Solanum tuberosum drought tolerance-related transcription factor gene stpif4 and use thereof

Abstract

Disclosed are a Solanum tuberosum drought tolerance-related transcription factor gene StPIF4 and the use thereof. A CDS sequence of StPIF4 has a length of 1554 bp, has a nucleotide sequence shown as SEQ ID No. 1, and encodes 517 amino acids, the amino acid sequence being shown as SEQ ID No. 2. The gene is located in a cell nucleus. The gene reduces the water loss of Solanum tuberosum seedlings under the drought stress by adjusting the opening degree of stomas, thus enhancing the drought tolerance of Solanum tuberosum. The present invention provides a new genetic material and a theoretical basis for analyzing the drought-tolerant molecular mechanism and drought-tolerant breeding improvement of Solanum tuberosum.

Description

Potato drought resistance-related transcription factor gene StPIF4 and its utilization Technical Field

[0001] This invention relates to the fields of plant genetic engineering and potato breeding technology, specifically to a potato drought-resistance-related transcription factor gene StPIF4 and its utilization. Background Technology

[0002] Plant growth and development are restricted by various environmental stresses in nature, and drought is one of the abiotic factors that hinder normal plant growth and development. After being subjected to drought stress, plant photosynthesis is inhibited, membrane structure and permeability are altered, and the original dynamic balance within the plant is disrupted; severe drought can lead to the cessation of photosynthesis and metabolic disorders, ultimately resulting in plant death.

[0003] Drought stress simultaneously affects gene expression in plants. Numerous drought stress response genes have been reported, primarily categorized into functional protein genes and regulatory protein genes. The former encodes key enzymes and metabolic proteins, such as LEA proteins, aquaporins, and heat shock proteins, whose encoded products directly exert protective functions within cells during water stress. The latter encodes various regulatory proteins, such as transcription factors, protein kinases, and phospholipases, whose encoded products mainly play a regulatory role. Five transcription factors in plants are involved in drought stress responses: AP2, MYB, NAC, bZIP, and WRKY. As important regulatory factors in plants, they act as terminal sensors in drought signal transduction, directly regulating the expression of a series of downstream genes through interactions with specific cis-elements in the promoter region. These interactions induce physiological and biochemical changes in plants through different drought stress response signaling pathways, ultimately enabling plants to adapt to drought adversity or enhance their drought resistance.

[0004] The plant phytochrome (PIF) family, belonging to the 15th subfamily of the basic helical-cyclic-helical (bHLH) transcription factor family, is a key factor connecting light, temperature, and other environmental signals. PIF4 has been reported to regulate photomorphogenesis in plants along with other members of the PIF family, while also influencing diurnal rhythms and participating in shade avoidance responses and flowering processes.

[0005] Although the functions of PIF4 have been studied in various plants, no reports have been found on its function in potato, particularly its role in regulating potato drought resistance. Therefore, exploring the function and mechanism of action of PIF4 in potato is of great significance for revealing the molecular mechanism of potato drought resistance. Technical issues

[0006] The aim is to provide a potato drought-resistant transcription factor gene, StPIF4, and its utilization. Technical solutions

[0007] This invention provides a potato drought resistance-related transcription factor gene StPIF4, whose full-length CDS is 1554 bp, and whose nucleotide sequence is shown in SEQ ID No. 1, encoding 517 amino acids, and whose amino acid sequence is shown in SEQ ID No. 2.

[0008] The present invention also provides a protein encoded by the potato drought-related transcription factor gene StPIF4, the above-mentioned protein having a conserved bHLH domain, and the amino acid sequence is shown in SEQ ID No. 2.

[0009] The present invention also provides a recombinant expression vector for the potato drought-resistant transcription factor gene StPIF4.

[0010] This invention also provides the application of the above-mentioned potato drought resistance-related transcription factor gene StPIF4, the protein encoded by the above-mentioned potato drought resistance-related transcription factor gene StPIF4, and the recombinant expression vector of the above-mentioned potato drought resistance-related transcription factor gene StPIF4 in potato drought resistance improvement breeding.

[0011] The invention process of this invention is as follows: The inventors screened StPIF4, a member of the potato PIFs family, from environmental stress transcriptome data in the potato PGSC database, which showed a significant response to high temperature, salt, and drought. This gene was highly expressed in leaves and lateral stems under drought stress, and subcellular localization results showed that it was located in the cell nucleus. Sequencing identification revealed that its full-length CDS is 1554 bp, with the nucleotide sequence shown in SEQ ID No. 1, encoding 517 amino acids, the amino acid sequence of which is shown in SEQ ID No. 2. Genetic transformation using Agrobacterium-mediated transformation was performed to obtain interfering transgenic lines. Drought treatment was applied to both the transgenic lines and the wild type, and the results showed that StPIF4 could enhance potato drought resistance. Stomatal observation results showed that the number of fully open stomata in the interfering lines was greater than that in the wild type, indicating that StPIF4 enhances potato drought resistance by regulating stomatal opening. Beneficial effects

[0012] This invention provides a potato drought-resistance-related transcription factor gene, StPIF4, with a CDS sequence of 1554 bp (as shown in SEQ ID No. 1) encoding 517 amino acids (as shown in SEQ ID No. 2). This gene is located in the cell nucleus and enhances potato drought resistance by regulating stomatal opening and reducing water loss in potato seedlings under drought stress. This provides new genetic material and theoretical basis for elucidating the molecular mechanisms of potato drought resistance and for drought-resistant breeding improvement. Attached Figure Description

[0013] Figure 1 shows the sequence characteristics analysis of StPIF4; where A is the phylogenetic tree analysis and B is the amino acid sequence alignment analysis.

[0014] Figure 2 shows the expression pattern analysis of StPIF4; where A is the qRT-PCR identification of StPIF4 tissue expression; and B is the qRT-PCR identification of StPIF4 expression induced by 20% PEG6000 simulated drought treatment.

[0015] Figure 3 shows the subcellular localization of StPIF4.

[0016] Figure 4 shows the identification of StPIF4 transgenic lines; Figure A shows the agarose gel electrophoresis identification of transgenic plants with vector primer pairs interfering with each other; Figure B shows the agarose gel electrophoresis identification of transgenic plants with StPIF4 gene primer pairs interfering with each other; Figure C shows the interference efficiency detection of StPIF4 interfering transgenic lines.

[0017] Figure 5 shows the phenotypes of WT and StPIF4 transgenic plants before and after drought treatment during the seedling stage.

[0018] Figure 6 shows the stomatal statistics of StPIF4 transgenic plants; Figure A shows stomata with different degrees of opening; Figure B shows the proportion of different types of stomata; and Figure C shows the stomatal density analysis.

[0019] Figure 7 shows the leaf temperature statistics of WT and StPIF4 transgenic plants; Figure A shows the leaf temperature analysis of WT and StPIF4 transgenic plants under normal conditions; Figure B shows the leaf temperature analysis of WT and StPIF4 transgenic plants under drought treatment. Embodiments of the present invention

[0020] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention. It should be noted that experimental materials whose source is not specified in the embodiments of the present invention are commercially available, and experimental methods whose specific conditions are not specified in the embodiments of the present invention are generally performed according to conventional experimental methods or methods recommended by the manufacturers of the experimental materials. Furthermore, it should be noted that the potato E3 mentioned in the present invention refers to Hubei potato variety 3, and StPIF4 represents the PIF4 gene of potato.

[0021] Example 1: Cloning and Sequence Characterization of StPIF4

[0022] StPIF4, a member of the potato PIFs family, was screened from the potato PGSC database's environmental stress transcriptome data. This gene showed a significant impact from drought and a clear response to high temperature, salt, and drought. It was highly expressed in leaves and lateral stems when induced by drought stress.

[0023] Using potato E3 leaf cDNA as a template, the sequence of StPIF4 was cloned and analyzed. The results showed that the CDS sequence of the StPIF4 gene was 1554 bp long, with the nucleotide sequence shown in SEQ ID No. 1, encoding 517 amino acids, the amino acid sequence of which is shown in SEQ ID No. 2. Phylogenetic analysis of StPIF4, compared with that of Arabidopsis thaliana, tomato (Solanum lycopersicum), pepper (Capsicum annuum), and eggplant (Solanum melongena), showed that the StPIF4 protein was genetically close to its homologous protein in the same genus as tomato (Figure 1A). Sequence alignment with homologous proteins in Arabidopsis thaliana and other related species revealed that the StPIF4 protein sequence contains a conserved bHLH domain (Figure 1B).

[0024] Example 2: StPIF4 Expression Pattern Analysis

[0025] qRT-PCR expression analysis was performed on different tissues of potato E3. The results showed that the expression level of StPIF4 was highest in leaves and lateral stems (Figure 2A). E3 tissue culture seedlings cultured for 14 days were treated with 20% PEG6000, and leaf samples were taken before and after treatment for qRT-PCR detection. The results showed that the transcription level of StPIF4 was induced and increased, reaching its maximum at 6 h (Figure 2B), indicating that StPIF4 may be involved in the potato drought stress response.

[0026] To investigate the cellular localization of the StPIF4 protein, the pCAMBIA1300-StPIF4-eGFP vector with a green fluorescent marker (eGFP) was transformed into Agrobacterium and then used to infect tobacco for transient expression. The results showed that the StPIF4 protein was located in the cell nucleus (Figure 3).

[0027] Example 3: Screening and Identification of StPIF4 Transgenic Lines

[0028] A StPIF4 interference expression vector was constructed using pCAMBIA1300 as the backbone. Using E3 as the recipient, genetic transformation was performed via Agrobacterium-mediated transformation to construct StPIF4 interference transgenic lines. Nineteen positive silencing lines were obtained after PCR identification. qRT-PCR detection of the interference lines showed that, except for Ri-6, the StPIF4 transcription level in all other interference lines was significantly downregulated compared to WT, indicating high interference efficiency (Figures 4A-C).

[0029] Example 4 Phenotypic Identification

[0030] (1) StPIF4 enhances the drought resistance of potato seedlings

[0031] To investigate whether StPIF4 regulates the drought tolerance of potato seedlings, wild-type and StPIF4-interfered transgenic plants were transplanted and subjected to drought treatment. After 25 days, the interfering transgenic plants showed significant wilting compared to the WT plants, and failed to recover or fully recover after rehydration. These results indicate that StPIF4 improves the drought tolerance of potato seedlings (Figure 5).

[0032] (2) StPIF4 enhances drought resistance by regulating the degree of stomatal opening.

[0033] Observation and statistical analysis of stomata in wild-type and StPIF4 interference transgenic lines showed no difference in stomatal density. However, the interference lines had more fully open and partially open stomata than the wild-type, and fewer fully closed stomata, resulting in greater water loss in the interference lines (Figures 6A-C). Leaf temperature analysis showed that under normal and drought conditions, the leaf temperature of StPIF4 interference transgenic plants was significantly lower than the WT (Figures 7A-B). These results suggest that the StPIF4 interference lines may exhibit weaker drought tolerance due to faster water loss and lower leaf temperature caused by increased stomatal opening.

[0034] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of a potato drought-resistance-related transcription factor gene StPIF4, the protein encoded by the potato drought-resistance-related transcription factor gene StPIF4, and a recombinant expression vector of the potato drought-resistance-related transcription factor gene StPIF4 in potato drought resistance improvement breeding, wherein the CDS sequence of the potato drought-resistance-related transcription factor gene StPIF4 is 1554 bp long, the nucleotide sequence is shown in SEQ ID No. 1, and it encodes 517 amino acids, the amino acid sequence is shown in SEQ ID No. 2.

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