A gene for improving the cadmium tolerance of potatoes and its application

Abstract

The present invention discloses a gene for improving the cadmium tolerance of potatoes and its application, which relates to the field of biotechnology. The nucleotide sequence of this gene is shown as SEQ ID NO.1. Through transcriptome and metabolome analysis, the present invention discovered a key gene, StAPR3 gene, which showed a significant response to cadmium stress. By verifying the function of this gene, it was found that overexpression of this gene could significantly improve the tolerance of transgenic yeast to Cd stress, thereby confirming that this gene is involved in the physiological process of potato cadmium tolerance and exhibits a positive regulatory effect. The present invention provides a new gene resource for cultivating potato varieties resistant to cadmium stress and has important practical application value.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a gene for improving the cadmium tolerance of potatoes and its application. Background Art

[0002] Cadmium in the soil can have an adverse effect on plants by reducing carbon assimilation, causing leaf chlorosis, inhibiting photosynthesis, and promoting oxidative stress. In addition, due to its high mobility, Cd can accumulate in crops, posing a toxic risk to humans and animals.

[0003] Cadmium is particularly easily absorbed by plants and transferred to different parts. This transport of Cd affects the physiological metabolism of plants by replacing essential cations at specific binding sites, resulting in the loss of the function of reactive oxygen species (ROS) in plant cells and the inhibition of the activities of several antioxidant enzymes. ROS are usually maintained at an acceptable level under the protection of the antioxidant system without causing oxidative damage. However, when plants are under heavy metal stress, the balance between the generation and decomposition of reactive oxygen species (ROS) in their bodies is disrupted, leading to a large accumulation of ROS in plant cells, causing oxidative stress. Excessive ROS can react with biological macromolecules such as proteins and nucleic acids in plant cells, disrupting cell signal transduction, inhibiting cell growth, causing cell damage, and affecting plant cell function. In severe cases, it can lead to the death of plant cells. Malondialdehyde (MDA) is usually used as a general indicator of lipid peroxidation induced by oxidative stress. An increase in MDA indirectly indicates an increase in ROS. Photosynthesis is also sensitive to Cd, and multiple studies have shown that Cd stress severely affects the synthesis of plant chlorophyll. Therefore, physiological parameters such as MDA, ROS, and chlorophyll can indirectly reflect the toxicity of Cd to plants. At the same time, plants have evolved a series of defense systems to cope with the stressful environment. One mechanism is stress avoidance. For example, plants can isolate heavy metal ions by increasing the cell wall thickness and changing other cell structures. Another mechanism is stress tolerance, which involves regulating the content of antioxidant enzymes and antioxidants in the body, eliminating ROS, reducing damage such as membrane lipid peroxidation, or maintaining the normal growth environment of cells by changing the content of osmoregulatory substances.

[0004] Potato (Solanum tuberosum L.) is the fourth largest food-demand crop in the world and is rich in starch and protein. It is necessary to explore its cadmium tolerance mechanism by clarifying the morphological, physiological, and molecular responses of potatoes under cadmium stress, so as to identify genes that can regulate the cadmium tolerance of potatoes, thereby providing technical support for cultivating new potato varieties resistant to cadmium stress. Summary of the Invention

[0005] The object of the present invention is to provide a gene for improving the cadmium tolerance of potatoes and its application, so as to solve the problems existing in the above-mentioned prior art. The present invention has confirmed that this gene is involved in the physiological process of potato cadmium tolerance and exhibits a positive regulatory effect. The present invention provides a new gene resource for cultivating potato varieties resistant to cadmium stress and has important practical application value.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a gene for improving the cadmium tolerance of potatoes, and its nucleotide sequence is as shown in SEQ ID NO.1.

[0008] The present invention also provides a recombinant plasmid containing the above gene.

[0009] Furthermore, the recombinant plasmid is obtained by constructing the gene into the pYES2 vector.

[0010] The present invention also provides a host cell containing the above recombinant plasmid.

[0011] Furthermore, the host cell is yeast.

[0012] The present invention also provides the application of the above gene or biological material in improving the cadmium tolerance of potatoes. The biological material is the substance described in the following (1) or (2):

[0013] (1) A recombinant plasmid containing the gene;

[0014] (2) A host cell containing the recombinant plasmid.

[0015] The present invention also provides a method for improving the cadmium tolerance of potatoes, including the step of constructing a transgenic potato plant overexpressing the gene by genetically transforming the above gene into a potato plant.

[0016] The present invention also provides the application of the above gene or biological material in improving the cadmium tolerance of yeast. The biological material is the substance described in the following (1) or (2):

[0017] (1) A recombinant plasmid containing the gene;

[0018] (2) A host cell containing the recombinant plasmid.

[0019] The present invention also provides a method for improving the cadmium tolerance of yeast, including the step of constructing a transgenic yeast overexpressing the gene by genetically transforming the above gene into yeast.

[0020] The present invention discloses the following technical effects:

[0021] Cadmium stress inhibited the growth of potato seedlings, promoted the production of reactive oxygen species (ROS) and the accumulation of cadmium, and ultimately led to cell death. Through transcriptome and metabolome analyses, 2995 differentially expressed genes (DEGs) and 302 differentially accumulated metabolites (DAMs) were found between the two control groups in the present invention. In addition, through the integrated analysis of transcriptome and metabolome, 8 significantly enriched metabolic pathways and 16 related DEGs were identified. Meanwhile, weighted gene co-expression network analysis (WGCNA) was performed to identify four highly significantly related gene modules, covering a total of 1119 key genes. By comprehensively analyzing the above results, the present invention verified the function of a key gene, StAPR3 gene, which showed a significant response to cadmium stress. The results of function verification showed that overexpression of the StAPR3 gene significantly improved the tolerance of transgenic yeast to Cd stress, thus confirming that the StAPR3 gene was involved in the physiological process of cadmium tolerance in potatoes and showed a positive regulatory effect.

[0022] The present invention provides new gene resources for cultivating potato varieties resistant to cadmium stress and has important practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0024] Figure 1 Effects of cadmium on the growth and development of potato seedlings; wherein, A is the potato seedling plants cultured in media with different cadmium concentrations; B is the statistical chart of plant height; C is the statistical chart of stem diameter; D is the statistical chart of fresh weight; E is the statistical chart of leaf number;

[0025] Figure 2 Detection results of root traits of potato seedlings under different cadmium concentration stresses; wherein, A is the statistical chart of total root length; B is the statistical chart of total root area; C is the statistical chart of total root volume; D is the statistical chart of root branching number; E is the statistical chart of root diameter; F is the statistical chart of root ratio;

[0026] Figure 3 Detection results of physiological parameters of potato seedlings under different cadmium concentration stresses; wherein, A is the statistical chart of chlorophyll content; B is the statistical chart of MDA content; C is the statistical chart of proline content; D is the statistical chart of POD activity; E is the statistical chart of SOD activity; F is the statistical chart of CAT activity;

[0027] Figure 4Detection results of physiological parameters of potato seedlings under Cd4 concentration stress; among them, A is the statistical chart of chlorophyll content; B is the statistical chart of MDA content; C is the statistical chart of proline content; D is the statistical chart of POD activity; E is the statistical chart of SOD activity; F is the statistical chart of CAT activity;

[0028] Figure 5 ROS accumulation level and cell wall condition of potato leaves under Cd4 concentration stress;

[0029] Figure 6 Statistical charts of leaf thickness (A) and cadmium content (B);

[0030] Figure 7 Observation results of the microstructure of leaves after cadmium stress treatment; among them, A is the microscopic observation diagram of the microstructure; B is the statistical chart of the palisade tissue thickness; C is the statistical chart of the spongy tissue thickness; D is the statistical chart of the epidermis thickness; in A, the scale bar in the first row is 100μm, and the scale bars in the second and third rows are 50μm;

[0031] Figure 8 Observation results of the transmission electron microscope of leaves after cadmium stress treatment; among them, A is the transmission electron microscope observation diagram; B is the statistical chart of the chloroplast area; C is the statistical chart of the mitochondrial area; D is the statistical chart of the cell wall thickness; in A, the scale bar in the first row is 5μm, and the scale bars in the second and third rows are 2μm;

[0032] Figure 9 Results of screening differentially expressed genes by transcriptome sequencing under cadmium stress; among them, A is the result of the clustering heat map analysis of all differential genes; B is the principal component analysis (PCA) diagram; C is the result of screening genes specifically responding to cadmium stress at 7 days; D is the result of screening genes specifically responding to cadmium stress at 21 days; E is the result of screening genes simultaneously responding to cadmium stress at 7 days and 21 days;

[0033] Figure 10 Clustering heat map of differentially expressed genes identified by transcriptome sequencing; among them, A is the result of the clustering heat map analysis of genes specifically responding to cadmium stress at 7 days; B is the result of the clustering heat map analysis of genes specifically responding to cadmium stress at 21 days; C is the result of the clustering heat map analysis of genes simultaneously responding to cadmium stress at 7 days and 21 days;

[0034] Figure 11 Results of screening differentially accumulated metabolites by metabolome sequencing under cadmium stress; among them, A is the result of the clustering heat map analysis of all differentially accumulated metabolites; B is the principal component analysis (PCA) diagram; C is the result of screening metabolites specifically responding to cadmium stress at 7 days; D is the result of screening metabolites specifically responding to cadmium stress at 21 days; E is the result of screening differentially accumulated metabolites simultaneously responding to cadmium stress at 7 days and 21 days;

[0035] Figure 12 Results of the integrated analysis of transcriptome and metabolome under cadmium stress; among them, A is the result of the nine-quadrant correlation analysis of differentially expressed genes and differentially expressed metabolites specifically responding to cadmium stress at 7 days; B is the result of the nine-quadrant correlation analysis of differentially expressed genes and differentially expressed metabolites specifically responding to cadmium stress at 21 days; C is the result of the correlation analysis between differentially expressed metabolites and their corresponding transcripts after 7 days of stress; D is the result of the correlation analysis between differentially expressed metabolites and their corresponding transcripts after 21 days of stress;

[0036] Figure 13 Is a dendrogram of the correlation coefficients of gene expression levels under different treatment conditions;

[0037] Figure 14 Is a figure showing the determination result of the soft threshold of the gene co-expression network; among them, A is the scale-free fitting index at different powers; B is the average connectivity (or degree of freedom) at different powers;

[0038] Figure 15 Is a clustering tree diagram obtained by hierarchical clustering of various matrices using the hclust function and then pruning using the dynamic tree cutting method;

[0039] Figure 16 Is a figure showing the relationship between different modules and various metabolites;

[0040] Figure 17 Is a statistical chart of the expression levels of genes (Soltu.DM.04G035270, Soltu.DM.05G009940, Soltu.DM.10G021250, Soltu.DM.04G021850, Soltu.DM.01G044770, Soltu.DM.03G010850, Soltu.DM.02G004130, Soltu.DM.05G009520, Soltu.DM.01G050280, and Soltu.DM.02G023850) before and after cadmium stress;

[0041] Figure 18 Is a double digestion verification diagram of the recombinant plasmid pYES2-StAPR3; among them, 1: original plasmid; 2: recombinant plasmid; 3: DNA Marker;

[0042] Figure 19 Is a figure showing the PCR identification result of positive colonies after plasmid transformation into yeast; among them, from left to right, the first lane is DNA Marker; the second to ninth lanes are transgenic yeast samples;

[0043] Figure 20 Is a figure showing the growth of yeast in media containing different concentrations of cadmium. Detailed implementation method

[0044] The various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and implementation schemes of the present invention.

[0045] It should be understood that the terms described in the present invention are only for describing specific embodiments and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0046] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0047] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are merely exemplary.

[0048] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0049] Example 1

[0050] 1. Materials and Methods

[0051] 1.1 Plant Cultivation and Experimental Design

[0052] The present invention uses Atlantic potatoes as research materials. These materials are cultured on 1 / 2 MS medium in a greenhouse (16 h light / 8 h dark, 23 - 25 °C, light intensity 400 - 600 μmol·m -2 ·s -1, relative humidity 60%-70%). After 21 days of normal growth, the seedlings with similar growth states were cut into segments with one bud and one leaf, each 2 cm long, and placed in a culture medium containing cadmium (Cd) for treatment. First, the present invention conducted screening to determine the optimal concentration of cadmium tolerance in potatoes. A total of 9 concentration gradients were set, namely 0, 10, 20, 50, 100, 150, 200, 300, and 400 μM (named CK, Cd1-8 experimental groups in sequence). After 21 days of stress treatment, the phenotypes and physiological indexes under various conditions were measured. Subsequently, transcriptome and metabolome analyses were performed on the samples at the maximum cadmium tolerance concentration (100 μM), and further analysis was carried out on the changes in the microscopic structure of potato leaves.

[0053] 1.2 Measurement of morphological parameters

[0054] Nine plants were selected under each treatment condition to measure their plant height, maximum root length, fresh weight, number of leaves, and root traits. Parameters such as the number of root tips, total root length, root surface area, root volume, average root diameter, and root length-volume ratio were analyzed using WinRHIZO 5.0 software. The experiment was repeated three times.

[0055] 1.3 Determination of physiological parameters

[0056] Solarbio detection kits (kit codes: BC0290, BC0030, BC0170, BC0090, and BC2700) were used to determine the accumulation of proline (Pro) and malondialdehyde (MDA) in the seedlings, as well as the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT).

[0057] Fresh samples (150 mg) from 21-day-old seedlings were used for analysis to extract and determine the chlorophyll content.

[0058] 1.4 Microscopic structure observation

[0059] Paraffin sections of the third top leaf were prepared and the cell structure was observed under an epifluorescence microscope (Carl Zeiss, Oberkochen, Germany).

[0060] 1.5 Determination of Cd content

[0061] The 21-day-old seedlings were washed with water and dried overnight in an oven at 70 °C for Cd content determination. After storing the samples at room temperature for 3 days, approximately 100 mg of the dried samples were weighed and placed in an HNO3-HClO4 solution with a volume ratio of 4:1 (HNO3:HClO4), and completely digested in a block digester at 200 °C. The digested samples were dissolved in distilled water and filtered through a 0.22 μm cellulose acetate filter. Detection was performed using inductively coupled plasma mass spectrometry (ICP-MS, Agilent 7500 USA).

[0062] 1.6 Transcriptomics analysis

[0063] Transcriptome sequencing was performed on the aerial parts of potato seedlings subjected to 100 μM Cd stress for 0, 7, and 21 days. The materials cultured in normal MS medium were used as controls. The samples were quickly collected and frozen in liquid nitrogen, then stored in dry ice and sent to Wuhan Maiwei Metabolic Biotechnology Co., Ltd. for transcriptome sequencing. RNA was extracted from the potato seedlings for sequencing. After filtering the raw data, the sequencing error rate and GC content distribution were examined to obtain clean reads for subsequent analysis. FPKM (Fragments Per Kilobase of exon per Million reads mapped) values were used to indicate the expression levels of transcripts or genes. Three biological replicates were set for each treatment.

[0064] 1.7 Metabolomics analysis

[0065] Simultaneously with transcriptome sequencing, untargeted metabolomics analysis was performed on the same samples. Sample preparation and data analysis for metabolomics analysis were carried out at Wuhan Metware Biotechnology Co., Ltd. (Wuhan, China) according to standard procedures. The prepared samples were ground and mixed for 1.5 minutes at a frequency of 30 Hz using a mixer mill (MM400, Retsch) equipped with zirconia beads. 100 mg of each sample was extracted overnight at 4 °C in 1.0 mL of 70% aqueous methanol. During the extraction process, vortex mixing was performed three times to improve the extraction efficiency.

[0066] 1.8 WGCNA analysis.

[0067] The "Weighted Gene Co-Expression Network Analysis (WGCNA)" package in R language was used to identify 32,933 genes from 15 samples. The Fragments Per Kilobase of exon per Million reads mapped (FPKM) values of these genes (denoted as R2) were used for co-expression network analysis. The eigengene values of each module were calculated to evaluate their correlations with 24 significantly different metabolites, and the interactive network was visualized using Cytoscape v3.7.1.

[0068] 1.9 Statistical analysis

[0069] One-way ANOVA (Duncan's post hoc test) and Student's t-test were performed using GraphPad Prism 9 and IBM SPSS Statistics 23.0 to evaluate the systematic variance under different treatment conditions (α = 0.05), respectively. All experiments were conducted with three biological replicates. Data were expressed as mean ± standard deviation (SD).

[0070] 2. Results

[0071] 2.1 Morphological Attribute Responses

[0072] This invention aims to explore the effects of cadmium on the growth and development of potato seedlings. First, potato tissue culture seedlings were stressed and induced with 8 different concentrations of cadmium. By screening growth phenotypes and detecting various physiological indexes, the maximum tolerance concentration of potato seedlings to cadmium was determined. The results showed that with the increase in Cd concentration, the growth and development of potato seedlings were inhibited to varying degrees ( Figure 1 ). At the Cd4 concentration, the plant height was significantly lower than that of the CK, only reaching 67.79% of the CK height. With the increase in Cd concentration, the inhibitory effect became more obvious. The fresh weight and leaf number showed a similar trend to the plant height, showing a significant decrease under the Cd2 condition. With the increase in Cd concentration, the degree of decrease gradually increased and reached the lowest at the Cd8 condition ( Figure 1 ). The stem diameter showed a different trend. From the Cd1 to Cd6 conditions, the stem diameter fluctuated slightly, but the difference was not significant. On the contrary, under the higher concentrations of Cd7 and Cd8 conditions, the stem diameter showed a significant increase compared with the control group ( Figure 2 ). In addition, the root index showed a different trend from the above-ground index ( Figure 2 ). Under the lowest level of Cd stress, all root parameters were significantly inhibited, and the inhibitory effect increased with the increase in concentration. When exposed to the Cd5 concentration, root growth was completely blocked. It should be noted that when root growth was not completely inhibited (Cd1 - Cd4), the root diameter increased with the increase in Cd concentration. Specifically, under the Cd3 and Cd4 conditions, the root diameters were 1.41 times and 1.46 times higher than that of the control group, respectively.

[0073] 2.2 Physiological Attribute Responses

[0074] To further clarify the effects of cadmium on the growth of potato seedlings, this invention additionally measured various physiological parameters related to photosynthesis, antioxidant system, and stress resistance. Different response patterns of each parameter under cadmium-induced stress were observed. At the Cd1 - Cd3 concentrations, the chlorophyll content showed no significant difference compared with the control. Nevertheless, a significant decrease was observed starting from the Cd4 concentration, and the lowest point was recorded at Cd5, only reaching 23.02% of the control level. Subsequently, the chlorophyll content tended to be stable (Figure 3 In A). Malondialdehyde (MDA) and proline are key physiological markers of the stress response. At the Cd2 concentration, the MDA content increased significantly compared to the control, with a peak 1.88 times that of the control level. Subsequently, the MDA content gradually decreased with the increase in Cd concentration, and no significant difference was observed compared to the control beyond the Cd7 concentration ( Figure 3 In B). Proline showed the opposite pattern. No significant difference was observed from Cd1 to Cd4; however, a significant increase in proline level relative to the control began at Cd5 and reached a peak at Cd8 ( Figure 3 In C). The activities of the three antioxidant enzymes showed a similar pattern, all showing significantly higher than the control starting from Cd4 ( Figure 3 In D - F). These activities gradually increased with the increase in Cd concentration and reached a peak at Cd8.

[0075] 2.3 Effects of the maximum Cd tolerance concentration on the microstructure

[0076] After comprehensively evaluating the phenotypic and physiological changes of potato seedlings under different Cd concentrations, Cd4 was determined as the maximum tolerance concentration of potato seedlings. Under this condition, all plant indices were significantly affected, but the plants managed to survive. When exceeding Cd4, the growth and development of plant roots were completely inhibited. It is well known that the phenotype, physiology, and microstructure of plants are affected under stress conditions. Therefore, the present invention further analyzed the changes in the plant microstructure under Cd4 conditions. Before this analysis, the present invention re - evaluated the key physiological indices. The observed trends of these changes were largely consistent with the previous findings of the present invention. After exposure to stress conditions, the levels of MDA and proline and the activities of antioxidant enzymes (POD, SOD, and CAT) increased significantly, while the chlorophyll content decreased significantly ( Figure 4 、 Figure 5 ). In addition, the present invention observed that under this treatment, compared to the control group, the leaf thickness increased by 45.71%, and the Cd accumulation in the plants reached up to 0.28 mg / g ( Figure 6 ).

[0077] The results of paraffin section showed that after growing for 21 days under normal conditions, the microstructure of the leaves remained basically unchanged ( Figure 7)。The epidermal tissue is arranged neatly, the palisade tissue is evenly distributed, the spongy tissue is uniform, and the chloroplasts are evenly distributed within the cells. However, significant changes in the leaf microstructure were observed after exposure to cadmium stress. The leaves showed an increase in thickness, gradual densification and thickening of the epidermal tissue, a decrease in cell size leading to crowding, an increase in the density of the palisade and spongy tissues, a decrease in chloroplast count, and irregular and dispersed intracellular morphology. Statistical analysis showed that under normal conditions, there were no significant changes in the palisade tissue, spongy tissue, and epidermal cells. On the contrary, under Cd stress, the palisade tissue, spongy tissue, and epidermal cells increased significantly compared to the control group, by 19.69%, 60.69%, and 68.64% respectively( Figure 7 )。

[0078] Meanwhile, transmission electron microscopy was used to analyze the changes in organelles( Figure 8 )。Under normal conditions, the ultrastructure of potato leaf cells appeared clear and complete. The chloroplasts were round or oval in shape, abundant in number, and the structural layers were obvious. The thylakoid layers were clearly visible and arranged orderly. The mitochondria were evenly distributed and had a compact structure. The starch grains were few and small, mainly located within the chloroplasts. The cell walls appeared clear, smooth, and dense. The nucleoli showed distinct colors and complete structures. After 21 days of stress, significant changes in the ultrastructure were observed. The chloroplasts became swollen, spherical in shape, and the membrane structure was blurred. The thylakoid structure underwent fragmentation, reduction, or complete disappearance, resulting in a lighter color and disordered layer arrangement. Large and plump starch grains appeared inside the chloroplasts, and the number increased sharply. The double membrane of the mitochondria dissolved, the cristae decreased or disappeared, and internal vacuolization occurred. The cell walls became thinner, lighter in color, the cells folded and deformed, and plasmolysis was observed. Statistical results showed that the chloroplast area decreased significantly by 75.21% after the stress treatment. The mitochondrial area showed a non-significant increase of 0.58%, while the cell wall thickness decreased significantly by 26.5%( Figure 8 )。

[0079] 2.4 Transcriptome profiling analysis

[0080] 2.4.1 Transcriptome sequencing and identification of differentially expressed genes

[0081] Since there were significant differences in growth phenotypes, physiological indices, and ultrastructures between CK and Cd4 treatments, transcriptome analysis was performed to explore the changes in potato gene expression under cadmium stress, aiming to further clarify the molecular mechanism of cadmium tolerance in this plant. A total of 687,348,902 raw reads were obtained from 15 samples, and 677,482,684 clean reads were retained after filtering using bioinformatics software. The final clean reads accounted for more than 98.41% of the total raw reads. The Q20 and Q30 values exceeded 99.52% and 95.77% respectively, while the GC content was between 52.14% and 56.14%, demonstrating the high reliability of the sequencing results.

[0082] The results of the heatmap analysis showed that the correlation coefficient between the two groups exceeded 0.8( Figure 9 in A). In addition, principal component analysis (PCA) highlighted the significant differences in single gene expression under Cd stress treatment( Figure 9 in B). Based on these findings, under Cd stress conditions, the gene expression within each treatment group was examined in detail at different time points. In addition, a fold change ≥ 2 and an adjusted P < 0.05 were established as the filtering criteria for paired comparison analysis to screen for DEGs (Differentially Expressed Genes) in different groups. In the paired comparisons of Group 1 (Cd7 vs. CK0), Group 2 (CK7 vs. CK0), Group 3 (Cd21 vs. CK0), and Group 4 (CK21 vs. CK0), 5503 (3106 up-regulated and 2397 down-regulated), 5066 (2760 up-regulated and 2306 down-regulated), 5756 (3265 up-regulated and 2491 down-regulated), and 4947 (2798 up-regulated and 2149 down-regulated) DEGs were identified respectively. To eliminate the DEGs affected by plant growth and development, further screening of DEGs was performed based on the grouping of Group1-Group2 and Group3-Group4. This led to the identification of 1454 and 1810 genes that specifically responded to Cd treatment for 7 days and 21 days, respectively. Subsequently, a Venny diagram analysis was performed on the genes that uniquely responded at 7 days and 21 days, and 1185 genes (Cd7-unique), 1541 genes (Cd21-unique), and 269 genes (Cd7 / 21-common) were found to show specific differential expression at 7 days, 21 days, and both time points respectively( Figure 9 in C-E). The expression of these genes under Cd stress was further analyzed by a clustering heatmap( Figure 10 ).

[0083] 2.4.2 GO analysis and KEGG pathway analysis

[0084] Gene Ontology (GO) was used to study gene clusters related to Cd stress tolerance. In the GO enrichment analysis, it was shown that in the Cd7-unique, Cd21-unique, and Cd7 / 21-common groups, 276, 319, and 268 annotated GO terms were significantly enriched (P-value < 0.05), respectively. In the Cd7-unique group, 70% of the Gene Ontology (GO) terms were classified as cellular components (CC), while biological processes (BP) and molecular functions (MF) accounted for only 20% and 10%, respectively. Among them, more than 80% of the GO terms were related to the photosynthesis process. In contrast, in the Cd21-unique group, 65% of the GO terms were related to BP, covering a wide range of processes such as sulfur metabolism (GO:0010438, GO:0006791, GO:0006792), secondary metabolism (GO:0019748), and glucose metabolism (GO:0010439, GO:1901071). CC and MF contributed only 20% and 15% to the enrichment, respectively. In the Cd7 / 21-common group, no differentially expressed genes (DEGs) were enriched in CC. Notably, 50% of the GO terms were related to metal ion transport or homeostasis, including iron ion homeostasis (GO:0055072), siderophore metabolic process (GO:0009237), and metal ion homeostasis (GO:0055065). The remaining terms were mainly related to various enzyme activities and secondary metabolism.

[0085] 2.4.3 KEGG Pathway Enrichment Analysis

[0086] As the main database of functional pathways, it can identify biological pathways involved in incompatible interactions. The most influential biochemical metabolism and signal transduction pathways related to DEGs can be characterized by the significant enrichment of pathways. In the Cd7-unique, Cd21-unique, and Cd7 / 21-common groups, 7, 5, and 4 pathways with P-value < 0.05 were identified, respectively. The metabolic pathways enriched in each group showed significant differences. For example, after 7 days of stress, the enriched pathways included photosystem (map00195), linoleic acid metabolism (map00591), and carotenoid biosynthesis (map00906); after 21 days of stress, the enriched pathways involved MAPK signaling (map04016) and plant-pathogen interaction (map04626). In addition, there were also pathways significantly enriched at different sampling time points, such as phenylpropanoid biosynthesis (map00940) and glutathione metabolism (map00480).

[0087] 2.5 Metabolome Profiling

[0088] 2.5.1 Quality Control of Metabolomics Data

[0089] To study the metabolic response mechanism of potatoes to cadmium stress, non-targeted metabolomics (LC-MS) analysis was used to reveal the metabolic network in the above-ground parts of potatoes under different treatments. A total of 1232 metabolites were detected in all samples. According to their characteristics, these metabolites were divided into 13 categories, among which the most prominent 5 categories were phenolic acids (17.97%), flavonoids (17.2%), alkaloids (14.07%), lipids (10.81%) and terpenoids (8.06%). Initially, multiple PCA analysis and OPLS-DA analysis were performed on the clustering information from each group. The results revealed a distinct separation between the control group and the Cd treatment group ( Figure 11 ). In addition, significant differences were observed in the OPLS-DA analysis between the Cd treatment groups at different time points. These results emphasized the significant differences in the metabolite profiles of potato seedlings subjected to cadmium stress of different durations. In addition, this study visually demonstrated the differentially accumulated metabolites (DAMs) and their accumulation patterns in each control group through a clustering heatmap ( Figure 11 ).

[0090] 2.5.2 Metabolite content

[0091] In this project, a fold change ≥ 2 or fold change ≤ 0.5 and VIP > 1 were established as the filtering criteria for paired comparison analysis to identify differentially accumulated metabolites from different treatment groups. The screening results of differentially accumulated metabolites between the Cd treatment group and the control group showed that in the paired comparisons of Group 1 (Cd7 vs CK0), Group 2 (CK7 vs CK0), Group 3 (Cd21 vs CK0) and Group 4 (CK21 vs CK0), a total of 630 differentially accumulated metabolites (498 up-regulated and 132 down-regulated), 593 differentially accumulated metabolites (473 up-regulated and 120 down-regulated), 650 differentially accumulated metabolites (498 up-regulated and 152 down-regulated) and 501 differentially accumulated metabolites (368 up-regulated and 133 down-regulated) were identified, respectively. To eliminate DAMs affected by plant growth and development, a method similar to that for transcriptome data analysis was used to further refine the DAMs specifically responsive to cadmium stress after 7 days and 21 days of exposure. The differentially accumulated metabolites specifically responsive to Cd treatment for 7 days and 21 days were isolated, including 630 and 650 differentially accumulated metabolites, respectively ( Figure 11 in C, D). Subsequently, Venn diagram analysis was performed on the genes showing specific responses at 7 days and 21 days, resulting in 73 unique DAMs for Cd7, 205 DAMs for Cd21 and 24 common DEMs for Cd7 / 21, which accumulated significantly differently at 7 days, 21 days and both time points ( Figure 11 in E).

[0092] 2.5.3 KEGG enrichment analysis

[0093] Through KEGG enrichment pathway analysis, the present invention studied the key metabolic pathways of the above-ground part of potato in response to cadmium toxicity. Subsequent analysis revealed a total of 63 key metabolic pathways involved in the response of potato buds to cadmium stress at different time points. Specifically, there are 18 pathways unique to Cd7, 35 pathways unique to Cd21, and 10 pathways common to Cd7 and Cd21. Based on this, the present invention further comprehensively considered the number of differential metabolites enriched in each pathway, the number of background metabolites, and the total amount of differential metabolites in the results. Subsequently, a significance analysis was performed on each pathway. Using a threshold of P<0.05, a total of 9 significantly enriched KEGG pathways were identified, including "2-oxocarboxylic acid metabolism", "amino acid biosynthesis", "pyruvate metabolism", "linoleic acid metabolism", "flavonoid biosynthesis", "biosynthesis of quercetin aglycone I / II", "biosynthesis of valine, leucine and isoleucine", and "glutathione metabolism". Finally, 34 different metabolites were enriched in these 9 metabolic pathways, highlighting their potential importance in the response of potato seedlings to cadmium stress.

[0094] 2.6 Joint analysis of transcriptome and metabolome

[0095] 2.6.1 Correlation between genes and metabolites

[0096] To explore the relationship between DEGs and DAMs in potato seedlings under cadmium treatment, a co-expression network joint analysis was performed on the previously identified DEGs and DAMs. The grouping criteria are as follows (Pearson correlation coefficient > 0.8, P<0.05): Cd7-uniqueG / M, Cd21-uniqueG / M, and Cd7 / 21-commonG / M. When comparing the Cd7 unique group and the Cd21 unique group, correlation analysis revealed significant correlations between multiple genes and metabolites ( Figure 12A and B). These findings suggest that the identified related genes may be involved in directly or indirectly regulating the accumulation of these metabolites. Subsequently, the present invention analyzed the importance of the pathways co-enriched by DEGs and DAMs. The present invention specifically selected the pathway information that is generally enriched under the condition of a significance level P < 0.05. In the Cd7-uniqueG / M group, a total of 10 pathways were enriched. Among them, the linoleic acid metabolism pathway (map00591) showed significant enrichment compared to other pathways. By simultaneously mapping DAMs and all DEGs to the KEGG pathway database, the present invention identified 5 pathways that are generally enriched by DEGs (P < 0.05) and DAMs (VIP > 1, P < 0.05), namely map00620, map00908, map00591, map00942, and map00290. In the Cd21-uniqueG / M group, more pathways were enriched, and 20 pathways simultaneously significantly enriched different genes and metabolites under the same threshold. In addition, in the Cd7 / 21-Common / M group, probably due to the small number of DAMs (only 24), only 3 common pathways were enriched, namely map00480, map04216, and map00290. On this basis, the correlation between the significantly enriched genes and metabolic pathways was examined, and a total of 17 pairs of significantly related gene-pathway relationships were identified ( Figure 12 C and D). Finally, the present invention identified 8 metabolic pathways and 16 highly related DEGs.

[0097] 2.6.2 WGCNA analysis

[0098] To comprehensively analyze the effects of cadmium on potato seedlings, 24 differential metabolites named M01-M24 selected from the Cd-7 / 21-common group were further analyzed using WGCNA. Considering the influence of outlier samples on network module analysis, it is crucial to perform outlier sample verification before constructing the network module to ensure the accuracy of the results. In this study, the correlation coefficients of gene expression levels under different treatment conditions were calculated, and the results were graphically presented through a dendrogram ( Figure 13 ). The results showed that there were no significant differences among the three replicates of each sample in different treatment groups, as they formed a unified cluster. Therefore, no outlier samples were detected in the present invention. In addition, to create a co-expression network, the correlation coefficients between gene pairs must first be determined, and then a gene similarity matrix is generated. Subsequently, the weighted gene co-expression network was established using the WGCNA package in R software. To ensure compliance with the scale-free network distribution characteristics, the pick soft threshold function in the WGCNA package was used to calculate the appropriate weight. As Figure 14 shown, a soft threshold of β = 7 was selected in the present invention to construct the co-expression network.

[0099] After determining the soft threshold as β = 7, the similarity matrix is converted into an adjacency matrix using the formula Amn = [(1 + Smn) / 2] × β. Subsequently, this adjacency matrix is further transformed into a topological overlap matrix (TOM). After this transformation, the inverse of the topological overlap matrix is obtained by performing diss Tom = 1 - TOM to derive a dissimilarity matrix. The next step involves using the hclust function to perform hierarchical clustering on various matrices, and then using the dynamic tree cut method to prune the resulting clustering tree. During this process, genes with similar expression patterns tend to cluster on the same branch, where each branch represents a co-expression module, distinguished by different colors, indicating different modules. The assignment of the number of genes within a module is based on their transcripts per million (TPM) values in the corresponding cluster, and genes assigned to the same module show a higher level of clustering ( Figure 15 ).

[0100] In the study of the present invention, the module is constructed through gene correlation analysis and integrated with the metabolite matrix. A total of 12 module heatmaps are generated, covering modules related to different metabolites. Modules showing strong correlations are screened, identified using the thresholds of R > 6 and P < 0.05, to examine the relationship and significance between each module and various metabolites. The research results show that not all modules show a significant association with metabolites, highlighting the significant differences between modules ( Figure 16 ). For example, the MEpink and MEgrey modules do not show a significant correlation with any metabolite; relatively few significant related metabolites are observed in the MEpurple, MEbrown, MEgreenyellow, MEyellow, and MEturquoise modules, which are 4, 6, 1, 1, and 5 respectively. In contrast, the MEblue, MEmagenta, MEblack, and MEred modules show a significant correlation with more metabolites, which are 14, 16, 22, and 16 respectively. These four modules represent the focus of subsequent research in the present invention.

[0101] Subsequently, the Cytoscape method is used to evaluate the contribution values of genes within each module, and the top 10 genes with the highest degree values in each module are designated as key focuses. Since these genes are from the correlation analysis with metabolites, their specific responses to Cd-induced stress remain uncertain. Therefore, the present invention conducts analyses at 7 days and 21 days after Cd stress treatment respectively. The results show that after 7 days of stress, two genes show significant expression differences, while after 21 days of stress, six genes show significant changes. Subsequently, based on the fold change in gene expression levels before and after stress ( Figure 17), and based on previous integrative transcriptomic and metabolomic analyses, a gene (Soltu.DM.02G004130) was selected for further functional verification. According to its sequence characteristics, this gene was named StAPR3 (Soltu.DM.02G004130).

[0102] The nucleotide sequence of the StAPR3 gene (SEQ ID NO.1) is as follows:

[0103]

[0104] Example 2

[0105] Verify the function of the StAPR3 gene in improving the cadmium tolerance of potatoes in yeast:

[0106] 1. Recombinant plasmid construction

[0107] After synthesizing the StAPR3 gene sequence, it was constructed into the pYES2 yeast expression vector to obtain the recombinant plasmid pYES2-StAPR3. After synthesis, double digestion verification was carried out, and the results are shown in Figure 17 .

[0108] 2. Transform the recombinant plasmid into the recipient bacterium YCF1

[0109] Transform the recombinant plasmid pYES2-StAPR3 into the yeast competent cell YCF1, and at the same time use the empty pYES2 vector as a control for transformation. The yeast transformation method is as follows:

[0110] (1) Pick a single colony of YCF1 from the YPDA plate and inoculate it into 4 mL of YPDA liquid medium. Incubate at 30 °C with shaking at 225 rpm for 18 h (overnight) until OD 600 > 1.5.

[0111] (2) Transfer to YPDA liquid medium with a culture volume of 50 mL to make the initial OD 600 = 0.2. Incubate at 30 °C with shaking at 225 rpm for 4 h until OD 600 = 0.6.

[0112] (3) Centrifuge to collect the bacteria at room temperature at 4000 rpm for 5 min.

[0113] (4) Resuspend the cells with 20 mL of sterile water, mix well, centrifuge to collect the bacteria at room temperature at 4000 rpm for 5 min, and discard the supernatant.

[0114] (5) Resuspend the cells with 5 mL of 0.1 M LiAc, mix well, centrifuge to collect the bacteria at room temperature at 4000 rpm for 5 min, and discard the supernatant.

[0115] (6) Resuspend the cells with 500 μL of 0.1 M LiAc, mix well, aliquot into 1.5 mL centrifuge tubes, 50 μL each (for each transformation), and set aside.

[0116] (7) Add 240 μL of 50% PEG3350, 36 μL of 1 M LiAc, 5 μL of ssDNA (10 mg / mL), and 5 μL of plasmid DNA to each 1.5 mL centrifuge tube in sequence. Mix well with a pipette tip or shake vigorously for about 1 min until completely mixed.

[0117] (8) Incubate in a 30 °C water bath for 30 min.

[0118] (9) Heat shock in a 42 °C water bath for 25 min.

[0119] (10) Resuscitate in a 30 °C water bath for 30 min.

[0120] (11) Centrifuge to collect bacteria at room temperature, 4000 rpm, for 5 min, and discard the supernatant.

[0121] (12) Suspend the bacterial cells with 200 μL of sterile water for each transformation, mix gently as much as possible, and spread on the corresponding defective screening plates.

[0122] (13) Incubate at 30 °C for 4 days.

[0123] The results of PCR identification of positive colonies after plasmid transformation into yeast are shown in Figure 19 .

[0124] 3. Yeast phenotype verification

[0125] Prepare SG-U (galactose substituted for glucose) solid media containing 0 μM, 40 μM, 50 μM, 60 μM, 80 μM, and 100 μM CdCl2 respectively. Take the single colonies verified correctly by PCR from the experimental group (pYES2-StAPR3) and the empty vector control group, and uniformly dilute the OD 600 value and spot plate onto SG-U plates containing 0 μM, 40 μM, 50 μM, 60 μM, 80 μM, and 100 μM CdCl2, and place them in an incubator at 30 °C for cultivation. Observe the colony growth after 10 d.

[0126] 4. Results

[0127] In this invention, the StAPR3 gene was overexpressed in yeast, and the growth status of transgenic yeast strains in media containing different concentrations of cadmium was observed to identify their cadmium tolerance function. The results are shown in Figure 20 . The results showed that transgenic yeast could grow normally in the control medium (0 μM), and the plaque concentration gradually decreased at different dilution ratios. However, under cadmium stress conditions, the transgenic yeast carrying the StAPR3 gene was only inhibited in growth at a concentration of 100 μM, while showing strong growth vitality at concentrations of 40, 50, 60, and 80 μM. These results indicate that the StAPR3 gene is involved in the physiological process of cadmium tolerance in potatoes and shows a positive regulatory effect.

[0128] The embodiments described above are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for improving the cadmium tolerance of yeast, characterized in that it includes the step of transforming a gene with a nucleotide sequence as shown in SEQ ID NO.1 into yeast by genetic transformation to construct a transgenic yeast overexpressing the gene.

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