Application of StRAP2.7 gene in regulation and control of potato leaf senescence

Abstract

The invention provides application of the StRAP2.7 gene in regulation and control of potato leaf senescence, the CDS sequence of the StRAP2.7 gene is as shown in SEQ ID No.1, the amino acid sequence of encoded protein is as shown in SEQ ID No.2, the gene is located in a cell nucleus, and leaf senescence is negatively regulated and controlled by promoting removal of ROS (reactive oxygen species). The overexpression of the StRAP2.7 in the potatoes can delay the senescence of potato leaves and prolong the growth period of the potatoes, and the reduction of the expression of the StRAP2.7 can accelerate the senescence of the potato leaves and shorten the growth period of the potatoes. A new genetic material and a theoretical basis are provided for analyzing a potato senescence regulation and control mechanism and potato improved breeding.

Description

Technical Field

[0001] This invention relates to the fields of plant genetic engineering and potato breeding technology, specifically to the application of the StRAP2.7 gene in regulating potato leaf senescence. Background Technology

[0002] Leaves are key organs for photosynthesis in plants, and leaf senescence is the final stage of leaf development, marking the beginning of its diminishing contribution to the plant body. However, this process is also an important pathway for plant material recycling and reuse, significantly impacting yield and nutritional value. Studies have shown that in major crops, delaying the senescence of later-stage functional leaves by one day can increase yield by 2-10%. Therefore, elucidating the mechanism of leaf senescence is of great significance for cultivating high-quality, high-yield new germplasm that delays senescence.

[0003] Leaf senescence is finely regulated by a variety of complex exogenous environmental signals, endogenous plant hormones, and leaf age. Chlorophyll degradation is the initiation marker of leaf senescence. Free chlorophyll (Chl) and its primary fluorescent metabolites (pFCCs), as upstream intermediates in chlorophyll catabolism, can act as photosensitizers in plants, leading to a significant increase in reactive oxygen species (ROS) levels, which may trigger cell damage and cell death. ROS are highly reactive molecules that can undergo oxidative reactions with cellular components such as proteins, nucleic acids, and lipids, leading to cell damage and functional degradation, disrupting the structure and function of leaf cells, and accelerating the leaf senescence process. Overall, plant leaf senescence involves multiple biochemical reactions and regulatory mechanisms and is a crucial step in plant developmental biology.

[0004] Potatoes are the world's third largest food crop after rice and wheat, playing a crucial role in ensuring food security. In most cases, potato varieties with later leaf senescence have longer photosynthetic cycles than those with earlier leaf senescence, leading to higher tuber yield and quality. Despite the long history of potato cultivation, the regulatory mechanisms of senescence and growth period are not fully understood. Early studies identified a series of senescence-activated genes, named senescence-associated genes (SAGs). Transcription factors are important regulators of gene expression levels, activating or inhibiting the transcriptional expression of target genes by binding to specific DNA sequences in the promoters of target genes. Current research indicates that numerous transcription factor families are crucial for regulating SAG expression during leaf senescence, with NAC, WRKY, and AP2 / ERF among those reported to participate in regulating gene expression changes during senescence. RAP2.7 (Related to APTALA 2.7) is a member of the AP2 / ERF transcription factor AP2 subfamily, also commonly referred to as TOE1 (Target of early activation tagged (EAT) 1). In Arabidopsis, studies have reported on RAP2.7's involvement in flowering, vegetative phase transitions, and stress responses. However, research on RAP2.7's regulation of leaf senescence has not yet been reported. Summary of the Invention

[0005] The inventors discovered that the expression level of the potato StRAP2.7 gene increases after leaf senescence. They further constructed StRAP2.7 transgenic lines with overexpression and interference expression of the gene for phenotypic identification. The results showed that StRAP2.7 can delay potato leaf senescence. Further reactive oxygen species (ROS) staining and related index measurements revealed that the overexpression lines had higher ROS scavenging capacity than the overexpression lines (WT), while the interference lines showed the opposite. This indicates that overexpression of StRAP2.7 can delay leaf senescence by promoting ROS scavenging.

[0006] In view of this, the purpose of this invention is to provide the application of the StRAP2.7 gene in regulating potato leaf senescence.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] This invention provides the application of the StRAP2.7 gene or its encoded protein in regulating potato leaf senescence. The CDS sequence of the StRAP2.7 gene is shown in SEQ ID No. 1, and the amino acid sequence of the encoded protein is shown in SEQ ID No. 2.

[0009] Specifically, the application involves overexpressing the StRAP2.7 gene in potatoes to delay leaf senescence and prolong the potato growth period; or knocking out, silencing, or reducing the expression of the StRAP2.7 gene in potatoes to accelerate leaf senescence and shorten the potato growth period.

[0010] The present invention also provides a method for delaying potato leaf senescence and extending potato growth period, the method comprising overexpressing StRAP2.7 in potatoes through genetic engineering techniques to delay potato leaf senescence and extend potato growth period.

[0011] The present invention also provides an agent for delaying potato leaf senescence, the agent comprising a reagent that can promote the overexpression of the StRAP2.7 gene.

[0012] The present invention also provides a method for accelerating potato leaf senescence and shortening potato growth period, the method comprising knocking out, silencing or reducing the expression of the StRAP2.7 gene in potatoes through genetic engineering techniques, thereby accelerating potato leaf senescence and shortening potato growth period.

[0013] The present invention also provides an agent for accelerating potato leaf senescence, the agent comprising an agent that can silence or reduce the expression of the StRAP2.7 gene.

[0014] This invention also provides the application of the StRAP2.7 gene, the StRAP2.7 gene-encoded protein, and the recombinant expression vector of the StRAP2.7 gene in potato growth period improvement breeding.

[0015] The present invention also provides a method for creating potatoes with extended growth period, the method comprising overexpressing StRAP2.7 in potatoes through genetic engineering techniques to obtain transgenic potato lines with delayed leaf senescence and extended growth period.

[0016] Furthermore, the method includes constructing a 35S::StRAP2.7 overexpression vector using the pRC19-eGFP vector as a backbone, and genetically transforming potatoes using Agrobacterium-mediated transformation to obtain transgenic potato lines with StRAP2.7 overexpression, delayed leaf senescence, and extended growth period.

[0017] The present invention also provides a method for creating potatoes with a shortened growth period, the method comprising knocking out, silencing or reducing the expression of the StRAP2.7 gene in potatoes through genetic engineering techniques to obtain transgenic potato lines with accelerated leaf senescence and shortened growth period.

[0018] Furthermore, the method includes constructing an RNAi interference vector for StRAP2.7 using the pHELLSGATE-8 vector as a backbone, and genetically transforming potatoes using Agrobacterium-mediated transformation to obtain transgenic potato lines with StRAP2.7 interference expression, accelerated leaf senescence, and shortened growth period.

[0019] The beneficial effects of this invention are as follows: This invention provides the application of the StRAP2.7 gene in regulating potato leaf senescence. The CDS sequence of the StRAP2.7 gene is shown in SEQ ID No. 1, and the amino acid sequence of the encoded protein is shown in SEQ ID No. 2. This gene is located in the cell nucleus and negatively regulates leaf senescence by promoting ROS scavenging. Overexpression of StRAP2.7 in potatoes can delay potato leaf senescence and prolong the potato growth period, while reducing the expression of StRAP2.7 in potatoes can accelerate potato leaf senescence and shorten the potato growth period. This provides new genetic materials and theoretical basis for elucidating the regulatory mechanism of potato senescence and for potato breeding improvement. Attached Figure Description

[0020] Figure 1 This analysis examines the expression patterns of StRAP2.7 in leaves at different developmental stages. A: Leaves at different developmental stages; YL: Young leaves; ML: Mature leaves; SL: Senescent leaves. B: Senescent potato leaves; T: Top; M: Middle part. B: Base; C: qRT-PCR detection of StRAP2.7 expression patterns in leaves at different developmental stages. D: qRT-PCR detection of StRAP2.7 expression patterns in different parts of senescent leaves.

[0021] Figure 2 Molecular characteristic information of StRAP2.7. A: Phylogenetic analysis of StRAP2.7b and Arabidopsis AP2 subfamily genes; B: Subcellular localization; C: StRAP2.7b gene structure diagram; D: Sequence alignment of StRAP2.7b with homologous genes of Arabidopsis and tomato, with the red line marking the conserved AP2 domain.

[0022] Figure 3 This describes the senescence of StRAP2.7 transgenic lines induced by continuous darkness. A: Growth status of each line at 0, 3, and 5 days after darkness induction. B: SPAD value determination of the top third leaf of different transgenic lines.

[0023] Figure 4This refers to leaf senescence under natural growth conditions of StRAP2.7. A: Growth status of plants at 60 d, 90 d, and 120 d. B: Chlorophyll content determination of leaves from different transgenic lines. C: Fv / Fm value determination of leaves from different transgenic lines.

[0024] Figure 5 StRAP2.7 negatively regulates leaf senescence by promoting ROS scavenging. A: DAB and NBT staining of different transgenic lines. B: Ion leakage rate determination of leaves of different transgenic lines; C: H2O2 content determination of leaves of different transgenic lines; D: Superoxide anion (OFR) determination of leaves of different transgenic lines; E: MDA content determination of leaves of different transgenic lines; F: CAT activity determination of leaves of different transgenic lines; G: POD activity determination of different transgenic lines; H: SOD activity determination of different transgenic lines. Detailed Implementation

[0025] 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 the experimental materials in the embodiments of the present invention whose source is not specified are all commercially available, and the experimental methods in the embodiments of the present invention whose specific conditions are not specified are generally carried out according to conventional experimental methods or according to the methods recommended by the experimental material manufacturers. It should be noted that potato E3 in this application document refers to the E'ma potato 3 variety, and WT represents wild type.

[0026] Example 1: Analysis of expression patterns and molecular characteristics of StRAP2.7 in leaves at different developmental stages

[0027] The expression patterns of StRAP2.7 in leaves at different developmental stages and in different parts of senescent leaves were detected using qRT-PCR. The results are as follows: Figure 1 As shown in AD, the results indicate that the expression level of StRAP2.7 increases continuously with leaf senescence, suggesting that StRAP2.7 can be induced by leaf senescence and that StRAP2.7 plays a corresponding role in regulating potato senescence.

[0028] Multiple sequence alignment revealed that the StRAP2.7b-encoded protein contains an AP2 domain and belongs to the AP2 / ERF transcription factor family. Figure 2 D). The phylogenetic tree shows that StRAP2.7b belongs to the same clade as the Arabidopsis flowering regulatory genes RAP2.7 (TOE1), TOE2, SMZ, SNZ, and AP2, and is the closest in genetic distance to RAP2.7. Figure 2A). Further analysis of the gene and protein sequences of StRAP2.7b revealed that it contains 9 exons, and the CDS sequence is shown in SEQ ID No. 1 ( Figure 2 StRAP2.7b (CD) encodes 407 amino acids, with the amino acid sequence shown in SEQ ID No. 2. It has an isoelectric point of 9.24 and is a hydrophilic protein. Subcellular localization results indicate that the protein encoded by StRAP2.7b is located in the cell nucleus (…). Figure 2 B).

[0029] Example 2: StRAP2.7 negatively regulates potato leaf senescence

[0030] A 35S::StRAP2.7 overexpression vector was constructed using the pRC19-eGFP vector as the backbone, and an RNAi interference vector for StRAP2.7 was constructed using the pHELLSGATE-8 vector as the backbone. 'E3' test-tube potatoes were genetically transformed using Agrobacterium-mediated transformation to obtain StRAP2.7 overexpression and interference transgenic lines. To identify whether StRAP2.7 is involved in the regulation of leaf senescence, continuous dark treatment was used to induce leaf senescence. Plants grown for 4 weeks were subjected to dark treatment, and representative plants and the top three leaves were photographed. The results showed that after 3 days of dark treatment, the leaves of the RNAi lines showed significant yellowing, with SPAD and Fv / Fm values ​​significantly lower than those of the wild type. The yellowing degree of the overexpression lines was not significantly different from that of the control. After 5 days of dark treatment, the control leaves also showed significant yellowing, but the yellowing degree of the overexpression lines was relatively milder than that of the wild type, and the SPAD and Fv / Fm values ​​were still significantly higher than those of the wild type. Figure 3 (AC) indicates that the overexpressed transgenic lines exhibited a phenotype of delayed leaf senescence under dark-induced senescence conditions.

[0031] To further clarify the function of StRAP2.7 in regulating senescence, WT and transgenic lines were planted in an artificial climate chamber (12 h light / 12 h dark, 22℃ / 18℃). The results showed that at 60 days of growth, compared with the wild type, the interference lines exhibited leaf yellowing, showing a clear premature senescence phenotype. At 90 days of growth, the wild type also showed leaf yellowing, but the interference lines had a shorter vegetative growth period and a higher degree of leaf yellowing, exhibiting a clear senescence phenotype, while the overexpression lines showed good leaf growth. At 120 days of growth, compared with the wild type, the aboveground parts of the interference lines were basically senescent and wilted, while the overexpression lines had a longer vegetative growth period and significantly delayed leaf senescence. Figure 4A) indicates that plants overexpressing StRAP2.7 exhibit delayed senescence and prolonged growth period, while interference with StRAP2.7 expression leads to premature senescence. Leaf senescence is often accompanied by a decrease in chlorophyll content and a reduction in photosynthetic rate. Therefore, the physiological indicators of the tenth leaf from the bottom of each line after 60 days of growth were further characterized by examining these indicators. The results showed that, compared with the wild type, the chlorophyll content and Fv / Fm value of the interference lines were significantly reduced ( Figure 4 (B, 4C) indicates that the senescence of the interfered strains is more severe, further proving that StRAP2.7 can delay leaf senescence.

[0032] Example 3: StRAP2.7 negatively regulates leaf senescence by promoting ROS scavenging.

[0033] During leaf senescence, increased lipid peroxidation leads to a higher rate of ROS production in plants, disrupting the original dynamic balance. Therefore, ROS is one of the key factors affecting plant senescence. To further investigate the effect of StRAP2.7 on ROS during senescence, DAB and NBT staining was performed on various lines. The results showed that compared with the wild type, the interference lines stained more deeply, while the overexpression lines stained less deeply and had lower levels of reactive oxygen species, indicating that overexpression of StRAP2.7 can inhibit the accumulation of reactive oxygen species during senescence. Figure 5 (AE). While ROS accumulates, plants, in order to protect themselves, increase the activity of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) to scavenge reactive oxygen species, thereby maintaining normal life activities. To further clarify the effect of StRAP2.7 on ROS metabolism, the activities of several key antioxidant enzymes in ROS metabolism were measured. The results showed that, compared with the wild type, the activities of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) were significantly increased in the overexpression lines; conversely, the activities of these antioxidant enzymes were decreased in the interference lines. Figure 5 (F, G, H). The above results indicate that overexpression of StRAP2.7 can delay leaf senescence by promoting ROS clearance.

[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 the StRAP2.7 gene or its encoded protein in regulating potato leaf senescence, characterized in that, The CDS sequence of the StRAP2.7 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 overexpressing the StRAP2.7 gene in potatoes to delay leaf senescence and prolong the potato growth period; or knocking out, silencing, or reducing the expression of the StRAP2.7 gene in potatoes to accelerate leaf senescence and shorten the potato growth period.

3. A method for delaying potato leaf senescence and extending the potato growth period, characterized in that, The method involves overexpressing StRAP2.7 in potatoes using genetic engineering techniques to delay leaf senescence and extend the potato's growth period.

4. A preparation for delaying the senescence of potato leaves, characterized in that, The formulation includes a reagent that promotes the overexpression of the StRAP2.7 gene.

5. A method for accelerating potato leaf senescence and shortening the potato growth period, characterized in that, The method involves using genetic engineering techniques to knock out, silence, or reduce the expression of the StRAP2.7 gene in potatoes, thereby accelerating leaf senescence and shortening the potato growth period.

6. An agent for accelerating potato leaf senescence, said agent comprising an agent capable of silencing or reducing the expression of the StRAP2.7 gene.

7. Application of StRAP2.7 gene, StRAP2.7 gene-encoded protein, and StRAP2.7 gene recombinant expression vector in potato growth period improvement breeding.

8. A method for creating potatoes with extended growing seasons, characterized in that, The method involves overexpressing StRAP2.7 in potatoes using genetic engineering techniques to obtain transgenic potato lines with delayed leaf senescence and extended growth period.

9. A method for creating potatoes with a shortened growth period, the method comprising obtaining transgenic potato lines with accelerated leaf senescence and shortened growth period by knocking out, silencing or reducing the expression of the StRAP2.7 gene in potatoes through genetic engineering techniques.

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