CN115873084A - Populus tremuloides metal transporter, gene and application thereof - Google Patents

Abstract

The invention belongs to the technical field of genetic engineering, and particularly relates to a populus tomentosa metal transporter, a gene and application thereof. The amino acid sequence of the protein is shown as SEQ ID NO.1, the nucleotide sequence of the coding gene is shown as SEQ ID NO.2, and experiments prove that the PcNRAMP1 gene overexpression can promote the absorption and accumulation of Cd from the environment by populus tremuloides ²⁺ Moreover, the PcNRAMP1 gene overexpression promotes the absorption and accumulation of manganese, iron, zinc and calcium by populus. The invention screens out important regulation and control effects on heavy metal ions such as Cd and the like by carrying out site-directed mutagenesis on different amino acid residues in the PcNAMP 1 geneThe used amino acid residue is beneficial to improving the restoring efficiency of the poplar to the Cd-polluted soil.

Description

Populus tremuloides metal transporter, gene and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a populus tomentosa metal transporter, a gene and application thereof.

Background

The heavy metal cadmium (Cd) is a non-essential element for the growth and development of plants, and the growth of the plants is inhibited due to the large accumulation of the Cd. Phytoremediation techniques have been widely used to remediate Cd contaminated soil. Populus trees (Populus) are considered to be ideal candidate plants for restoring Cd-contaminated soil. In the process of absorption, transportation and accumulation of Cd by plants, metal ion transporters play an important role. Natural Resistance-associated Macrophage proteins (NRAMPs), are a class of transport proteins for heavy metal Cd and other divalent cations.

NRAMPs are a class of transporters associated with metal ion transport that are widely found in plants. AtNRAMP1 localizes to the cytoplasmic membrane and is expressed mainly in roots; heterologous expression of AtNRAMP1 in yeast enhances the sensitivity to Cd, and simultaneously improves the Cd content in yeast cells. Under the conditions of low temperature and lack of Mn, the conserved histidine in the AtNRAMP1 protein regulates the growth and development of plants. AtNRAMP6 is positioned on an endomembrane system of an intracellular vesicle of Arabidopsis thaliana and is used as a metal transporter in a cell to influence the distribution of Cd in different subcellular structures in the cell. OsNRAMP1 promotes Cd transport to the overground part in the roots of rice plants. OsNRAMP3 is localized in plasma membrane and nucleus, highly expressed in the nodes of rice leaf, stem and ear junctions, and can supply necessary Mn to developing tissues. OsNRAMP3 is involved in Mn transport in yeast cells, and is not related to the transport of Fe and Cd.

However, the reports that the NRAMPs protein is related to the metal transport function in the woody plant poplar are not found.

Disclosure of Invention

In order to make up for the blank of the prior art, the invention provides a populus griseus metal transporter, a gene and application thereof.

The first purpose of the invention is to provide a populus griseus metal transporter, and the amino acid sequence is shown in SEQ ID NO. 1.

The second purpose of the invention is to provide the coding gene of the protein, and the nucleotide sequence is shown as SEQ ID NO. 2.

The third object of the present invention is to provide a vector containing the gene.

The fourth purpose of the invention is to provide a genetically engineered bacterium containing the vector.

The fifth purpose of the invention is to provide the application of the protein or the gene in regulating and controlling the metal transport capacity of plants, wherein the metals comprise cadmium, manganese, iron, zinc and calcium.

In a sixth aspect of the present invention, there is provided a method for constructing a transgenic plant, comprising the steps of:

the protein is over-expressed in the target plant, so that the capability of the target plant for transporting metals is improved, wherein the metals comprise cadmium, manganese, iron, zinc and calcium.

Further, the method for over-expressing the protein in the target plant comprises the following steps: and introducing the gene into a target plant to obtain a transgenic plant.

Further, the plant includes populus tomentosa.

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

the invention proves that the populus griseus PcNMARMP 1 gene is a functional gene involved in metal transport and accumulation for the first time.

Experiments prove that PcNRAMP1 gene overexpression can obviously promote absorption and accumulation of Cd from environment of populus griseus ²⁺ Moreover, pcNRAMP1 gene overexpression promotes the absorption and accumulation of Mn and Fe by Populus sieboldii.

The invention also screens out amino acid residues with important regulation and control effects on heavy metal ions such as Cd by carrying out site-directed mutagenesis on different amino acid residues in the PcNRAMP1 gene, thereby being beneficial to improving the remediation efficiency of poplar on Cd-polluted soil.

Drawings

FIG. 1 shows the results of PcNRAMP1 gene full-length cDNA identification, M: DL5000 marker; lanes 2 and 3 are clones with stem and root as templates, respectively.

FIG. 2 shows the DNA level (a) and RNA level (b) characterization of PcNAMP 1 overexpression in Populus tremula.

FIG. 3 is a phenotype of over-expressing PcNAMP 1 and wild type populus tremuloides, (a) over-expressing PcNAMP 1 and wild type populus tremula whole plant phenotype; (b) Over-expressing PcNRAMP1 populus and wild type populus griseus leaf (LPI = 8) phenotype.

FIG. 4 shows the phenotype of plants of Populus tremuloides overexpressing PcNAMP 1 and wild-type Populus tremula responding to Cd stress (a) the phenotype of whole plants of wild-type Populus tremula and Populus tremula overexpressing PcNAMP 1 without (0 μ M) or with (100 μ M) treatment with Cd (-Cd); (b) Wild type populus tomentosa and a stem Cd poison phenotype of over-expressed PcNAMP 1 populus tomentosa; (c) Wild type ash and over-expressing PcNRAMP1 ash leaves (LPI = 7-9) Cd poison phenotype.

FIG. 5 shows photosynthetic properties of plants overexpressing PcNAMP 1 and wild type populus griseus under Cd stress, (a) net photosynthetic rate, (b) stomatal conductance, and (c) transpiration rate.

FIG. 6 shows the pigment content of plants of PcNAMP 1 Populus tremula and wild type Populus tremula under Cd stress, (a) chlorophyll content, and (b) carotenoid content.

FIG. 7 shows the Cd expression of PcNAMP 1 and wild type ash plants at different sites from the root tip ²⁺ Flow rate.

FIG. 8 shows the overexpression of PcNAMP 1 and the wild type plant Cd of Buxus sinica ²⁺ Absorption tendency (a) and absorption rate (b).

FIG. 9 shows the Cd contents of the roots (a) and aerial parts (b) of short-term Cd-treated PcNAMP 1 and wild-type ash plants.

FIG. 10 shows Cd content in different tissues of plants overexpressing PcNRAMP1 and wild type populus siensis after long-term Cd treatment.

FIG. 11 shows the total Cd content (a) and the biological enrichment factor (b) of plants overexpressing PcNAMP 1 Buxus sinica and wild type Buxus sinica.

FIG. 12 is a graph of Mn and Fe content of short-term Cd-treated PcNAMP 1 Populus tremula and wild-type Populus tremula plants.

FIG. 13 shows the growth of the yeast cells after expression of the empty vector, pcNRAMP1 and point mutations thereof.

FIG. 14 shows the growth state (c) and Cd content (d) of PcNRAMP1 protein structure (a), the ratio to the homologous protein sequence (b), and the expression of empty vector in yeast cells, and the point mutation of PcNRAMP1.

FIG. 15 shows the growth status and the corresponding metal content of Mn (a), fe (b) -treated yeast cells after point mutation of PcNRAMP1.

Detailed Description

The invention is described in detail below with reference to the figures and the specific embodiments, but the invention should not be construed as being limited thereto.

The following examples relate to experimental materials:

1) Plant material

The ash poplar (Populus x canescens) used in the test is preserved in the key laboratory of China forest genetic breeding institute of forestry science and research.

2) Strains and vectors

21 ) strains of bacteria

The mutant yeast strain Δ ycf1, the manganese-uptake deficient mutant yeast strain Δ smf1, the manganese-sensitive mutant yeast strain Δ pmr1, the zinc-sensitive mutant yeast strain Δ zrc and the iron-sensitive mutant yeast strain Δ ccc1 were all stored in the applicant's laboratory.

Δ ycf1 is disclosed in: li ZS, lu YP, zhen RG, szczypka M, thiele DJ, rea PA. A new path for vacuolar cadmium sequencing in Saccharomyces cerevisiae YCF1-catalyzed transport of bis (glutamaterino) cadmium. Proc Natl Acad Sci U S A.1997Jan 7;94 (1):42-7.

Δ smf1 and Δ pmr1 are disclosed in: maeda T, sugiura R, kita A, saito M, deng L, he Y, yabin L, fujita Y, takegawa K, shuntoh H, kuno T.Pmr1, a P-type ATPase, and Pdt1, an Nramp homologue, cooperativity regulation cell morphogenesis in mission year: the import of Mn2+ hoseostasis cells.2004Jan;9 (1):71-82.

Δ zrc is disclosed in: miyabe S, IZawa S, inoue Y.the Zrc is involved in zinc transport system between vacuum and cytosol in Saccharomyces cerevisiae Biochem Biophys Commun.2001Mar 23;282 (1):79-83.

Δ ccc1 is disclosed in: li L, chen OS, mcVey Ward D, kaplan J.CCC1 is a transporter which is media vacuolar iron storage in yeast.J Biol chem.2001Aug3;276 (31):29515-9.

22 ) the carrier

Gateway technology entry vectors pDDOR 222 and pDDOR 207, yeast expression vector pYES2-DEST52, plant overexpression vector pK2GW7 are commercially available.

3) Preparing a reagent:

SD-U Medium/SG-U Medium (solid, 1L): SD-U powder 8g + agar 20g + agar 950mL ultrapure water, pH 6, autoclaved at 121 ℃ for 15min, and then 50mL sterilized 40% glucose/galactose was added.

SD-U Medium/SG-U Medium (liquid, 1L): SD-U powder, 8g, 42 mL ultrapure water, is autoclaved at 121 ℃ for 15min, and then 50mL sterilized 40% glucose/galactose is added.

Poplar subculture medium (1L): MS powder 2.2g, sucrose 20g, agar powder 7g, pH 5.8, and high temperature and high pressure sterilizing at 121 deg.C for 20min.

Poplar co-culture medium (1L): MS powder 2.2g, sucrose 20g, agar powder 7g, pH 5.8, and high temperature and high pressure sterilizing at 121 deg.C for 20min.

Poplar selection medium (1L): MS powder 2.2g, sucrose 20g, agar powder 7g, pH 5.8, autoclaving at 121 deg.C for 20min, and adding thidiazuron 0.0022mg/L, cefadriamycin 100mg/L, timentin 100mg/L, and kanamycin 50 mg/L.

Poplar rooting medium (1L): MS powder 2.2g, sucrose 20g and agar powder 7g, pH 5.8, high temperature and high pressure sterilizing at 121 deg.C for 20min, and adding 100mg/L of cefamycin, 100mg/L of timentin and 50mg/L of kanamycin.

Example 1: cloning of populus griseus PcNRAMP1 gene, over-expression plasmid and construction of recombinant agrobacterium

1. Cloning of Populus tremula PcNRAMP1 Gene

The RNA of tissue culture seedlings of Populus tremula was extracted by the CTAB method, followed by the use of Primescript ^TM The 1st strand cDNA synthesis kit reverse transcribes RNA digested by DNase to obtain cDNA as a template. Specific primers PcNRAMP1-F (SEQ ID NO. 3) and PcNRAMP1-R (SEQ ID NO. 4) are designed for gene cloning, an amplified band of about 1600bp (shown in figure 1) is subjected to gel recovery, a pMD19-T vector is connected, then sequencing is carried out, and the sequenced sequence is compared with the sequence in a genome. The length of the coding region of the cloned PcNRAMP1 gene is 1626bp (SEQ ID NO. 2), and the coding region is codedCode amino acid 541aa (SEQ ID NO. 1) having a molecular weight of 58.65kDa. The sequencing positive plasmid was designated pM-PcNRAMP1.

2. Construction of overexpression plasmid and transformation of agrobacterium

The method comprises the steps of carrying out BP reaction on pM-PcNRAMP1 and pDONR222 to obtain pDONR222-PcNRAMP1 plasmids, connecting the pDONR222-PcNRAMP1 plasmids with a plant expression vector pK2GW7 through LR reaction to obtain PcNRAMP1-pK2GW7, transforming the PcNRAMP1-pK2GW7 into DH5 alpha escherichia coli competence, picking up single colonies which grow normally to sequence, and transforming positive plasmids into agrobacterium to obtain recombinant agrobacterium.

Example 2: functional analysis of PcNAMP 1 gene in Populus tremula

1 method of experiment

1.1 genetic transformation procedure of Populus tremuloides

(1) The recombinant Agrobacterium obtained in example 1 was activated and single colonies were picked and grown to OD ₆₀₀ And = 0.2-0.8. Adding 10 μ M acetosyringone (As) and culturing on a shaker at 28 deg.C for 30min.

(2) Tissue culture seedling of populus tomentosa growing for 6 weeks in a culture room is taken as a material. The leaves and petioles were cut, the stem segments were cut into segments (the nodes between stem ends were removed), and 1/2 of the length was cut along the stem segments.

(3) All explants were transferred to the Agrobacterium culture of step (1) and incubated at 28 ℃ and 120rpm for 30min.

(4) Transferring the explants to a poplar co-culture medium, and co-culturing for 2-3d in the dark.

(5) After the co-cultivation phase is completed, the explants are washed.

(6) The washed explants were transferred to a petri dish of poplar selection medium and cultured in the dark for about 6-8 weeks. After callus and buds grow on the longitudinal cut wound of the stem section, transferring the germinated stem section to a new poplar selective culture medium, and transferring the stem section to a weak light for further growth.

(7) Transferring the explant with the bud to a poplar rooting culture medium for continuous culture. After the bud grows to 1-2cm, cutting off the bud and transferring the bud into a rooting culture medium.

1.2 identification and screening of overexpression Populus tremuloides lines

Extracting total DNA and RNA of over-expressed and wild populus griseus plant leaves by using a CTAB method.

The transgenic plant is identified by PCR using the leaf DNA as a template and 35s-PcNRAMPs-F (SEQ ID NO. 5) and 35s-PcNRAMPs-R (SEQ ID NO. 6) as primers. The positive control is recombinant vector plasmid, and the negative control is wild type populus tomentosa plant.

And carrying out semi-quantitative PCR by taking cDNA of the transgenic populus griseus and the wild type populus griseus as templates and PcNAMP 1-RT-F/R. And identifying the relative expression quantity of different transgenic lines of the populus griseus. The control was wild type plants. PcActin is an internal reference gene.

1.3 Populus tremula Cd treatment

Subcultured wild type aspen (empty vector) and transgenic aspen were cultured for 4-6 weeks in the culture room. Plants with similar growth were selected for hydroponic culture in 1/4Hoagland (pH 5.8). After two months of hydroponics, wild type populus griseus (empty vector) and three lines of over-expressed populus griseus were divided into four groups. Meanwhile, each group is further divided into two subgroups (each subgroup has 4 populus tomentosa plants). The Populus tremula in each subgroup is treated with 0 or 100. Mu.M CdCl ₂ The treatment was carried out for 2 weeks.

1.4 Populus Cd ²⁺ Flow rate measurement

White fine roots of populus sieboldii treated with Cd and without Cd in 1/4Hoagland nutrient solution are taken as growth test materials, and the Cd at the root tip of the populus tree is detected by adopting NMT ²⁺ Flow rate. The specific method comprises the following steps: the electrodes are first calibrated. Selecting white fine roots with similar size and thickness of poplar, and placing the white fine roots in the test solution for balancing for 15min. Subsequently, the white fine roots after equilibration were transferred to a test solution for measurement. Each line tested 6-8 white fine roots, each root assayed for 5 minutes. And collecting data information by utilizing IM-FLUX software.

1.5 photosynthetic assay and sample harvesting

Before harvesting the populus tomentosa, the photosynthetic indexes of the populus tomentosa leaves (LPI = 6-9) are measured by using a LiCOR-6400 portable photosynthetic instrument. After the Cd ion current and the photosynthetic index are detected, 5 tissues of different tissues of the populus tomentosa, including roots, barks, woods, young leaves and mature leaves, are harvested. At harvest, the root of Buxus sinica Linn is placed in 50mM CaCl ₂ Washing with water for 1 timeThen transferring the mixture into distilled water and washing the mixture for 3 times. The fresh weight of roots, bark, wood, young and mature leaves of each plant was recorded. The harvested samples were immediately frozen in liquid nitrogen. The frozen samples were ground to a fine powder using a ball mill and stored in a-80 ℃ freezer for further analysis. Fresh powder (about 100 mg) was dried at 80 ℃ for 72h to calculate the plant dry-to-wet ratio and biomass.

1.6 determination of content of Metal elements in poplar

Weighing appropriate amount of root, bark, wood and leaf of populus tomentosa in a 5mL test tube, drying at 80 ℃ to constant weight, weighing and recording the dry weight. And (3) measuring the contents of elements such as Cd, mn and Fe by using an ICP-MS method, and counting the biological enrichment coefficient of Cd.

1.7 Total Cd in plant tissue and biological enrichment coefficient

The total Cd of the populus plant tissues and the biological enrichment coefficient are calculated according to a conventional method.

1.8 measurement of pigment content

20mg of the ground populus tomentosa leaf powder is added with 1mL of 80% acetone for extraction. Mixing by turning upside down. Placing in a refrigerator at-20 deg.C for about 30min, and mixing by reversing every 10 min. After centrifugation, the supernatant was aspirated. The absorbance values at 663nm,645nm and 470nm were determined.

2 results of the experiment

2.1PcNRAMP1 overexpression Populus tremula strain identification and phenotype analysis

As can be seen from FIG. 2a, no band was detected in wild type ash (WT), a band was detected in the recombinant plasmid PcNAMP 1-pK2GW7, and bands of the same size as the recombinant plasmid band were detected in the ash overexpression lines PcNAMP 1-OE 1-PcNAMP 1-OE10. As a result, 10 lines were obtained by overexpressing the PcNAMP 1 gene in Populus tremula and designated PcNAMP 1-OE1 to PcNAMP 1-OE10.

Through the expression amount identification, 6 strains with higher expression amount were identified, namely PcNAMP 1-OE1, pcNAMP 1-OE3, pcNAMP 1-OE4, pcNAMP 1-OE5, pcNAMP 1-OE8 and PcNAMP 1-OE10 (FIG. 2 b). And (4) subculturing the strains with higher expression quantity into a 1/2MS solid culture medium without antibiotics for propagation.

Selecting 3 overexpression strains (PcNMAR 1-OE5, pcNMAR 1-OE8 and PcNMAR 1-OE 9) with higher expression after screening and wild type populus tremuloides (WT) and transferring to 1/4Hoagland nutrient solution for water culture. As shown in fig. 3, after 1 month of hydroponics, mature leaves of over-expressed aspen (LPI = 8) appeared wrinkled and a yellowing phenotype between leaf veins compared to wild-type aspen.

2.2 Effect of Cd stress on PcNRAMP1 overexpression Poplar phenotype, photosynthesis, pigment content and Biomass

As can be seen from FIG. 4, in the case of no Cd or Cd, the growth conditions of the overexpression strain and the wild type strain are not different, and the growth of the populus tremuloides plant can be significantly inhibited by Cd treatment (FIG. 4 a). Under the condition of no Cd, the over-expression populus tomentosa and wild-type stems have no Cd poisoning phenotype, and the stem thickening is inhibited after Cd treatment (figure 4 b).

As shown in FIG. 4c, in the presence of Cd, there were more black spots on the stems of 3 lines of PcNAMP 1 Populus tremula overexpressing (Cd poison phenotype). In the absence of Cd, overexpressing populus griseus and wild type leaves (LPI = 7-9) did not show a Cd poisoning phenotype; in the case of Cd, it was found that more red spots (Cd poisoning phenotype) appeared on the leaves of the overexpressed populus tremuloides.

Through the determination of the net photosynthetic rate of the populus tremuloides of different strains under the condition of containing Cd or not containing Cd, the net photosynthetic rate of the overexpression and wild type strains is obviously reduced after Cd treatment compared with the net photosynthetic rate of the populus tremuloides without Cd treatment. Cd treatment resulted in a 25.36%,29.26% and 25.69% decrease in net photosynthetic rate of PcNRAMP1-OE5, pcNRAMP1-OE8 and PcNRAMP1-OE9, respectively, relative to the net photosynthetic rate of wild type aspen after treatment (FIG. 5 a). The determination of the stomatal conductance and the transpiration rate of different strains of poplars under the condition of containing Cd or not containing Cd proves that: the porosity conductance and transpiration rate of each line of populus tomentosa plants which are not subjected to Cd treatment are not different. Cd treatment significantly reduced stomatal conductance (FIG. 5 b) and transpiration rate (FIG. 5 c) for both the over-expressed and wild-type strains, but the stomatal conductance difference between the over-expressed and wild-type ash strains was not significant.

In order to further explore the influence of Cd treatment and PcNAMP 1 on Populus tremula photosynthesis, the pigment content of different strains was determined. In the absence of Cd treatment, the total amount of chlorophyll over-expressed and wild-type was consistent and did not differ significantly, with the content of chlorophyll a and chlorophyll b not differing significantly (fig. 6 a). After Cd treatment, the over-expression and wild-type total chlorophyll content was reduced. In the case of Cd treatment, both the over-expressed ash lines chlorophyll a and chlorophyll b were significantly reduced compared to the total amount of chlorophyll in wild-type ash. In the case of Cd treatment, the carotenoid content was significantly reduced compared to the untreated content.

(FIG. 6 b).

The above results indicate that heavy metal Cd can affect populus griseus plant photosynthesis. Under Cd treatment, overexpression of PcNAMP 1 can exacerbate the damage of plant photosystems.

2.3PcNRAMP1 overexpression of Cd in Populus tremula ²⁺ Flow rate, cd content and enrichment coefficient

(1) PcNRAMP1 overexpression of Cd in populus griseus root ²⁺ Flow rate of flow

As shown in FIG. 7, cd was absorbed by both overexpressed and wild type Populus tremula roots ²⁺ . However, different segments of over-expressed and wild-type white rootlets were specific for Cd ²⁺ The net absorption rate is different. In the selected 8 sweep-point sections, cd ²⁺ The maximum net absorption rate is achieved at 600 μm, so 600 μm is selected as the probe for Cd couple ²⁺ Measurement point of absorption flow rate of (2), determination of Cd within 5 minutes ²⁺ The net rate of absorption. Transferring the white fine roots without Cd treatment to a test solution containing Cd, and overexpressing three strains of PcNRAMP1 gene and wild type plants presenting Cd ²⁺ Absorption tendency (fig. 8 a). The average net uptake rates for PcNRAMP1-OE5, pcNRAMP1-OE8, and PcNRAMP1-OE9 were increased by 40.62%,38.52%, and 52.30%, respectively, compared to wild-type (FIG. 8 b). The above results demonstrate that overexpression of PcNAMP 1 enhances the Cd pairing of populus griseus roots ²⁺ The absorption capacity of (1).

(2) PcNAMP 1 overexpression of Cd content in populus griseus roots

As can be seen from FIG. 9, after Cd treatment, the Cd content in different tissues of over-expressed and wild type populus sieboldii was: root > aboveground part. After Cd treatment, the Cd content in Populus tremula roots over-expressing the PcNRAMP1 gene was increased by 28.86%,26.54% and 27.70% respectively compared with the wild type (FIG. 9 a). After Cd treatment, the difference between Cd content in the overground part of the over-expressed populus griseus plant and Cd content in the wild type overground part was smaller (FIG. 9 b).

(3) Long-term treatment of Cd content, total Cd and BCF of PcNRAMP1 overexpression Populus sieboldii

As can be seen from FIG. 10, after Cd treatment, the total Cd in different tissues of over-expressed and wild type populus griseus are as follows:

bark, root, wood and leaf.

The total Cd content in the over-expressed plant root is obviously higher than that of the wild type, and is respectively increased by 35.48%,20.15% and 25.48%. The total Cd content of the overground part of the Populus tremula plants over-expressing PcNRAMP1 is obviously higher than that of the wild type, and is respectively increased by 28.98%,24.65% and 29.15%. Wherein, the difference between total Cd in the over-expression plant wood and wild type is not large; the total Cd content in the over-expression plant bark is obviously higher than that of the wild type, and is respectively increased by 41.33 percent, 29.57 percent and 30.37 percent; the total Cd content in leaves of three lines of PcNRAMP1 Populus tremula overexpressed was significantly higher than that of the wild type, and increased by 48.60%,40.75% and 70.50%, respectively (FIG. 11 a).

As shown in fig. 11b, after Cd treatment, the BCF was significantly higher in the roots of the different ash lines than in the aerial parts. The over-expressed plant root BCF is obviously higher than that of a wild type plant, and is respectively improved by 14.29 percent, 15.46 percent and 19.32 percent. The BCF on the overground part of the over-expressed PcNAMP 1 populus tremula plant is obviously higher than that of the wild type, and is respectively improved by 34.41 percent, 36.00 percent and 41.50 percent.

2.4PcNRAMP1 overexpression absorption analysis of populus griseus on other nutrient elements

In order to explore the influence of PcNRAMP1 overexpression on the absorption of other metal elements by Buxus sinica, different Buxus sinica strains were subjected to short-term Cd treatment, and the Mn and Fe contents of the roots and aerial parts of the Buxus sinica were determined. As can be seen from fig. 12, the Mn and Fe contents of populus griseus overexpressing PcNRAMP1 after Cd treatment were significantly higher in the roots than in the wild type. In the aerial part, after Cd treatment, the Mn content of Populus tremula overexpressing PcNAMP 1 was significantly higher than that of the wild type.

In order to explore the absorption of PcNAMP 1 overexpression plants to other nutrients under long-term Cd treatment, 3 transgenic ash plants which overexpress PcNAMP 1 and wild-type ash plants which are treated with Cd and without Cd and are used as materials are used to determine the contents of Mn, fe, zn, ca and Mg in different tissues of different plants, and the results are shown in tables 1 and 2.

TABLE 1PcNRAMP1 overexpression of Mn, fe, zn, ca and Mg content in roots and wood of Populus sieboldii and wild type Populus sieboldii plants

TABLE 2 Mn, fe, zn, ca and Mg contents in bark and leaves of Populus tremula plants overexpressing PcNRAMP1 and wild type Populus tremula plants

Note: metal ion content unit: μ g g ^-1 DW, data are shown as mean ± SE (n = 4), with different letters after the same column of values indicating significant differences. The P values of genotype (G), cd and the interaction between them (GxCd) are shown by two-way anova<0.05；**:P<0.01；***:P<0.001；****:P<0.0001; ns is not significant.

Example 3: functional analysis of PcNRAMP1 amino acid residues

1 method

1.1 Yeast vector construction

The pDONR222-PcNRAMP1 plasmid and a yeast expression vector pYES2-DEST52 are connected through LR reaction to obtain a yeast recombinant plasmid containing a target gene PcNRAMP 1: pYES2-PcNRAMP1.

1.2 site-directed mutagenesis

According to the design rule of point mutation primers, designing primers aiming at amino acids needing mutation, taking plasmid DNA as a template, and carrying out PCR reaction on the primers with mutation sites and the plasmid DNA.

Reaction system

Plasmid DNA, 1. Mu.L; forward and reverse mutation primers, 0.5. Mu.L each; primeSTAR Max DNA Polymerase, 25. Mu.L; ddH ₂ O，23μL。

Reaction procedure

Pre-denaturation at 98 deg.C for 2min; denaturation, 98 ℃,20s; annealing at 55 ℃ for 10s; extension at 68 ℃ and 2kb/min; final extension, at 68 ℃ for 5min; storage at 16 deg.C and infinity.

Wherein the denaturation-extension process requires 18 cycles.

1.3 Yeast transformation

mu.L of DpnI enzyme was added to 50. Mu.L of PCR product after the reaction, which was then transferred to a 37 ℃ metal bath for digestion for 1-1.5h. After digestion, the digested PCR product was transformed into E.coli DH 5. Alpha. The plate was inverted and incubated at 37 ℃ for 12-16h. Sequencing, amplifying and propagating the bacterial solution with correct sequencing comparison, and extracting plasmids. Finally, the positive mutant plasmids are respectively transferred into corresponding yeast mutant strains.

2 results

2.1PcNRAMP1 transmembrane domain conserved amino acid residues on Cd transport

The mutant proteins of pYES2-PcNRAMP1 and PcNRAMP1 after point mutation are transferred into a Cd-sensitive yeast mutant strain delta ycf1 for expression. The obtained yeast strains carrying the natural PcNAMP 1 and the mutant PcNAMP 1 are treated with 30 μ M Cd, and the influence of different amino acid residue mutations on Cd affinity of the transgenic yeast cells is identified.

In the absence of Cd ²⁺ In the case of (2), an empty vector, pcNAMP 1 and 5 point mutations PcNAMP 1s (PcNAMP 1) were expressed ^D61A 、PcNRAMP1 ^G63A 、PcNRAMP1 ^N64A 、PcNRAMP1 ^Q70D And PcNRAMP1 ^S340A ) The growth of yeast cells was consistent (FIG. 13). In the presence of 30 μ M Cd ²⁺ The SG-U solid medium of (1) shows that the growth state of yeast cells expressing the above proteins is inhibited to different degrees. Growth pattern of yeast cells expressing empty vector and native PcNAMP 1In contrast, expression of PcNRAMP1 ^D61A ，PcNRAMP1 ^G63A ，PcNRAMP1 ^N64A ，PcNRAMP1 ^Q70D And PcNRAMP1 ^S340A The growth of yeast cells is more significantly inhibited.

As can be seen from FIG. 13, no Cd was added ²⁺ In the case of (2), an empty vector, pcNRAMP1 and 5 point mutations PcNRAMP1s (PcNRAMP 1) were expressed ^D68A 、PcNRAMP1 ^E129D 、PcNRAMP1 ^M236A 、PcNRAMP1 ^P237A And PcNAMP 1 ^P405A ) The growth conditions of the yeast cells are consistent. In the presence of 30. Mu.M Cd ²⁺ The SG-U solid medium of (1) shows that the growth state of yeast cells expressing the above proteins is inhibited to a different extent as compared with that of the empty vector. Expression of PcNRAMP1 compared to growth status of yeast expressing PcNRAMP1 ^D68A ,PcNRAMP1 ^E129D ，PcNRAMP1 ^M236A ，PcNRAMP1 ^P237A And PcNRAMP1 ^P405A The growth state of yeast cells was inhibited less by Cd treatment than PcNRAMP1, suggesting that mutation of amino acid residues at positions D68, E129, M236, P237 and P405 in PcNRAMP1 to alanine (A) and aspartic acid (D) results in PcNRAMP1 acting on Cd ²⁺ The absorption capacity of (b) is reduced.

To verify the Cd selectivity of yeast cells mutated at different amino acid residues in one step. The SWILL MODEL website was used to MODEL the PcNRAMP1 protein structure. The three-dimensional structure of the protein of PcNRAMP1 obtained by modeling is similar to the crystal structure of EcoDMT, and the similarity is 34.96%. Both have 12 transmembrane domains, and the protein N-and C-termini are on the same side of the plasma membrane (fig. 14 a). PcNRAMP1 was selected from FIG. 13 ^D61A ,PcNRAMP1 ^G63A ，PcNRAMP1 ^M236A And PcNRAMP1 ^P405A Further studies were carried out. From FIG. 14b, it was found that the 4 amino acid residues D61, G63, M236 and P405 are conserved in different species and are located in the 1,6, 10 transmembrane domain, respectively.

PcNRAMP1 point mutation yeast cells (PcNRAMP 1) which cause the enhancement of Cd sensitivity or Cd insensitivity of the yeast cells after PcNRAMP1 mutation are selected ^D61A ,PcNRAMP1 ^G63A ，PcNRAMP1 ^M236A And PcNRAMP1 ^P405A ) And (5) determining the content of Cd. As a result, they found that PcNAMP 1-expressing yeast cellsCompared with Cd content in the product, pcNRAMP1 ^D61A ,PcNRAMP1 ^G63A ，PcNRAMP1 ^M236A And PcNRAMP1 ^P405A The Cd content in 4 yeast cells was reduced, and PcNRAMP1 ^M236A And PcNRAMP1 ^P405A Significantly decreased (fig. 14 d).

2.2PcNRAMP1 transmembrane domain conserved amino acid residue mutations on other metal affinity effects

The mutant proteins of PcNRAMP1-pYES2 and PcNRAMP1 after point mutation were transformed into Mn sensitive mutant strain Δ pmr1 and Fe sensitive yeast mutant strain Δ ccc1 for expression. The yeast mutants obtained carrying the PcNRAMP1 and PcNRAMP1 muteins were treated with 1mM Mn and 8mM Fe to identify the effect of different amino acid residue mutations on Mn and Fe affinity of the transgenic yeast cells.

As can be seen from FIG. 15a, in normal Mn ²⁺ In the case of (2), the empty vector, natural PcNRAMP1 and PcNRAMP1 are expressed ^D61A ,PcNRAMP1 ^G63A ，PcNRAMP1 ^M236A And PcNRAMP1 ^P405A The growth conditions of the yeast cells of the PcNRAMP1 protein after the 4-point mutation are consistent. In the presence of 1mM Mn ²⁺ The growth state of yeast cells of the protein after point mutation of PcNRAMP1 in SG-U solid medium of (1) was inhibited to various degrees. The inhibition of growth of yeast cells in which the amino acid residues at positions D61, G63, M236 and P405 of PcNRAMP1 protein were mutated to alanine (A) was significant as compared with the growth of yeast cells expressing the empty vector, and the mutation of D61 resulted in a decrease in Mn in yeast cells as compared with PcNRAMP1 ²⁺ Sensitivity.

As can be seen from FIG. 15b, in the absence of Fe ²⁺ In the case of (2), empty vectors, pcNRAMP1 and PcNRAMP1 are expressed ^D61A ,PcNRAMP1 ^G63A ，PcNRAMP1 ^M236A And PcNRAMP1 ^P405A The growth conditions of the yeast cells of the PcNRAMP1 protein after the 4-point mutation are consistent. In the presence of 8mM Fe ²⁺ The growth state of yeast cells of the protein after PcNRAMP1 point mutation was inhibited to various degrees in SG-U solid medium (2). The growth status of yeast cells with mutations in the amino acid residues at positions M236 of the PcNRAMP1 protein is consistent with the expression of the empty vector. Mutation M236 resulted in yeast cells compared to PcNRAMP1For Fe ²⁺ It is not sensitive.

It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.

Claims (8)

1. A populus griseus metal transporter is characterized in that the amino acid sequence is shown as SEQ ID NO. 1.

2. The gene encoding the protein according to claim 1, wherein the nucleotide sequence is represented by SEQ ID No. 2.

3. A vector comprising the gene of claim 2.

4. A genetically engineered bacterium comprising the vector of claim 3.

5. Use of the protein of claim 1 or the gene of claim 2 for modulating metal transport capacity in a plant, wherein the metals include cadmium, manganese, iron, zinc and calcium.

6. A method of constructing a transgenic plant comprising the steps of:

over-expression of the protein of claim 1 in a plant of interest results in increased plant capacity to transport metals including cadmium, manganese, iron, zinc and calcium.

7. The method of claim 6, wherein the method for over-expressing the protein of claim 1 in the plant of interest is: a transgenic plant obtained by introducing the gene of claim 2 into a plant of interest.

8. The method of claim 7, wherein the plant comprises populus tremuloides.

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