Dinreet1 protein of dinitus lanceolatus, gene encoding the same and application

Abstract

The application discloses a Dlnreel1 protein, a coding gene and application thereof. A protein with rare earth transport capacity is screened and identified from a plant Dlnreel1, and the gene sequence coding the protein is cloned. The protein can improve the enrichment capacity of fungi or plants to rare earth elements by overexpressing the protein in the fungi or plants, and the protein can be used for cultivating transgenic plants with rare earth repair functions, and then used for rare earth element contaminated soil treatment and plant mining. The Dlnreel1 protein and the coding gene thereof have important significance for the repair and resource recovery of rare earth contaminated soil.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology. More specifically, it relates to a DlNREET1 protein from *Dictyophora indica*, its encoding gene, and its applications. Background Technology

[0002] Rare earth elements (REEs) are a collective term for 17 elements, including the lanthanides and scandium and yttrium. They can be divided into light rare earth elements and heavy rare earth elements and are important strategic resources. China is a major rare earth resource country, ranking first in the world in both reserves and mining volume. However, extensive mining activities have not only led to a continuous decline in my country's rare earth reserves but have also left behind large areas of rare earth tailings. These tailings contain large amounts of rare earth elements, threatening ecosystem security and causing significant losses of rare earth resources. Furthermore, improper mining methods have resulted in large amounts of rare earth elements entering surrounding rivers, leading to rare earth pollution of farmland around mining areas. Therefore, remediating these rare earth tailings and contaminated soils, reducing their environmental risks, and recovering the rare earth resources within them are of great significance.

[0003] Phytomining is a green and environmentally friendly phytoremediation technology that utilizes hyperaccumulating plants in rare earth-contaminated sites to harvest their aboveground biomass, thereby recovering target elements from contaminated soil. This provides a solution for the ecological restoration of contaminated soil and the recycling of rare earth resources in mining areas. To date, only 21 species of rare earth hyperaccumulating plants have been reported in academia; among them, *Dicranopteris linearis* exhibits the strongest rare earth accumulation capacity, with its aboveground parts containing up to 0.7% of the plant's dry weight, making it a potential material for developing rare earth phytomining technology. Despite its many advantages, *Dicranopteris linearis* has a relatively small biomass and is difficult to propagate artificially, significantly limiting the feasibility of directly utilizing it for rare earth phytomining. However, its exceptionally strong rare earth accumulation capacity indicates the existence of a highly efficient rare earth absorption system within its root system. Therefore, exploring the molecular mechanisms by which *Dicranopteris linearis* roots absorb rare earth elements and mining related molecular resources can lay a solid foundation for cultivating phytomining materials with large biomass and strong rare earth accumulation capabilities. However, no studies have yet reported the presence of proteins or genes in plants that are related to rare earth element transport. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the defects and shortcomings of existing rare earth plant mining technology, and to provide a DlNREET1 protein of *Dictamnus dasycarpus* with rare earth element transport capabilities, its encoding gene and its applications.

[0005] The first objective of this invention is to provide a DlNREET1 protein.

[0006] A second objective of this invention is to provide the gene encoding the DlNREET1 protein of the gentian.

[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 DlNREET1 protein, the encoding gene, or the recombinant expression vector in enhancing the ability of fungi to enrich rare earth elements.

[0010] A sixth objective of this invention is to provide the application of the DlNREET1 protein of *Dictyophora indica*, the encoding gene, the recombinant expression vector, or the recombinant engineered bacteria in enhancing the ability of plants to enrich rare earth elements.

[0011] A seventh objective of this invention is to provide the application of the DlNREET1 protein of *Dictyophora inermis*, the encoding gene, the recombinant expression vector, or the recombinant engineered bacteria in the cultivation of transgenic plants with rare earth remediation functions.

[0012] The above-mentioned objective of this invention is achieved through the following technical solution:

[0013] This invention screened and identified a protein with rare earth element transport capabilities from the rare earth hyperaccumulator *Dicranopteris linearis*, named DlNREET1 (Dicranopteris linearis NRAMP Rare Earth Element Transporter1), whose amino acid sequence is shown in SEQ ID NO. 2. This invention also obtained the gene encoding the *Dicranopteris linearis* DlNREET1 protein, the nucleotide sequence of which is shown in SEQ ID NO. 1. By overexpressing the *Dicranopteris linearis* DlNREET1 protein in fungi and plants, this invention found that it can enhance the transport capacity of fungi and plants for rare earth elements, thereby enriching rare earth elements. Therefore, the *Dicranopteris linearis* DlNREET1 protein can be used to enhance the accumulation capacity of fungi or plants for rare earth elements or to cultivate transgenic plants with rare earth remediation functions.

[0014] This invention provides a DlNREET1 protein, the amino acid sequence of which is shown in SEQ ID NO.2.

[0015] The present invention also provides the encoding gene of the DlNREET1 protein of the gentian.

[0016] Specifically, the nucleotide sequence of the encoding gene is shown in SEQ ID NO.1.

[0017] The present invention also provides a recombinant expression vector containing the gene encoding the DlNREET1 protein of *D. spp.*

[0018] The present invention also provides a recombinant engineered bacterium containing the above-mentioned recombinant expression vector.

[0019] This invention utilizes yeast and the model plant Arabidopsis thaliana to demonstrate that overexpression of the *DlNREET1* protein can enhance the ability of yeast and plants to accumulate rare earth elements. Therefore, this invention applies for protection of the *DlNREET1* protein, the encoding gene, and the recombinant expression vector in enhancing the ability of fungi to accumulate rare earth elements.

[0020] This invention also applies for protection of the application of the DlNREET1 protein of *Dictyophora indica*, the encoding gene, the recombinant expression vector, or the recombinant engineered bacteria in improving the ability of plants to enrich rare earth elements.

[0021] Specifically, the ability of plants to accumulate rare earth elements is enhanced by expressing the DlNREET1 protein in plants.

[0022] This invention also seeks to protect the application of the DlNREET1 protein of *Dictyophora indica*, the encoding gene, the recombinant expression vector, or the recombinant engineered bacteria in the cultivation of transgenic plants with rare earth remediation functions.

[0023] As an alternative implementation, the plant is Arabidopsis thaliana.

[0024] The present invention has the following beneficial effects:

[0025] This invention screened and identified a protein with rare earth element transport capabilities from the plant *Miscanthus sinensis*, named it DlNREET1, and cloned the gene sequence encoding this protein. This invention demonstrated that overexpression of this protein in fungi or plants can enhance their ability to accumulate rare earth elements. This protein can be used to cultivate transgenic plants with rare earth remediation capabilities, and further for the remediation and treatment of rare earth element contaminated soil. The *Miscanthus sinensis* DlNREET1 protein and its encoding gene described in this invention are of great significance for the remediation and resource recovery of rare earth contaminated soil. Attached Figure Description

[0026] Figure 1To verify the rare earth element transport function of the DlNREET1 protein of *Saccharomyces cerevisiae* BY4741; Figure A shows the sensitivity test results of yeast containing and without the DlNREET1 gene to rare earth elements; Figure B shows the determination of rare earth element content in yeast containing and without the DlNREET1 gene after culturing in a medium containing rare earth elements; * indicates significant difference, P < 0.05.

[0027] Figure 2 The results show the determination of rare earth element content in the aboveground parts and roots of Arabidopsis thaliana overexpressing DlNREET1 and wild-type Arabidopsis thaliana; * in the figure indicates significant difference, P<0.05. Detailed Implementation

[0028] 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.

[0029] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0030] Example 1: Cloning of the DlNREET1 gene from *Dendrobium nobile*

[0031] This invention screened and identified a protein with rare earth transport capabilities from a three-generation full-length transcriptome database of the rare earth hyperaccumulator *Dicranopteris linearis*. This protein was named DlNREET1 (Dicranopteris linearis NRAMPRare Earth Element Transporter 1), and its amino acid sequence is shown below (SEQ ID NO.2):

[0032]

[0033] Based on this sequence, the present invention cloned the gene encoding the DlNREET1 protein of *D. cyperus*, namely the *D. cyperus* DlNREET1 gene, and the cloning method is as follows:

[0034] 1. RNA extraction and reverse transcription:

[0035] Fresh root tissues of *Dictamnus dasycarpus* cultured in hydroponics were collected. Total RNA was extracted from the *Dictamnus dasycarpus* tissues using an RNA extraction kit (Sigma-Aldrich). cDNA was obtained by reverse transcription using the HiScriptII OneStep RT-PCR kit (Vazyme Biotech). The specific steps were performed according to the kit instructions.

[0036] 2. Cloning primers:

[0037] The cloning primers for the DlNREET1 gene of *Dictyophora indica* are shown below:

[0038] Front primer F: GGATGCAAAACGATGCTTTA

[0039] Terminal primer R: ACCATTACAATGGCATCACT

[0040] 3. PCR amplification:

[0041] Using the reverse-transcribed *Dictamnus dasycarpus* cDNA as a template, high-fidelity enzyme I-5 was employed. TM PCR amplification was performed using 2× High-Fidelity MasterMix (MCLAB, I5HM-200). 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, 2 μL of end primer R, and ddH2O to make up the remainder. 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℃.

[0042] 4. PCR product verification:

[0043] 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 *DlNREET1* gene is as follows (SEQ ID NO.1):

[0044]

[0045]

[0046] Example 2

[0047] This invention utilizes *Saccharomyces cerevisiae* BY4741 to verify the function of the *DlNREET1* protein described in Example 1, as follows:

[0048] 1. Construction of the DlNREET1 yeast expression vector:

[0049] Using the cloning vector of the *DlNREET1* gene constructed in Example 1 as a template, cloning primers with homologous arm sequences were designed using Novizan's CEDesign software, and the homologous arm DlNREET1 sequence was amplified and cloned using PCR. After gel purification, the PCR product was homologously recombined between the Kpn I and BamHI restriction enzyme sites of the pYES2 expression vector using a one-step rapid cloning kit to obtain the recombinant expression vector pYES2::DlNREET1 containing DlNREET1. The constructed recombinant expression vector was retransformed into *E. coli* DH5α competent cells, and the sequence in the vector was verified according to the method described in Example 1 to ensure that DlNREET1 was correctly ligated into the recombinant expression vector pYES2::DlNREET1.

[0050] 2. Transformation and identification of yeast competent cells:

[0051] Using a rapid yeast transformation kit, the validated pYES2::DlNREET1 recombinant yeast expression vector and the empty vector pYES2 were transformed into the Saccharomyces cerevisiae BY4741 strain, respectively. The transformed yeast was cultured on SD-U selective medium to screen for positive colonies. Plasmids of positive monoclonal yeast strains were extracted and transformed into Escherichia coli DH5α for secondary sequencing verification.

[0052] 3. Yeast sensitivity test:

[0053] The validated single-clone yeast containing the pYES2::DlNREET1 recombinant expression vector and the pYES2 empty vector (i.e., yeast containing the DlNREET1 gene and blank control) were placed in SD-U liquid medium and cultured overnight (30℃, 200rpm); OD 600 When the value is 0.8 to 1.0, follow the 10 -1 10 -2 10 -3 10 -4 Perform serial dilutions and spot the samples on solid culture media containing 0 μM and 20 μM of the rare earth element lanthanum (La), respectively; incubate the media upside down for 3–7 days, and then observe and photograph the samples.

[0054] The results of the sensitivity test for rare earth elements in yeasts containing and without the DlNREET1 gene are as follows: Figure 1 As shown in A, by Figure 1As shown in A, yeasts containing the DlNREET1 gene and yeasts without the DlNREET1 gene exhibit the same growth pattern on a medium without rare earth elements. However, when both types of yeasts are inoculated onto a medium containing La, the growth of yeasts containing the DlNREET1 gene is significantly inhibited, indicating that they may be poisoned due to absorbing more rare earth element La.

[0055] 4. Yeast rare earth enrichment experiment:

[0056] To further verify the function of the DlNREET1 gene, yeast containing and without the DlNREET1 gene were transferred to SD-U liquid medium and cultured until the yeast reached the OD level. 600 When the concentration was 1.0, the yeast cells were centrifuged and resuspended, and then transferred to yeast culture media containing light rare earth elements (10 μM LaCl3, 10 μM NdCl3) and heavy rare earth elements (10 μM YbCl3) respectively (the culture medium was free of phosphorus and the pH was 4.2). After enriching rare earth elements by culturing in a shaker at 30°C for 2 h, the yeast cells were collected. The collected yeast cells were first cleaned three times with 20 mM MES-10 mM EDTA to remove the rare earth elements adsorbed on the surface of the yeast cells, and then washed three times with sterile water. The yeast cells were then dried in an oven at 60°C for 3 days. The dried yeast cells were weighed, digested, and the rare earth content in the cells was determined by ICP-MS.

[0057] The results of rare earth element content determination in yeast containing and without the DlNREET1 gene are as follows: Figure 1 As shown in B, by Figure 1 As shown in B, the yeast expressing the DlNREET1 gene (yeast containing the DlNREET1 gene) showed significantly higher enrichment capacity for the three rare earth elements (La, Nd, and Yb) than the control yeast pYES2 strain (which does not contain the DlNREET1 gene). This indicates that the DlNREET1 protein encoded by the DlNREET1 gene has the ability to transport rare earth elements, and its ability to transport light rare earth elements is stronger, making it suitable for the enrichment of rare earth elements.

[0058] Example 3

[0059] This invention utilizes the model plant Arabidopsis thaliana to verify the function of the DlNREET1 protein from *Dictamnus dasycarpus* described in Example 1, as follows:

[0060] 1. Construction of DlNREET1 plant expression vector:

[0061] Using the cloning vector of the *DlNREET1* gene constructed in Example 1 as a template, cloning primers with homologous arm sequences were designed using Novizan's CEDesign software, and the homologous arm DlNREET1 sequence with homologous arm sequences was amplified and cloned by PCR. After the PCR product was purified by gel extraction, DlNREET1 was homologously recombined 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. The recombinant vector was retransformed into *E. coli* DH5α competent cells, and the sequence was verified according to the method described in Example 1.

[0062] 2. Agrobacterium infection and transformation of Arabidopsis thaliana:

[0063] The pSN1305 vector containing the DlNREET1 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; immerse healthy Arabidopsis thaliana flower stamens in infection solution containing Agrobacterium for 45s; each Arabidopsis thaliana flower stamen 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.

[0064] 3. Functional validation of Arabidopsis thaliana DlNREET1 overexpression lines:

[0065] To verify whether the rare earth element transport gene DlNREET1 in *Arabidopsis thaliana* can function in other plants, this invention selected the identified T2 generation homozygous Arabidopsis thaliana lines Ex2 and Ex10, and aseptically incubated them together with wild-type Arabidopsis thaliana WT on 1 / 2 MS medium. Neodymium (Nd) was selected to represent rare earth elements. Arabidopsis thaliana plants with uniform growth were transferred to media containing 1 μM Nd and 5 μM Nd, respectively, and cultured under the same conditions for 7 days before harvesting. The plants were divided into aboveground parts and roots. The roots were analyzed 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 element content in different tissues of the plants.

[0066] The results of rare earth element content determination in the aboveground parts and roots of Arabidopsis thaliana overexpressing DlNREET1 and wild-type Arabidopsis thaliana are as follows: Figure 2 As shown, by Figure 2It was found that the concentrations of rare earth element (Nd) in the roots and shoots of Arabidopsis thaliana lines Ex2 and Ex10, which overexpressed the DlNREET1 gene, were significantly higher than those in wild-type Arabidopsis thaliana (WT). Furthermore, with increasing rare earth element treatment concentrations, the concentrations of rare earth elements in the shoots of Ex2 and Ex10 lines increased significantly, while the increase in the shoots of the WT line was not significant. When the rare earth element content increased to 5 μM, the rare earth element content in the shoots of Arabidopsis thaliana lines overexpressing the DlNREET1 gene was three times that of wild-type Arabidopsis thaliana, indicating that the transgenic plant material constructed using the DlNREET1 gene has a strong rare earth element enrichment capacity.

[0067] 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. A DlNREET1 protein from *Dictamnus dasycarpus*, characterized in that, The amino acid sequence of the protein is shown in SEQ ID NO.

2.

2. The gene encoding the DlNREET1 protein of claim 1.

3. The encoding gene according to claim 2, characterized in that, The nucleotide sequence of the encoding gene is shown in SEQ ID NO.

1.

4. A recombinant expression vector, characterized in that, It contains the gene encoding the DlNREET1 protein as described in claim 2 or 3.

5. A recombinant engineered bacterium, characterized in that, It contains the recombinant expression vector as described in claim 4.

6. The application of the *DlNREET1* protein of claim 1, the encoding gene of claim 2 or 3, or the recombinant expression vector of claim 4 in enhancing the ability of fungi to enrich rare earth elements, characterized in that... The fungus is Saccharomyces cerevisiae, and the rare earth elements are La, Nd, and / or Yb.

7. The application of the *DlNREET1* protein of claim 1, the encoding gene of claim 2 or 3, the recombinant expression vector of claim 4, or the recombinant engineered bacteria of claim 5 in enhancing the ability of plants to accumulate rare earth elements, characterized in that... The plant is Arabidopsis thaliana, and the rare earth element is Nd.

8. The application according to claim 7, characterized in that, The ability of plants to accumulate rare earth elements is enhanced by expressing the DlNREET1 protein in plants.

9. The application of the *DlNREET1* protein of claim 1, the encoding gene of claim 2 or 3, the recombinant expression vector of claim 4, or the recombinant engineered bacteria of claim 5 in cultivating transgenic plants with remediation functions for rare earth element contaminated soil, characterized in that, The plant is Arabidopsis thaliana, and the rare earth element is Nd.

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