Use of pveil3a in improving plant tolerance to heavy metal stress

Abstract

The application provides application of PvEIL3a in improving heavy metal stress tolerance of plants, and relates to the technical field of genetic engineering of energy forage plants.In the application, the trait modification of switchgrass is realized by regulating a key gene of a sulfur assimilation pathway, namely, the switchgrass PvEIL3a gene; in the application, the PvEIL3a protein is overexpressed in switchgrass by using a genetic engineering method, and a switchgrass strain with enhanced nutrient transformation efficiency and enhanced heavy metal resistance is obtained, which will provide high-quality materials for the modification and efficient utilization of marginal land and the improvement of heavy metal contaminated land, and has important reference significance for improving the utilization of energy plants.

Description

Technical Field

[0001] This application relates to the field of genetic engineering technology for energy forage plants, and in particular to the application of PvEIL3a in improving the tolerance of plants to heavy metal stress. Background Technology

[0002] In the field of heavy metal contaminated soil remediation, phytoremediation is an advanced bioremediation technology that has emerged in recent years and is widely used to reduce the various negative effects of human activities on the environment, especially soil and water pollution. Compared with traditional physical and chemical remediation technologies, phytoremediation technology has the advantages of simple processes, low cost, environmental friendliness, and no secondary pollution. Currently, how to create new, highly efficient remediation plant resources has always been a difficult and hot topic in the field of soil phytoremediation in China.

[0003] Switchgrass is a perennial C4 tall herbaceous plant belonging to the genus Millet in the family Poaceae. It is highly resistant to adverse conditions and can be planted in barren, arid, or saline-alkali marginal lands, making it an excellent plant for environmental restoration. Summary of the Invention

[0004] To address the aforementioned problems, this invention utilizes a full-length cDNA library of switchgrass for cloning. PvEIL3a Genes and their encoded products regulate sulfur assimilation in plants, improve nutrient utilization efficiency and heavy metal resistance. Based on this, molecular design and genetic improvement of switchgrass were carried out to create new germplasm resources with high nutrient utilization and strong stress resistance. Its tolerance and biomass in nutrient-deficient environments, especially sulfur-deficient, nitrogen-deficient and heavy metal stress environments, were comprehensively evaluated, providing high-quality materials for the transformation and efficient utilization of marginal land.

[0005] On the one hand, this application provides the application of PvEIL3a protein or its encoding gene in improving plant nutrient utilization efficiency, promoting plant growth and / or enhancing plant tolerance to heavy metal stress.

[0006] The gene sequence number of the PvEIL3a protein is Pavir.6KG367600.

[0007] Furthermore, improving plant nutrient utilization efficiency includes improving the utilization efficiency of nitrogen and / or sulfur elements in plants.

[0008] The transgenic switchgrass obtained in this application ultimately improved sulfur utilization by 15.8% and nitrogen utilization by 17.4% compared to the wild type.

[0009] Further, the heavy metal is cadmium ion; preferably, the concentration of cadmium ion is 0-200 μM; more preferably, the concentration of cadmium ion is 0-100 μM.

[0010] The concentration of cadmium ions can be selected from any one of the following concentrations: 0, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 20 μM, 25 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, 100 μM, and 200 μM.

[0011] Furthermore, the plant in question is switchgrass.

[0012] In a preferred embodiment, this application uses switchgrass (Alamo) as an example for experimental verification. Those skilled in the art will understand that the specific switchgrass variety has no impact on the experimental results of this application, and readily available switchgrass varieties can be used to complete the technical solution described in this application.

[0013] Further, the amino acid sequence of the PvEIL3a protein is as shown in SEQ ID No. 1 or has at least 98% identity with SEQ ID No. 1; preferably, the gene sequence encoding the PvEIL3a protein is the nucleotide sequence shown in SEQ ID No. 2 or has at least 98% identity with SEQ ID No. 2.

[0014] The amino acid sequence of the PvEIL3a protein is as shown in SEQ ID No. 1 or has 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity with SEQ ID No. 1.

[0015] The gene sequence encoding the PvEIL3a protein is the nucleotide sequence shown in SEQ ID No. 2 or has 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity with SEQ ID No. 2.

[0016] Furthermore, the promotion of plant growth includes promoting plant growth in heavy metal stress environments.

[0017] Preferably, the heavy metal in the heavy metal stress is cadmium ion; preferably, the concentration of cadmium ion is 0-200 μM; more preferably, the concentration of cadmium ion is 0-100 μM.

[0018] In a preferred embodiment, promoting plant growth under abiotic stress conditions refers to promoting the growth of plant roots, stems, and leaves, specifically including increasing the length of the taproot, lateral roots, plant height, and leaf length. This has demonstrated that the transgenic switchgrass constructed using the method of this application exhibits better stress resistance and growth compared to wild-type switchgrass.

[0019] On the other hand, this application also provides a method for improving plant nutrient utilization efficiency, promoting plant growth and / or tolerance to heavy metal stress, the method comprising the step of increasing the expression level of PvEIL3a protein or the mRNA transcription level in plants.

[0020] Furthermore, the method includes the step of introducing the PvEIL3a protein into the plant; preferably, the introduction of the PvEIL3a protein is achieved by introducing a gene sequence encoding the PvEIL3a protein.

[0021] In a preferred embodiment, the gene sequence encoding the PvEIL3a protein is introduced into the plant via Agrobacterium genetic transformation. The Agrobacterium genetic transformation method can be any commonly used method.

[0022] In the above process, those skilled in the art can select known plasmids to construct recombinant vectors according to the actual situation and perform routine optimization and modification on the constructed recombinant vectors. Alternatively, they can use commonly used Agrobacterium and its universal transformation methods to complete the above genetic transformation process. This application does not impose mandatory limitations on the types of plasmids and Agrobacterium.

[0023] The parts of switchgrass used as material for genetic transformation can be seeds, leaves, stems, roots, etc., as long as the genetic transformation process can be completed. This application does not impose too many restrictions on the specific genetic transformation methods or the specific parts of the genetic transformation material.

[0024] Further, the amino acid sequence of the PvEIL3a protein is shown in SEQ ID No. 1; preferably, the gene sequence encoding the PvEIL3a protein is the nucleotide sequence shown in SEQ ID No. 2 or a nucleotide sequence having at least 98% identity with SEQ ID No. 2.

[0025] Furthermore, the plant in question is switchgrass.

[0026] This application focuses on the key role of EIL3a, a member of the EIL family of transcriptional regulators, in plant responses to abiotic stresses. It delves into the potential regulatory mechanisms by which transcription factor PvEIL3a regulates sulfur assimilation, improves nutrient utilization efficiency, and enhances heavy metal resistance. Based on this, using identified transcription factors as targets, molecular design and genetic improvement of switchgrass are conducted to create novel germplasm resources with high nutrient utilization and strong stress resistance. A comprehensive evaluation of its tolerance and biomass under sulfur deficiency and heavy metal stress environments is conducted, providing high-quality materials for the transformation and efficient utilization of marginal lands. This has significant guiding significance for the genetic breeding of energy grasses and the improvement of marginal lands.

[0027] The present invention has the following beneficial effects:

[0028] This invention is the first to regulate the key gene of switchgrass (Swiss millet) in the sulfur assimilation pathway. Panicum virgatum L.) PvEIL3a Genes enable the modification of traits in switchgrass;

[0029] This invention uses genetic engineering techniques to overexpress the PvEIL3a protein in switchgrass, obtaining switchgrass lines with enhanced nutrient conversion efficiency and heavy metal resistance. This will provide high-quality materials for the transformation and efficient utilization of marginal land and the improvement of heavy metal-contaminated land, and has important reference significance for improving the utilization of energy plants. Attached Figure Description

[0030] The accompanying drawings, which are included to provide a further understanding of this application and form part of this application, illustrate exemplary embodiments and are used to explain this application, but do not constitute an undue limitation of this application. In the drawings:

[0031] Figure 1 This is a flowchart of genetic transformation of switchgrass, where (A) is embryogenic callus regeneration, (B) is positive callus selection, (C) is positive callus differentiation, (D) is clustered shoot formation, and (E) is rooting of tissue culture seedlings.

[0032] Figure 2 This is an image showing the electrophoresis results of a positive identification of a transgenic plant.

[0033] Figure 3 This is an elemental analysis chart of transgenic plants, with the left chart showing sulfur content and the right chart showing nitrogen content.

[0034] Figure 4 This is a schematic diagram of the treatment of genetically modified plants with the heavy metal cadmium.

[0035] Figure 5These are heavy metal content and phenotypic diagrams of transgenic plants. In the diagram, a is a statistical graph of cadmium content in the leaves and roots of wild-type and transgenic plants treated with 20 μM cadmium; b is a statistical graph of cadmium content in the leaves and roots of wild-type and transgenic plants treated with 50 μM cadmium; c is a statistical graph of physiological indicators of wild-type and transgenic plants treated with cadmium; and d is a phenotypic photograph of wild-type and transgenic plants treated with cadmium. Detailed Implementation

[0036] To more clearly illustrate the overall concept of this application, a detailed description is provided below with reference to the accompanying drawings and embodiments. Numerous specific details are set forth in the following description to provide a more thorough understanding of the invention. However, it will be apparent to those skilled in the art that the invention can be practiced without one or more of these details. In other instances, certain technical features well-known in the art have not been described to avoid confusion with the invention.

[0037] Unless otherwise specified in the examples, the conditions shall be performed under conventional conditions or conditions recommended by the manufacturer. Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention shall employ conventional techniques in the fields of microbiology, biochemistry, analytical chemistry, cell culture, and related areas.

[0038] Among them, the switchgrass uses the Alamo variety.

[0039] Unless otherwise specified, in the following embodiments, reagents or instruments whose manufacturers are not indicated are all conventional products that can be purchased commercially.

[0040] In addition, the "water" mentioned in this invention includes any feasible water that can be used in the art, such as deionized water, distilled water, ion-exchanged water, double-distilled water, high-purity water, and purified water.

[0041] In the following embodiments, unless otherwise specified, % means wt%, i.e., weight percentage.

[0042] The culture media involved in the following examples include:

[0043] Transformation inoculum (SM3) (per liter): MS powder 4.43 g, sucrose 30 g, 2,4-D 3 mg, 6-BA 0.15 mg, pH adjusted to 6.0 with 2 M KOH solution, autoclaved at 121°C for 15 min.

[0044] Subculture medium (SM5) (per liter): MS powder 4.43 g, sucrose 30 g, 2,4-D 5 mg, 6-BA 0.15 mg, pH adjusted to 6.0 with 2 M KOH solution, agar 7.8 g, autoclaved at 121℃ for 15 min.

[0045] Screening medium (SM3) (per liter): MS powder 4.43 g, sucrose 30 g, 2,4-D 3 mg, 6-BA 0.15 mg, pH adjusted to 6.0 with 2 M KOH solution, agar 7.8 g, autoclaved at 121℃ for 15 min. After cooling the medium to approximately 60℃, add Tim to 400 mg / L and Hyg to 30 mg / L.

[0046] Differentiation medium (MSBK) (per liter): MS powder 4.43 g, sucrose 30 g, 6-BA 0.5 mg, KT 1 mg, pH adjusted to 6.0 with 2M KOH solution, agar 7.8 g, autoclaved at 121℃ for 15 min. After cooling the medium to approximately 60℃, add Tim to 400 mg / L and Hyg to 5 mg / L.

[0047] Rooting medium (MS0) (per liter): 2.215 g MS powder, 10 g sucrose, pH adjusted to 6.0 with 2 M KOH solution, 7.8 g agar, autoclaved at 121°C for 15 min. After cooling the medium to approximately 60°C, add Tim to 300 mg / L and Hyg to 15 mg / L.

[0048] 1. Construction of a full-length cDNA library of switchgrass

[0049] Young leaves of *Stichopebus parvifolia* seedlings were collected, and total RNA was extracted using the TransZol method (TransGen Biotech, catalog number: ET101-01). The content and purity of total RNA were detected using a nucleic acid analyzer. The total RNA was then used for reverse transcription. The kit used was [kit name missing]. EasyScript ® One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotech, Catalog No.: AE311-02). The specific steps are as follows:

[0050] 1.1 Homogenization

[0051] Take tissue samples of switchgrass stored at -80℃, transfer them to a mortar pre-cooled with liquid nitrogen, and grind the plant tissue continuously with a pestle until the sample is ground into powder. Transfer the powder to a 1.5 mL pre-cooled centrifuge tube. Remove the liquid nitrogen from the tube to prevent cap breakage. Take 50-100 mg of the ground sample (approximately 1 / 5 of the tube volume), add 1 mL of TransZol, and vortex to mix, obtaining a homogenized sample.

[0052] 1.2 Layering

[0053] Place the homogenized sample on ice for 5 minutes to allow complete separation of nucleoproteins. Add 200 mL of chloroform, tighten the cap, shake vigorously for 15 seconds, and place on ice for 3 minutes. Centrifuge at high speed at 4°C for approximately 15 minutes to separate the phases, which will consist of a colorless supernatant, a white protein layer, and a red organic lower phase.

[0054] 1.3 RNA precipitation

[0055] At room temperature, transfer approximately 500-650 mL of the supernatant to a new, pre-chilled 1.5 mL centrifuge tube. Add an equal volume of isopropanol to the supernatant, gently invert to mix, and incubate at -20°C for 30 min. Centrifuge at 4°C on high speed for 10 min, and discard the supernatant to remove the isopropanol.

[0056] Once completed, repeat the above steps once more.

[0057] 1.4 RNA washing

[0058] Add at least 1 mL of 75% ethanol, vortex to mix, and centrifuge at 7500 g for 5 min at 4°C.

[0059] 1.5 RNA Redissolution

[0060] Discard the supernatant, absorb the ethanol, and let it air dry at room temperature for 10 minutes after opening the lid.

[0061] Add 30-40 μL of RNase-free ddH2O to dissolve the RNA for 2-3 min.

[0062] 1.6 The content and purity of total RNA were determined using agarose gel electrophoresis and a nucleic acid analyzer.

[0063] 1.7 RNA reverse transcription

[0064] The total RNA obtained using the above method was used for reverse transcription. Specifically, the RNA was added to microcentrifuge tubes according to the system in Table 1 below and mixed thoroughly. First, the RNA template, Anchored Oligo (dT), and RNase-free water were mixed and incubated at 65°C for 5 min, then placed on ice for 2 min. Next, the other reaction components were added, gently mixed, and incubated at 42°C for 15 min. The TransScript RT and gDNA Remover were then inactivated by heating at 85°C for 5 s to obtain switchgrass cDNA.

[0065] Table 1

[0066]

[0067] 2 PvEIL3a Gene cloning and overexpression vector construction

[0068] 2.1 PvEIL3a Gene cloning

[0069] Using switchgrass cDNA as a template, and employing forward primer (F) and reverse primer (R):

[0070] F: 5′-ATGGACCATCTTGGTATTCTTG-3′,

[0071] R: 5′-TCATGTGCCCAGGTATGGCATC-3′,

[0072] PCR amplification was performed, and 5 μL of the amplification product was added to 1 μL of loading buffer for electrophoresis. After 10 min, the gel image was observed using a gel imaging system. The amplified fragment was approximately 1100 bp, yielding switchgrass. PvEIL3a The fragment (amino acid sequence as shown in SEQ ID No. 1, nucleotide sequence as shown in SEQ ID No. 2) was recovered. The reaction system was scaled up, and the gene fragment was recovered using a gel extraction kit.

[0073] In the PCR amplification reaction, the reaction system was 20 μL: 10 μL of 2×Phanta enzyme, 1 μL of forward primer, 1 μL of reverse primer, 6 μL of water, and 2 μL of cDNA.

[0074] The PCR amplification program reaction conditions were as follows: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 15 s, 57℃ annealing for 15 s, 72℃ extension for 2 min, for a total of 35 cycles, 72℃ extension for 10 min, and cooling at 16℃.

[0075] 2.2 Construction of intermediate carrier

[0076] Simultaneously utilizing restriction endonucleases Ahd I. The pGWC vector was digested with enzymes at 37℃ for 1 h, loading buffer was added, and agarose gel electrophoresis was performed. The gel image was observed, and the empty vector fragment was recovered using a recovery kit. The reaction system for digesting the intermediate vector was as follows: Ahd I 1 μL, 2×buffer 2 μL, carrier 10 μL, water 7 μL.

[0077] Using the infusion method to extract switchgrass PvEIL3a The fragment was ligated into the pGWC vector. The reaction was carried out at 37°C for 30 min using Exnase II. The ligation product was then transformed into *E. coli* DH5α using a heat shock method, followed by plating. The reaction mixture consisted of: 2 μL pGWC vector, 1 μL enzyme, 1 μL fragment, and 1 μL 5×CE II buffer.

[0078] 2.3 Identification of intermediate carriers

[0079] Single clones were selected and cultured in LB medium for bacterial PCR detection. The 20 μL reaction mixture contained: 10 μL buffer, 1 μL each of forward and reverse primers, 1 μL template, and 7 μL ddH2O. Reaction conditions were: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 2 min, for a total of 29 cycles, followed by a final extension at 72℃ for 10 min, and cooling at 16℃. The PCR products were detected by agarose gel electrophoresis. PCR-positive single clones were sequenced; if the sequencing was correct, plasmids were extracted from the bacterial culture, and the intermediate vector was named pGWC-. PvEIL3a .

[0080] 2.4 Construction of overexpression vectors

[0081] The intermediate vector and the final vector were subjected to an LR reaction at 25°C for 4 hours to deliver the target gene. PvEIL3a The intermediate vector was ligated into the PANIC6B vector, followed by transformation of *E. coli* DH5α. The reaction mixture consisted of 1 μL of intermediate vector, 0.5 μL of final vector, 0.5 μL of LR enzyme, and 3 μL of water. The final product was... pANIC6B - PvEIL3a The carrier.

[0082] 3 PvEIL3a -OE transgenic plants obtained

[0083] Using 2 pANIC6B - PvEIL3aThe target gene was introduced into the embryogenic callus cell line of wild-type switchgrass using Agrobacterium-mediated genetic transformation (EHA105). After tissue culture, resistance selection and seedling differentiation, transgenic plants were obtained (Genetic transformation method reference: 2009, Agrobacterium-mediated transformation of switchgrass and inheritance of the transgenes. BioEnergy Research 2: 275–283).

[0084] Preparation of embryogenic callus cell lines: Surface disinfection was performed on the nodes of switchgrass containing young spikelets. Initial surface disinfection was carried out in 75% alcohol for 30 min, followed by rinsing three times with sterile water and blotting dry with sterile filter paper. The stems were cut open with a blade, and the young spikelets were removed. The spikelets were placed on SM5 medium, ensuring full contact with the medium. The medium was then cultured in the dark at 24℃ for 8-12 weeks to obtain embryogenic callus cell lines.

[0085] Preparation of Agrobacterium: pANIC6B - PvEIL3a Agrobacterium strain EHA105 was introduced for gene transformation. Single Agrobacterium colonies were transferred to liquid Luria-Bertani medium containing 50 mg / L kanamycin and 25 mg / L rifampin. The cultures were incubated at 28°C and 10,000 rpm on a shaker until OD (outcome limit) was reached. 600 The concentration reached 0.8-1.0. A 100 μM acetylsuccinone solution was prepared and added to the culture, with shaking continued for 1 h. Then, the cells were centrifuged at 2,400 × g for 15 min and resuspended in transformation medium (SM3). The Agrobacterium density (OD) of the suspension was determined. 600 The value was adjusted to approximately 0.5.

[0086] Genetic transformation: Embryogenic callus cells of switchgrass were immersed in a culture vessel containing Agrobacterium suspension. The culture vessel was placed in a vacuum chamber and evacuated for 10 min. After releasing the vacuum, the callus tissue and Agrobacterium were sonicated for 5 min, followed by another 10 min of evacuation. After releasing the vacuum, the infection solution was discarded, and the transformed callus tissue was transferred to filter paper and co-cultured in an empty culture dish at 25°C in the dark.

[0087] Resistance screening: After co-culturing for 2 days, filter paper carrying transformed callus was transferred to selection medium (SM5) for further culture. Two weeks later, the callus was transferred to a new selection medium (SM3) for continued selection.

[0088] Seedling differentiation: After 5-8 weeks of screening, the resistant callus tissue obtained was transferred to the differentiation medium MSBK basal medium. The culture dish was placed under light to ensure that the callus could differentiate into buds smoothly.

[0089] Rooting of seedlings: Transfer the regenerated shoots or plants to plastic containers containing rooting medium (MSO). All regenerated seedlings were kept at a temperature of 25°C and a light intensity of 140 μE / m². 2 s 1 Under suitable conditions, the plants were grown in a growing chamber with a photoperiod of 16 hours. After 4-5 weeks, plants with well-developed root systems were transplanted into soil and grown in a greenhouse (16 hours photoperiod, 390 μM). 2 s 1 It grows in the environment.

[0090] Genetic transformation process such as Figure 1 As shown.

[0091] Positive transgenic plants of the target gene were obtained by PCR identification (i.e., PvEIL3a Transgenic switchgrass plants (or switchgrass overexpression plants) were transplanted into soil and grown in a greenhouse. Specific identification methods: Genomic DNA extracted from wild-type and transgenic switchgrass using the 2×CTAB method was used as templates. The upstream and downstream primers (hph3+hph4) for the hygromycin resistance gene, as well as the universal vector primer ZmUbq-F and the downstream primer PvEIL3a-R for the target gene, were used to detect whether the plants contained the hygromycin resistance selection gene and the target gene, respectively. The primer sequences involved are shown in Table 2, and the identification results are as follows: Figure 2 As shown.

[0092] Reaction conditions: 95°C pre-denaturation for 5 min; 95°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 30 s, 29 cycles; 72°C extension for 10 min, cooling at 16°C.

[0093] Table 2

[0094]

[0095] 4 PvEIL3a Elemental analysis of transgenic plants

[0096] The three preparations obtained PvEIL3aLeaves from the I3 region of the E4 stage were selected from OE transgenic switchgrass plants (switchgrass overexpression plants). (Reference: 2013 Standardization of switchgrass sample collection for cell wall and biomass trait analysis. Bioenergy Research 6, 755–762). After freeze-drying, the samples were milled to homogeneity using a ball mill. The samples were washed with ethanol and water for 2 days, dried, and then accurately weighed (1-2 mg). The content of C, H, S, and other elements in the samples was analyzed using a vario EL cube. Untransgenic common switchgrass materials (wild-type switchgrass plants) were treated in the same way as a control group. The results are shown in Table 3. Figure 3 As shown.

[0097] Table 3

[0098]

[0099] As shown in Table 3 and Figure 5 The results showed that the S and N contents in the leaves of overexpressing plants were significantly increased, indicating that... PvEIL3a Overexpression can improve the plant's utilization of nutrients.

[0100] 5 PvEIL3a Observation of heavy metal resistance in transgenic plants

[0101] Wild-type switchgrass plants (K1) and switchgrass overexpression plants ( PvEIL3a The phenotype and tolerance of switchgrass were observed under different cadmium ion concentrations (0, 25 μM, 50 μM, 100 μM) after heavy metal treatment with cadmium ions (-OE).

[0102] The differentiated K1 and PvEIL3a -OE plantlets were placed in vials and cultured for a period of time. Afterward, the roots were uniformly trimmed, and the plants were transferred to tissue culture vials containing different heavy metal concentrations of MS0 medium. The growth of the tissue culture seedlings was recorded by photographing them weekly. Figure 4 As shown, leaf and root samples were collected from tissue culture seedlings over a period of 3 to 4 weeks to detect the cadmium content. ICP-MS was used for heavy metal content detection. First, the samples were vacuum freeze-dried. 0.2 g of sample (accurate to 0.001 g) was placed in a polytetrafluoroethylene digestion vessel, 5 mL of nitric acid was added, and digestion was performed according to microwave digestion parameters. After digestion, the sample was removed at 115℃, and the solution was transferred to a 50 mL volumetric flask, allowed to stand or filtered, and then transferred to the instrument. The detection results are shown in Tables 4 and 5. Figure 5 As shown.

[0103] Table 4. Cadmium content in transgenic plants at 28 days (μg / g / DW)

[0104]

[0105] Table 5. Cadmium content in wild-type plants after 28 days (μg / g / DW)

[0106]

[0107] The results showed that the overexpressing plants contained more cadmium compared to the wild-type plants. Physiologically, the overexpressing plants were taller, had longer taproots and lateral roots, and exhibited no pathological characteristics, indicating that the overexpressing plants had significant tolerance to cadmium ions. PvEIL3a Gene overexpression can enhance the heavy metal resistance of switchgrass.

[0108] The above description is merely an embodiment of this application and is not intended to limit the scope of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the scope of the claims of this application.

Claims

1. Application of overexpression of PvEIL3a protein or its encoding gene in improving plant nutrient utilization efficiency, promoting plant growth and / or enhancing plant tolerance to heavy metal stress; Improving plant nutrient utilization efficiency includes improving the utilization efficiency of nitrogen and sulfur elements in plants; The promotion of plant growth includes promoting plant growth in heavy metal stress environments, which includes promoting the increase of lateral root length and / or plant height. The heavy metal is cadmium ions; The plant in question is *Symplocos serrata*. The amino acid sequence of the PvEIL3a protein is shown in SEQ ID No.

1.

2. Use according to claim 1, characterized in that, The concentration of cadmium ions is 1-200 μM.

3. Use according to claim 2, characterized in that, The concentration of cadmium ions is 1-100 μM.

4. Use according to claim 1, characterized in that, The gene sequence encoding the PvEIL3a protein is shown in SEQ ID No.

2.

5. A method for improving nutrient use efficiency, promoting plant growth and / or tolerance to heavy metal stress in plants, characterized in that, The method includes the step of increasing the expression level of PvEIL3a protein in plants; Improving plant nutrient utilization efficiency includes improving the utilization efficiency of nitrogen and sulfur elements in plants; The promotion of plant growth includes promoting plant growth in heavy metal stress environments, which includes promoting the increase of lateral root length and / or plant height. The heavy metal is cadmium ions; The plant in question is *Symplocos serrata*. The amino acid sequence of the PvEIL3a protein is shown in SEQ ID No.

1.

6. The method of claim 5, wherein, The expression level of PvEIL3a protein in plants can be increased by introducing the gene sequence encoding PvEIL3a protein.

7. The method according to claim 6, characterized in that, The gene sequence encoding the PvEIL3a protein is shown in SEQ ID No. 2.

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