PaNIP2, a protein from the American pokeweed, its encoding gene, and its applications.

Abstract

This invention discloses a pokeweed protein, PaNIP2, its encoding gene, and its applications. A protein, named PaNIP2, was identified from the American pokeweed (Phytolacca Americana L.) and associated with its ability to hyperaccumulate rare earth elements, particularly heavy rare earth elements. Overexpression of PaNIP2 in fungi or plants can enhance their ability to accumulate rare earth elements, especially heavy rare earth elements. This protein can be used to cultivate transgenic plants with large biomass and the ability to accumulate rare earth elements, which can then be used to remediate soil contaminated with rare earth elements. This invention not only enriches the molecular resources related to plant accumulation of rare earth elements but also facilitates the remediation of rare earth-contaminated soil and the resource recovery of rare earth elements.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and rare earth pollution control technology. More specifically, it relates to a pokeweed protein PaNIP2, its encoding gene, and its applications. Background Technology

[0002] Rare earth elements (REEs) are a collective term for 17 elements, including the lanthanides, scandium, and yttrium, and can be divided into light rare earth elements and heavy rare earth elements. In recent years, due to strong market demand, my country's rare earth resources have been consumed at an accelerated rate, leading to a sharp decrease in rare earth reserves. Heavy rare earth elements are even scarcer due to their extremely low abundance. Of the five rare earth elements in the world with the most severe supply shortages (dysprosium, terbium, europium, neodymium, and yttrium), three are heavy rare earth elements. Furthermore, large amounts of rare earth elements remain in rare earth tailings left behind due to outdated and crude mining methods. This not only wastes rare earth resources but also pollutes the surrounding ecological environment, endangering ecosystem security. Exploring methods for enriching and recovering the rare earth elements remaining in rare earth tailings can both utilize wasted rare earth resources and restore the ecological environment polluted by rare earth elements. Using rare earth hyperaccumulating plants for phytomining is a green and environmentally friendly method for recycling rare earth resources and remediating contaminated soil.

[0003] Of the 21 rare earth hyperaccumulators reported to date, the vast majority prefer to accumulate light rare earth elements (REEs), with only the aboveground parts of *Phytolacca Americana* L. showing a preference for accumulating heavy REEs. *Phytolacca Americana* L. is a perennial plant widely distributed worldwide, possessing advantages such as large biomass and rapid growth rate, making it an excellent material for accumulating REEs, especially heavy REEs. However, *Phytolacca Americana* L. is an invasive species, and the entire plant is poisonous, posing a certain ecological risk. To avoid its harm, molecular resources related to REE accumulation can be explored, laying a good foundation for cultivating non-toxic plants with large biomass and strong REE accumulation capabilities. Currently, there are no reports on *Phytolacca Americana* L. proteins or genes related to REE transport. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the defects and deficiencies of the existing technologies and to provide a PaNIP2 protein from the American pokeweed, its encoding gene and its applications.

[0005] The first objective of this invention is to provide a protein from American pokeweed, PaNIP2.

[0006] A second objective of this invention is to provide a gene encoding the American pokeweed protein PaNIP2.

[0007] A third objective of this invention is to provide a recombinant expression vector.

[0008] The fourth objective of this invention is to provide a recombinant engineered bacterium.

[0009] A fifth objective of this invention is to provide the application of the American pokeweed protein PaNIP2, the gene, or the recombinant expression vector in enhancing the ability of fungi to enrich rare earth elements.

[0010] The sixth objective of this invention is to provide the application of the PaNIP2 protein of Phytolacca acinosa, the gene, the recombinant expression vector, or the recombinant engineered bacteria in improving the plant's ability to enrich rare earth elements.

[0011] A seventh objective of this invention is to provide the application of the PaNIP2 protein of Phytolacca acinosa, the gene, the recombinant expression vector, or the recombinant engineered bacteria in the cultivation of transgenic plants with rare earth enrichment function.

[0012] The eighth objective of this invention is to provide a method for improving the rare earth element enrichment capacity of plants.

[0013] The above-mentioned objective of this invention is achieved through the following technical solution:

[0014] This invention provides a pokeweed protein, PaNIP2, the amino acid sequence of which is shown in SEQ ID NO.2.

[0015] The PaNIP2 protein described in this invention was screened and identified from a three-generation full-length transcriptome database of Phytolacca Americana L., and it was found to be related to the rare earth enrichment capacity of Phytolacca Americana L.

[0016] The present invention also provides a gene encoding the American pokeweed protein PaNIP2.

[0017] Specifically, the nucleotide sequence of the gene encoding the American pokeweed protein PaNIP2 described in this invention is shown in SEQ ID NO.1.

[0018] The present invention also provides a recombinant expression vector containing a gene sequence encoding the American pokeweed protein PaNIP2.

[0019] As an optional implementation, the expression vector used in this invention is pYES2 or pSN1305 (pCAMBIA1305). pYES2 can be used to overexpress the gene encoding the American pokeweed protein PaNIP2 in fungi, and pSN1305 (pCAMBIA1305) can be used to overexpress the gene encoding the American pokeweed protein PaNIP2 in plants.

[0020] The present invention also provides a recombinant engineered bacterium containing the recombinant expression vector described in the present invention.

[0021] Given that overexpression of the gene encoding the pokeweed protein PaNIP2 in fungi or plants can enhance their ability to accumulate rare earth elements, this invention seeks protection for the use of the pokeweed protein PaNIP2, the gene encoding the pokeweed protein PaNIP2, or the recombinant expression vector in enhancing the ability of fungi to accumulate rare earth elements.

[0022] Specifically, the fungus is Saccharomyces cerevisiae.

[0023] Specifically, the rare earth element is a heavy rare earth element.

[0024] More specifically, the heavy rare earth element is ytterbium (Yb).

[0025] The present invention also claims protection for the application of the pokeweed protein PaNIP2, the gene encoding the American pokeweed protein PaNIP2, the recombinant expression vector, or the recombinant engineered bacteria in improving the enrichment capacity of plants for rare earth elements.

[0026] Specifically, the enhancement of plants' ability to accumulate rare earth elements is achieved by overexpressing the American pokeweed protein PaNIP2 or overexpressing the gene encoding the American pokeweed protein PaNIP2 in plants.

[0027] Specifically, the plant in question is Arabidopsis thaliana.

[0028] Specifically, the rare earth element is a heavy rare earth element.

[0029] The present invention also claims protection for the use of the pokeweed protein PaNIP2, the gene encoding the American pokeweed protein PaNIP2, the recombinant expression vector, or the recombinant engineered bacteria in the cultivation of transgenic plants with rare earth enrichment function.

[0030] The present invention also provides a method for improving the rare earth element enrichment capacity of plants, wherein the method comprises: introducing a gene encoding the pokeweed protein PaNIP2 described in the present invention into a recipient plant to obtain a transgenic plant.

[0031] Specifically, the recipient plant is Arabidopsis thaliana.

[0032] This invention also seeks protection for the application of the transgenic plants prepared by the above method in the remediation of rare earth contaminated soil or the recycling of rare earth resources.

[0033] Specifically, the rare earth elements are heavy rare earth elements.

[0034] The present invention has the following beneficial effects:

[0035] This invention identifies a protein, named PaNIP2, from *Phytolacca Americana* L., associated with its ability to hyperaccumulate rare earth elements (REEs), particularly heavy REEs. Overexpression of PaNIP2 in fungi or plants can enhance their ability to accumulate REEs, especially heavy REEs. This protein can be used to cultivate transgenic plants with high biomass, rapid growth, and the ability to accumulate REEs, which can then be used to remediate REE-contaminated soil. This invention not only enriches molecular resources related to plant accumulation of REEs but also facilitates the remediation of REE-contaminated soil and the resource recovery of REEs. Attached Figure Description

[0036] Figure 1 The figures show the results of ytterbium (Yb) content determination in Saccharomyces cerevisiae expressing PaNIP2 and Saccharomyces cerevisiae not expressing PaNIP2 after culturing in a medium containing the heavy rare earth element ytterbium (Yb); * in the figure indicates significant difference, P < 0.05.

[0037] Figure 2 The figures show the results of Nd and Yb content determination in Arabidopsis thaliana overexpressing PaNIP2 and wild-type Arabidopsis thaliana after culturing in mediums containing the light rare earth element neodymium (Nd) and the heavy rare earth element ytterbium (Yb), respectively. In the figure, A represents the relative expression level of the PaNIP2 gene in different Arabidopsis thaliana lines overexpressing PaNIP2; B represents the results of light rare earth element content determination in Arabidopsis thaliana; and C represents the results of heavy rare earth element content determination in Arabidopsis thaliana. Different letters indicate significant differences, P < 0.05.

[0038] Figure 3 The figures show the total rare earth element content and the ratio of light to heavy rare earth element concentrations in the aboveground parts of Arabidopsis thaliana overexpressing PaNIP2 and wild-type Arabidopsis thaliana; A in the figure represents the total rare earth element content in the aboveground parts of Arabidopsis thaliana; B in the figure represents the ratio of light to heavy rare earth element concentrations. Detailed Implementation

[0039] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0040] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0041] Example 1: American pokeweed protein PaNIP2 and its encoding gene

[0042] This invention screened and identified a protein with rare earth transport capabilities from a three-generation full-length transcriptome database of Phytolacca Americana L., namely a protein associated with the rare earth hyperenrichment capacity of Phytolacca Americana L., named PaNIP2, whose amino acid sequence is shown below (SEQ ID NO.2):

[0043]

[0044] This invention also cloned the gene encoding the American pokeweed protein PaNIP2 (PaNIP2), and the cloning process is as follows:

[0045] 1. RNA extraction and reverse transcription:

[0046] Root tissues from freshly hydroponically cultured American pokeweed were collected, and total RNA was extracted using an RNA extraction kit (Sigma-Aldrich). cDNA was then obtained by reverse transcription using the HiScriptII OneStep RT-PCR kit (Vazyme Biotech). The specific steps were performed according to the kit instructions.

[0047] 2. Cloning primer design:

[0048] The cloning primers for the gene encoding the American pokeweed protein PaNIP2 are shown below:

[0049] Front primer F: ACTCTCTTACTTCTGTGTGTC

[0050] Terminal primer R: GTTACAAGCATCATAGTGCG

[0051] 3. PCR amplification:

[0052] Using American pokeweed cDNA obtained by reverse transcription as a template, high-fidelity enzyme I-5 was used... TM PCR amplification was performed using 2× High-FidelityMaster Mix (MCLAB, I5HM-200). The total volume of the PCR amplification reaction system was 50 μL, including 25 μL of I5mix, 1 μL of cDNA template, 2 μL of front primer F (10 pmol / μL), 2 μL of end primer R (10 pmol / μL), and the remainder made up with ddH2O. The PCR amplification reaction program was as follows: 98℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s; 58℃ annealing for 15 s; 72℃ extension for 30 s; 35 cycles; 72℃ extension for 5 min; and incubation at 4℃.

[0053] 4. PCR product verification:

[0054] PCR products were purified using a gel extraction kit and then ligated into the TOPO cloning vector to obtain a recombinant cloning vector. The ligation product was then transformed back into *E. coli* DH5α competent cells. Positive transformants were screened on a medium containing ampicillin, and the positive single clones were sent to a biotechnology company for sequencing. The sequencing results showed that the nucleotide sequence of the gene encoding the American pokeweed protein PaNIP2 is as follows (SEQ ID NO.1):

[0055]

[0056] Example 2 Expression of PaNIP2 in Saccharomyces cerevisiae

[0057] In this embodiment, the function of PaNIP2, the American pokeweed protein described in Example 1, was detected using Saccharomyces cerevisiae BY4741. The process is as follows:

[0058] 1. Construction of recombinant expression vectors:

[0059] Primers for cloning the PaNIP2 gene, containing homologous arm sequences from the pYES2 expression vector, were designed using Novizan's CE Design software. The primers are shown below:

[0060] Front primer F: gggaatattaagcttggtaccATGGGCGAAATTGCAAGGG

[0061] Terminal primer R: gcggccgttactagtggatccCTAGCGCTGTGAGCCATTGTT

[0062] Using the recombinant cloning vector of the PaNIP2 gene constructed in Example 1 as a template, the PaNIP2 gene with homologous arm sequences was obtained by PCR amplification. The total volume of the PCR amplification reaction system was 50 μL, including 25 μL of I5 mix, 1 μL of cDNA template, 2 μL of front primer F (10 pmol / μL), 2 μL of end primer R (10 pmol / μL), and ddH2O to make up the balance. The PCR amplification reaction program was as follows: 98℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s; 58℃ annealing for 15 s; 72℃ extension for 30 s; 35 cycles; 72℃ extension for 5 min; and incubation at 4℃.

[0063] The PCR products obtained by gel electrophoresis were purified by gel recovery. Homologous recombination was then used to homologously recombine the PaNIP2 gene into the Kpn I and BamHI restriction sites of the pYES2 expression vector using a one-step rapid cloning kit, resulting in the recombinant expression vector pYES2::PaNIP2 containing PaNIP2. This recombinant expression vector was then retransformed into *E. coli* DH5α competent cells. Positive transformants were screened, and the positive single-clone strains were sent to a biotechnology company for sequencing to ensure that PaNIP2 was correctly ligated into the recombinant expression vector pYES2::PaNIP2.

[0064] 2. Transformation and identification of yeast competent cells:

[0065] Using a rapid yeast transformation kit (SK2400, Beijing Coolapk Technology Co., Ltd.), the validated pYES2::PaNIP2 recombinant yeast expression vector and the empty vector pYES2 were transformed into Saccharomyces cerevisiae BY4741 strain, respectively. The transformed Saccharomyces cerevisiae were cultured on SD-U selective medium (containing 0.67 g / 100 mL of amino acid-free yeast nitrogen source, catalog number Y0626, Sigma; 0.192 g / 100 mL of yeast auxotrophic medium supplement, catalog number Y1501, Sigma; and 2 g / 100 mL of galactose, catalog number G0750, Sigma) to screen for positive colonies. Plasmids of positive monoclonal Saccharomyces cerevisiae strains were extracted and transformed into Escherichia coli DH5α for secondary sequencing verification.

[0066] 3. Rare earth enrichment experiment:

[0067] To further verify the function of the PaNIP2 gene, *Saccharomyces cerevisiae* expressing the PaNIP2 gene and *Saccharomyces cerevisiae* containing the empty vector pYES2 were transferred to SD-U liquid selective medium and cultured until the *Saccharomyces cerevisiae* reached the OD value. 600 When the concentration was 1.0, the brewing yeast was centrifuged and resuspended, and then transferred to yeast culture medium containing heavy rare earth elements (10 μM YbCl3), citric acid-bound heavy rare earth elements (10 μM YbCl3 and 30 μM Cit), and malic acid-bound heavy rare earth elements (10 μM YbCl3 and 30 μM Mal) (main components as above, but without P, ​​pH 4.2). After culturing in a shaker at 30℃ for 1 h, the brewing yeast was collected. The collected brewing yeast was first washed three times with 20 mM MES-10 mM EDTA cleaning agent to remove the rare earth elements adsorbed on the surface of the brewing yeast, and then washed three times with sterile water. It was then placed in a 60℃ oven and dried for 3 days. The dried yeast cells were weighed, digested, and the content of heavy rare earth elements in the cells was determined by ICP-MS.

[0068] The ytterbium (Yb) content in *Saccharomyces cerevisiae* expressing PaNIP2 and *Saccharomyces cerevisiae* not expressing PaNIP2 was determined after culturing in a medium containing the heavy rare earth element ytterbium (Yb). The results are as follows: Figure 1 As shown in the figure; * indicates a significant difference, P < 0.05. Figure 1 It can be seen that the enrichment concentrations of citric acid-bound and malic acid-bound heavy rare earth element ytterbium (Yb) in Saccharomyces cerevisiae expressing the PaNIP2 gene are higher than those in Saccharomyces cerevisiae not expressing PaNIP2. This indicates that the PaNIP2 protein encoded by the PaNIP2 gene has the ability to transport heavy rare earth elements, and prefers to transport organic acid-bound heavy rare earth elements rather than ionic rare earth elements.

[0069] Example 3: Expression of PaNIP2 in Arabidopsis thaliana

[0070] In this embodiment, the function of the American pokeweed protein PaNIP2 described in Example 1 was detected using the model plant Arabidopsis thaliana. The process is as follows:

[0071] 1. Construction of recombinant expression vectors:

[0072] Primers for cloning the PaNIP2 gene, containing homologous arm sequences from the plant overexpression vector pSN1305 (pCAMBIA1305), were designed using Novizan's CE Design software. The primers are shown below:

[0073] Front primer F: gaacgataggagctcggtaccATGGGCGAAATTGCAAGGG

[0074] Terminal primer R: caggtcgactctagaggatccCTAGCGCTGTGAGCCATTGTT

[0075] Using the recombinant cloning vector of the PaNIP2 gene constructed in Example 1 as a template, the PaNIP2 gene with homologous arm sequences was obtained by PCR amplification. The total volume of the PCR amplification reaction system was 50 μL, including 25 μL of I5 mix, 1 μL of cDNA template, 2 μL of front primer F (10 pmol / μL), 2 μL of end primer R (10 pmol / μL), and ddH2O to make up the balance. The PCR amplification reaction program was as follows: 98℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s; 58℃ annealing for 15 s; 72℃ extension for 30 s; 35 cycles; 72℃ extension for 5 min; and incubation at 4℃.

[0076] The PCR products obtained by gel electrophoresis were purified by gel recovery and then homologous recombination was used to homologously recombine the PaNIP2 gene into the plant overexpression vector pSN1305 (pCAMBIA1305) using a one-step rapid cloning kit. (This vector carries a strong CaMV 35S promoter, which can express the target gene in large quantities in plants.) This yielded the recombinant expression vector pSN1305::PaNIP2 containing PaNIP2. The constructed recombinant expression vector was then retransformed into E. coli DH5α competent cells, and positive transformants were screened. The positive single clones were sent to a biotechnology company for sequencing to ensure that PaNIP2 was correctly ligated into the recombinant expression vector pSN1305::PaNIP2.

[0077] 2. Agrobacterium infection and transformation of Arabidopsis thaliana:

[0078] The recombinant expression vector pSN1305::PaNIP2 containing the PaNIP2 gene was transformed into Agrobacterium GV3101, and plasmids were extracted and transduced into E. coli for secondary sequence verification. The verified Agrobacterium was cultured in LB broth containing rifampicin and kanamycin resistance to achieve optimal viability (OD). 600 =1.0), centrifuge to obtain Agrobacterium and resuspend Agrobacterium in infection solution (5% sucrose solution, 0.05% Silwet L-77); immerse the stamens of healthy Arabidopsis thaliana flowers in the infection solution containing Agrobacterium for 45s; each Arabidopsis thaliana flower needs to be infected for three consecutive weeks, once a week; after infection, harvest Arabidopsis thaliana seeds (T0 generation), screen positive seedlings from T0 generation seeds and cultivate them in soil to T1 generation; after harvesting T1 generation seeds, continue to screen positive seedlings and cultivate to T2 generation, screen and identify T2 generation homozygous positive seedlings, and conduct rare earth enrichment experiments.

[0079] 3. Rare earth enrichment experiment:

[0080] To investigate whether the rare earth element transporter PaNIP2 in *Phytolacca americana* can function in other plants, this invention selected identified T2 generation homozygous *Arabidopsis thaliana* lines Ex5, Ex6, and Ex14 (the relative expression abundance of PaNIP2 in these *Arabidopsis thaliana* lines is shown in Figure 1). Figure 2 As shown in Figure A), the *Arabidopsis thaliana* (WT) and wild-type *Arabidopsis thaliana* were aseptically germinated on 1 / 2 MS medium. Neodymium (Nd) was selected to represent light rare earth elements, and ytterbium (Yb) was selected to represent heavy rare earth elements. *Arabidopsis thaliana* plants with uniform growth were transferred to media containing 30 μM Nd and 30 μM Yb, respectively, and harvested after culturing under the same conditions for 7 days. The plants were divided into aboveground parts and roots. The roots were desorbed with 5 mM CaCl2 for 15 min and then washed three times with ultrapure water. The aboveground parts were washed three times with ultrapure water, weighed, and digested to measure the rare earth content in different tissues of the plants.

[0081] After culturing in mediums containing the light rare earth element neodymium (Nd) and the heavy rare earth element ytterbium (Yb), the contents of neodymium (Nd) and ytterbium (Yb) in Arabidopsis thaliana overexpressing PaNIP2 and wild-type Arabidopsis thaliana were determined as follows: Figure 2 As shown. Figure 2 In the figure, A represents the relative expression level of the PaNIP2 gene in different Arabidopsis lines overexpressing PaNIP2, derived from... Figure 2 As can be seen from A in the figure, the T2 generation homozygous Arabidopsis strains Ex5, Ex6 and Ex14 selected in this invention are all Arabidopsis strains that overexpress PaNIP2. Figure 2 B in the figure represents the result of the determination of light rare earth element content in Arabidopsis thaliana; Figure 2 In the figure, C represents the result of heavy rare earth element content determination in Arabidopsis thaliana; from Figure 2 As shown in B and C, the contents of light rare earth elements (LREEs) and heavy rare earth elements (HREEs) in the roots and shoots of Arabidopsis thaliana lines Ex5, Ex16, and Ex14, which overexpress the PaNIP2 gene, were significantly higher than those in wild-type Arabidopsis thaliana. This indicates that overexpression of the PaNIP2 gene can significantly improve the enrichment capacity of Arabidopsis thaliana lines for LREEs and HREEs, and the enrichment capacity for HREEs is even higher than that for LREEs.

[0082] Example 4

[0083] To further investigate the effect of PaNIP2 gene overexpression on the enrichment capacity of Arabidopsis thaliana for mixed rare earth elements, the enrichment capacity of the T2 generation homozygous Arabidopsis thaliana lines Ex5, Ex6, and Ex14 described in Example 2 for mixed rare earth elements was experimentally tested. The process is as follows:

[0084] The T2 generation homozygous Arabidopsis thaliana lines Ex5, Ex6, and Ex14 described in Example 2 were cultured together with seeds of wild-type Arabidopsis thaliana WT in sand culture. After germination, they were transferred to hydroponics. Hydroponic experiments were conducted using 14 lanthanide rare earth elements. Arabidopsis thaliana plants with uniform growth were transferred to hydroponic environments containing 14 rare earth elements, namely lanthanum (La), cerium (Ce), praseodymium (Pr), neodymium (Nd), promethium (Pm), samarium (Sm), and europium. The nutrient solution containing Eu, Gd, Tb, Dysprosium, Ho, Erbium, Tm, Yb, and Lu had a concentration of 5 μM for each rare earth element and a total rare earth element concentration of 70 μM. The plants were harvested after being cultured for 7 days under the same conditions. The aboveground parts of the plants were washed three times with ultrapure water, the fresh weight of the plants was weighed, and digestion was performed to measure the rare earth element content of the aboveground parts of the plants.

[0085] The total rare earth element content and the ratio of light to heavy rare earth element concentrations in the aboveground parts of Arabidopsis thaliana overexpressing PaNIP2 and wild-type Arabidopsis thaliana are as follows: Figure 3 As shown; Figure 3 In this context, A represents the total rare earth element content of the aboveground parts of Arabidopsis thaliana; Figure 3 In this context, B represents the concentration ratio of light to heavy rare earth elements. Figure 3 As shown in A, the total rare earth element (REE) content in the shoots of Arabidopsis thaliana lines overexpressing PaNIP2 was significantly higher than that in wild-type Arabidopsis, indicating that overexpression of PaNIP2 can enhance the enrichment capacity of Arabidopsis for REEs. Furthermore, the ratio of light REEs to heavy REEs in the shoots of Arabidopsis lines Ex5, Ex16, and Ex14 was lower than that in the shoots of wild-type Arabidopsis, suggesting that overexpression of PaNIP2 not only enhances the enrichment capacity of Arabidopsis for REEs but also enhances its enrichment capacity for heavy REEs, meaning that the PaNIP2 protein prefers to transport heavy REEs.

[0086] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The application of PaNIP2, a protein from the American pokeweed, in enhancing the enrichment capacity of Saccharomyces cerevisiae for the rare earth element ytterbium (Yb), wherein the amino acid sequence of the protein is shown in SEQ ID NO.

2.

2. The application of a gene encoding the American pokeweed protein PaNIP2 in improving the enrichment capacity of the rare earth element ytterbium (Yb) in Saccharomyces cerevisiae, wherein the nucleotide sequence of the gene encoding the American pokeweed protein PaNIP2 is shown in SEQ ID NO.

1.

3. The application of a recombinant expression vector in improving the enrichment capacity of the rare earth element ytterbium (Yb) in Saccharomyces cerevisiae, wherein the recombinant expression vector contains a gene sequence encoding the American pokeweed protein PaNIP2, and the gene sequence encoding the American pokeweed protein PaNIP2 is shown in SEQ ID NO.

1.

4. Application of American pokeweed protein PaNIP2 in enhancing the enrichment capacity of Arabidopsis thaliana for rare earth elements, wherein the rare earth elements are light rare earth element neodymium (Nd) and / or heavy rare earth element ytterbium (Yb), and the amino acid sequence of the protein is shown in SEQ ID NO.

2.

5. Application of the gene encoding the American pokeweed protein PaNIP2 in enhancing the enrichment capacity of Arabidopsis thaliana for rare earth elements, wherein the rare earth elements are the light rare earth element neodymium (Nd) and / or the heavy rare earth element ytterbium (Yb), and the nucleotide sequence of the gene encoding the American pokeweed protein PaNIP2 is shown in SEQ ID NO.

1.

6. Application of recombinant expression vector in improving the enrichment capacity of rare earth elements in Arabidopsis thaliana, wherein the rare earth elements are light rare earth element neodymium (Nd) and / or heavy rare earth element ytterbium (Yb), and the recombinant expression vector contains a gene sequence encoding the American pokeweed protein PaNIP2, the gene sequence encoding the American pokeweed protein PaNIP2 being shown in SEQ ID NO.

1.

7. Application of recombinant engineered bacteria in enhancing the enrichment capacity of Arabidopsis thaliana for rare earth elements, wherein the rare earth elements are the light rare earth element neodymium (Nd) and / or the heavy rare earth element ytterbium (Yb), the recombinant engineered bacteria contain a recombinant expression vector, the recombinant expression vector contains a gene sequence encoding the American pokeweed protein PaNIP2, the gene sequence encoding the American pokeweed protein PaNIP2 is shown in SEQ ID NO.

1.

8. The application according to any one of claims 4 to 7, characterized in that, The enhancement of rare earth element enrichment in Arabidopsis thaliana was achieved by overexpressing the gene encoding the American pokeweed protein PaNIP2 in Arabidopsis thaliana.

9. Application of the gene encoding the American pokeweed protein PaNIP2 in the cultivation of transgenic Arabidopsis thaliana with rare earth enrichment function, wherein the rare earth is the light rare earth element neodymium (Nd) and / or the heavy rare earth element ytterbium (Yb), and the nucleotide sequence of the gene encoding the American pokeweed protein PaNIP2 is shown in SEQ ID NO.

1.

10. Application of recombinant expression vector in the cultivation of transgenic Arabidopsis thaliana with rare earth enrichment function, wherein the rare earth is neodymium (Nd) and / or ytterbium (Yb) (a light rare earth element), and the recombinant expression vector contains a gene sequence encoding the American pokeweed protein PaNIP2, the gene sequence encoding the American pokeweed protein PaNIP2 being shown in SEQ ID NO.

1.

11. Application of recombinant engineered bacteria in the cultivation of transgenic Arabidopsis thaliana with rare earth enrichment function, wherein the rare earth is the light rare earth element neodymium (Nd) and / or the heavy rare earth element ytterbium (Yb), the recombinant engineered bacteria contains a recombinant expression vector, the recombinant expression vector contains a gene sequence encoding the American pokeweed protein PaNIP2, and the gene sequence encoding the American pokeweed protein PaNIP2 is shown in SEQ ID NO.

1.

12. A method for improving the rare earth element enrichment capacity of Arabidopsis thaliana, characterized in that, The method is as follows: a gene encoding the American pokeweed protein PaNIP2 is introduced into the recipient Arabidopsis thaliana to obtain transgenic Arabidopsis thaliana. The rare earth element is the light rare earth element neodymium (Nd) and / or the heavy rare earth element ytterbium (Yb). The sequence of the gene encoding the American pokeweed protein PaNIP2 is shown in SEQ ID NO.1.

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