Application of ramie BnWRKY50 gene in increasing cadmium accumulation in plant roots and enhancing cadmium tolerance of plants

Abstract

The invention discloses application of a ramie BnWRKY50 gene in increasing cadmium accumulation in plant roots and enhancing cadmium tolerance of plants, and belongs to the technical field of plant genetic engineering. The invention provides application of a ramie BnWRKY50 gene in increasing cadmium accumulation in plant roots and enhancing cadmium tolerance of plants. The CDS nucleotide sequence of the ramie BnWRKY50 gene is as shown in SEQ ID NO. 1. The specific functions of the ramie BnWRKY50 gene in cadmium accumulation and cadmium tolerance regulation are determined for the first time, and after the ramie BnWRKY50 gene is over-expressed in plants, the cadmium tolerance of the plants is enhanced, the cadmium accumulation amount of plant roots is remarkably increased, and the cadmium accumulation amount of overground parts is reduced.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, specifically to the application of the ramie BnWRKY50 gene in increasing cadmium accumulation in plant roots and enhancing plant cadmium tolerance. Background Technology

[0002] Cadmium (Cd) is a non-essential heavy metal element for plant growth, but it is also a highly toxic environmental pollutant. It is difficult to degrade in soil and has strong mobility, causing irreversible toxicity to crop growth and development. Furthermore, Cd can accumulate in the human body through the food chain, posing a serious threat to human health. Ramie (Boehmerianivea L.) is a perennial bast fiber crop native to my country, with a well-developed root system, rapid growth, strong tolerance and accumulation capacity for heavy metals, and does not enter the food chain, making it considered an ideal crop for remediating Cd-contaminated soil. Field trials for Cd-contaminated soil remediation have shown that after four years of ramie planting, soil Cd content decreased from 1.8 mg / kg to 0.8-1.0 mg / kg, a reduction of 44%-55%. Hunan Province has also included ramie in its industrial restructuring plan for severely heavy metal-contaminated farmland as one of the preferred remediation crops. However, there is currently a lack of suitable ramie varieties for remediation, and there is an urgent need to cultivate new ramie varieties with high fiber yield, excellent quality, strong cadmium tolerance, and high accumulation. Molecular breeding methods can accelerate the breeding process of perennial ramie, but the current lack of available Cd tolerance-related genes seriously affects the development of molecular breeding work for Cd pollution remediation varieties.

[0003] WRKY transcription factors (TFs) are one of the largest and most important families of transcription factors in plants. They contain a conserved WRKYGQK domain at their N-terminus and play a crucial role in plant growth, development, and responses to biotic and abiotic stresses. Numerous studies have shown that WRKY proteins regulate cadmium tolerance from multiple dimensions, including the uptake, transport, compartmentalization, regulation of chelating agent synthesis, and antioxidant systems. PyWRKY71 regulates the scavenging of reactive oxygen species (ROS), improving plant resistance to cadmium; PyWRKY48 regulates the expression of glutathione (GSH) and phytochelate peptides (PC), increasing Cd... 2+ Chelation reduces cadmium toxicity; ZmWRKY64 and AtWRKY33 can regulate the expression of cadmium transporters HMA2 and NRAMP1, reducing Cd influx and improving plant tolerance. There are no reports on the specific functions of WRKY family genes in ramie under cadmium stress. Discovering genes that increase cadmium accumulation and enhance cadmium tolerance has significant theoretical and practical implications for the breeding of cadmium-remediation plant varieties. Summary of the Invention

[0004] The purpose of this invention is to overcome the above-mentioned technical deficiencies and provide an application of the ramie BnWRKY50 gene in increasing cadmium accumulation in plant roots and enhancing cadmium tolerance in plants, thereby solving the technical problem of how to increase cadmium accumulation and enhance cadmium tolerance in plants in the prior art.

[0005] To achieve the above-mentioned technical objectives, the present invention provides an application of the ramie BnWRKY50 gene in increasing cadmium accumulation in plant roots and enhancing cadmium tolerance in plants. The CDS nucleotide sequence of the ramie BnWRKY50 gene is shown in SEQ ID NO.1.

[0006] In any embodiment, the amino acid sequence of the protein encoded by the ramie BnWRKY50 gene is shown in SEQ ID NO.2.

[0007] In any implementation, by upregulating the expression level of the BnWRKY50 gene in plants or increasing the content of the protein encoded by the BnWRKY50 gene in plants, cadmium accumulation in plant roots and cadmium tolerance are increased.

[0008] In any embodiment, the plant includes Arabidopsis thaliana and ramie.

[0009] In addition, the present invention also proposes a primer pair for amplifying the above-mentioned ramie BnWRKY50 gene, the base sequences of which are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

[0010] Furthermore, this invention also proposes a method for increasing cadmium accumulation in plant roots and enhancing cadmium tolerance in plants. The above-mentioned ramie BnWRKY50 gene is introduced into recipient plants to obtain transgenic plants that overexpress the BnWRKY50 gene. The transgenic plants have increased cadmium accumulation in their roots and enhanced cadmium tolerance.

[0011] In any embodiment, an overexpression vector containing the BnWRKY50 gene is constructed, the overexpression vector is transformed into Agrobacterium, and the BnWRKY50 gene is introduced into the recipient plant via Agrobacterium-mediated inflorescence staining.

[0012] In any embodiment, the overexpression vector is obtained by ligating the BnWRKY50 gene to the XbaⅠ and BamHI restriction sites of pEGOEP35S-G418 through homologous recombination based on pEGOEP35S-G418; the primer pair base sequences for amplifying the BnWRKY50 gene fragment for homologous recombination are shown in SEQ ID NO.5 and SEQ ID NO.6.

[0013] In any embodiment, the method further includes: screening and identifying the obtained transgenic plants, wherein the primer pairs used for identification have base sequences as shown in SEQ ID NO.7 and SEQ ID NO.8.

[0014] Furthermore, this invention also proposes the application of the aforementioned ramie BnWRKY50 gene in the cultivation of plant varieties for cadmium-contaminated soil remediation.

[0015] Compared with the prior art, the beneficial effects of the present invention include: the present invention clarifies for the first time the specific function of the ramie BnWRKY50 gene in the regulation of cadmium accumulation and cadmium tolerance, and that overexpression in plants enhances cadmium tolerance, significantly increases cadmium accumulation in plant roots, and reduces cadmium accumulation in aboveground parts.

[0016] Plants overexpressing BnWRKY50 showed significantly better growth under cadmium stress than wild-type plants, effectively alleviating the inhibition of root growth by cadmium. At the same time, they significantly increased the activity of antioxidant enzymes such as SOD, POD, and CAT, reduced the content of malondialdehyde (MDA) and hydrogen peroxide (H2O2), reduced the accumulation of reactive oxygen species, and enhanced the plant's defense against oxidative damage caused by cadmium stress.

[0017] Overexpression of BnWRKY50 significantly increased cadmium accumulation in plant roots and reduced cadmium accumulation in aboveground parts. Non-invasive microtesting confirmed that the net cadmium ion influx rate in the roots of transgenic plants was significantly increased. At the same time, this gene reduced cadmium transport to aboveground parts by inhibiting the expression of cadmium transporter genes AtHMA2, AtHMA4, AtNRAMP3 and AtNRAMP4, thereby achieving cadmium enrichment in the roots.

[0018] For crops used to remediate cadmium-contaminated soil, an increase in cadmium content in the roots can improve soil remediation efficiency and shorten the remediation period; a decrease in cadmium content in the aboveground parts makes it possible to safely utilize the fiber, feed, and other products of the remediation plants, thus expanding the range of applications for these plants.

[0019] Overexpression of BnWRKY50 significantly improves the cadmium tolerance of plants, enabling remediation plants to be planted in heavily cadmium-polluted areas and mining areas, and enhancing the remediation and coverage capacity of plants for cadmium-polluted soil.

[0020] The BnWRKY50 gene provided by this invention offers an important gene resource for molecular breeding of plants for cadmium pollution remediation. The constructed overexpression vector and screening and identification methods provide a complete technical system for cultivating cadmium-tolerant and cadmium-accumulating remediation plant varieties, which has significant application value and market prospects in the field of phytoremediation of cadmium-contaminated soil. Attached Figure Description

[0021] Figure 1 This is a diagram showing the amplification results of the gene BnWRKY50 in Example 1 of the present invention.

[0022] Figure 2 This is a diagram showing the amplification results of the BnWRKY50 fragment with homologous arms in Example 2 of the present invention.

[0023] Figure 3 This is a diagram showing the construction results of the BnWRKY50 overexpression vector in Example 2 of the present invention.

[0024] Figure 4 This is a diagram showing the screening and identification results of Arabidopsis thaliana overexpressing BnWRKY50 in Example 4 of the present invention. Figure 4 Figure A in the graph represents the resistance screening results using G418. Figure 4 B in the figure represents the PCR identification results of the transgenic Arabidopsis thaliana.

[0025] Figure 5 This is a graph showing the results of detecting the expression level of the BnWRKY50 gene in plants overexpressing the gene in Example 5 of this invention.

[0026] Figure 6 This is a phenotypic diagram of Arabidopsis thaliana under cadmium stress in Example 6 of the present invention, wherein... Figure 6 In the diagram, A represents the root length phenotypic observation of the WT and BnWRKY50-OE lines. Figure 6 B in the figure represents a quantitative analysis of root length, fresh weight, and root length inhibition rate.

[0027] Figure 7 This is a diagram showing the phenotypic and physiological changes of Arabidopsis thaliana under soil cultivation conditions in Example 6 of the present invention. Figure 7 In the diagram, A represents the DAB and NBT staining pattern. Figure 7 The graphs showing BG represent the results of measurements of antioxidant enzyme activities (SOD, POD, CAT), MDA and H2O2 content, and SPAD values. Figure 7 H in the figure represents the phenotypic observation of WT and BnWRKY50-OE plants after Cd treatment under soil culture conditions. The top row shows the BnWRKY50-OE1 (left) and BnWRKY50-OE2 (right) lines, and the bottom row shows the WT plants.

[0028] Figure 8 This is a graph showing the results of the analysis of expression levels of relevant functional genes in Arabidopsis thaliana in Example 6 of the present invention.

[0029] Figure 9 This is a graph showing the cadmium content detection results in Arabidopsis thaliana in Example 7 of the present invention.

[0030] Figure 10 This is a graph showing the results of cadmium flow rate measurement in Arabidopsis thaliana roots in Example 7 of the present invention. Detailed Implementation

[0031] The "range" disclosed in this application is defined by a lower limit and an upper limit. A given range is defined by selecting a lower limit and an upper limit, which define the boundaries of a particular range. Ranges defined in this way can include or exclude endpoints and can be arbitrarily combined; that is, any lower limit can be combined with any upper limit to form a range. For example, if ranges of 60~120 and 80~110 are listed for a specific parameter, it is also expected that ranges of 60~110 and 80~120 are also included. Furthermore, if minimum range values ​​of 1 and 2 are listed, and if maximum range values ​​of 3, 4, and 5 are listed, then the following ranges are all expected: 1~3, 1~4, 1~5, 2~3, 2~4, and 2~5. In this application, unless otherwise stated, the numerical range "a~b" represents a shortened representation of any combination of real numbers between a and b, where a and b are real numbers. For example, the numerical range "0~5" indicates that all real numbers between "0~5" have been listed in this article; "0~5" is simply a shortened representation of these numerical combinations. Furthermore, when a parameter is stated as an integer ≥2, it is equivalent to disclosing that the parameter is, for example, an integer such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0032] Unless otherwise specified, the terms "comprising" and "including" as used in this application can be open-ended or closed-ended. For example, "comprising" and "including" can mean that other components not listed may also be included, or that only the listed components may be included.

[0033] Unless otherwise specified, the term "or" is inclusive in this application. For example, the phrase "A or B" means "A, B, or both A and B". More specifically, the condition "A or B" is satisfied by any of the following conditions: A is true (or exists) and B is false (or does not exist); A is false (or does not exist) and B is true (or exists); or both A and B are true (or exist).

[0034] This specific embodiment provides an application of the ramie BnWRKY50 gene in increasing cadmium accumulation in plant roots and enhancing cadmium tolerance in plants. The CDS nucleotide sequence of the ramie BnWRKY50 gene is shown in SEQ ID NO.1; the amino acid sequence of the protein encoded by the ramie BnWRKY50 gene is shown in SEQ ID NO.2. By upregulating the expression level of the BnWRKY50 gene in plants or increasing the content of the protein encoded by the BnWRKY50 gene in plants, cadmium accumulation in plant roots and enhanced cadmium tolerance are achieved.

[0035] In some embodiments, the plants include Arabidopsis thaliana and ramie.

[0036] This specific embodiment also proposes a primer pair for amplifying the ramie BnWRKY50 gene described above, the base sequences of which are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

[0037] This specific embodiment also proposes a method to increase cadmium accumulation in plant roots and enhance cadmium tolerance in plants. The method involves introducing the aforementioned ramie BnWRKY50 gene into recipient plants to obtain transgenic plants overexpressing the BnWRKY50 gene. The transgenic plants exhibit increased cadmium accumulation and enhanced cadmium tolerance in their roots. Further, in some embodiments, an overexpression vector containing the BnWRKY50 gene is constructed, the overexpression vector is transformed into Agrobacterium, and the BnWRKY50 gene is introduced into recipient plants via Agrobacterium-mediated inflorescence staining. In some embodiments, the overexpression vector is based on pEGOEP35S-G418, and the BnWRKY50 gene is ligated between the XbaⅠ and BamHI restriction sites of pEGOEP35S-G418 via homologous recombination. The primer pair sequences for amplifying the BnWRKY50 gene fragment used for homologous recombination are shown in SEQ ID NO. 5 and SEQ ID NO. 6.

[0038] In some embodiments, the method further includes screening and identifying the obtained transgenic plants, using primer pairs with base sequences as shown in SEQ ID NO.7 and SEQ ID NO.8.

[0039] This specific embodiment also proposes the application of the above-mentioned ramie BnWRKY50 gene in the cultivation of plant varieties specifically for the remediation of cadmium-contaminated soil.

[0040] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0041] In this invention, the terms "some embodiments," "this embodiment," and examples are used to describe a subset of all possible embodiments. However, it is understood that "some embodiments" can be the same subset or different subsets of all possible embodiments and can be combined with each other without conflict.

[0042] If the application documents contain similar descriptions such as "first / second", the following explanation shall be added: In the following description, the terms "first / second / third" are used only to distinguish similar objects and do not represent a specific ordering of objects. It is understood that "first / second / third" may be interchanged in a specific order or sequence where permitted, so that the embodiments described herein can be implemented in an order other than that illustrated or described herein.

[0043] In this embodiment, the term "and / or" is merely a description of the relationship between related objects, indicating that there can be three relationships. For example, object A and / or object B can represent three situations: object A exists alone, object A and object B exist simultaneously, and object B exists alone.

[0044] The following describes embodiments of this application. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially.

[0045] Example 1: Obtaining the BnWRKY50 gene

[0046] Ramie lateral branches were propagated by hydroponics, and roots cultured for 7 days after rooting were used as material. Total RNA was extracted from the roots, and using the RNA as a template, cDNA was reverse transcribed into cDNA using the Evo M-MLV Plus cDNA synthesis kit. The amplification system is shown in Table 1. The first reaction conditions were 65℃ for 5 min; storage at 12℃. The second reaction conditions were 30℃ for 10 min; 42℃ for 60 min; 95℃ for 5 min; storage at 12℃. Using the synthesized cDNA as a template, the target gene fragment was amplified using BnWRKY50 amplification primers and high-fidelity enzyme. The amplification system is shown in Table 2. The PCR reaction conditions were 94℃ for 1 min, 98℃ for 10 s, 56℃ for 5 s, 72℃ for 5 s / kb, 35 cycles; storage at 12℃. After the reaction, the PCR products were detected by electrophoresis on a 1% agarose gel. Figure 1 After the gel imaging system displays the correct bands, the target fragment is purified using a gel extraction and recovery kit.

[0047] Table 1 Reverse transcription reaction system

[0048]

[0049] Table 2 BnWRKY50 gene amplification system

[0050]

[0051] Example 2 Construction of overexpression vector

[0052] The pEGOEP35S-G418 expression vector was selected with two restriction enzyme sites, XbaⅠ and BamHI. The target fragment BnWRKY50 with specific homologous arms was amplified using primers SEQ ID NO.5 and SEQ ID NO.6. Figure 2The recombinant product was ligated into the pEGOEP35S-G418 expression vector using homologous recombination, and the amplification system is shown in Table 3. The reaction conditions were 50℃ for 15 min and storage at 12℃. After transformation into *E. coli* DH5α competent cells, single colonies were picked for PCR identification. Agarose gel electrophoresis results showed... Figure 3 After expanding the bacterial culture to match the target band of exactly the expected size and sending it for comparison, the pEGOEP35S-G418-BnWRKY50 recombinant plasmid was extracted and transformed into Agrobacterium GV3101 competent cells for Arabidopsis genetic transformation.

[0053] Table 3. Vector Construction PCR Amplification System

[0054]

[0055] Example 3 Genetic transformation of Arabidopsis thaliana overexpression

[0056] Remove GV3101 Agrobacterium competent cells and thaw them on ice. Add pEGOEP35S-G418-BnWRKY50 plasmid and gently mix. Incubate on ice for 5 min, then in liquid nitrogen for 5 min, then in a 37°C water bath for 5 min, and finally in an ice bath for 5 min. Then add 700 µL of antibiotic-free sterile YEB liquid medium, mix well, and incubate at 28°C and 200 rpm for 2-3 h to recover. Spread the recovered cells evenly onto YEB solid medium (containing Kan and Rif dual antibiotics) and incubate upside down at 28°C for 2-3 days.

[0057] Add 1 mL of sterile water to a 1.5 mL centrifuge tube and place an appropriate amount of wild-type Arabidopsis seeds in the tube. Incubate at 4°C for 2 days for vernalization treatment. First, wash with 75% ethanol for 1 min, then with mercuric chloride for 3 min, and finally wash 4-5 times with sterile water. Sow evenly on 1 / 2 MS medium. Transplant after 4 true leaves have emerged. After the Arabidopsis bolts and flowers, use the pEGOEP35S-G418-BnWRKY50 overexpression vector to transgenerate Arabidopsis using Agrobacterium-mediated inflorescence infection. The specific steps are as follows:

[0058] (1) Add the correctly sequenced Agrobacterium culture to 50 mL of YEB liquid medium (containing Kan and Rif), and incubate overnight at 28°C and 200 rpm.

[0059] (2) Centrifuge at 8000 rpm for 10 minutes and collect the bacterial cells, discarding the supernatant. Prepare a resuspension (50 mL / L MES, 2 g / L MgCl2) to resuspend the bacterial cells, so that the OD600 value is around 0.8. Add 200 μM acetylsuccinone and let stand at 25℃ for 2 h.

[0060] (3) Place the Arabidopsis inflorescence in the infection solution for 30s ~ 1min, and then culture it in the dark for 24 hours before normal culture. After one week, infect it again to improve the infection efficiency.

[0061] (4) After the Arabidopsis thaliana matures, the plants are divided and the seeds are collected. The resulting seeds are recorded as generation T0.

[0062] Example 4: Screening of overexpression plants

[0063] After vernalization at 4℃, T0 generation Arabidopsis seeds were washed and evenly sown on 1 / 2 MS medium (20 mg / L 418 antibiotic) for selection. After 1-2 weeks, lines with a survival-to-death ratio of 3:1 were selected for transplanting. Once the seedlings had grown, genomic DNA was extracted from several Arabidopsis leaves using the CTAB method. PCR verification was performed using primers SEQ ID NO.7 and SEQ ID NO.8. After correct sequencing, the final positive plants were confirmed and harvested as T1 generation. Results are as follows: Figure 4 As shown.

[0064] Example 5: Detection of BnWRKY50 gene expression level in overexpressing plants

[0065] When the harvested T1 generation overexpressing Arabidopsis seeds were cultured to the T3 generation, several overexpression lines were randomly selected and total RNA was extracted from Arabidopsis samples using the SteadyPure Plant RNA Extraction Kit. The RNA was then reverse transcribed into cDNA using the SynScript® Ⅲ RT SuperMix for qPCR (+gDNA Remover). The reverse transcription system is shown in Table 4. Gene expression levels were detected using primers SEQ ID NO. 9 and SEQ ID NO. 10, with the AtActin gene used as an internal control (SEQ ID NO. 11 and SEQ ID NO. 12). qRT-PCR was performed using ArtiCanATM SYBR qPCR Mix on a Bio-Rad CFX96 real-time PCR instrument using a two-step amplification method with three biological replicates. The reaction system is shown in Table 5, with the reaction conditions as follows: 95℃ for 1 min; 95℃ for 10 s, 60℃ for 20 s, 40 cycles; 72℃ for 30 s. After the reaction, the relative expression levels of the genes were calculated using the 2-ΔΔCt method. Two lines with significantly higher BnWRKY50 expression levels than WT were selected and designated as BnWRKY50-OE1 and BnWRKY50-OE2. The results are shown in Figure 5.

[0066] Depend on Figure 5It can be seen that BnWRKY50 is hardly expressed in WT lines, but its expression level is significantly higher in T3 overexpression transgenic lines than in WT.

[0067] Table 4 Reverse transcription reaction system

[0068]

[0069] Table 5 Real-time quantitative PCR reaction system

[0070]

[0071] Example 6: Detection of cadmium tolerance in plants overexpressing the BnWRKY50 gene

[0072] Wild-type (WT) and homozygous transgenic BnWRKY50 overexpression lines of Arabidopsis thaliana seeds were vernalized, cleaned and disinfected, and sown on 1 / 2 MS solid medium. After germination and seed emergence, the seeds were transferred to 1 / 2 MS medium containing 0, 25, 50, 75, and 100 μM cadmium for vertical culture for 2 weeks. Root length and fresh weight were then statistically analyzed. Additionally, Arabidopsis thaliana seedlings that reached the four-leaf stage on 1 / 2 MS medium were transplanted to nutrient soil and cultured conventionally for 1-2 weeks. Then, they were treated with 0 mM, 0.5 mM, 1 mM, and 2 mM cadmium solutions, respectively. Phenotypic observation was performed 7-10 days later. Enzyme activities were measured using SOD, POD, CAT, MDA, and H2O2 kits, and leaf SPAD values ​​were measured using a SPAD-502 chlorophyll content analyzer (Konica Minolta, Japan). Meanwhile, Arabidopsis thaliana leaves treated with 0.5 mM were selected for DAB and NBT histochemical staining. Another Arabidopsis thaliana sample treated with the same concentration of cadmium was treated with liquid nitrogen and stored at -80℃ for RNA extraction. qPR-PCR was performed on AtSOD (SEQ ID NO.13 and SEQ ID NO.14), AtPOD ​​(SEQ ID NO.15 and SEQ ID NO.16), AtCAT (SEQ ID NO.17 and SEQ ID NO.18), AtAPX1 (SEQ ID NO.19 and SEQ ID NO.20), AtHMA2 (SEQ ID NO.21 and SEQ ID NO.22), AtHMA4 (SEQ ID NO.23 and SEQ ID NO.24), AtNramp3 (SEQ ID NO.25 and SEQ ID NO.26), and AtNramp4 (SEQ ID NO.27 and SEQ ID NO.28).

[0073] Depend on Figure 6It was found that root growth in Arabidopsis thaliana was gradually inhibited at a cadmium concentration of 50 μM, with the root length inhibition rate of the WT line reaching as high as 41.23%, while that of the BnWRKY50-OE line was only 26.13%. However, the root length (average approximately 7.39 cm) and fresh weight (average approximately 8.93 mg per plant) of the overexpression line were increased by approximately 24.62% and 56.67% respectively compared to the WT line. This indicates that overexpression of BnWRKY50 can alleviate the inhibitory effect of cadmium on root growth.

[0074] Depend on Figure 7 It was found that the activities of SOD, POD, and CAT antioxidant enzymes in the BnWRKY50-OE strain under cadmium stress were significantly higher than those in the WT strain, being 1.32 times, 1.48 times, and 1.65 times that of WT, respectively, and the SPAD value was 1.43 times that of WT. However, the contents of malondialdehyde (MDA) and hydrogen peroxide (H2O2) were 0.60 times and 0.85 times that of WT, respectively. The results of DAB and NBT staining were also consistent with the enzyme activity assay results. Under cadmium stress, the levels of hydrogen peroxide (H2O2) and superoxide anion (O2) in the leaves of the BnWRKY50-OE strain were significantly higher. 2- The accumulation of CdCl2 was significantly lower in WT than in WT. After treatment with 1mM and 2mM CdCl2, WT leaves showed obvious yellowing and curling, while BnWRKY50-OE plants showed milder symptoms.

[0075] Depend on Figure 8 It was found that the relative expression levels of AtSOD, AtPOD, AtCAT, and AtAPX1 in the BnWRKY50-OE strain were approximately 3-4 times higher than those in the WT strain, thus enhancing Arabidopsis' defense against oxidative damage. Conversely, BnWRKY50 significantly inhibited the expression of multiple cadmium transporter genes. Specifically, the relative expression levels of AtHMA2 and AtHMA4 decreased by approximately 50%–70% and 60%–70%, respectively, compared to WT; the expression levels of AtNRAMP3 and AtNRAMP4 also decreased by approximately 70%–80% and 80%–85%, respectively.

[0076] These results indicate that BnWRKY50 significantly improves Arabidopsis thaliana's tolerance to cadmium stress by inhibiting the expression of related cadmium transporter genes.

[0077] Example 7: Determination of Cadmium Content in Plants Overexpressing the BnWRKY50 Gene

[0078] The aboveground and underground parts of Arabidopsis thaliana, after cadmium treatment, were washed with deionized water and thoroughly ground into a fine powder. The powder was then placed in a 105℃ oven for 20 minutes to kill the green color, and dried at 80℃ to constant weight. The cadmium content of the aboveground and underground parts of each strain was determined by inductively coupled plasma mass spectrometry (ICP-MS). Net Cd in the roots of Arabidopsis thaliana was determined using non-destructive micro-testing (NMT). 2+ Flux.

[0079] Depend on Figure 9 It can be seen that the cadmium content in the roots of the BnWRKY50-OE strain was about 61.13% higher than that in the WT strain, while the cadmium content in the aboveground parts was about 35.73% lower than that in the WT strain (17.63 mg / kg).

[0080] Depend on Figure 10 It can be seen that the net cadmium ion influx rate of transgenic Arabidopsis roots is significantly higher than that of WT, averaging about 18.1 times that of WT.

[0081] The above results indicate that transgenic BnWRKY50 Arabidopsis plants accumulate more cadmium in their root tissues, while cadmium accumulation in their aboveground parts is relatively low.

[0082] The BnWRKY50 gene provided by this invention, when overexpressed in plants, can reduce cadmium accumulation in the aboveground parts of the plant, increase cadmium accumulation in the roots, and enhance the plant's cadmium tolerance. For cadmium remediation crops, the improved cadmium tolerance allows for the expansion of the plant's planting range to heavily polluted areas and mining areas; the increased cadmium content in the roots improves remediation efficiency and shortens the remediation period; and the reduced cadmium content in the aboveground parts facilitates the safe utilization of the plant for fiber and feed products. Therefore, the BnWRKY50 gene will have significant application value in phytoremediation of cadmium-contaminated soil.

[0083] The sequences in the above embodiments are shown below:

[0084] SEQ ID NO.1 Gene CDS Sequence

[0085] ATGATGAAGTACCAGCAGAGCTTGTTTGATCAGGAGAGTAATAATATTGATGATCATGATCATGATCAAGATCAAGATCAAGATCAAGCTGATCATCAAACCCCATCACAAATGGGTTTCTTTTCTTTTCCTCCAAACAACATGACCCTAATCAATAATCCCCCAATATTGGGATGCCTCAAATCCTTTCCCTCATCATCAACAACAACTACAATCTCAGCCTTGTTGCCTTGTTCCCTTTCAATATCTGATCATCACCACACTTTGCCTTCCTCTATCAACAGCAATAATAGTAGCAATAATACTAATAATAATAACCTCTCATCAGAAACTCTTTTAGTCTCATCTCTGGGTATTAATAGCAATATTATTCCTTCTAATAACAAGCATAGAGATCAGGACTTCACTACTGCTGATTATATTGGAGCTGGAACAACCCAACTTCTTTCCTTGCAAAGATCAACCTCAAATCTCTGGGCATGGGGTGAAGTGAGTGGTACTTGCATGGGAAACAAGAGGTTAATTAGTAGTGGGATTAGTATTGGCGACGAAGATCATGATAACCATGATAATGATTATGATGAGGATCATAACCATAACAATCATAATCTTCATCATCATCATCATCATAATCATAATCATATTGGGGTTTCAACGATGAAGATGAAGAAGATAAAGGGAAGAAGAAAGGTGAGAGAGCCAAGGTTTTGCTTCAAGACAATGAGCGAAGTGGATGTGCTTGATGATGGATACAAGTGGAGGAAGTACGGACAGAAAGTGGTGAAGAACACACAGCATCCCAGAAGCTATTACCGTTGCACGCAAGATAACTGTCGCGTGAAGAAACGCGTGGAGCGGTTAGCGGAGGACCCGAGGATGGTGATAACGACGTACGAAGGAAGACATGTTCACTCTCCGTCCCACGATTTGGAGACTGATTCACAAGCTCCGTCTCCCTTGAGTAACTTCTTCTGGTAG。

[0086] SEQ ID NO.2 Gene amino acid sequence

[0087] MMKYQQSLFDQESNNIDDHDQDQDQDQADHQTPSQMGFFSFPPNNMTLINNPPILGCLKSFPSSSTTTTISALLPCSLSISDHHHTLPSSINSNNSSNNTNNNNLSSETLLVSSLGINSNIIPSNNKHRDQDFTTADYIGAGTTQLLSLQRSTSNLWAWGE VSGTCMGNKRLISSGISIGDEDHDNHDNDYDEDHNHNNHNLHHHHHHNHNHIGVSTMKMKKIKGRRKVREPRFCFKTMSEVDVLDDGYKWRKYGQKVVKNTQHPRSYYRCTQDNCRVKKRVERLAEDPRMVITTYEGRHVHSPSHDLETDSQAPSPLSNFFW.

[0088] SEQ ID NO.3 Pre-primer for gene amplification

[0089] BnWRKY50-F:ATGATGAAGTACCAGCAGAGC.

[0090] SEQ ID NO.4 Primers after gene amplification

[0091] BnWRKY50-R:CTACCAGAAGAAGTTACTCAAGGG.

[0092] SEQ ID NO.5 Pre-primer for gene ligation vector amplification

[0093] 35S-BnWRKY50-F:GAGAACACGGGGGACTCTAGAATGATGAAGTACCAGCAGAGC.

[0094] SEQ ID NO.6 Primers after gene ligation vector amplification

[0095] 35S-BnWRKY50-R:GGACTGACCACCCGGGGATCCCTACCAGAAGAAGTTACTCAAGGG.

[0096] Primers for validation of overexpressing plants (SEQ ID NO.7)

[0097] 35S-F:AGAACACGGGGGACTCT.

[0098] Primers for validation of SEQ ID NO.8 overexpressing plants

[0099] NOS-R:ACCGGCAACAGGATTCAA.

[0100] Primers for gene expression level detection (SEQ ID NO.9)

[0101] BnWRKY50-qF:TCCTCCAAACAACATGACCCT.

[0102] SEQ ID NO.10 Gene expression level detection primers

[0103] BnWRKY50-qR:GGAACAAGGCAACAAGGCTG.

[0104] SEQ ID NO.11 Internal reference Actin preprime

[0105] AtActin-qF:GCACCACCTGAAAGGAAGTACA.

[0106] SEQ ID NO.12 Internal reference Actin post-primer

[0107] AtActin-qR:CGATTCCTGGACCTGCCTCATC.

[0108] SEQ ID NO.13 Pre-primer for gene amplification

[0109] AtSOD-qF:AGGAAACATCACTGTTGGAGAT.

[0110] SEQ ID NO.14 Primers after gene amplification

[0111] AtSOD-qR:GAGTTTGGTCCAGTAAGAGGAA.

[0112] SEQ ID NO.15 Pre-primer for gene amplification

[0113] AtPOD-qF:CGTGCCCTTCATATTGTTGG.

[0114] SEQ ID NO.16 Primers after gene amplification

[0115] AtPOD-qR:GACGCCATCAACAACGAGTC.

[0116] SEQ ID NO.17 Pre-gene amplification primers

[0117] AtCAT-qF:CTTGTGGTTCCTGGAATCTACT.

[0118] SEQ ID NO.18 Primers after gene amplification

[0119] AtCAT-qR:AGGATCAAACTTTGAGGGGTAG.

[0120] SEQ ID NO.19 Pre-primer for gene amplification

[0121] AtAPX1-qF:CTCATGGAGCCAACAGTGGT.

[0122] SEQ ID NO.20 Primers after gene amplification

[0123] AtAPX1-qR: ACACCAGCAAGCTGATGGAA.

[0124] SEQ ID NO.21 Pre-primer for gene amplification

[0125] AtHMA2-qF:AAGATTCGACGGTTTGGGCT.

[0126] SEQ ID NO.22 Primers after gene amplification

[0127] AtHMA2-qR:CATCTTCGCAACCACGCAAT.

[0128] SEQ ID NO.23 Pre-primer for gene amplification

[0129] AtHMA4-qF:GAGCACGACGAAAGTTGTT.

[0130] SEQ ID NO.24 Primers after gene amplification

[0131] AtHMA4-qR:TGACCAGCAAATGCCAAAGC.

[0132] SEQ ID NO.25 Pre-primer for gene amplification

[0133] AtNRAMP3-qF:AAGAAACGAAGAGGAGGACGAT.

[0134] SEQ ID NO.26 Primers after gene amplification

[0135] AtNRAMP3-qR:AAAAACCCAGGTCCGGTGAAT.

[0136] SEQ ID NO.27 Pre-gene amplification primers

[0137] AtNRAMP4-qF:TCGTGGCGCTTGCTTTGA.

[0138] SEQ ID NO.28 Primers after gene amplification

[0139] AtNRAMP4-qR:AAGGAGTGGGGATCACAGCG.

[0140] The specific embodiments of the present invention described above do not constitute a limitation on the scope of protection of the present invention. Any other corresponding changes and modifications made in accordance with the technical concept of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. The application of a ramie BnWRKY50 gene in increasing cadmium accumulation in plant roots and enhancing cadmium tolerance in plants, characterized in that, The CDS nucleotide sequence of the ramie BnWRKY50 gene is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, The amino acid sequence of the protein encoded by the ramie BnWRKY50 gene is shown in SEQ ID NO.

2.

3. The application according to claim 1, characterized in that, By upregulating the expression level of the BnWRKY50 gene in plants, or increasing the content of the protein encoded by the BnWRKY50 gene in plants, cadmium accumulation in plant roots and cadmium tolerance can be increased.

4. The application according to any one of claims 1-3, characterized in that, The plants mentioned include Arabidopsis thaliana and ramie.

5. A primer pair for amplifying the ramie BnWRKY50 gene as described in claim 1, characterized in that, The base sequences of the primer pairs are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

6. A method for increasing cadmium accumulation in plant roots and enhancing plant cadmium tolerance, characterized in that, The ramie BnWRKY50 gene of claim 1 is introduced into a recipient plant to obtain a transgenic plant that overexpresses the BnWRKY50 gene, wherein the transgenic plant has increased cadmium accumulation in its roots and enhanced cadmium tolerance.

7. The method according to claim 6, characterized in that, An overexpression vector containing the BnWRKY50 gene was constructed, the overexpression vector was transformed into Agrobacterium, and the BnWRKY50 gene was introduced into recipient plants via Agrobacterium-mediated inflorescence staining.

8. The method according to claim 6, characterized in that, The overexpression vector was obtained by ligating the BnWRKY50 gene to the XbaⅠ and BamHI restriction sites of pEGOEP35S-G418 through homologous recombination based on pEGOEP35S-G418; the primer pair base sequences for amplifying the BnWRKY50 gene fragment used for homologous recombination are shown in SEQ ID NO.5 and SEQ ID NO.

6.

9. The method according to any one of claims 6-8, characterized in that, It also includes: screening and identifying the obtained transgenic plants, with the primer pairs used for identification having the base sequences shown in SEQ ID NO.7 and SEQ ID NO.

8.

10. The application of the ramie BnWRKY50 gene as described in claim 1 in the cultivation of plant varieties for cadmium-contaminated soil remediation.

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