Sudangrass ssHIPP6 gene and its anti-cadmium application

Abstract

The present application belongs to the technical field of plant genetic engineering, and relates to a heavy metal related isoprenylated plant protein gene SsHIPP6 of sudangrass and application thereof. The sequence of the sudangrass SsHIPP6 gene is selected from the nucleotide sequence shown in SEQ ID NO:1, a complementary nucleotide sequence or a variant having >95% homology, and when overexpressed in plants, the variant can reduce the cadmium accumulation in the aboveground part by >26.9% and the cadmium accumulation in the root system by >27.4%. The gene can effectively improve the resistance of plants to cadmium stress. By overexpressing the gene, the cadmium accumulation in the aboveground part of the plant can be reduced by >26.9%, and the cadmium accumulation in the root system can be reduced by >27.4%, thereby enhancing the survival rate and growth ability of the plant in cadmium contaminated soil, providing a possibility for planting high-quality forage grass on heavy metal contaminated land, and having important significance for guaranteeing the ecological safety of agriculture and the sustainable development of animal husbandry.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology and relates to the isolation and application of SsHIPP6, a heavy metal-related isopreneated plant protein gene from Sudan grass. Background Technology

[0002] Sudan grass (Sorghum sudanense (Piper) Stapf.) is a world-renowned annual high-yield C4 grass, prized forage for its high biomass yield, rich nutritional value, and wide adaptability. It is widely cultivated globally and plays a vital role in the development of herbivorous livestock farming. However, its global population is approximately 2.35 × 10⁻⁶. 12 m 2 Trace element pollution in farmland soil seriously affects agricultural production and ecological security, threatening the survival of plants and animals and human life.

[0003] Cadmium is a highly carcinogenic heavy metal that can cause toxic reactions even at low concentrations. High concentrations of cadmium are toxic to soil organisms and easily enter the food chain. In forage grasses grown on cadmium-contaminated soil, cadmium accumulates in their bodies and is subsequently transferred through the food chain, harming livestock health and ultimately impacting human health. Therefore, exploring resistance-related genes, understanding the response mechanisms and molecular pathways of plants to cadmium stress, and using genetic engineering methods to cultivate cadmium-tolerant forage grass varieties are of great significance for ensuring agricultural ecological security and the sustainable development of animal husbandry.

[0004] Traditional breeding methods struggle to quickly and effectively cultivate forage varieties with high cadmium resistance. However, advancements in genetic engineering offer a new approach to addressing this issue. By identifying and utilizing functional genes associated with cadmium resistance in plants, targeted improvements in plant cadmium resistance can be achieved.

[0005] Current methods for improving cadmium resistance in forage grasses face the following limitations:

[0006] 1. Sudan grass species have low genetic diversity and a lack of Cd-resistant germplasm resources;

[0007] 2. The hybridization breeding cycle is long (≥8 years), and resistance is negatively correlated with yield traits;

[0008] 3. It has been reported that most Cd resistance genes originate from crops, and their heterologous expression efficiency in forage grasses is low;

[0009] 4. The cloning and functional verification of endogenous Cd resistance genes in Sudan grass are lacking, and the resistance mechanism is unclear.

[0010] Therefore, there is an urgent need to further explore resistance-related genes, understand the potential mechanisms of stress response and molecular pathways, and thus accelerate forage breeding using genetic engineering methods such as overexpression. Summary of the Invention

[0011] Therefore, the present invention aims to provide a nucleotide sequence, encoded protein, recombinant expression vector of a key gene closely related to cadmium resistance, and its application in improving the resistance of plants to heavy metal cadmium (Cd) stress, especially suitable for the genetic improvement of gramineous forage grasses in cadmium-contaminated soil.

[0012] Through long-term exploration and experimentation, and continuous reform and innovation, the inventors have provided a technical solution to solve the above-mentioned technical problems: a Sudan grass SsHIPP6 gene, wherein the SsHIPP6 gene sequence is selected from any of the following:

[0013] The nucleotide sequence shown in SEQ ID NO:1;

[0014] Nucleotide sequence complementary to SEQ ID NO:1;

[0015] A variant that has ≥95% homology with SEQ ID NO:1 and whose overexpression in plants reduces cadmium accumulation in aerial parts by ≥26.9% and cadmium accumulation in roots by ≥27.4%.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0017] The SsHIPP6 gene of Sudan grass provided by this invention can effectively improve the plant's resistance to cadmium stress. By overexpressing this gene, the cadmium accumulation in the aboveground parts of the plant can be reduced by ≥26.9% and the cadmium accumulation in the roots can be reduced by ≥27.4%, thereby enhancing the survival rate and growth capacity of the plant in cadmium-contaminated soil. This provides the possibility of planting high-quality forage grass on heavy metal contaminated land and is of great significance for ensuring agricultural ecological security and the sustainable development of animal husbandry.

[0018] The present invention also provides a protein encoded by the said gene, the amino acid sequence of which is shown in SEQ ID NO.2.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0020] Overexpression of the SsHIPP6 gene can effectively enhance the plant's resistance to cadmium stress, significantly reducing cadmium accumulation in the aboveground parts and roots. This provides key gene resources and protein targets for breeding cadmium-tolerant forage varieties, and is of great significance for improving the plant's growth capacity on cadmium-contaminated land and ensuring agricultural ecological security.

[0021] The present invention also provides a recombinant expression vector, characterized in that it contains the SsHIPP6 gene and expression regulatory elements.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0023] The recombinant expression vector provided by this invention achieves efficient expression of the SsHIPP6 gene in plants by combining it with expression regulatory elements, thereby significantly enhancing the plant's resistance to cadmium stress. It provides a convenient and effective tool for genetic improvement of cadmium tolerance in plants and has important application value.

[0024] Based on the above technical solution, the present invention can be further improved as follows:

[0025] Further: The SsHIPP6 gene was amplified using the following method: first upstream primer F: 5'-ggatcttccagagatATGTATGCAGGCTTTCAGCCC-3' and first downstream primer R: 5'-ctgccgttcgacgatCTAGGGAGCCTGCAAAATCTCTG-3'.

[0026] The KpnI-XbaI digestion product was ligated into the pCAMBIA1300-35 vector.

[0027] Compared with the existing technology, the beneficial effects of adopting the above-mentioned further technical solution are as follows:

[0028] By using specific primer amplification and enzyme digestion ligation, recombinant expression vectors can be constructed efficiently and accurately, ensuring the stable expression of the SsHIPP6 gene in plants. This provides a reliable technical means for improving cadmium resistance in plants and significantly improves the success rate and practicality of vector construction.

[0029] The present invention also provides a recombinant host cell comprising the recombinant expression vector described above.

[0030] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0031] This invention can efficiently express the SsHIPP6 gene of Sudan grass, enhance the plant's cadmium resistance, provide a powerful tool for cultivating cadmium-tolerant plants, and help grow high-quality forage grass on polluted land.

[0032] Based on the above technical solution, the present invention can be further improved as follows:

[0033] Furthermore, the host cell is *Saccharomyces cerevisiae*, *Arabidopsis thaliana* cells, or *Sudangrass* cells.

[0034] Compared with the existing technology, the beneficial effects of adopting the above-mentioned further technical solution are as follows:

[0035] By adopting the above-mentioned further technical solutions, the application scope of the SsHIPP6 gene has been expanded, making it not limited to specific biological systems, but providing flexibility and convenience for conducting gene function research and improving plant cadmium resistance in different host cells.

[0036] As a eukaryotic single-cell biological model, *Saccharomyces cerevisiae* cells offer advantages such as simple genetic manipulation, rapid growth, and low cost. Introducing recombinant expression vectors into *Saccharomyces cerevisiae* allows for rapid preliminary screening and validation of the SsHIPP6 gene function, providing an efficient platform for further in-depth research into its molecular mechanisms. Simultaneously, the *Saccharomyces cerevisiae* system helps elucidate the conserved biological functions of this gene in response to cadmium stress, providing a theoretical basis for cross-species applications.

[0037] Arabidopsis thaliana is a model plant for plant molecular biology research, characterized by its complete genome information, abundant mutant resources, and mature genetic manipulation system. Expressing the SsHIPP6 gene in Arabidopsis cells allows for in-depth exploration of its mechanism of action within the plant, including gene expression regulation, protein-protein interactions, and signal transduction pathways, utilizing mature genetic and molecular biology tools. This contributes to a comprehensive understanding of the molecular basis for the SsHIPP6 gene's enhancement of cadmium resistance in plants, providing detailed theoretical guidance for its application in other plants.

[0038] Using Sudan grass cells as the target plant cells, the introduction of recombinant expression vectors into Sudan grass cells is a key step in realizing the application value of this invention. By overexpressing the SsHIPP6 gene in Sudan grass cells, its effect on enhancing cadmium resistance in plants can be directly observed and evaluated, providing practical evidence for breeding cadmium-tolerant forage varieties. Simultaneously, research using Sudan grass cells can further optimize the gene expression system, making it more adapted to the physiological characteristics of the target plant and improving the success rate and stability of genetic engineering improvement.

[0039] The present invention also provides a method for producing cadmium-resistant Sudan grass cells, comprising:

[0040] (1) The SsHIPP6 gene was amplified using the SsHIPP6F / SsHIPP6R primer pair. The primer sequences are as follows:

[0041] SsHIPP6F: 5'-ATGTATGCAGGCTTTCAGCCC-3',

[0042] SsHIPP6R: 5'-CTAGGGAGCCTGCAAAATCTCTG-3';

[0043] (2) Insert the amplification product into the expression vector;

[0044] (3) Transformation of Sudan grass cells via Agrobacterium-mediated transformation.

[0045] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0046] Through genetic engineering techniques such as specific primer amplification, vector construction, and Agrobacterium-mediated transformation, the SsHIPP6 gene of Sudan grass was efficiently and stably expressed in Sudan grass cells, significantly improving the cadmium resistance of Sudan grass cells. This provides an efficient and reliable technical approach for breeding cadmium-tolerant Sudan grass varieties and strongly promotes the application of plant genetic engineering in addressing soil heavy metal pollution.

[0047] The present invention also provides a method for improving resistance to Sudan cadmium stress, comprising:

[0048] (1) The SsHIPP6 gene was amplified using the SsHIPP6F / SsHIPP6R primer pair. The primer sequences are as follows:

[0049] SsHIPP6F: 5'-ATGTATGCAGGCTTTCAGCCC-3',

[0050] SsHIPP6R: 5'-CTAGGGAGCCTGCAAAATCTCTG-3';

[0051] (2) Insert the SsHIPP6 gene into the expression vector;

[0052] (3) The recombinant vector was introduced into Sudan grass cells;

[0053] (4) Screening plants with enhanced expression of the SsHIPP6 gene.

[0054] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0055] This invention achieves efficient expression of the SsHIPP6 gene in Sudan grass, significantly enhancing its resistance to cadmium stress and reducing cadmium accumulation in the aboveground parts and roots. It provides a practical technical solution for planting high-quality Sudan grass in cadmium-contaminated soil, effectively solving the problem that traditional breeding methods are difficult to rapidly cultivate cadmium-tolerant forage varieties. It has significant innovation and practicality.

[0056] The present invention also provides the application of the SsHIPP6 gene in improving cadmium resistance in plants, which is achieved by overexpressing the gene in plants.

[0057] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0058] This invention effectively enhances the tolerance of plants to cadmium stress and reduces the accumulation of cadmium in plants, providing an effective solution for planting plants in cadmium-contaminated soil.

[0059] This invention also provides the application of the protein shown in SEQ ID NO:2 as a biomarker of cadmium stress in plants.

[0060] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0061] This biomarker can effectively reflect the physiological state of plants under cadmium stress, providing a key monitoring indicator for plant cadmium resistance research, helping to understand the plant response mechanism to cadmium stress, and providing important technical support for breeding cadmium-tolerant plant varieties. Attached Figure Description

[0062] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained from these drawings without creative effort.

[0063] Figure 1 This is an electrophoresis image of the SsHIPP6 gene amplification product.

[0064] Figure 2 This is the result of an experiment on the resistance of the SsHIPP6 gene to cadmium stress in Saccharomyces cerevisiae.

[0065] Figure 3 This is a comparison of the growth of wild-type Arabidopsis thaliana and Arabidopsis thaliana overexpressing the SsHIPP6 gene under cadmium stress treatment. Detailed Implementation

[0066] The following description is based on specific embodiments.

[0067] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to represent selected embodiments of the present invention.

[0068] In this invention, unless otherwise specified, all equipment and raw materials are available from the market or commonly used in the industry. Unless otherwise specified, the methods in the following embodiments are conventional methods in the art.

[0069] Example 1

[0070] This embodiment describes the construction of the SsHIPP6 gene, a heavy metal-related isopreneated plant protein from Sudan grass, and its expression vector.

[0071] The experimental material was the Sudan grass variety 'Chuansu No. 1', which was grown in the greenhouse of the Institute of Agricultural Resources and Environment, Sichuan Academy of Agricultural Sciences.

[0072] Total RNA was extracted from young leaves. The plant total RNA extraction kit from Tiangen (Beijing) Biochemical Technology Co., Ltd. was used for RNA extraction, and the procedure was performed according to the included instructions. After extraction, RNA integrity was assessed using 1% agarose gel electrophoresis, and RNA concentration and purity were determined using a micro spectrophotometer. Reverse transcription was performed using the PrimeScript II 1ststrand cDNA synthesis kit from TaKaRa, and the procedure was performed according to the included instructions.

[0073] Using the Sudan grass reference genome as a template, amplification was performed using full-length primers (SsHIPP6F / SsHIPP6R) with cDNA as a template. Amplification was performed using the TaKaRa PrimeSTAR Max DNA Polymerase kit; the procedure was followed according to the included instructions. The PCR amplification system is shown in Table 1. Primer sequences are as follows:

[0074] SsHIPP6F: 5'-ATGTATGCAGGCTTTCAGCCC-3';

[0075] SsHIPP6R: 5'-CTAGGGAGCCTGCAAAATCTCTG-3'.

[0076] The reaction conditions were: 98℃ pre-denaturation for 4 min; 98℃ denaturation for 10 s, 55℃ annealing for 5 s, 72℃ extension for 30 s, 35 cycles; and a final run at 72℃ for 10 min. PCR products were detected by 0.8% agarose gel electrophoresis. (See [link to relevant documentation]). Figure 1 .

[0077] Table 1 PCR amplification system

[0078]

[0079] The gel was cut under UV light, and the target fragment was recovered and purified using the TaKaRa Mini BEST Agarose Gel DNA Extraction Kit. For specific instructions, please refer to the included manual.

[0080] An "A" tail was added to the 3' end of the target DNA fragment using the TaKaRa DNAA-Tailing Kit. After completion, 4 μl of the above DNA solution was taken, and 1 μl of pMD18-T vector and 5 μl of Solution (containing ligase) were added and mixed well. The mixture was incubated at 16°C for 30 min. After the reaction, the above solution was added to 100 μl of DH5α competent cells, incubated on ice for 30 min, heated at 42°C for 45 s, and then placed on ice for 1 min.

[0081] The transformed competent cells were added to 890 μl of SOC medium and incubated at 37°C for 60 minutes. The cells were then plated onto LB medium containing ampicillin (Amp) and incubated upside down overnight. After incubation, single colonies were selected for culture and bacterial PCR was used to verify successful insertion of the target fragment. The target band was amplified by bacterial PCR and subjected to paired-end sequencing using primer M13 to verify successful cloning.

[0082] The full-length SsHIPP6 gene fragment of Sudan grass is 447 bp, and its sequence is shown in SEQ ID NO. 1. The protein it encodes contains 148 amino acids, and its sequence is shown in SEQ ID NO. 2.

[0083] Example 2

[0084] This embodiment describes the expression verification of SsHIPP6 Saccharomyces cerevisiae.

[0085] To preliminarily identify the function of SsHIPP6, the previously cloned SsHIPP6 fragment was digested with KpnI-XbaI as the restriction site. The digested fragment was then ligated into the linearized pYES2 yeast expression plasmid using the ClonExpress Ultra Onestep Cloning kit from Novizan Biotech (Nanjing). The recombinant pYES2-DgMYM1 plasmid was transformed into the *Saccharomyces cerevisiae* strain (INVScI, MATahis3Δ1leu2 trp1-289ura3-52 / MATαhis3Δ1leu2 trp1-289ura3-52) via Carrier DNA (Coolaber, China), with a blank pYES2 plasmid as a control. The transformed *Saccharomyces cerevisiae* was cultured in SC-Ura containing 20 mg / mL glucose at 28℃ for 48 h. Single clones were selected for PCR confirmation. Primers used were:

[0086] F: 5'-ggatcttccagagatATGTATGCAGGCTTTCAGCCC-3';

[0087] R: 5'-ctgccgttcgacgatCTAGGGAGCCTGCAAAATCTCTG-3'.

[0088] Positively transformed yeast was cultured in liquid SC-Ura medium containing 2 mg / mL galactose, centrifuged at 150 rpm, and then diluted 10 times. The diluted yeast suspensions were then plated on SD-Ura medium containing 20 μM and incubated at 28°C.

[0089] The experiment included three treatment groups: PYES2 / Δycf1 (empty vector), SsHIPP6-PYES2 / Δycf1, and a positive control. Each group of yeast culture was serially diluted to 10⁻⁶ ppm. -1 10 -2 10 -3 and 10 -4 See also Figure 2 The results showed that with increasing dilution, the number of colonies in the empty vector group decreased significantly, while the yeast strain overexpressing the SsHIPP6 gene could still form more colonies at high dilutions, indicating that the SsHIPP6 gene can significantly enhance the resistance of Saccharomyces cerevisiae to cadmium stress. This verifies the tolerance of this gene to cadmium stress in a heterologous expression system and provides preliminary evidence for its future application in plants.

[0090] Example 3

[0091] This example describes a validation experiment of SsHIPP6 overexpression in Arabidopsis thaliana.

[0092] To transform Arabidopsis thaliana, we cloned the CDS (SEQ ID NO.1) of SsHIPP6 from the cDNA of *Sultania sulphureus* leaves and ligated it into the pCAMBIA1300-35 vector under the control of the CaMV 35S promoter, which was then used to transform *Agrobacterium tumefaciens* GV3101. Transformation was performed using the Arabidopsis thaliana col-0 flower immersion method. Positive transgenic plants were selected using hygromycin, and after self-pollination for homozygous selection, further analysis was conducted up to the T3 generation.

[0093] After disinfection with 4% sodium hypochlorite, 30 seeds from each line of transgenic and wild-type Arabidopsis thaliana were sown on MS medium at 0.5 times its concentration and vernalized at 4℃ for 3 days. When the seedlings had 4 true leaves after germination, the transgenic and wild-type Arabidopsis thaliana plants with consistent growth were transferred to square pots. When they reached the 8-leaf stage, both transgenic and wild-type Arabidopsis thaliana plants were treated with 100 μmol / L CdCl2 to simulate cadmium stress for 35 days. Cadmium content in the aboveground parts and roots was measured 21 days after treatment. Figure 3This study compares the growth of wild-type Arabidopsis thaliana and Arabidopsis thaliana overexpressing the SsHIPP6 gene under cadmium stress. The results showed that wild-type Arabidopsis thaliana began to yellow and die after 21 days of cadmium stress, while the SsHIPP6-transgenic Arabidopsis thaliana remained healthy until 35 days when the wild-type completely died and stopped growing. The transgenic lines still retained some green leaves, indicating that the SsHIPP6-transgenic Arabidopsis thaliana exhibited significantly better resistance to cadmium than the wild-type. The experiment demonstrates that overexpression of the SsHIPP6 gene in plants can effectively enhance plant resistance to cadmium stress and reduce the inhibitory effect of cadmium on plant growth, validating the practical application value of this gene in improving plant cadmium resistance.

[0094] Table 2 shows the cadmium content and transfer coefficient in different tissues of wild-type and SsHIPP6 overexpression lines after cadmium stress.

[0095] Table 2. Cadmium content in different tissues of Arabidopsis thaliana overexpressing SsHIPP6 after cadmium stress.

[0096]

[0097] Note: Data in the table are "mean ± standard deviation"; ** indicates that the difference between this data and the wild type is highly significant at the P < 0.01 level.

[0098] As can be seen from Table 2:

[0099] Aboveground parts: The cadmium content of the SsHIPP6 overexpression line was 1.22±0.14 mg / g, which was significantly lower than that of the wild type (1.67±0.23 mg / g). This indicates that overexpression of the SsHIPP6 gene can effectively reduce the accumulation of cadmium in the aboveground parts of plants. This is because the expression product of this gene plays a role in the absorption, transport, or detoxification of cadmium in the plant, thereby reducing the cadmium content in the aboveground parts.

[0100] Root system: The cadmium content in the roots of the SsHIPP6 overexpressing line was 2.41±0.18 mg / g, which was significantly lower than that in the wild type (3.32±0.17 mg / g). This indicates that overexpression of the SsHIPP6 gene can also inhibit the absorption and accumulation of cadmium in the roots. This is of great significance for preventing the translocation of cadmium to the aboveground parts through the roots and reducing the accumulation of cadmium in the entire plant.

[0101] The transfer coefficient reflects the plant's ability to translocate cadmium from the roots to the aboveground parts. The transfer coefficients for the wild-type and SsHIPP6-overexpressing lines were 0.51±0.05 and 0.49±0.04, respectively, with no significant difference between the two. This indicates that overexpression of the SsHIPP6 gene mainly affects the uptake and accumulation of cadmium in plants, but does not significantly alter the relative translocation efficiency of cadmium between the roots and aboveground parts. In other words, the mechanism of action of this gene may not involve significant regulation of cadmium translocation, or its effect on translocation may not have reached a detectable level under the experimental conditions.

[0102] Overall, overexpression of the SsHIPP6 gene can significantly reduce cadmium accumulation in the aboveground parts and roots of plants, thereby improving plant resistance to cadmium stress. This has important practical significance for planting plants in cadmium-contaminated soil, helping to reduce the absorption and accumulation of cadmium by plants, reducing the risk of cadmium entering organisms through the food chain, and thus improving their resistance to the heavy metal cadmium. At the same time, it also provides potential genetic resources for the remediation of cadmium-contaminated soil using plants.

[0103] Through experiments in Examples 2 and 3, we further clarified the application potential of the protein represented by sequence ID NO:2 as a biomarker for cadmium stress in plants. In the *Saccharomyces cerevisiae* experiment, yeast strains overexpressing the SsHIPP6 gene exhibited significantly enhanced cadmium resistance, indicating that the protein encoded by this gene has a responsive and detoxifying function in cellular cadmium stress. In the *Arabidopsis thaliana* experiment, lines overexpressing the SsHIPP6 gene showed significantly reduced cadmium accumulation in both the aboveground parts and roots under cadmium stress. This indicates that the protein represented by sequence ID NO:2 can effectively enhance plant resistance to cadmium stress. This ability to enhance cadmium resistance in different biological systems suggests that the protein represented by sequence ID NO:2 can serve as a key indicator of plant cadmium stress response. It can reflect the degree of plant sensitivity to cadmium stress under different growth environments, providing important information for studying the mechanisms of cadmium stress in plants. In practical applications, the degree of cadmium stress experienced by plants can be determined by detecting changes in the expression level of this protein in the plant. This provides an important means of assessing the growth status of plants in polluted soil environments and can also serve as a reference standard for screening and cultivating cadmium-resistant plant varieties, thereby promoting the application of plants in the remediation of heavy metal-polluted soils and safe agricultural production.

[0104] In the description of this invention, it should be understood that "-" and "~" represent a range between two values, and this range includes the endpoints. For example, "AB" represents a range greater than or equal to A and less than or equal to B. "A~B" represents a range greater than or equal to A and less than or equal to B.

[0105] In the description of this invention, the term "and / or" is merely a description of the relationship between related objects, indicating that there can be three relationships. For example, A and / or B can represent three cases: A exists alone, A and B exist simultaneously, and B exists alone.

[0106] In the description of the invention, the numerical values ​​of time, temperature, ratio, and mass involved can be based on actual measurements, standard equipment parameters, simplified rounding results, or within an acceptable error range, ensuring the practicality and repeatability of the invention.

[0107] In the description of this invention, the terms “about” or “approximately” are used to express approximate values ​​or ranges, allowing for a certain degree of error to ensure the flexibility and practicality of the description, while remaining within an acceptable range of error, with the maximum error not exceeding 10% of the corresponding value or range.

[0108] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be considered as limitations on the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. For those skilled in the art, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for producing cadmium-resistant Arabidopsis thaliana cells, characterized in that, include: (1) The SsHIPP6 gene was amplified using primers SsHIPP6F and SsHIPP6R. The nucleotide sequence of the SsHIPP6 gene is shown in SEQ ID NO:1, and the primer sequences are as follows: SsHIPP6F: 5'-ATGTATGCAGGCTTTCAGCCC-3', SsHIPP6R: 5'-CTAGGGAGCCTGCAAAATCTCTG-3'; (2) Insert the amplification product into the expression vector; (3) Transformation of Arabidopsis thaliana cells via Agrobacterium-mediated transformation.

2. A method for improving the cadmium stress resistance of Arabidopsis thaliana, characterized in that, include: (1) The SsHIPP6 gene was amplified using primers SsHIPP6F and SsHIPP6R. The nucleotide sequence of the SsHIPP6 gene is shown in SEQ ID NO:1, and the primer sequences are as follows: SsHIPP6F: 5'-ATGTATGCAGGCTTTCAGCCC-3', SsHIPP6R: 5'-CTAGGGAGCCTGCAAAATCTCTG-3'; (2) Insert the SsHIPP6 gene into the expression vector; (3) The recombinant vector was introduced into Arabidopsis cells; (4) Obtain plants with enhanced expression of the SsHIPP6 gene.

3. The application of the SsHIPP6 gene in improving cadmium resistance in Arabidopsis thaliana, characterized in that, This was achieved by overexpressing the gene in Arabidopsis thaliana, and the nucleotide sequence of the SsHIPP6 gene is shown in SEQ ID NO:1.

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