Application of StPIF4 gene in regulation and control of salt tolerance of potatoes
By knocking out or silencing the expression of the potato StPIF4 gene through genetic engineering techniques, the accumulation of reactive oxygen species was regulated, which solved the problem of potato's sensitivity to salt stress, enhanced its salt tolerance, and provided new breeding targets and theoretical basis.
Patent Information
- Application Number
- CN202511645649.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-11
- Publication Date
- 2026-01-23
AI Technical Summary
Potatoes are sensitive to salt stress, and existing technologies lack effective regulatory mechanisms to enhance their salt tolerance. The expression and regulatory mechanisms of the PIF4 gene under salt stress are not yet fully understood.
By using genetic engineering techniques to knock out or silence the expression of the potato StPIF4 gene, the StPIF4 gene can negatively regulate the accumulation of reactive oxygen species, affecting cell membrane integrity and thus enhancing the salt tolerance of potatoes.
This study effectively alleviates oxidative damage under salt stress and enhances the salt tolerance of potatoes, providing new targets and theoretical basis for salt-tolerant breeding.
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Abstract
Description
TECHNICAL FIELD
[0001] The application relates to the field of plant genetic engineering technology and potato breeding technology, and particularly relates to application of a StPIF4 gene in regulating salt tolerance of potatoes. BACKGROUND
[0002] The growth and development of plants are limited by various environmental stresses in nature, and soil salinization is one of the main abiotic stresses that restrict crop yield and threaten food security. Under salt stress conditions, plants suffer from osmotic stress and ion toxicity, leading to inhibition of photosynthesis, damage to the membrane system, imbalance of ion homeostasis, and disturbance of normal physiological metabolism of plants. In severe cases, leaf yellowing, growth stagnation, and even plant death can occur. Potato (Solanum tuberosum L.) is the fourth largest food crop in the world, and it has moderate sensitivity to salt. Therefore, it is of great significance to develop more salt-tolerant potato varieties.
[0003] The most classic adaptive strategy of plants to salt stress is the calcium signal-dependent salt overly sensitive (SOS) signal pathway. When plants encounter salt stress, the intracellular calcium signal is rapidly triggered to increase, which is then sensed by the calcium-binding protein SOS3 and forms a complex with SOS2, which is a serine / threonine protein kinase. SOS2 can phosphorylate and activate SOS1, which transports Na+ from the cytoplasm to the outside of the cell, thereby improving the salt tolerance of plants.
[0004] Besides triggering physiological changes at the plasma membrane level, salt stress also induces alterations at the overall transcriptome level, thereby adjusting plant metabolism and developmental adaptations. The plant phytochrome interaction factor (PIF) family belongs to the basic helical-loop-helical (bHLH) transcription factor family and typically accumulates in dark environments, inhibiting photomorphogenesis. Under salt stress, PIF4 can negatively regulate the expression of salt tolerance-related genes, reducing plant salt tolerance. For example, in Arabidopsis, it inhibits salt stress by directly binding to the JUB1 promoter. Salt stress can also activate SOS2 kinase, which interacts with PIF4 in the nucleus, phosphorylating and promoting PIF4 degradation, thus relieving the inhibition of salt-responsive gene expression by PIF4 and enhancing plant salt tolerance. Salt stress induces the formation of "halobodies" in the nucleus through liquid-liquid phase separation. SSN1 interacts with PIF4 and recruits the SOS2-PIF4 complex into the halobodies, promoting PIF4 degradation and accelerating plant adaptation to salt stress. Regulation of PIF4 Salt Response by Light Signaling: Light-activated phytochromes (such as PhyA and PhyB) can enhance the kinase activity of SOS2, indirectly promoting the phosphorylation and degradation of PIF4. Furthermore, light signals influence the role of PIF4 in salt stress response by regulating its expression and stability. For example, under light conditions, PIF4 accumulation decreases, while under salt stress, PIF4 degradation accelerates, helping plants adapt to saline environments.
[0005] In summary, PIF4 is a key dynamic regulator in plant salt response, and its function is finely regulated by multiple mechanisms, including salt stress signaling pathways, light signals, and intranuclear phase separation. Although the function of PIF4 in various plants has been explored, systematic research on its specific role and regulatory mechanisms in potato, particularly in salt stress response, remains lacking. Therefore, elucidating the expression pattern and regulatory network of the StPIF4 gene in potato under salt stress is of significant theoretical and applied value for revealing the molecular mechanisms of potato salt tolerance and breeding new salt-tolerant potato varieties. Summary of the Invention
[0006] The inventors screened StPIF4, a member of the potato PIFs family, from environmental stress transcriptome data in the potato PGSC database, and found that its transcriptional level decreased under salt stress. Genetic transformation using Agrobacterium-mediated transformation yielded interfering transgenic lines. Salt stress treatment of both transgenic and wild-type lines showed that the StPIF4 gene negatively regulates reactive oxygen species accumulation, thereby affecting cell membrane integrity. Silencing StPIF4 effectively alleviated oxidative damage under salt stress, thus enhancing the salt tolerance of potatoes.
[0007] In view of this, the purpose of this invention is to provide the application of the StPIF4 gene in regulating potato salt tolerance.
[0008] To achieve the above object, the present application provides the following technical solutions.
[0009] The present application provides the application of StPIF4 gene or its coded protein in regulating the salt tolerance of potato, the CDS sequence of the StPIF4 gene is shown as SEQ ID No. 1, and the amino acid sequence of the coded protein is shown as SEQ ID No. 2.
[0010] Specifically, the application is to enhance the salt tolerance of potato by knocking out the StPIF4 gene or silencing or reducing the expression amount of the StPIF4 gene through genetic engineering technical means.
[0011] The present application also provides a method for enhancing the salt tolerance of potato, which comprises knocking out the StPIF4 gene or silencing or reducing the expression amount of the StPIF4 gene through genetic engineering technical means to enhance the salt tolerance of potato.
[0012] The present application also provides a preparation for enhancing the salt tolerance of potato, which comprises a reagent capable of reducing the expression amount of the StPIF4 gene.
[0013] The present application also provides the application of StPIF4 gene, StPIF4 gene coded protein and StPIF4 gene recombinant expression vector in the breeding of potato with improved salt tolerance.
[0014] The present application also provides a method for creating potato with salt tolerance, which comprises knocking out the StPIF4 gene or silencing or reducing the expression amount of the StPIF4 gene through genetic engineering technical means.
[0015] Further, the method comprises cloning the CDS specific segment of the StPIF4 gene into the pHELLSGATE-8 vector to obtain an interference vector, and then using Agrobacterium-mediated genetic transformation of potato slices to obtain potato mutants with StPIF4 interference expression and enhanced salt tolerance.
[0016] The present application has the beneficial effects that the present application provides the application of StPIF4 gene in regulating the salt tolerance of potato, the CDS sequence of the StPIF4 gene is 1554 bp long, the sequence is shown as SEQ ID No. 1, it encodes 517 amino acids, the sequence is shown as SEQ ID No. 2, the gene is located in the nucleus, it negatively regulates the salt tolerance of potato, and it affects the integrity of cell membrane by negatively regulating the accumulation of active oxygen. The silencing can effectively alleviate the oxidative damage under salt stress, thereby enhancing the salt tolerance of potato. This provides a new target gene and theoretical basis for analyzing the molecular mechanism of potato salt tolerance and breeding improvement. BRIEF DESCRIPTION OF DRAWINGS
[0017] Figure 1 For StPIF4 sequence feature analysis; wherein A is phylogenetic tree analysis; B is amino acid sequence alignment analysis.
[0018] Figure 2 For StPIF4 expression pattern analysis under 200mM NaCl treatment.
[0019] Figure 3 For StPIF4 subcellular localization.
[0020] Figure 4 For StPIF4 transgenic line identification; wherein (a) is agarose gel electrophoresis identification of vector primers on interference transgenic plants; (b) is StPIF4 interference transgenic line interference efficiency detection; (c) is agarose gel electrophoresis identification of vector primers on overexpression transgenic plants; (d) is StPIF4 overexpression transgenic line overexpression efficiency detection.
[0021] Figure 5 For wild type and StPIF4 transgenic line phenotype under normal conditions and after 20d salt treatment; wherein (a) is wild type and StPIF4 transgenic line phenotype under normal conditions and after 20d 200mM NaCl treatment, bar = 5cm; (b) is StPIF4 interference line height under normal conditions and 200mM NaCl treatment; (c) is StPIF4 overexpression line height under normal conditions and 200mM NaCl treatment.
[0022] Figure 6 For wild type and StPIF4 interference transgenic plant physiological index determination before and after salt treatment under salt stress; wherein (a) is superoxide anion content; (b) is hydrogen peroxide content; (c) is proline content; (d) is malondialdehyde (MDA) content; (e) is hydrogen peroxidase activity; (f) is StPIF4 gene interference line DAB staining; (g) is StPIF4 gene interference line NBT staining; (h) is StPIF4 gene interference line Evans Blue staining. All index determinations set salt stress (200mM NaCl) and control (no salt treatment) two treatments, each treatment set 3 biological replicates. Data is mean ± standard deviation, different letters or asterisks indicate significant differences between treatments (* represents P<0.05, ** represents P<0.01, *** represents P<0.001). E3 is wild type control, RNAi-2, RNAi-4, RNAi-19 is StPIF4 gene interference line.
[0023] Figure 7Figure 6. Physiological indexes of wild type and StPIF4 overexpression lines before and after salt treatment. Figure 6(a) is the superoxide anion content; Figure 6(b) is the hydrogen peroxide content; Figure 6(c) is the proline content; Figure 6(d) is the malondialdehyde (MDA) content; Figure 6(e) is the catalase activity; Figure 6(f) is the DAB staining of StPIF4 overexpression lines; Figure 6(g) is the NBT staining of StPIF4 overexpression lines; Figure 6(h) is the Evans Blue staining of StPIF4 overexpression lines. All indexes were measured under salt stress (200 mM NaCl) and control (no salt treatment), and each treatment was set with 3 biological replicates. Data are mean ± standard deviation, and different letters or asterisks indicate significant differences between treatments (* represents P < 0.05, ** represents P < 0.01, *** represents P < 0.001). AT is the wild type control, and OE-6, OE-20, and OE-24 are StPIF4 overexpression lines. DETAILED DESCRIPTION
[0024] The technical solutions of the present application will be further described in detail below in combination with specific examples. It should be understood that the following examples are only illustratively described and explained the present application, and should not be interpreted as limiting the scope of protection of the present application. Any technology realized based on the above description of the present application is covered within the scope of the present application intended to protect. It should be noted that the experimental materials not mentioned in the experimental materials source in the examples of the present application are commercially available, and the experimental methods not mentioned in the specific conditions in the examples of the present application are usually carried out according to the conventional experimental methods or according to the experimental material manufacturer's recommended method. It should be noted that the potato E3 in the present application is the No. 3 potato variety in Hubei, and AT is the Atlantic potato variety, and WT represents the wild type.
[0025] Example 1 Cloning and sequence characteristic analysis of StPIF4
[0026] The inventors screened StPIF4, a member of the PIFs family in potato that has a significant response to salt stress, from the potato PGSC database of environmental stress transcriptome data, and its transcription level decreased under salt stress.
[0027] The StPIF4 gene CDS sequence is 1554 bp long, the nucleotide sequence is shown as SEQ ID No. 1, and it encodes 517 amino acids, and the amino acid sequence is shown as SEQ ID No. 2. Phylogenetic tree analysis of StPIF4 shows that the genetic distance between StPIF4 protein and the homologous protein of Solanum lycopersicum, which belongs to the same genus, is relatively close Figure 1 A). Sequence alignment with the homologous proteins of Arabidopsis thaliana and the same genus shows that the StPIF4 protein sequence has a conserved bHLH domain Figure 1 B).
[0028] Example 2: Analysis of StPIF4 expression pattern
[0029] In order to explore the potential function of StPIF4 in salt stress response, 200 mM NaCl was used to treat wild type tissue culture seedlings after 14 days of cultivation. The leaves of the control group and before and after treatment were sampled and subjected to qRT-PCR detection, and the results showed that the transcription level of StPIF4 decreased after salt stress treatment Figure 2 ), indicating that StPIF4 may be involved in potato salt stress response.
[0030] In order to explore the cell localization of StPIF4 protein, pCAMBIA1300-StPIF4-eGFP vector with green fluorescent marker (eGFP) was transformed into Agrobacterium and then infected tobacco for transient expression, and the experimental results showed that StPIF4 protein was localized in the nucleus Figure 3 ).
[0031] Example 3: Screening and identification of StPIF4 transgenic lines
[0032] To further analyze the biological function of StPIF4 in the response of potato to salt stress, the StPIF4 overexpression vector was constructed, the CDS of StPIF4 was cloned into the pCAMBIA1300 vector to obtain the 35S::StPIF4-GFP recombinant vector, and the CDS of StPIF4 was cloned into the pHELLSGATE-8 vector to obtain the interference vector. Using E3 and AT as the receptors, three RNAi lines (RNAi-2, RNAi-4, and RNAi-19) and three overexpression lines (OE-6, OE-20, and OE-24) were obtained through Agrobacterium-mediated potato slice genetic transformation, and positive identification and efficiency identification were performed through PCR and qRT-PCR, respectively Figure 4 a-d).
[0033] Example 4 StPIF4 negatively regulates the salt tolerance of potato seedlings
[0034] (1) Phenotypic identification
[0035] To further explore whether StPIF4 regulates the salt tolerance of potato seedlings, wild-type and StPIF4 transgenic plants were treated with 200 mM NaCl, and 20 days later, the plant height of the interference transgenic plants was significantly higher than that of WT, while the height of the overexpression transgenic plants was significantly lower than that of WT Figure 5 a-c).
[0036] (2) Effect of StPIF4 gene on active oxygen and osmotic adjustment substances under salt stress
[0037] Further determination of the changes of related physiological indexes before and after salt stress treatment showed that under 200 mM NaCl salt stress, the active oxygen (ROS) related indexes of the interference and overexpression lines of StPIF4 gene showed significant differences compared with the control group (E3 or AT). For the content of superoxide anion (O2 - ), the O2 - content of the interference line was significantly lower than that of E3 Figure 6 a), while the O2 - content of the overexpression line was significantly higher than that of AT Figure 7 a); the change trend of hydrogen peroxide content was consistent with that of O2 - , the H2O2 content of the interference line was significantly lower than that of E3 Figure 6 b), and the H2O2 content of the overexpression line was significantly higher than that of AT Figure 7 b). In terms of osmotic adjustment, the proline content of the interference line was significantly higher than that of E3 Figure 7 c), and the proline content of the overexpression line was significantly lower than that of AT Figure 7 c), indicating that StPIF4 gene was involved in the proline-mediated osmotic adjustment process.
[0038] (3) Effects of the StPIF4 gene on membrane lipid peroxidation and antioxidant enzyme activity under salt stress
[0039] The content of malondialdehyde (MDA), a membrane lipid peroxidation product, was significantly lower in the interference line under salt stress than in E3. Figure 6 d), the overexpression lines were significantly higher than AT( Figure 7 d); Catalase (CAT) activity in the interference lines under salt stress was significantly higher than that in E3. Figure 6 e), the overexpression lines were significantly lower than AT( Figure 7 e) This suggests that the StPIF4 gene influences the degree of membrane lipid peroxidation by regulating the activity of antioxidant enzymes, thereby participating in the salt stress response.
[0040] (4) Histochemical analysis of the effects of StPIF4 gene on reactive oxygen species accumulation and cell membrane damage under salt stress
[0041] This section uses histochemical staining (DAB, NBT, Evans Blue) to visually demonstrate the effect of the StPIF4 gene on reactive oxygen species (H2O2, O2) under salt stress. - ) accumulation and regulation of cell membrane damage.
[0042] DAB staining revealed H2O2 content; under salt stress, the brown staining of leaves from the StPIF4 gene-interfered line was significantly lighter than that of the control E3. Figure 6 f), while the brown staining of leaves from the overexpressing strains was significantly deeper than that of the control AT (f). Figure 7 f). This indicates that under salt stress, silencing the StPIF4 gene inhibits H2O2 accumulation, while overexpression promotes H2O2 accumulation.
[0043] NBT staining detection of O2 - Under salt stress, the blue staining depth of the leaves of the interference line was significantly lower than that of E3. Figure 6 g); the blue staining intensity of leaves from overexpressing lines was significantly higher than that of AT ( Figure 7 g). This indicates that silencing the StPIF4 gene reduces O2. - The production of O2, and its overexpression, exacerbates the problem. - The accumulation of.
[0044] Evans Blue staining was used to detect the degree of cell membrane damage. Under salt stress, the blue staining of the leaves of the interference line was significantly lighter than that of E3. Figure 6 h); the blue staining of leaves from overexpressing lines was significantly deeper than that of AT (h). Figure 7 This indicates that silencing the StPIF4 gene alleviates cell membrane damage under salt stress, while overexpression exacerbates cell membrane damage.
[0045] In summary, StPIF4 gene can regulate the metabolism of reactive oxygen species, osmotic regulation, antioxidant enzyme activity of potato multiple pathways, through negative regulation of reactive oxygen accumulation and affect the integrity of the cell membrane, StPIF4 gene silencing can effectively alleviate the oxidative damage under salt stress, thereby enhancing the salt tolerance of potato.
Claims
1. The application of the StPIF4 gene or its encoded protein in regulating potato salt tolerance, wherein the CDS sequence of the StPIF4 gene is shown in SEQ ID No. 1, and the amino acid sequence of the encoded protein is shown in SEQ ID No.
2.
2. The application according to claim 1, characterized in that, The application involves using genetic engineering techniques to knock out or silence the StPIF4 gene, or to reduce the expression level of the StPIF4 gene, thereby enhancing the salt tolerance of potatoes.
3. A method for enhancing the salt tolerance of potatoes, characterized in that, The method includes using genetic engineering techniques to knock out the StPIF4 gene, silence its expression, or reduce its expression level, thereby enhancing the salt tolerance of potatoes.
4. A preparation for enhancing the salt tolerance of potatoes, characterized in that, The formulation includes a reagent that can reduce the expression level of the StPIF4 gene.
5. Application of the StPIF4 gene, the StPIF4 gene-encoded protein, and the recombinant expression vector of the StPIF4 gene in potato salt tolerance improvement breeding.
6. A method for creating salt-tolerant potatoes, characterized in that, The method includes obtaining potato mutants with enhanced salt tolerance by knocking out or silencing the expression of the StPIF4 gene or reducing the expression level of the StPIF4 gene through genetic engineering techniques.
7. The method according to claim 6, characterized in that, The method involves cloning the CDS-specific region of the StPIF4 gene into the pHELLSGATE-8 vector to obtain an interference vector, and then using Agrobacterium-mediated genetic transformation of potato chips to obtain a potato mutant with StPIF4 interference expression and enhanced salt tolerance.