of the CcNRAMP2 in saccharomycetes and camphor tree leaves for promoting Cd absorption and accumulation is beneficial to understanding the biological function of the camphor tree NRAMP gene family in heavy metal ion absorption and transfer, can be used as an important candidate gene for carrying out Cd pollution repair on woody plants, and provides scientific basis for cultivating novel Cd enriched forest germplasm by utilizing a modern biotechnology.
3. The camphor tree CcNRAMP2 has 1632bp nucleotide length, encodes 543 amino acids, has protein molecular weight of 59.05kD, contains 33 acidic amino acids and 40 basic amino acids, has theoretical isoelectric point pI of 8.85, has lipid solubility coefficient of 119.80 and has protein hydrophobicity coefficient of 0.604. The CcNRAMP2 protein contains 53.59% alpha-helix, 12.89% beta-sheet, 2.03% beta-turn and 31.49% random coil. In addition, the NRAMP domain in the CcNRAMP2 protein exists at 62-424 sites and contains 12 transmembrane regions, which is beneficial to the transmission and communication of information inside and outside the cell membrane.
Drawings
FIG. 1 is a phylogenetic tree analysis of camphor tree CcNRAMP2;
FIG. 2 shows the expression pattern of the camphor tree CcNRAMP2 gene; (A) the expression level of CcNRAMP2 in the roots, stems and leaves of camphor tree; (B) CcNRAMP2 at 60. Mu.M CdCl 2 Expression level under treatment;
FIG. 3 is a constructed expression vector; the microbial expression vector pYES2-CcNRAMP2 constructed in (A); the plant expression vector pCAMBIA3300-CcNRAMP2 constructed in (B);
FIG. 4 shows Cd in yeast from camphor tree CcNRAMP2 2+ And (5) carrying out a transfer function complementation experiment. The Cd transporter mutant strain was Δycf1 (Matα; his3 Δ1; leu2Δ0; lys2Δ0; met15Δ0; ura3 Δ0; YDT 35c: kanMX4);
FIG. 5 is a CdCl 2 Treating a growth curve of the camphorwood CcNRAMP2 in yeast;
FIG. 6 shows the expression of camphor tree CcNRAMP2 vs Cd in yeast 2+ Is characterized by (A) and (C) NRAMP2 over-expressing Cd in camphor tree leaves 2+ Is a component (B).
Detailed Description
The following examples are further illustrative of the invention and are not intended to be limiting thereof.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified. The following examples do not identify specific experimental procedures and may be carried out according to conventional methods. Conditions as described in J.Sam Brooks et al, guidelines for molecular cloning experiments, F.Osbert et al, guidelines for precision-compiled molecular biology experiments, or according to the instructions of the manufacturer of the product used.
1. Experimental materials
Camphor tree (Cinnamomum camphora (Linn) Presl) used in the examples was planted in the Zhejiang national institute of forestry sciences germplasm nursery (N40°01', E116°25'; hangzhou, china).
The polysaccharide polyphenol plant RNA extraction kit is purchased from Beijing Hua Vietnam biotechnology Co., ltd (product number: 0416-50).
Reverse transcriptase PrimeScrip TM RT reagent Kit with gDNA Eraser from Takara (cat# RR 047Q).
SMARTer TM RACE cDNA Amplification Kit from Clontech (cat# 634923).
pMD18-T Vector was purchased from Takara corporation (cat# D101A).
SYBR Premix Ex Taq TM Kit was purchased from Takara corporation (cat# DRR 420A).
HD Cloning Kit was purchased from Takara (cat# 639648).
GeneJET Plasmidminiprep Kit from Thermo Scientific (cat# K0502).
High-fidelity enzyme premix 2X HieffPCR Master Mix was purchased from YEASEN (cat# 10136ES 01).
LB and MS culture media are common culture media in the field, and the formula of the culture media is referred to J.Sam Brookfield et al, molecular cloning experiment guidelines.
2. Experimental procedure
(1) Extraction of total RNA of synthetic camphor tree leaves and cDNA first chain
100mg of camphor tree leaves are cut and ground into powder under liquid nitrogen. And extracting total RNA of camphor tree leaves by using a polysaccharide polyphenol plant RNA extraction kit.
Total RNA content and purity of camphor tree leaves was 359.39 + -6.05 ng/. Mu.L, A, as measured using a NanoDropTM 2000c ultra micro spectrophotometer (Thermo Scientific, wisconsin, U.S.) and a 1.0% agarose gel electrophoresis apparatus (Biorad, calif.), A 260nm /A 280nm ＝1.98±0.35，A 260nm /A 230nm ＝2.16±0.11。
Mu.g of purified total RNA was taken according to the reverse transcriptase PrimeScrip TM RT reagent Kit with gDNA Eraser instructions for the synthesis of the first strand of cDNA were performed and the reaction product was diluted to a concentration of 50 ng/. Mu.L and stored in a refrigerator at-80 ℃.
(2) Amplification and sequence analysis of camphor tree CcNRAMP2 Gene
Based on the annotation sequences in published camphor tree genome data (Shen T, qi H, luan X, xu W, yu F, zhong Y, xu M. The chromosomeme-level genome sequence of the camphor tree provides insights into Lauraceae evolution and terpene biosynthesis. Plant Biotechnol J.2022, 20:244-246), nested PCR was used with camphor tree leaf cDNA obtained by reverse transcription as template, using Primer Premier 5.0 softPrimers SEQ ID NO.3 and SEQ ID NO.4 were designed by 2 XHieff (Premier Biosoft Co., calif., U.S.A.)PCR Master Mix (YEASEN Co., shanghai, china) amplified to obtain camphor tree CcNRAMP2 gene.
PCR reaction System (25. Mu.L):
reverse transcription template 1.0 μl;
2×HieffPCR Master Mix 12.5μL；
1.25. Mu.L of the upstream primer (SEQ ID NO. 3);
1.25. Mu.L of the downstream primer (SEQ ID NO. 4);
sterile water 9 μl.
PCR reaction procedure: pre-denaturation at 98℃for 3min followed by 35 cycling reactions (98℃10s,60℃20s,72℃1 min) and final extension at 72℃for 5min.
PCR products were detected using a 1.0% agarose gel electrophoresis apparatus (Biorad, california, U.S.A.) and recovered using GeneJET Plasmidminiprep Kit (Thermo Scientific, wisconsin, U.S.A.), ligated to pMD18-Tv actor, and the ligation product was transformed into E.coli DH 5. Alpha. (Shanghai Weidi Biotechnology Co., shanghai, china) and coated with a gel containing 100. Mu.g.mL -1 Ampicillin on LB plates, 37℃overnight. And selecting a single colony as a template, performing colony PCR verification, and sending positive clones to Beijing qingke biotechnology Co.
The nucleotide sequence of the obtained CcNRAMP2 is shown in SEQ ID NO. 1. The nucleotide length is 1632bp, 543 amino acids are encoded, and the amino acid of the encoded protein is shown as SEQ ID NO. 2. The molecular weight of the CcNRAMP2 protein is about 59.05kD, which contains 33 acidic amino acids, 40 basic amino acids, has a theoretical isoelectric point of 8.85, a lipid solubility coefficient of 119.80, and a protein hydrophobicity coefficient of 0.604. The CcNRAMP2 protein contains an alpha helix of 53.59%, a beta sheet of 12.89%, a beta turn of 2.03% and a random coil of 31.49%.
TMHMM-2.0 software analysis showed that the CcNRAMP2 protein contains 12 transmembrane regions, which are important channels linking the extramembranous environment with the intracellular environment. The NRAMP domain is conserved at positions 62-424 of the CcNRAMP2 protein.
(3) Evolutionary tree analysis
A systematic evolutionary tree is constructed by adopting a neighbor method (N-J) in MEGA7.0 software, and an experimental result is shown in a figure 1, and shows that camphor tree CcNRAMP2 and rice OsNRAMP3 (LOC_Os06g 46310) have closest relatives and are clustered on the same branch with arabidopsis AtNRAMP1 (AT 1G 80830) and AtNRAMP6 (AT 1G 15960).
(4) Analysis of the expression Pattern of camphor Tree CcNRAMP2 Gene
Collecting different parts (root, stem and leaf) of camphor tree, extracting RNA and performing reverse transcription reaction according to the method (1). Fluorescent quantitative PCR (polymerase chain reaction) is carried out by respectively designing primers SEQ ID NO.5 and SEQ ID NO.6 according to CcNRAMP2 gene and adopting SYBR Premix Ex Taq TM Kit was subjected to fluorescent quantitative PCR amplification.
The reaction procedure was 95℃denaturation for 2min, followed by 40 cycles of reaction (95℃15s,60℃1 min). The reference gene is an action gene.
Reaction in480 instruments real-time fluorescence quantitative PCR (Roche diagnostics Co., mannheim, germany) was run to obtain data, using 2 -ΔΔCT The method calculates the relative expression data of each sample. The DoEF-1 alpha primer is SEQ ID NO.7 and SEQ ID NO.8.
FIG. 2 shows the expression pattern of the camphor tree CcNRAMP2 gene; (A) the expression level of CcNRAMP2 in the roots, stems and leaves of camphor tree; (B) CcNRAMP2 at 60. Mu.M CdCl 2 Expression level under treatment. R represents root, S represents stem, L represents leaf, CK represents control group, each group bar represents + -standard error (n=6), statistical analysis is comparison of each treatment and control, single factor variance analysis is adopted, p<The 0.05 difference is statistically significant. As shown in FIG. 2, the CcNRAMP2 gene was expressed most in camphor tree roots, followed by leaves and stems (FIG. 2A). In addition, cadmium chloride (CdCl) 2 ) Treatment induced significant upregulation of expression of CcNRAMP2 (figure2B) A. The invention relates to a method for producing a fibre-reinforced plastic composite Thus, the camphor tree CcNRAMP2 gene is closely related to Cd uptake and accumulation.
(4) Absorption of heterologously expressed CcNRAMP 2-assisted Cd in Saccharomyces cerevisiae
(1) Construction of a microbial expression vector for CcNRAMP2
FIG. 3 is a constructed expression vector; the microbial expression vector pYES2-CcNRAMP2 constructed in (A); the plant expression vector pCAMBIA3300-CcNRAMP2 constructed in (B). According to pYES2 microorganism expression vector (figure 3A) and camphor tree CcNRAMP2 sequence, designing primers SEQ ID NO.9 and SEQ ID NO.10 at BamH I and Hind III enzyme cutting sites respectively, taking camphor tree cDNA as a template, amplifying with high fidelity to obtain CcNRAMP2 sequence, purifying amplified products, and recovering target fragments. pYES2 was digested with BamHI and HindIII. By usingThe HD Cloning Kit constructs a CcNRAMP2 microorganism expression vector, and the specific operation method is shown in the specification. The camphor tree CcNRAMP2 microorganism expression vector is constructed and named pYES2-CcNRAMP2.
(2) Transformation of recombinant plasmid pYES2-CcNRAMP2 into Yeast Δycf1
The recombinant plasmid pYES2-CcNRAMP2 is transformed into saccharomycete BY4717 BY a heat shock method, and the specific method is as follows:
mu.g of recombinant plasmid pYES2-CcNRAMP2 was mixed with 100. Mu.L of yeast BY4717 competent cells, placed on ice for 30min, placed in a water bath at 42℃for 90s, and rapidly transferred to ice bath for 2min. 900. Mu.L of LB medium without antibiotics was added and incubated at 37℃on a shaking table at 150rpm for 45min. The culture was plated on 25mL LB plates (50 mg. ML in medium) -1 Ampicillin). The plates were placed in an incubator at 37℃and incubated until colonies developed (about 12-16 h). And (5) picking the monoclonal to perform colony PCR identification. Yeast into which the recombinant plasmid pYES2-CcNRAMP2 had been transferred was selected as a positive clone, and designated BY4717-pYES2 with pYES2 empty vector as a control.
Similarly, the recombinant plasmid pYES2-CcNRAMP2 was transformed into yeast Δycf1 by heat shock, and yeast transformed with the recombinant plasmid pYES2-CcNRAMP2 was obtained as a positive clone, designated as Δycf1-CcNRAMP2, and designated as Δycf1-pYES2 with no-load pYES2 as a control.
(3) Cd from camphor tree CcNRAMP2 in yeast 2+ Transfer function complementation experiment
Yeast containing BY4717-pYES2, Δycf1-pYES2 and Δycf1-CcNRAMP2 after sequential activation was picked up as single colony in 100mL LB liquid medium (50 mg. ML) -1 Ampicillin), shaking culture at 37℃and 220rpm overnight (about 16 hours) to OD 600nm =0.8 to 1.0. Respectively diluting the bacterial liquid to 10 -2 mu.L of the yeast was plated on a solid medium containing 30. Mu.M SC-Ura (20% galactose), incubated at 30℃for 3 days, photographed and the growth of the yeast was recorded.
FIG. 4 shows Cd in yeast from camphor tree CcNRAMP2 2+ And (5) carrying out a transfer function complementation experiment. The Cd transporter mutant strains were Δycf1 (Matα; his 3. DELTA.1; leu2Delta0; lys2Delta0; met15Delta0; ura3Delta0; YDT 35c: kanMX4), as shown in FIG. 4, the growth of yeasts transformed with either Δycf1 or Δycf1-CcNRAMP2 was inhibited to varying degrees relative to the yeast BY4717, with increased Cd concentration (FIG. 4). The yeast transformed with delta ycf1 has smaller colony, poorer growth vigor and smaller quantity, and compared with the yeast transformed with delta ycf1, the yeast transformed with delta ycf1-CcNRAMP2 is recovered, so that the heterologous expression of the camphor tree CcNRAMP2 gene increases the tolerance of the yeast to Cd.
(4) Growth curve of camphor tree CcNRAMP2 in yeast under Cd treatment
To clarify the growth curves of different positive recombinant plasmids under Cd treatment, single colonies BY4717-pYES2, deltaycf 1-pYES2 and Deltaycf 1-CcNRAMP2 were sequentially picked and activated in a liquid medium of SC-Ura (20% galactose) to OD 600nm =0.6, transferred to SC-Ura (20% galactose) liquid medium containing 10 μm Cd, OD values were detected and yeast growth curves were drawn.
FIG. 5 is a CdCl 2 The growth curve of the camphorwood CcNRAMP2 in yeast was treated. As shown in FIG. 5, the Cd treatment significantly inhibited the growth of yeast, and compared with BY4717, the growth of Cd-sensitive yeast Δycf1 was significantly inhibited, and the growth of yeast transformed into camphor tree CcNRAMP2 was improved (graph5). The above results further confirm that the heterologous expression of the camphor tree CcNRAMP2 gene effectively enhances the Cd tolerance of the recombinant yeast.
(5) Determination of Cd content in recombinant plasmids of different yeasts
And sequentially picking BY4717-pYES2, delta ycf1-pYES2 and delta ycf1-CcNRAMP2, respectively transferring into SC-Ura (20% galactose) liquid culture medium containing 10 mu m mCd, shake culturing at 30 ℃ and 220rpm for 72 hours, collecting saccharomycetes liquid, centrifuging to obtain saccharomycetes, drying at 55 ℃, and measuring the Cd content in the saccharomycetes containing different recombinant plasmids.
The result shows that the Cd concentration in the camphor tree CcNRAMP2 gene transferred yeast is obviously higher than that in the empty transferred yeast, and the statistical analysis shows that the Cd concentrations of the camphor tree CcNRAMP2 gene and the empty transferred yeast have extremely obvious difference (figure 6A), so that the camphor tree CcNRAMP2 gene effectively enhances the Cd absorption capacity of the yeast.
(5) Overexpression of CcNRAMP2 promotes accumulation of Cd in camphor tree leaves
(1) Construction of camphor tree CcNRAMP2 overexpression vector
According to the sequences on pCAMBIA3300 plant expression vector (figure 3B) and camphor tree CcNRAMP2, primers SEQ ID NO.11 and SEQ ID NO.12 are respectively designed at BamH I and Hind III enzyme cutting sites, camphor tree cDNA is used as a template, ccNRAMP2 sequences are obtained through high-fidelity amplification, and target fragments are recovered after amplification products are purified. pCAMBIA3300 was digested with BamHI and HindIII. By usingThe HD Cloning Kit (Takara Corp., chinese company) constructs a plant expression vector, and the specific operation method is shown in the specification. The pCAMBIA3300 vector contains a CaMV 35S promoter, the CcNRAMP2 is kept with ATG, a stop codon is removed, and the vector is connected with the 35S promoter to construct a CcNRAMP2 overexpression vector, and the plant recombinant expression vector is named pCAMBIA3300-CcNRAMP2.
(2) Recombinant plasmid pCAMBIA3300-CcNRAMP2 was transformed into Agrobacterium GV3101 (pSoup-p 19)
The recombinant plasmid pCAMBIA3300-CcNRAMP2 was transformed into Agrobacterium GV3101 (pSoup-p 19) by heat shock, as follows:
mu.g of recombinant plasmid pCAMBIA3300-CcNRAMP2 was mixed with 100. Mu.L of Agrobacterium GV3101 (pSoup-p 19) competent cells, placed on ice for 5min, snap frozen with liquid nitrogen for 5min, rapidly transferred to a 37℃water bath for 5min, and then placed on ice for 5min. 700. Mu.L of LB medium without antibiotics was added and incubated for 2-3h at 100rpm on a shaking table at 28 ℃. The culture was plated on 25mL LB plates (50 mg. ML in medium) -1 Kanamycin). The plates were placed in an incubator at 28℃upside down until colonies grew out (about 2 days). And (5) picking the monoclonal to perform colony PCR identification. Agrobacterium (pSoup-p 19) into which the recombinant plasmid pCAMBIA3300-CcNRAMP2 had been transferred was selected as a positive clone.
(3) Transient overexpression of CcNRAMP2 Gene in camphor tree leaves
The Agrobacterium of pCAMBIA3300-CcNRAMP2 was activated and single colonies were picked up in 100mL LB liquid medium (containing 50 mg. Mu.mL) -1 Kanamycin), 28℃and 180rpm, overnight (about 16 hours) to OD 600 =0.6 to 0.8. And (5) centrifuging at room temperature of 5000 Xg for 5min to collect bacterial liquid. 100mL of permeation buffer (0.2 mM acetosyringone, 10mM Cl) 2 10mM MES, pH 5.7) the precipitated Agrobacterium was resuspended, centrifuged at 5000 Xg for 5min, the supernatant discarded and the OD adjusted with permeation buffer 600 And (3) carrying out standing and activation at 25 ℃ for 2 hours at room temperature in dark place. And selecting a second leaf from top to bottom of camphor tree seedlings, sucking activated bacterial liquid by using a 1mL syringe, and injecting the activated bacterial liquid into leaf backs until the bacterial liquid just oozes out. Soaking camphor tree leaves in the bacterial liquid, vacuumizing, maintaining for 2min, and airing. After the tissue culture room is dark-cultured for 24 hours at the temperature of 23+/-2 ℃, the tissue culture room is cultured for 2 days under the illumination condition, and positive leaves are screened. Semi-quantitative PCR amplification was performed according to conventional PCR amplification.
The PCR reaction conditions were: pre-denaturation at 98℃for 3min followed by 30 cycling reactions (98℃10s,60℃20s,72℃1 min) and final extension at 72℃for 5min.
The PCR products were detected electrophoretically on a 1% agarose gel. And selecting camphor tree leaves which over express CcNRAMP2 for subsequent Cd content detection.
FIG. 6 shows the expression of camphor tree CcNRAMP2 vs Cd in yeast 2+ Is characterized by (A) and (C) NRAMP2 over-expressing Cd in camphor tree leaves 2+ Is a component (B). Each of whichGroup bar represents ± standard error (n=6), p<The 0.05 difference is statistically significant.
The results show that after the camphor tree CcNRAMP2 is transferred into yeast, the capacity of the yeast strain for absorbing and accumulating Cd is obviously enhanced, 153.47% is increased compared with a control group (figure 6A), the expression of the CcNRAMP2 gene in camphor tree leaves subjected to transient over-expression treatment is obviously up-regulated, and the accumulation of Cd content in the leaves is obviously increased (figure 6B). Thus, either heterologously expressed in yeast or transiently overexpressed in camphor tree, camphor tree CcNRAMP2 promotes the absorption and accumulation of Cd in cells or plants.
The foregoing describes in detail preferred embodiments of the present invention. It should be understood that numerous modifications and variations can be made in accordance with the concepts of the invention by one of ordinary skill in the art without undue burden. Therefore, all technical solutions which can be obtained by logic analysis, reasoning or limited experiments based on the prior art by the person skilled in the art according to the inventive concept shall be within the scope of protection defined by the claims.
Claims (4)
1. The application of camphor tree CcNRAMP2 in promoting cadmium absorption and accumulation is characterized in that the nucleotide sequence of camphor tree CcNRAMP2 is shown as SEQ ID NO.1, and the amino acid sequence is shown as SEQ ID NO. 2.
2. A recombinant vector comprising camphor tree CcNRAMP2 according to claim 1, wherein the recombinant vector is a microbial expression vector pYES2-CcNRAMP2 and/or a plant expression vector pCAMBIA3300-CcNRAMP2.
3. An engineering bacterium comprising the recombinant vector of camphor tree CcNRAMP2 according to claim 2, wherein the engineering bacterium is a yeast comprising a recombinant vector of pYES2-CcNRAMP2 and/or an agrobacterium comprising pCAMBIA3300-CcNRAMP2.
4. The application of camphor tree CcNRAMP2 in promoting the absorption and accumulation of yeast or camphor tree leaf cadmium is characterized in that the nucleotide sequence of camphor tree CcNRAMP2 is shown as SEQ ID NO.1, and the amino acid sequence is shown as SEQ ID NO. 2.
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