Application of RsERF109a gene in regulating and controlling yellowing and senescence of radish sprouting vegetable leaves

Abstract

The invention discloses an application of an RsERF109a gene in regulation and control of yellowing and aging of radish sprouting vegetable leaves, and is characterized in that a nucleotide sequence of the RsERF109a gene is shown as SEQ ID NO.1, an amino acid sequence of protein encoded by the RsERF109a gene is shown as SEQ ID NO.2, aging of the radish sprouting vegetable leaves is delayed by inhibiting expression of the RsERF109a gene, and the application in plant breeding comprises construction of overexpressed RsERF109a radish sprouting vegetable. The invention also provides an application of the RsERF109a gene in preparation of a chlorophyll degradation related gene expression promoter. The RsERF109a gene has the advantages that the key function of the radish AP2 / ERF family transcription factor RsERF109a in regulating and controlling postharvest yellowing and senescence of the radish sprouting vegetable is identified and verified for the first time, postharvest yellowing of cotyledons of the radish sprouting vegetable can be remarkably accelerated by overexpressing the RsERF109a gene in the radish sprouting vegetable, and the postharvest fruit and vegetable quality can be accurately regulated and controlled through a molecular means.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology engineering, and in particular relates to the application of the RsERF109a gene in regulating the yellowing and senescence of radish sprout leaves. Background Technology

[0002] Radish sprouts are an important type of sprout vegetable, widely popular due to their rich nutrition and rapid growth. However, like many leafy green vegetables, radish sprouts are highly susceptible to yellowing and senescence during post-harvest storage, characterized by rapid chlorosis of the cotyledons and chlorophyll degradation. This leads to a sharp decline in their edible quality and commercial value, and a significantly shortened shelf life. Yellowing caused by chlorophyll degradation is a key issue limiting the storage, transportation, and sales of fresh vegetables such as radish sprouts. Therefore, elucidating the molecular mechanisms regulating post-harvest yellowing and developing technologies to delay it has significant industrial value.

[0003] The ripening and senescence of fruits and vegetables are regulated by a variety of environmental factors and genes. Identifying the key factors regulating chlorophyll metabolism through the cross-interactions of different plant hormone signaling molecules during postharvest ripening and senescence is of significant industrial importance for maintaining the color and quality of fruits and vegetables after harvest.

[0004] Transcription factors (TFs) are a class of proteins located in the cell nucleus that can specifically interact with cis-acting elements in the non-coding regions of genes, thereby directly regulating the expression of target genes. For example, members such as VvERF17 in grapes, MdERF17 in apples, and BcERF109 in Chinese cabbage can directly activate the expression of chlorophyll catabolism genes such as PAO, NYC1, and SGR1, thereby driving the postharvest yellowing process in fruits and vegetables. Treatment with the ethylene inhibitor 1-MCP can effectively delay yellowing by inhibiting the expression of ERF transcription factors such as BcERF109 in Chinese cabbage. In addition to ethylene, other hormones such as gibberellic acid (GA) can also affect senescence by regulating the expression of chlorophyll degradation-related genes. However, current research mainly focuses on model plants or fruit crops such as Arabidopsis thaliana, grapes, apples, and tomatoes. In vegetable crops such as radishes, key transcription factors that can integrate different signaling pathways and directly regulate postharvest yellowing and senescence are rarely reported, which limits our ability to precisely regulate the quality of harvested fruits and vegetables through molecular means. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide an application of the RsERF109a gene in regulating the yellowing and senescence of radish sprout leaves.

[0006] The technical solution adopted by the present invention to solve the above-mentioned technical problems is: the application of the RsERF109a gene in regulating the yellowing and senescence of radish sprout leaves, wherein the nucleotide sequence of the RsERF109a gene is shown in SEQ ID NO.1.

[0007] Furthermore, the amino acid sequence of the protein encoded by RsERF109a is shown in SEQ ID NO.2.

[0008] Furthermore, inhibiting the expression of the RsERF109a gene delayed the senescence of radish sprout leaves.

[0009] Furthermore, the application includes applications in plant breeding.

[0010] Furthermore, the plant breeding applications include the construction of radish sprouts overexpressing RsERF109a.

[0011] Furthermore, the nucleotide sequence of the upstream amplification primer for overexpressing the RsERF109a gene used in the construction of the RsERF109a overexpressing radish sprouts is shown in SEQ ID NO.18: tttggagaggacacgctcgagATGCATTATCCTCACAACAGACCC, and the nucleotide sequence of the downstream amplification primer for overexpressing the RsERF109a gene is shown in SEQ ID NO.19: tcattaaagcaggactctagaTCACTGGAACATATCAGCAATAGTGT.

[0012] This invention also provides the application of the RsERF109a gene in the preparation of chlorophyll degradation-related gene expression promoters, wherein the chlorophyll degradation-related genes include RsPPH1, RsPPH4, RsRCCR1, RsRCCR2, RsPAO, RsNYC1, RsNOL and RsSGR1 .

[0013] The present invention also provides an inhibitor of RsERF109a gene expression, wherein the inhibitor is nano-selenium.

[0014] Compared with the prior art, the advantages of this invention are as follows: The application of the RsERF109a gene in regulating the yellowing and senescence of radish sprout leaves is demonstrated by cloning the RsERF109a gene from radish sprouts and proving through yeast one-hybrid and dual-luciferase reporter gene experiments that the RsERF109a transcription factor can directly bind to key genes involved in chlorophyll degradation. RsSGR1 It significantly activates the (Stay-Green 1) promoter (proSGR1) region and enhances its activation. RsSGR1 The transcriptional activity of the gene. This indicates that RsERF109a directly positively regulates... RsSGR1 The expression of key genes in the chlorophyll degradation pathway is increased, thereby promoting chlorophyll degradation and driving the chlorosis process. Meanwhile, treatment of radish sprouts with nano-selenium (Nano-Se) can significantly delay chlorosis. RsERF109aHigh expression of the gene effectively delays the yellowing of cotyledons after harvesting radish sprouts.

[0015] In summary, this invention is the first to identify and verify the key role of the radish AP2 / ERF family transcription factor RsERF109a in regulating postharvest yellowing and senescence of radish sprouts, and to demonstrate its effectiveness in overexpressing this transcription factor in radish sprouts. RsERF109a The gene can significantly accelerate postharvest chlorosis of its cotyledons, accompanied by chlorophyll degradation-related genes (such as...). RsSGR1 The expression of AP2 / ERF was upregulated. This discovery not only enriches the transcriptional regulatory network of plant senescence, especially postharvest senescence, and provides new theoretical and experimental evidence for the functional study of transcription factors AP2 / ERFs, but also has important application potential. Attached Figure Description

[0016] Figure 1 The results of agarose gel electrophoresis analysis of the ERF109a gene in Specific Example 1; Figure 2 In specific embodiment two, the yeast one-hybrid verification of RsERF109a and proRsSGR1 Interaction analysis was performed, where A shows that the minimum inhibitory concentration of 3-AT in the yeast one-hybrid system was determined to be 20 mM; B shows that the yeast one-hybrid experiment showed that RsERF109a can bind to... RsSGR1 Promoter fragment; Figure 3 For the in vivo imaging analysis of the transient expression system in tobacco leaves in Specific Embodiment 3, RsERF109a was used. RsSGR1 The effect of RsERF109a on promoter activity and its influence on the growth rate of promoters. RsSGR1 Quantitative analysis of promoter activity; Figure 4 For the overexpression in specific embodiment four RsERF109a Identification of radish sprout plants, where A represents overexpression. RsERF109a After cotyledon harvest RsERF109a Gene expression level, B represents overexpression RsERF109a Image showing how to promote yellowing of cotyledons in radish sprouts; Figure 5 For overexpression in specific embodiment five RsERF109a The effects of storage on the photosynthetic pigment content of radish sprouts, including: A) total chlorophyll content; B) chlorophyll a content; C) chlorophyll b content; and D) carotenoid content. Figure 6 For overexpression in specific embodiment six RsERF109a The effects of A, B, C, and D on the relative expression levels of genes related to chlorophyll degradation in postharvest radish sprouts. RsPPH1 B. RsPPH4 C. RsRCCR1 D. RsRCCR2 E,RsPAO F, RsNYC1 G, RsNOL H, RsSGR1 ; Figure 7 The effect of nano-selenium treatment on the cotyledon phenotype of radish sprouts during storage in Specific Example 7. RsERF109a The effect of nano-selenium treatment on cotyledon phenotype of radish sprouts, where A is the phenotype of nano-selenium treatment on cotyledon phenotype of radish sprouts, and B is the effect of nano-selenium treatment on cotyledon phenotype of radish sprouts. RsERF109a The impact of expression levels. Detailed Implementation

[0017] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0018] Specific Example 1: Cloning and Sequence Analysis of Transcription Factor RsERF109a Gene.

[0019] Step 1: Extract total RNA from radish sprouts and use the extracted RNA as a reactant for reverse transcription to obtain a cDNA template. Total RNA was extracted from radish sprouts using the Vazyme RC411-01 Plant Total RNA Extraction Kit. Approximately 0.1 g of fresh sample was weighed, thoroughly ground into a fine powder under liquid nitrogen, and quickly transferred to a 1.5 mL RNase-free EP tube. RNA extraction was then performed according to the kit instructions. The quality and concentration of the extracted RNA were determined by measuring the A260 / A280 ratio. Qualified samples were stored at -80 ℃ for later use. Based on the RNA concentration, reverse transcription was performed using the HiScript Q RTSuperMiX for qPCR (+gDNA wiper) (Vazyme) kit to synthesize full-length cDNA. The reverse transcription product cDNA was stored at -80 ℃ for subsequent cloning and related experimental analysis.

[0020] Step 2, PCR amplification: The RsERF109a gene amplification product was obtained by PCR amplification. The PCR amplification reaction system was as follows: 2 μL cDNA, 12.5 μL 2× Phanta Max Master Mix, 1 μL each of the upstream and downstream primers for RsERF109a gene amplification, and 8.5 μL ddH2O. The PCR amplification program was as follows: denaturation at 95℃ for 15s, annealing at 62℃ for 15s, extension at 72℃ for 1min, for 35 cycles. The nucleotide sequence of the upstream primer for RsERF109a gene amplification is shown in SEQ ID NO.3: ATGCATTATCCTCACAACAGACCC; the nucleotide sequence of the downstream primer for RsERF109a gene amplification is shown in SEQ ID NO.4: TCACTGGAACATATCAGCAATAGTGT.

[0021] Step 3: Analyze the amplification products by agarose gel electrophoresis, obtaining a band of approximately 831 bp, as shown below. Figure 1As shown, the target band and brightness meet the requirements. After further verification by PCR and sequencing, the RsERF109a gene was obtained, and its nucleotide sequence is as shown in SEQ ID NO.1: ATGCATTATCCTCACAACAGACCCGAATTCGTCGGAGCTCCACCTTCGGATCGCGATTTGACCCGGTATCATCATCGGGAGCAGTTGTCACCGGAGCAAGAACTATCCGTTATGGTCTCCGCTTTGCAGCACGTGATCTCAGGGGAAAACGAAACGGCGCCGTATCAGGGCTTCTCCAGTACCGATAGCACGGTGATAAGCGCGGGAATGCCTCGGTCGGATTCCGACACTTGCCAAGTGTGCAGGATCGACGGTTGTCTCGGCTGCAACTACTTTTTCGCGCCAAATCAGAGAATCGAAAACAGAGTTGAAGAAGAAGGGATTAGTAGTAGTAGTAGAGGAAGAGAGAGTCCCGTGGCGACGGGGAAGAAAGCAGCGGAAGGAGGAGGGAAAGTGAGGAAGAGGAAGAACAAGAAGAACGGGTACAGAGGAGTTAGGCAGAGACCTTGGGGTAAATTTGCAGCTGAGATCAGAGATCCTAAGAGAGCGACACGTGTCTGGCTCGGCACTTTCGAGACAGCCGAGGACGCAGCTCGAGCATACGATAGAGCAGCTATTGGGTTCCGTGGGCCGCGGGCTAAACTCAACTTCCCGTTTATGGATTACACTTCTTCCCCTGTGGCTGCTGACAATGTAGGAACGAGTGCGAGTGTGAATGCAAGTGCGAGTGCGAGTACGAGCGTAAGCGCAGGGGATTCTGCGGAAACAGAGCAATGGCGTGGAGGAGGAGATTGTGATATGGATGAATATTTGAAGATGATGATGATGATGGATTTTGGGAGTGGAGATTCTTCTGATTCAGGGAACACTATTGCTGATATGTTCCAGTGA。

[0022] The amino acid sequence of the protein encoded by RsERF109a is shown in SEQ ID NO.2: MHYPHNRPEFVGAPPSDRDLTRYHHREQLSPEQELSVMVSALQHVISGENETAPYQGFSSTDSTVISAGMPRSDSDTCQVCRIDGCLGCNYFFAPNQRIENRVEEEGISSSSRGRESPVATGKKAAEGGGKVRKRKNKKNGYRGVRQRPWGKFAAEIRDPKRATRVWLGTFETAEDAARAYDRAAIGFRGPRAKLNFPFMDYTSSPVAADNVGTSASVNASASASTSVSAGDSAETEQWRGGGDCDMDEYLKMMMMMDFGSGDSSDSGNTIADMFQ.

[0023] Specific Example 2: The interaction between the RsERF109a transcription factor and the proRsSGR1 promoter was verified using a yeast one-hybrid system.

[0024] Step 1: Construct the pGADT7-RsERF109a vector: The RsERF109a gene was constructed into the pGADT7 vector using homologous recombination.

[0025] The nucleotide sequence of the upstream primer for amplifying the yeast mono-hybrid RsERF109a gene is shown in SEQ ID NO.5: gtgggcatcgatacgggatccATGCATTATCCTCACAACAGACCC, and the nucleotide sequence of the downstream primer for amplifying the yeast mono-hybrid RsERF109a gene is shown in SEQ ID NO.6: acgattcatctgcagctcgagTCACTGGAACATATCAGCAATAGTGT. After PCR amplification, the target fragment of the RsERF109a gene with restriction enzyme sites was purified and recovered by 1% agarose gel electrophoresis for subsequent recombination.

[0026] The pGADT7 vector was digested using a BamHI and Xhol double enzyme digestion system. The digestion reaction mixture was as follows: 1 µL BamHI enzyme, 1 µL Xhol enzyme, 5 µL NEB Buffer, 1 µL plasmid, and up to 50 µL RNase-free water. The reaction program was set to incubate at 37°C for 15 min, followed by heat inactivation at 65°C for 20 min. The linearized vector fragment was successfully obtained by agarose gel electrophoresis and purified for recombination. The target fragment and vector were purified using a kit (SanPrep DNA Gel Extraction Kit, Sangon Biotech (Shanghai) Co., Ltd.). After nucleic acid concentration determination, the purified product was stored at -20°C.

[0027] Following the instructions of the ClonExpress® II One Step Cloning Kit, the linearized vector fragment, the target RsERF109a gene fragment with restriction enzyme sites, 5×CE II buffer, and Exnase II were added to a 200 µL enzyme-free centrifuge tube. After thorough mixing by pipetting, the tube was incubated at 37°C for 30 min to complete the recombination reaction and construct the pGADT7-RsERF109a recombinant vector.

[0028] Step 2: Construct the pHIS2-proRsSGR1 vector The nucleotide sequence of the proRsSGR1 promoter is shown in SEQ ID NO.7: TTCCATCAACACACACATTTGTTGCTCATAGTGATTCGGGTGAGGCTTCTCGGTTTCACCAGGTCATGTCATTAAACTTGAGCCACATTG. The pHIS2 vector has two restriction enzyme sites: SmaI and sacll. The upstream primer sequence for proRsSGR1 gene amplification is shown in SEQ ID NO.8: actatagggcgaattcccgggTGCTGTTTTGTCCATAAATATAGGATC, and the downstream primer sequence for proRsSGR1 gene amplification is shown in SEQ ID NO.9: ccaggaatttctagaccgcggTTATAACCAAACACCAAAGTACTAAGCC. The target fragment of the proRsSGR1 gene containing the restriction enzyme sites was constructed into the pHIS2 vector using homologous recombination, resulting in the pHIS2-proRsSGR1 recombinant vector.

[0029] Step 3: Transform E. coli with the pGADT7-RsERF109a and pHIS2-proRsSGR1 recombinant vectors, respectively. The specific steps are as follows: (1) Take 10 μL of the two recombinant vectors obtained in step 2 and add them to Escherichia coli DH5α competent cells respectively, place them on ice for 30 min; gently shake the cells, heat shock them in a water bath at 42℃ for 45 s, and then quickly place them on ice for 2 min. (2) Then add 800 μL of antibiotic-free LB liquid medium to each and mix gently. Then incubate at 37°C and shake at 220 rpm for 1 h. The composition of LB liquid medium is: yeast extract 5 g / L, tryptone 10 g / L, sodium chloride 10 g / L, and water as solvent. (3) Take 100 μL of the mixture obtained in step (2) and add pGADT7-RsERF109a and pHIS2-proRsSGR1 to LB solid medium containing ampicillin (Amp) antibiotic. Spread evenly until the bacterial solution is completely dry on the plate. Seal with sealing film, mark, and incubate overnight at 37℃ for 12 h. (4) Remove the overnight cultured plates from the incubator and observe the colony count on the plates. If there are single colonies, pick 8 single colonies from each plate on a clean bench and culture them in 1 mL of LB liquid medium containing the corresponding antibiotic. After incubating at 220 rpm for 3 h in a 37℃ constant temperature shaker, take the bacterial solutions for bacterial PCR identification. The upstream primer sequence for pGADT7-RsERF109a bacterial solution identification is shown in SEQ ID NO.10: AGGGATGTTTAATACCACTAC. The downstream primer sequence for pGADT7-RsERF109a bacterial solution identification is shown in SEQ ID NO.11: AGATGGTGCACGATGCACAG. The upstream primer sequence for pHIS2-proRsSGR1 bacterial solution identification is shown in SEQ ID NO.12: TTCGCTATTACGCCAGCTG. The downstream primer sequence for pHIS2-proRsSGR1 bacterial solution identification is shown in SEQ ID NO.12: TTCGCTATTACGCCAGCTG. As shown in NO.13: GTTTATCTTGCCTGCTCATT, PCR identification was performed. The bacterial culture PCR reaction system was as follows: 2 μL bacterial culture, 1 μL each of upstream and downstream primers, 12.5 μL of 2× Rapid Taq Master Mix, and 8.5 μL of ddH2O. The bacterial culture PCR reaction system was 95℃ for 3 min; 95℃ for 15 s, 55℃ for 15 s, 72℃ for 1 min, for 35 cycles; annealing at 72℃ for 5 min; 4℃, infinity. (5) Perform gel electrophoresis on the obtained PCR products to check the results. Take the samples with positive bands for expansion culture (100 μL of bacterial culture and 4 mL of LB liquid medium containing ampicillin (Amp) antibiotic) and use Plasmid Mini Kit II plasmid extraction kit to extract plasmids to obtain pGADT7-RsERF109a prey vector and pHIS2-proRsSGR1 bait vector.

[0030] Step 4: Conversion of bait carrier pHIS2-RsSGR1 Y187 yeast strain was activated by streaking on YPDA plates, and single colonies were picked and inoculated into liquid YPDA medium with shaking culture. Yeast competent cells were prepared, and the constructed bait vector pHIS2-RsSGR1 was transformed into the competent cells. Transformants were screened using SD / -Trp plates to obtain the corresponding bait yeast strain Y187-pHIS2-RsSGR1.

[0031] Step 5: Self-activation detection SD / -His / -Trp large plates (φ 90 mm) containing different concentrations of 3-AT (0, 10, 20, 40, 60, 80, 100, 120 mM) were prepared. The culture of the transformed bait strain Y187-pHIS2-RsSGR1 was diluted to the single-clone level (OD600 < 0.0002) and plated onto the corresponding plates. The optimal working concentration of 3-AT required to inhibit the self-activation of each bait strain was screened. The optimal concentration was characterized by significantly limited and sparse colony growth compared to the control, with no significant growth even after prolonged incubation. Results are as follows: Figure 2 As shown in A, the minimum inhibitory concentration of 3-AT in the yeast one-hybrid system was determined to be 20 mM.

[0032] Step 6: Interaction Filtering (1) Three bait strains (Y187-pHIS2-RsSGR1) were cultured in liquid SD / -Trp medium and their competent cells were prepared. The prey vector pGADT7-RsERF109a and empty pGADT7 were transferred into the competent cells of the above bait strains. (2) For the co-conversion product, take 10 μL and dilute it 10 times, 100 times, 1000 times and 10000 times respectively, and spread it on an SD / -Leu / -Trp plate for calculating the conversion efficiency; spread all remaining conversion products on SD / -His / -Leu / -Trp plates (including the optimal 3-AT concentration obtained for screening this bait) and incubate at 30℃ for 3–5 days; (3) Select clones grown on SD / -His / -Leu / -Trp plates for PCR and sequencing identification; the specific PCR method is the same as step 3 (4) above. Perform point-to-point verification experiments. The experimental results are as follows. Figure 2 As shown in B, RsERF109a can effectively bind to the promoter region of RsSGR1 and regulate RsSGR1 expression. Experimental group: pGADT7-RsERF109a+pHIS2-RsSGR1; Negative control: pGADT7+pHIS2-RsSGR.

[0033] Specific Example 3: The interaction between the RsERF109a transcription factor and the proRsSGR1 promoter was verified using a dual-luciferase assay.

[0034] Step 1: Using homologous recombination, RsERF109a was constructed into the pGreenII 62-sk vector, and proRsSGR1 was constructed into the pGreenII 0800 vector. For detailed steps of homologous recombination, please refer to Example 2. The dual-luciferase RsERF109a gene has two restriction enzyme sites: BamhI and KpnI. The sequence of the upstream primer for the dual-luciferase RsERF109a gene is shown in SEQ ID NO.14: cgctctagaactagtggatccATGCATTATCCTCACAACAGACCC, and the sequence of the downstream primer is shown in SEQ ID NO.15: tcagcgtaccgaattggtaccTCACTGGAACATATCAGCAATAGTGT. The double restriction enzyme sites of the pGreenII 0800-proRsSGR1 gene are KpnI and BamhI. The sequence of the upstream primer of the pGreenII 0800-proRsSGR1 gene is shown in SEQ ID NO.16: ctatagggcgaattgggtaccTTCCATCAACACACACATTTGTTG. The sequence of the downstream primer of the pGreenII 0800-proRsSGR1 gene is shown in SEQ ID NO.17: cgctctagaactagtggatccCAATGTGGCTCAAGTTTAATGACAT.

[0035] Step 2: Transform all the constructed recombinant plasmids into Agrobacterium competent cells GV3101 (pSoup-p19). The Agrobacterium transformation steps are as follows: (1) Take out the competent Agrobacterium cells stored at -80℃ and thaw them on ice; add 1 μg of plasmid DNA to each 100 μL of competent cells and mix well, then place on ice for 5 min, in liquid nitrogen for 5 min, at 37℃ for 5 min, and in an ice bath for 5 min in sequence; (2) Add 700 μL of antibiotic-free LB liquid medium and incubate at 28°C with shaking for 2–3 h; (3) Centrifuge at 5000 rpm for three minutes to collect bacteria, and take about 50 μL of supernatant to gently aspirate and resuspend the bacterial block. Spread it on LB plates containing the corresponding antibiotics, and invert it in an incubator at 28℃ for 2 to 3 days. (4) Select a single colony for culture and identification. For each 300 μL of correctly identified bacterial solution, add 700 μL of 50% glycerol and store at -80℃.

[0036] Step 3: Mix the bacterial cultures of pGreenII 0800-proRsSGR1 and pGreenII 62-sk-RsERF109a at a ratio of 1:9 (OD). 600 =0.5), and then the bacterial culture was transferred to osmotic culture medium (containing 10 mM MES, 10 mM MgCl2, and 200 µM acetylsylcholine), and vigorously shaken to mix thoroughly. Four-week-old *Nicotiana benthamiana* plants were selected, and the undersides of tobacco leaves were infected using the needle of a 1 mL syringe. Three biological replicates were set up for each group. The infected tobacco plants were first incubated in a dark room for about 12 h, then incubated under light. Observations were performed after 48 h.

[0037] Step 4: Select leaves injected with different genes and, according to the Promega Dual-Glo® Luciferase Assay System manual, determine the activities of firefly luciferase (LUC) and Renilla luciferase (REN), and finally calculate their ratio. The experimental results are as follows: Figure 3 As shown, RsERF109a and RsSGR1 promoter-driven LUC are co-expressed in tobacco leaves, indicating that RsERF109a can significantly upregulate the expression of RsSGR1.

[0038] Specific Implementation Example 4: Obtaining and Identifying Radish Sprouts with Transient Overexpression of RsERF109a.

[0039] Step 1: Construct the pART-CAM-RsERF109a vector The double restriction enzyme sites for overexpressing the RsERF109a gene were designed as Xhol and Xbal. The nucleotide sequence of the upstream amplification primer for overexpressing the RsERF109a gene is shown in SEQ ID NO.18: tttggagaggacacgctcgagATGCATTATCCTCACAACAGACCC. The nucleotide sequence of the downstream amplification primer for overexpressing the RsERF109a gene is shown in SEQ ID NO.19: tcattaaagcaggactctagaTCACTGGAACATATCAGCAATAGTGT. The PCR amplification product was purified and recovered by 1% agarose gel electrophoresis and then ligated into the pART-CAM vector to obtain the overexpression vector pART-CAM-RsERF109a.

[0040] Step 2: Transform the overexpression vector pART-CAM-RsERF109a into E. coli. For specific methods, please refer to Example 2.

[0041] Step 3: Transform the recombinant plasmid extracted from the positive strain obtained in Step 2 into Agrobacterium, and refer to Specific Example 3 for the specific method.

[0042] Step 4: Agrobacterium briefly infects radish cotyledons. Transfer the pART-CAM-RsERF109a bacterial culture to osmotic culture medium (containing 10 mM MES, 10 mM MgCl2, and 200 µM acetylsuccinone), shake vigorously to mix thoroughly, and then adjust the OD. 600 The concentration is 0.8. After standing in the dark for 2-3 hours, it can infect the cotyledons of radish sprouts.

[0043] Radish sprouts (seeds of "Yanghua Radish" purchased from Nanjing Lvling Seed Industry Co., Ltd.) were selected after 7 days of hydroponics followed by 3 days of soil cultivation. The radish seeds were disinfected with 4% sodium hypochlorite for 5 minutes, rinsed three times with deionized water, and then soaked for 6-8 hours to allow them to fully absorb water and swell. They were then placed in a damp gauze seedling tray and cultured in the dark for 36 hours to promote germination. The seedlings were then transferred to a nutrient solution containing 1 / 4 Hoagland, with a light / dark ratio of 16 hours / 8 hours (temperature 25±3℃), and cultured for 4 days before being transplanted into soil for 3 days of cultivation. The underside of the cotyledons was infected using the needle of a 1 mL syringe. Three biological replicates were set up for each group. The infected sprouts were first cultured in a dark room for about 12 hours, then cultured under light for 48 hours before sampling.

[0044] Step 5: Determine the expression level of the overexpressing plants. RsERF109a Gene expression level The samples obtained above were subjected to RNA extraction and cDNA reverse transcription according to Specific Example 1. qRT-PCR primer sequences for RsERF109a were designed using primer design software Primer 5.0, as follows: the upstream quantitative primer sequence for qRsERF109a is SEQ ID NO.20: TTCGGATCGCGATTTGACCC, and the downstream quantitative primer sequence for qRsERF109a is SEQ ID NO.21: ATCGGTACTGGAGAAGCCCT. RsRPII was used as an internal reference gene, denoted as qRsActin. The upstream quantitative primer for qRsActin (abbreviated as...) qRsActin F The sequence is SEQ ID NO.22: ATCACGCTAAATGGTCTCCT, qRsActin downstream quantitative primer (abbreviated as...). qRsActin R The sequence is SEQ ID NO.23: GCTCTCAATCAAGTCAATC. The expression level of the RsERF109a gene in overexpressing plants was measured daily during constant temperature storage. qRT-PCR was performed according to the TransStart Green qPCR SuperMixUDG (TransGen) instruction manual. Three technical replicates were set for each cDNA sample, and three biological replicates were set for each biological sample, using 2... -ΔΔCT Relative expression levels were calculated using a method similar to that used in the differential analysis. One-way ANOVA was employed for the analysis of differences. p <0.05 indicates a significant difference.

[0045] The experimental results are shown in Figure 4A. Overexpression RsERF109a Later plants RsERF109a Gene expression levels were significantly higher than those of the empty vector, indicating that RsERF109a Transiently overexpressing radish sprouts were introduced. The cotyledons of the overexpressing plants were observed under constant temperature storage (25±3℃). As shown in Figure 4B, the cotyledons of the transiently overexpressing plants exhibited significantly higher yellowing during storage than the control group, with the most pronounced yellowing phenotype occurring on day 2.

[0046] Specific Implementation Case 5: Determination of photosynthetic pigment content in cotyledons of overexpressed plants.

[0047] 0.1 g of radish cotyledons were extracted with 95% ethanol in the dark for 48 h. After the leaves turned completely white, the absorbance of the solution was measured at 665 nm, 649 nm, and 470 nm for the determination of photosynthetic pigment content. The specific calculation formula is as follows: C a =13.95A 665nm -6.88A 649nm C b =24.96A 649nm -7.32A 665nm

[0048] C car =(1000×A 470nm -3.27× C a -104× C b ) / 299, In the formula, C a Chlorophyll a mass concentration; C b Chlorophyll b mass concentration; C chl Total chlorophyll concentration (mg / L); C car Carotenoid content; V : Extraction liquid volume (L); N Dilution factor; m Sample mass (g).

[0049] The experimental results are shown in Figure 5 (AD). In the cotyledons of the overexpressing plants, the contents of total chlorophyll, chlorophyll a, and carotenoids all showed a gradual decreasing trend with the extension of storage time, and were significantly lower than the control at each time point; while the contents of chlorophyll b generally showed an increasing trend, but its level was still lower than the control.

[0050] Specific Implementation Case 6: Determination of the expression level of chlorophyll degradation genes in overexpressed plants.

[0051] Step 1, RNA extraction and reverse transcription: The method is the same as in Specific Example 1.

[0052] Step 2, Primer Design: Use primer design software Primer 5.0 to design chlorophyll degradation-related genes, such as... RsPPH1 , RsPPH4, RsRCCR1 , RsRCCR2 , RsPAO , RsNYC1, RsNOL and RsSGR1 The qRT-PCR primers, with RsRPII as the internal reference gene, are denoted as qRsActin. All qRT-PCR sequences are shown in Table 1 below.

[0053] Table 1 Primer sequences for qPCR of chlorophyll degradation genes

[0054] Step 3, qRT-PCR The expression levels of chlorophyll degradation genes in overexpressing plants were determined daily during constant-temperature storage. qRT-PCR assays were performed according to the instructions for the TransStartGreen qPCR SuperMix UDG (TransGen). The results are as follows: Figure 6 In AH, genes related to chlorophyll degradation (including...) in overexpressed plants... RsPPH1, RsPPH4, RsRCCR1, RsRCCR2, RsPAO, RsNYC1, RsNOL and of RsSGR1) Expression levels reached their peak on the second day of storage, and were significantly higher than those in the control group. Among them, RsPPH4, RsRCCR1, RsRCCR2 and RsSGR1 On day 0 of storage, the expression level in the overexpressing plants was already higher than that in the control group. These results indicate that overexpression... RsERF109a It may promote the yellowing of cotyledons in radish sprouts by continuously upregulating the expression of genes related to chlorophyll degradation.

[0055] Specific Implementation Case 7: Effects of nano-selenium treatment on the phenotype and ERF109a expression level of radish sprouts.

[0056] The seeds of Green Collar "Flowering Radish" were purchased from Nanjing Green Collar Seed Industry Co., Ltd. After disinfecting with 4% sodium hypochlorite for 5 minutes, the radish seeds were rinsed three times with deionized water, followed by soaking for 6-8 hours to allow them to fully absorb water and swell. They were then placed in damp gauze seedling trays for dark cultivation for 36 hours to promote germination. The seedlings were then transferred to a 1 / 4 Hoagland nutrient solution containing 7.5 mg / L nano-selenium and cultured in the dark for 48 hours, followed by light cultivation for 48 hours (temperature 25±3℃). The seedlings were stored in a constant temperature environment (25±3℃), with samples taken on days 0, 1, 3, and 5, and then stored at -80℃ for later use. Phenotypic observation of the radish sprouts was conducted according to Case Study 4. RsERF109a Gene expression level determination is performed in accordance with Case Study 4.

[0057] The results are as follows Figure 7 As shown in Figure A, nano-selenium treatment significantly delayed the yellowing process of radish sprouts. Throughout the storage period, the cotyledons of the nano-selenium-treated sprouts remained green, with minimal yellowing, indicating that nano-selenium treatment effectively inhibits chlorophyll degradation, thereby maintaining the color and quality of the sprouts. Figure 7 As shown in B, in the control group RsERF109a The gene showed high expression levels early on, but selenium treatment significantly delayed the increase in gene expression, with high expression mainly occurring on day 3, consistent with the delayed cotyledon chlorosis. This indicates that selenium treatment delayed the expression of the gene. RsERF109a Increased gene expression effectively slowed down the leaf yellowing process.

[0058] The foregoing description is not intended to limit the invention, nor is the invention limited to the examples given. Any changes, modifications, additions, or substitutions made by those skilled in the art within the scope of the invention should also be considered within the protection scope of the invention.

Claims

1. The application of the RsERF109a gene in regulating the yellowing and senescence of radish sprout leaves, characterized by: The nucleotide sequence of the RsERF109a gene is shown in SEQ ID NO.

1.

2. The application of the RsERF109a gene according to claim 1 in regulating the yellowing and senescence of radish sprout leaves, characterized in that: The amino acid sequence of the protein encoded by RsERF109a is shown in SEQ ID NO.

2.

3. The application of the RsERF109a gene according to claim 1 in regulating the yellowing and senescence of radish sprout leaves, characterized in that: The applications include those in plant breeding.

4. The application of the RsERF109a gene according to claim 3 in regulating the yellowing and senescence of radish sprout leaves, characterized in that: The plant breeding applications include the construction of radish sprouts overexpressing RsERF109a.

5. The application of the RsERF109a gene according to claim 4 in regulating the yellowing and senescence of radish sprout leaves, characterized in that: The nucleotide sequence of the upstream amplification primer for overexpressing the RsERF109a gene used in the construction of the RsERF109a overexpression radish sprouts is shown in SEQ ID NO.18: tttggagaggacacgctcgagATGCATTATCCTCACAACAGACCC, and the nucleotide sequence of the downstream amplification primer for overexpressing the RsERF109a gene is shown in SEQ ID NO.19: tcattaaagcaggactctagaTCACTGGAACATATCAGCAATAGTGT.

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