Use and method of cyccl1 protein or its encoding gene in regulating plant leaf senescence

Abstract

The application belongs to the field of genetic engineering and relates to a new function and application of a gene. CycC1;1 The application discloses a protein or a coding gene thereof, an application and a method for regulating leaf senescence of a plant, and a transgenic plant with delayed leaf senescence. CycC1;1 The application discloses a protein or a coding gene thereof, an application and a method for regulating leaf senescence of a plant, and a transgenic plant with delayed leaf senescence. CycC1;1 The application discloses a protein or a coding gene thereof, an application and a method for regulating leaf senescence of a plant, and a transgenic plant with delayed leaf senescence. CycC1;1 The application discloses a protein or a coding gene thereof, an application and a method for regulating leaf senescence of a plant, and a transgenic plant with delayed leaf senescence. CycC1;1 The application discloses a protein or a coding gene thereof, an application and a method for regulating leaf senescence of a plant, and a transgenic plant with delayed leaf senescence. CycC1;1 CycC1;1 The application discloses a protein or a coding gene thereof, an application and a method for regulating leaf senescence of a plant, and a transgenic plant with delayed leaf senescence.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering and relates to a new function and application of a gene. Background Technology

[0002] Leaves, as the organs of photosynthesis in plants, have a significant impact on plant growth and development. Leaf senescence is the final process in plant leaf growth and development. During this process, chlorophyll degrades, reactive oxygen species accumulate, senescence gene expression is upregulated, and nutrients are transferred from the leaves to the seeds. Therefore, leaf senescence plays a crucial role in the recycling and reuse of nutrients. Timely senescence is beneficial to plant adaptability and reproduction, but premature senescence significantly reduces crop yield and quality. Therefore, identifying key genes regulating senescence and precisely controlling the leaf senescence process is of great significance to agricultural production.

[0003] The senescence process is precisely regulated by multiple factors. Internal factors influencing leaf senescence include plant hormones such as jasmonic acid, while external environmental factors such as drought, osmotic stress, and cold stress can accelerate leaf senescence. Previous research has shown that MYC2 is a key transcription factor in the jasmonic acid signaling pathway. MYC2 The mutant plant exhibits delayed leaf senescence, while MYC2 Overexpression of AHL11 gene in plants leads to premature leaf senescence. Application CN115058436A discloses the application of AHL11 gene in regulating plant leaf senescence, which mainly regulates the senescence process by controlling ethylene accumulation and senescence signaling. Application CN111471692A discloses the application of AHL9 and AHL11 genes in regulating plant leaf senescence; both genes regulate the senescence process by controlling ethylene accumulation and senescence signaling. Plant leaves are regulated by multiple mechanisms. To further explore senescence-related genes, the mediator complex, a class of conserved transcriptional coactivators that can connect transcription factors to RNA polymerase II (RNAPII), has been investigated. Whether this type of activator is related to senescence has not been reported. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention proposes an application and method for CycC1;1 protein or its encoding gene in regulating plant leaf senescence.

[0005] The technical solution of this invention is implemented as follows:

[0006] On the one hand, this application provides the application of CycC1;1 protein or its encoding gene in regulating plant leaf senescence.

[0007] Furthermore, the sequence of the CycC1;1 protein described above has at least 80% homology with the amino acid sequence shown in SEQ ID No. 1.

[0008] Preferably, the CycC1;1 protein has 80%, 90%, 95% or 99% or more homology with the amino acid sequence shown in SEQ ID No. 1, and encodes a nucleotide sequence that regulates leaf senescence.

[0009] It can also be a nucleotide sequence that hybridizes to the sequence described in a) or b) under strict conditions and encodes a protein that regulates leaf senescence.

[0010] The aforementioned regulation includes both delaying and promoting leaf senescence in plants. Therefore, the CycC1;1 protein or its encoding gene can be used for:

[0011] ① Delaying plant leaf senescence; ② Promoting plant leaf senescence; ③ Breeding plant varieties with delayed senescence; ④ Breeding plant varieties with advanced senescence.

[0012] The aforementioned delay in plant leaf senescence is achieved by increasing the content of CycC1;1 protein or overexpressing its encoding gene.

[0013] The aforementioned promotion of plant leaf senescence is achieved by reducing the content of CycC1;1 protein or silencing its encoding gene.

[0014] Furthermore, the aforementioned plants are monocotyledonous or dicotyledonous plants, preferably plants of the Brassicaceae family or plants of the Solanaceae family that are closely related.

[0015] Preferably, the above-mentioned plant is any one of Arabidopsis thaliana, rice, corn, wheat, soybean, Chinese cabbage, lettuce and tomato.

[0016] In a second aspect, a recombinant vector is provided, comprising an expression vector overexpressing the CycC1;1 encoding gene or an interference vector overexpressing the CycC1;1 encoding gene; the nucleotide sequence of the CycC1;1 encoding gene is shown in SEQ ID No. 2.

[0017] Thirdly, a method for delaying plant leaf senescence is provided, the steps of which are: transforming the expression vector of the above-mentioned overexpression of the CycC1;1 encoding gene into the plant to be improved.

[0018] Thirdly, a method for promoting leaf senescence in plants is provided, the steps of which are: transforming the interference vector of the CycC1;1 encoding gene into the plant to be improved.

[0019] Propagation material or offspring of improved plants bred using the methods of this application can inherit the delayed or premature leaf senescence phenotype.

[0020] The beneficial effects of this invention are as follows:

[0021] 1. This invention provides a method for regulating plant leaf senescence, the method being achieved by increasing or decreasing the levels of certain nutrients in the plant. CycC1;1 This is achieved through gene expression levels; among them, increasing... CycC1;1 Gene expression or protein activity can delay leaf senescence and reduce [the risk of senescence]. CycC1;1 Gene expression can promote leaf senescence. CycC1;1 The nucleotide sequence of the gene is shown in SEQ ID No. 2.

[0022] 2. This invention provides the application of the CycC1;1 gene or protein in regulating plant leaf senescence. The application involves constructing a structure containing... CycC1;1 Gene expression vectors are used to transform plants, resulting in transgenic plants with delayed leaf senescence; or by knocking out, knocking down, or mutating endogenous genes in plants. CycC1;1 Genes were used to obtain transgenic plants with premature leaf senescence. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 The diagram shows the in vitro and in vivo interactions between CycC1 and MYC2 proteins; where A is yeast two-hybrid (Y2H); B is bimolecular fluorescence complementation (BiFC); and C is GST protein pull-down.

[0025] Figure 2 For aging genes SAGs in wild type (WT), CycC1;1 Loss-of-function mutants ( cycc1;1 ), CycC1;1 The qPCR analysis of expression in overexpression (OE) plants is shown in the figure; where A is... SAG12 B is SAG13, C is SAG29 D is SAG113 .

[0026] Figure 3 This demonstrates the effectiveness of the dual-luciferase reporter gene assay (Dual-LUC); where A represents the reporter gene (SAG29pro::LUC), effector gene (MYC2-GFP), and internal control (REN) used in the experiment, and B represents the ratio of LUC activity to REN activity, reflecting the degree to which SAG29pro::LUC is activated.

[0027] Figure 4 This is to demonstrate the effectiveness of chromatin immunoprecipitation (ChIP) experiments; where A represents the primer design position for chromatin immunoprecipitation-quantitative PCR (Chip-qPCR) experiments and... CycC1 Proteins can bind directly to SAG29 The promoter region of a gene; B represents the region of a gene promoter. cycc1;1 In the mutant, RNA polymerase II (Pol II) in SAG29 The degree of enrichment on the promoter.

[0028] Figure 5 This is a phenotypic diagram of plant leaf senescence; where A represents the senescence condition under natural conditions. cycc1;1 The mutant plants exhibited a premature aging phenotype (leaves turned yellow earlier); B is... CycC1;1 Overexpression (OE) plants exhibit a distinct delayed senescence phenotype (leaves remain green for a longer period). Detailed Implementation

[0029] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0030] Unless otherwise specified, the experimental methods used in the following experimental examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0031] This application is based on MYC2 Based on research into key genes regulating aging, the inventor provided... CycC1;1 The novel application of gene regulation of MYC2 transcriptional activity to control plant leaf senescence provides a new approach for breeding new varieties from the perspective of molecular genetic engineering in the future.

[0032] During yeast library screening of CycC1;1 interacting proteins, the inventors discovered that a core aging transcription factor, MYC2, interacts with CycC1;1. Subsequent BiFC and GST-pull-down experiments further confirmed the interaction between MYC2 and CycC1;1. Further qPCR analysis revealed… CycC1;1 T-DNA insertion mutant cycc1;1 Age-related genes (SAGs, such as...) SAG12, SAG29, SAG113 The expression level of ) was significantly upregulated; while CycC1;1In overexpression (OE) plants, the expression of these SAGs was suppressed. Subsequently, a dual-luciferase reporter gene assay (Dual-LUC) demonstrated that CycC1;1 could inhibit MYC2 expression. SAG29 The promoter-driven activation of the Luciferase reporter gene (SAG29pro::LUC). To further confirm the role of CycC1;1 in... MYC2 The mechanism by which transcriptional activity is affected was investigated using ChIP-qPCR experiments. The results showed that CycC1;1 binds to MYC2. SAG29 The promoter region, and CycC1;1 interferes with RNAPII by interacting with MYC2. SAG29 Recruitment of gene promoter regions. Finally, [the process was examined]. cycc1;1 Senescence phenotypes in mutants and OE overexpression materials were found. cycc1;1 It exhibits a premature aging phenotype, while OE shows delayed aging.

[0033] The specific steps are as follows:

[0034] Example 1: MYC2 and CycC1;1 interact.

[0035] Search for CycC1;1 and MYC2 on the Tair website (http: / / www.arabidopsis.org / ). Use the yeast two-hybrid (Y2H) system to clone the MYC2 coding sequence into the pGADT7 vector (AD vector). Primers were designed as follows:

[0036] pGAD-MYC2-F:

[0037] 5'-GGGCATCGATACGGGGATGACTGATTACCGGCTACAAC-3';

[0038] pGAD-MYC2-R:

[0039] 5'-AGCTCGAGCTCGATGTTAACCGATTTTTGAAATCAAACTTGC-3';

[0040] In addition, our research group previously disclosed the co-transformation of pGBK-CycC1;1 into yeast AH109 cells. Yeast transformation and culture experiments were performed using the Matchmaker system (Clontech), following the manufacturer's instructions. After culturing on a Trp / Leu-deficient medium, interaction was verified by drop plate testing on a Trp / Leu / His / Ade-deficient medium. Figure 1 The results showed that the co-transformed groups of AD-MYC2 and BD-CycC1;1 could grow on a four-deficient medium, proving that there is a direct interaction between the two.

[0041] To further determine the interaction between MYC2 and CycC1;1, we used a bimolecular fluorescence complementation assay. The coding sequence of CycC1;1 was cloned into the 1300-35S::YCE vector, and the coding sequence of MYC2 was cloned into the 1300-35S::YNE vector. The primers were designed as follows:

[0042] CycC1;1-YCE-F:

[0043] 5'-GGCGCGCCACTAGTGGATCCATGTTCCTGATTGATTCGTTTTG-3';

[0044] CycC1;1-YCE-R:

[0045] 5'-AGTACTATCGATGGATCCTATGGGTTTGTAGCAAGTTTGT-3';

[0046] MYC2-YNE-F:

[0047] 5'-CGGGGATCCTCTAGAGTCATGACTGATTACCGGCTACAAC-3';

[0048] MYC2-YNE-R:

[0049] 5'-GATCAGCTTCTGCTCGTCACCGATTTTTGAAATCAAACTTGC-3';

[0050] Using H2B-mCherry as the nuclear localization marker, the Agrobacterium tumefaciens culture of combination B was collected by centrifugation and resuspended in transformation medium (10 mM MES, 10 mM MgCl2, 0.2 mM acetylsylcholine, pH 5.6), adjusting the OD value to 1.0. Then, the culture was injected into tobacco leaves via a 1 mL syringe through the abaxial surface, with an injection volume of approximately 0.25 mL. The tobacco leaves were then incubated in a light incubator for 2-3 days. A small number of leaves were then cut off, and the interaction was observed in the GFP and mCherry fluorescence channels using a Zeiss LSM980 confocal microscope. Figure 1 As shown in Figure B, in the simultaneous injection of the combination of 1300-35S::CycC1;1-YCE and 1300-35S::MYC2-YNE, there was a significant green light signal in the cell nucleus, and the green light signal co-localized with the red nuclear localization signal, proving that the two interact directly in the cell nucleus of the tobacco system.

[0051] We also determined the in vitro interaction between MYC2 and CycC1;1. The full-length CDS sequences of MYC2 and CycC1;1 were cloned into the pGEX4T and pPSUMO (6×His-SUMO) vectors, respectively, using the following primer sequences:

[0052] pGEX4T-MYC2-F:

[0053] 5'-GGATTCTGGTTCCGCGTATGACTGATTACCGGCTACAAC-3';

[0054] pGEX4T-MYC2-R:

[0055] 5'-CGGGAATTCCGGGGATCCACCGATTTTTGAAATCAAACTTGC-3';

[0056] pPSUMO-CycC1;1-F:

[0057] 5'-CAGAGAACAGATTGGTGGAATGGCTGCCAATTTCTGGAATTC-3';

[0058] pPSUMO-CycC1;1-R:

[0059] 5'-GACGGAGCTCGAATTCGGACAGCTTCAAAGCCAATTTGC-3';

[0060] We expressed and purified GST-MYC2 and 6×His-SUMO-CycC1;1 from *E. coli*. The mixture was incubated on ice for 1 hour with GST-MYC2 or GST alone, followed by washing with glutathione-S-transferase fusion protein purification beads at 4°C for 3 hours. Detection was performed using anti-His and anti-GST antibodies, respectively. Figure 1 As shown in Figure C, after the addition of GST-MYC2, 6×His-SUMO-CycC1;1 can also be pulled down by the glutathione-S-transferase fusion protein purification magnetic beads, which proves the direct interaction between the two in vitro.

[0061] Example 2: cycc1;1 Upregulation of genes in the middle and late stages of aging

[0062] because MYC2 As one of the core transcription factors in the aging pathway, we selected wild-type (WT) and wild-type (WT) transcription factors. cycc1;1Leaves from 21-day-old mutant (SALK_053291) and overexpression (OE) plants were treated with filter paper soaked in liquid 1 / 2 MS medium containing 10 uM MeJA. After 4 days of treatment, total RNA was extracted from the leaves, reverse transcribed into cDNA, and then analyzed by qPCR. Actin2-8 Genes were used as internal references, and the relative expression levels of genes were calculated using the 2^(-ΔΔCt) method.

[0063] SAG12, SAG13, SAG29, SAG113 The quantitative primer design is as follows:

[0064] SAG12-qF: 5'-GCTTTGCCGGTTTCTGTTG-3';

[0065] SAG12-qR: 5'-GTTTCCCTTTCTTTTATTTGTGTTG-3';

[0066] SAG13-qF: 5'-CCCATTGTTAAAAGCCTCTGGC-3';

[0067] SAG13-qR: 5'-GAAAAAATCGTTACTTAAAGGAG-3';

[0068] SAG29-qF: 5'-CATAGCCATGTTCTCGCTTAC-3';

[0069] SAG29-qR: 5'-CTTTGTCTTTATCACACGAGCC-3';

[0070] SAG113-qF: 5'-GAATGGAGATCCGGAGGTTTAAG-3';

[0071] SAG113-qR: 5'-CGTCTTCTTCCACAGACTGAAGCG-3'.

[0072] The results are as follows Figure 2 The display shows that, compared to WT, cycc1;1 In mutants SAG12 , SAG13 , SAG29, SAG113 The expression levels of these genes were significantly increased in OE plants (p<0.01); while in OE plants, the expression levels of these genes were significantly decreased (p<0.01). This demonstrates... CycC1;1 Negative regulation of SAG expression.

[0073] Example 3: CycC1;1 inhibition MYC2 transcriptional activity

[0074] Based on the commercially available pGreenII 0800-LUC vector, using Sac Using I as the multiple cloning site, the following specific primers were used to obtain pGreen0800-SAG29. pro::LUC Recombinant expression transformants were obtained using the commercial vector pCAMBIA1300-35S::GFP and the following specific primers to obtain the recombinant expression vectors 1300-35S::CycC1;1-GFP and 1300-35S::MYC2-GFP.

[0075] 0800-SAG29pro-F:

[0076] 5'-CAGCCCGGGGGAGGGATTAAAACAGGAGACGAAG-3';

[0077] 0800-SAG29pro-R:

[0078] 5'-CCGCTCTAGAACTAGTGGATTTCTATAGCAATTGAGAAAACTTC-3';

[0079] 35S::CycC1;1-F:

[0080] 5'-GGCGCGCCACTAGTGGATCCATGTTCCTGATTGATTCGTTTTG-3';

[0081] 35S::CycC1;1-R:

[0082] 5'-AGTACTATCGATGGATCCTATGGGTTTGTAGCAAGTTTGT-3';

[0083] 35S::MYC2-F:

[0084] 5'-CGGGGATCCTCTAGAGTCATGACTGATTACCGGCTACAAC-3';

[0085] 35S::MYC2-R:

[0086] 5'-GATCAGCTTCTGCTCGTCACCGATTTTTGAAATCAAACTTGC-3'.

[0087] Will Figure 3Agrobacterium bifidum culture of combination A was injected into tobacco as described in Example 1. Approximately 25 mg of tobacco leaves were cut, flash-frozen in liquid nitrogen, and then rapidly ground into powder in a mortar. The powder was transferred to an EP tube, and 100-150 μL of cell lysis buffer was added. The cells were lysed at 4°C for at least 30 min. After centrifugation at 14,000 rpm for 10 min at 4°C, 50 μL of protein supernatant was transferred to the wells of a black microplate reader. At least three replicates were made for each combination. Then, an equal volume of 50 μL of firefly luciferase reaction solution was added to the wells. The plate was placed in the microplate reader, shaken to mix, and the LUC fluorescence value was detected at 560 nm. The microplate was then removed, and another 50 μL of Renilla luciferase reaction solution was added to the wells. The plate was placed in the microplate reader, shaken to mix, and the REN fluorescence value was detected at 480 nm. The LUC / REN value was calculated.

[0088] The results are as follows Figure 3 As shown in Figure B, co-injection of CycC1;1-GFP and SAG29pro::LUC does not activate [the virus / organization]. SAG29 When MYC2-GFP was co-injected with SAG29pro::LUC, MYC2 expression was significantly activated. SAG29 Expression; when MYC2 is simultaneously injected with MYC2-GFP, CycC1;1-GFP and SAG29pro::LUC, MYC2 expression is affected. SAG29 Transcriptional activation is suppressed.

[0089] Example 4: CycC1;1 combination SAG29 The promoter depends on MYC2, CycC1;1 to inhibit Pol II recruitment.

[0090] Will myc2(SALK_017005) and homozygous Arabidopsis thaliana seeds were sown with WT on 1 / 2 MS medium and cultured for 3 weeks. Approximately 300 mg of seedling material was weighed and placed in a 50 mL centrifuge tube containing 20 mL fixation buffer (0.4 M Sucrose, 1 M Tris-HCl pH 8.0, 20% Triton X-100, formaldehyde). The mixture was then vacuum-crosslinked for 5 min. 5 mL of glycine buffer (2 M Glycine) was added, mixed thoroughly, and vacuum-crosslinked for 10 min to terminate the crosslinking. The sample was carefully washed 3-4 times with distilled water, and excess water was absorbed with absorbent paper. The sample was then rapidly ground into powder in a mortar and transferred to a 50 mL centrifuge tube. 25 mL of pre-cooled extraction buffer 1 (0.4 M Sucrose, 1 M Tris-HCl pH 8.0, 0.5 M EDTA, 5 mM β-mercaptoethanol, 1 mM...) was added. Add PMSF to a test tube and mix thoroughly; filter through Miracloth (Millipore, CAT #475855-1R), collect the filtrate into a 50 mL centrifuge tube, and centrifuge at 2,500 g for 10-15 min at 4°C; discard the supernatant, add 2 mL of pre-chilled extraction buffer 2 (0.25 M Sucrose, 0.5 M EDTA, 1 M Tris-HCl pH 8.0, 1 M MgCl2, 20% Triton X-100, 5 mM β-mercaptoethanol, 1 mM PMSF) to a centrifuge tube, resuspend by pipetting, and transfer the resuspended solution to a 2 mL EP tube; centrifuge at 12,000 g for 10 min at 4°C; discard the supernatant, add 1 mL of pre-chilled nucleilysis buffer (1 M Tris-HCl, 0.5 M EDTA, 10% SDS, 1 mM PMSF) to the precipitate, and mix by pipetting; resuspended solution 1 Divide the mL into 4 tubes and break them for 10, 15, 20, and 30 cycles respectively; centrifuge at 16,000 g for 10 min, and take 1 mL of supernatant into a new EP tube; 900 μL of pre-cooled ChIP dilution buffer (1 M Tris-HCl pH 8.0, 5 M NaCl, 0.Equilibrate 5 M EDTA, 20% Triton X-100, and 1 mM PMSF in 100 μL of sepharose beads; centrifuge at 2,000 g for 5 min at 4 °C and discard the supernatant; add 900 μL of pre-cooled ChIP dilution buffer and 100 μL of broken chromatin sample, with or without 2 μL of CycC1;1 antibody or 2 μL of Pol II antibody, and incubate overnight at 4 °C on a shaker. The samples were washed sequentially with 900 μL of wash buffer (Low-salt: 5 M NaCl, 10% SDS, 20% Triton X-100, 0.5 M EDTA, 1 M Tris-HCl pH 8.0, 1 mM PMSF; High-salt: 5 M NaCl, 10% SDS, 20% Triton X-100 LiCl, 0.5 M EDTA, 1 M Tris-HCl pH 8.0, 1 mM PMSF; LiCl: 0.25 M LiCl, 20% NP-40, Sodium dcoxycholate, 0.5 M EDTA, 1 M Tris-HCl pH 8.0, 1 mM PMSF; and TE: 1 M Tris-HCl pH 8.0, 0.5 M EDTA, 1 mM MPMSF), each time with a shaker at 4°C for 5 min. After washing, the samples were centrifuged at 2,000 g for 5 min at 4°C, the supernatant was discarded, and then 300 μL of fresh solution was added. The sample was briefly vortexed with buffer (10% SDS, 0.1 M NaHCO3) and incubated at 65°C with shaking in a metal bath (0.2 g agitation) for 1 h. It was then centrifuged at 16,000 g for 5 min, and the supernatant was transferred to a new EP tube. 13 μL of 5 M NaCl was added, and the tube was incubated overnight at 65°C to decrosslink. Finally, DNA was extracted to obtain the sample.

[0091] The obtained samples were subjected to real-time PCR. The Chip primer design position is shown in Figure A, and the primer sequences are as follows:

[0092] SAG29-500-F: 5'-gttagatctttaatcattctcacctagactcc-3';

[0093] SAG29-500-R: 5'-gttagatctttaatcattctcacctagactcc-3';

[0094] SAG29-TATA-F: 5'-gttagatctttaatcattctcacctagactcc-3';

[0095] SAG29-TATA-R: 5'-gttagatctttaatcattctcacctagactcc-3';

[0096] SAG29-CDS-F:5'-gttagatctttaatcattctcacctagactcc-3';

[0097] SAG29-CDS-R:5'-gttagatctttaatcattctcacctagactcc-3';

[0098] SAG29-TER-F:5'-gttagatctttaatcattctcacctagactcc-3';

[0099] SAG29-TER-R:5'-cacaaaactttatgcaagtagtacatcatc-3';

[0100] The results are as follows Figure 4 As shown in Figure A, in WT, CycC1;1 can bind to a -500 bp site, which is... SAG29 On the promoter, and myc2 In the middle, CycC1;1 cannot bind to SAG29 On the promoter. For example Figure 4 As shown in Figure B, in WT, Pol II can bind to the TATA box, while... cycc1;1 In this process, the binding capacity increases.

[0101] Example 5: cycc1;1 Premature aging, delayed aging (OE)

[0102] WT, cycc1;1 , OE3#6 After 45 days of cultivation, leaves were harvested and soaked in 95% ethanol, then incubated overnight at 4°C. Tissue fragments were removed by centrifugation, and the absorbance of the supernatant at 645 nm and 663 nm was measured using a spectrophotometer. The chlorophyll concentration was calculated using the following formula:

[0103] Chlorophyll a content (Ca) = 12.72 × OD663 - 2.59 × OD645;

[0104] Chlorophyll b content (Cb) = 22.88 × OD645 - 4.65 × OD663;

[0105] Total chlorophyll content (Ct) = Ca + Cb;

[0106] like Figure 5 As shown in A and B, compared to WT, cycc1;1 The chlorophyll content in the leaves decreased, while OE#6 The higher chlorophyll content indicates that under natural growth conditions, cycc1;1 Mutants experience premature aging, while overexpressed materials... OE#6 Delayed aging.

[0107] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made 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 CycC1;1 protein regulating plant leaf senescence by binding to the promoter region of SAG29 via MYC2, characterized by: The amino acid sequence of the CycC1;1 protein is shown in SEQ ID No. 1; senescence refers to yellowing of plant leaves and a decrease in chlorophyll. The regulation is achieved by reducing the content of CycC1;1 protein or silencing its encoding gene to promote plant leaf senescence. The plant in question is either Arabidopsis thaliana or tobacco.

2. A method for promoting yellowing of plant leaves, characterized in that, The steps are as follows: construct an interference vector encoding the CycC1;1 gene, and then transfer it into the target plant to promote leaf senescence and yellowing. The nucleotide sequence of the CycC1;1 encoding gene is shown in SEQ ID No. 2; the plant is Arabidopsis thaliana or tobacco.

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