Application of strawberry transcription factor FaWRKY21 in postharvest resistance to botrytis cinerea of strawberry

Abstract

This invention discloses the application of the strawberry transcription factor FaWRKY21 in postharvest resistance to gray mold in strawberries. The key feature is that the nucleotide sequence of the strawberry postharvest gray mold-related gene FaWRKY21 is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2. It also provides a method to improve postharvest resistance to gray mold in strawberries by increasing the expression level of the FaWRKY21 gene in strawberry cells. Specifically, a recombinant vector containing the encoding gene of the strawberry transcription factor FaWRKY21 is transferred into strawberries using Agrobacterium tumefaciens, resulting in transgenic strawberry fruits overexpressing the FaWRKY21 encoding gene. The advantage is that it can significantly activate the strawberry's disease resistance defense response, effectively inhibit the incidence of disease, reduce the diameter of lesions, and thus improve the strawberry's resistance to postharvest gray mold.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to the application of a strawberry transcription factor FaWRKY21 in enhancing the postharvest resistance of strawberry fruits to gray mold. Background Technology

[0002] strawberry( Fragaria × ananassa As a fruit with extremely high economic value, strawberries are loved by consumers for their unique flavor, vibrant color, and rich nutritional value. However, strawberries are a typical non-climacteric fruit, meaning they have vigorous post-harvest physiological activity, extremely thin skin, delicate tissue, and high water content. During post-harvest storage, transportation, and sales, they are highly susceptible to mechanical damage and pathogen infection, leading to rot and spoilage, and causing huge economic losses.

[0003] Among the many postharvest diseases, those caused by Botrytis cinerea (Glaucus gracilis) Botrytis cinerea Gray mold, caused by *Botrytis cinerea*, is one of the most serious and damaging diseases affecting strawberries. *Botrytis cinerea* is a necrotrophic fungus with an extremely wide distribution. Its conidia can infect fruit through pre-harvest incubation or post-harvest wounds. After infection, the mycelium spreads rapidly within the fruit tissue, secreting cell wall-degrading enzymes and toxins, leading to water-soaked, browned, softened, and rotten fruit, and the production of a typical gray mold layer. The disease has a short incubation period and develops rapidly; under normal temperature and high humidity conditions, it can cause the entire box of fruit to rot completely within 2-3 days, resulting in complete loss of commercial value. Statistics show that post-harvest losses of 30%-50% in strawberries due to gray mold can be even higher in some cases, severely impacting the economic benefits and sustainable development of the strawberry industry.

[0004] Currently, the control of postharvest gray mold in strawberries mainly relies on chemical fungicides. However, long-term use can easily lead to problems such as pathogen resistance, pesticide residues, and environmental pollution. Although physical methods such as low temperature and ultraviolet irradiation, and biological control methods such as antagonistic microorganisms, can be used as supplements, the former is costly and has limited effectiveness, while the latter suffers from unstable control effects and complex processes. Therefore, finding safe and efficient alternative control strategies is urgent. Utilizing the strawberry's own disease-resistant genes and cultivating highly disease-resistant new strawberry varieties through genetic engineering is an important way to achieve green and sustainable disease control.

[0005] WRKY transcription factors are an important class of transcriptional regulators in plants, widely involved in plant responses to biotic and abiotic stresses. They play a crucial role in plant immunity by specifically recognizing W-box elements (TTGAC / C / T) in target gene promoters. WRKY proteins typically contain one or two highly conserved WRKY domains and zinc finger structures, and can be divided into three main classes based on their structural characteristics. Numerous studies have shown that WRKYs regulate plant defense responses to Botrytis cinerea in a single, synergistic, or complex manner. Although strawberry genomics research has identified a large number of WRKY transcription factor family members, the functions of most members remain unclear. Therefore, in-depth exploration and elucidation of the function of the FaWRKY21 gene not only has significant theoretical value but also provides direct genetic resources and theoretical support for developing new technologies and strategies for postharvest disease control in strawberries. This is of great significance for overcoming industry bottlenecks, reducing dependence on chemical pesticides, and promoting the high-quality development of the strawberry industry. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide an application of the strawberry transcription factor FaWRKY21 in postharvest resistance to gray mold in strawberries. Overexpression of the FaWRKY21 gene can significantly improve the postharvest resistance of strawberries to gray mold.

[0007] The technical solution adopted by the present invention to solve the above-mentioned technical problems is: the application of a strawberry transcription factor FaWRKY21 in improving the postharvest resistance of strawberries to gray mold, wherein the FaWRKY21 gene is the nucleotide sequence shown in SEQ ID NO.1.

[0008] Furthermore, the amino acid sequence of the protein encoded by the FaWRKY21 gene is shown in SEQ ID NO.2.

[0009] Furthermore, the application of overexpression of the FaWRKY21 gene in improving postharvest resistance to gray mold in strawberries.

[0010] The present invention also provides a method for improving the postharvest resistance of strawberries to gray mold, which is achieved by increasing the expression level of the FaWRKY21 gene in strawberry cells; the nucleotide sequence of the FaWRKY21 gene is shown in SEQ ID NO.1.

[0011] Furthermore, the recombinant vector containing the strawberry transcription factor FaWRKY21 encoding gene was transferred into strawberries using Agrobacterium to construct transgenic strawberry fruits that overexpress the strawberry transcription factor FaWRKY21 encoding gene.

[0012] Compared with existing technologies, the advantages of this invention are as follows: This invention is the first to discover and confirm that overexpression of the FaWRKY21 gene can significantly improve postharvest resistance to gray mold in strawberries, clarifying the positive regulatory function of this gene. Experiments showed that after inoculation with gray mold, strawberry fruits overexpressing FaWRKY21 exhibited significantly inhibited lesion area, significantly smaller lesion diameter, and significantly lower disease incidence than the control group, resulting in a substantial improvement in disease resistance. This invention demonstrates that FaWRKY21 can be used as a target gene to improve postharvest disease resistance in strawberries, providing a new molecular target and application strategy for the breeding of postharvest resistant strawberry varieties and the development of green biological preservation technologies. Attached Figure Description

[0013] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings required in the embodiments 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.

[0014] Figure 1 The pART-CAM-his-FaWRKY21 vector was constructed; (A) is a schematic diagram of the pART-CAM-his-FaWRKY21 vector construction; (B) is a plasmid map of the pART-CAM-his-FaWRKY21 vector construction.

[0015] Figure 2 To identify the disease resistance of strawberry fruits overexpressing FaWRKY21; (A) is a schematic diagram of the time between FaWRKY21 transient transformation into Agrobacterium and inoculation with Botrytis cinerea; (B) is the quantitative verification of transient overexpression of the FaWRKY21 gene in strawberry fruits; (C) is the phenotype of control and FaWRKY21 overexpressing strawberry fruits after inoculation, with a scale bar of 1 cm; (D) is the disease incidence of control and FaWRKY21 overexpressing strawberry fruits after inoculation; (E) is the diameter of lesions of control and FaWRKY21 overexpressing strawberry fruits after inoculation.

[0016] Figure 3 To determine the JA content in strawberry fruits by overexpressing FaWRKY21;

[0017] Figure 4 The relative expression levels of key genes in the JA pathway in strawberry fruits overexpressed with FaWRKY21 are shown in Figure 1, where (A) represents the expression level of the gene. FaOPR3Ⅱ (B) is FaJMT (C) is FaAOS-C ;

[0018] In the above figure, EV-OE represents the control group; FaWRKY21-OE represents the experimental group; the asterisks above the bars indicate significant differences, *: P < 0.05; **: P < 0.01; ***: P < 0.001. Detailed Implementation

[0019] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0020] Example 1: Construction and validation of a strawberry system with transient overexpression of the FaWRKY21 gene.

[0021] 1. Acquisition of the FaWRKY21 gene

[0022] The FaWRKY21 gene (Gene ID: maker-Fvb4-1-augustus-gene-169.65) was obtained from Phytozome (plant genome database). The CDS sequence of the FaWRKY21 gene is shown in SEQ ID NO.1.

[0023] ATGGACGGCGACAAGGACACACGCGATCCGGGACTCACCGACTTCCCGAGCTGGGACAACGCTTACTTCAGTGAATTCGGCTGGAATTTCAACCCGGACGACCCGATCGGCACCGACTACAGGTCTGATTTGGCGGGAAGCAGCAATACTACAACTCTGCAGGCTGAACAAGTTCCCGAAAGAAGCAGCAATACTAGCTCCCGATCCGACCCTGCCCCCGTGGTCGGATCTGTCTCGTCAAGCTCAACTGAGGATCCGCCAGAGAGGTCTACTGGCTCCGGCGGCACGCCACCGCTCGAAACACCGAGTAGCACTACGAAGGTGAACAAAAAGAAGGGGCAGAAGCGAATCCGGCAGCCAAGATTTGCGTTTATGACTAAGAGTGAAGTTGATCATCTTGAGGATGGCTACCGTTGGCGGAAATATGGACAGAAGGCTGTCAAGAATAGCCCATATCCAAGGAGCTACTATCGGTGCACAAATAGCAAATGCATTGTGAAGAAAAGGGTTGAACGTTCCTCAGAAGATCCGACGACAGTAATCACGACGTATGAAGGGCAACACTGCCATCATACGGTTACGTTTCCCCGCGGCGGTGGCGGGGGAGTCAATATCATTAGTCATGACCAATCTGGCTTTGCCACAGGAAGCCATCATCAGTTAATGCAGCTTCCTCCAGTCTCACAGTTCATGCATTTTCCAACTTCAATTCAACCCAGATCACAAATTAGTCCAACTCATAATTTGACAATAGGAGGGTCGCAGTCGCATCGATTAGTCCCGGCTGGTGGTACTCATCACCATTATGATGAAGCAGCTGGAGGATCCCAGTGCATTACTAATCAAAACCATGAGCCACTCCCAGTTCCTACAGATGAAGGGCTTCTTGGGGACATTGTACCTCCTGGAATGCGTAATCCGTTCGATGGATAA。

[0024] The amino acid sequence of the protein encoded by the FaWRKY21 gene is as SEQ ID Shown in NO.2: MDGDKDTRDPGLTDFPSWDNAYFSEFGWNFNPDDPIGTDYRSDLAGSSNTTTTLQAEQVPERSSNTSSRSDPAPVVGSVSSSSTEDPPERSTGSGGTPPLETPSSTTKVNKKKGQKRIRQPRFAFMTKSEVDHLEDGYRWRKYGQKAVKNSPY PRSYYRCTNSKCIVKKRVERSSEDPTTVITTYEGQHCHHTVTFPRGGGGGVNIISHDQSGFATGSHHQLMQLPPVSQFMHFPTSIQPRSQISPTHNLTIGGSQSHRLVPAGGTHHHYDEAAGSQCITNQNHEPLPVPTDEGLLGDIVPPGMRNPFDG*.

[0025] 2. Preparation of strawberry cDNA

[0026] Total RNA extraction from strawberry fruit: The Hongyan strawberry variety was selected. Equatorial region pulp was cut from the strawberry fruit and quickly placed in liquid nitrogen for thorough grinding. Total RNA was extracted using the Promega Eastern Super Total RNA Extraction Kit. After determining the RNA concentration using a Nano-300 micro-spectrophotometer, the RNA was reverse transcribed into cDNA using the HiScript II QRT SuperMix for qPCR (+gDNA wiper) kit.

[0027] 3. Construction of an overexpression vector for strawberry transcription factor FaWRKY21

[0028] Based on the CDS sequence information of the target gene FaWRKY21, forward and reverse amplification primers for the FaWRKY21 gene were designed and synthesized using Novizan's online website. Appropriate restriction enzyme sites were added according to the sequence and information on the pART-CAM-his vector. Strawberry cDNA was used as the amplification template for specific amplification. The relevant primers are as follows:

[0029] The nucleotide sequence of the forward amplification primers for the FaWRKY21 gene is shown in SEQ ID NO.3: 5'-ATGGACGGCGACAAGGACA-3'; the nucleotide sequence of the reverse amplification primers for the FaWRKY21 gene is shown in SEQ ID NO.4: 5'-TCCATCGAACGGATTACGCA-3'. The PCR system for amplifying the target fragment consisted of 3 uL strawberry DNA, 25 uL 2×PhantaMax Master Mix, 2 uL each of forward and reverse primers, and 18 uL ddH2O. The PCR reaction program was as follows: pre-denaturation at 95℃ for 3 min; denaturation at 95℃ for 15 s; annealing at 58℃ for 15 s; extension at 72℃ for 1 min; and final extension at 72℃ for 5 min (the denaturation, annealing, and extension steps were repeated 35 times).

[0030] Meanwhile, the pART-CAM-his vector plasmid was double-digested with FastDigest XhoI and FastDigest XbaI. The PCR reaction system consisted of 1 μL each of FastDigest XhoI and FastDigest XbaI, 2 μL of 10× FastDigest Buffer, 2 μL of vector plasmid, and 16 μL of ddH2O. The PCR reaction program was: 37℃ for 30 min; 80℃ for 5 min.

[0031] After gel recovery of the target fragment, the target fragment, linearization vector, 5× CE II Buffer, and Exnase® II were mixed according to the manufacturer's instructions using Novizan's ClonExpress® II One Step Cloning Kit. The mixture was incubated at 37°C for 30 min, then transferred to 4°C for homologous recombination of the target fragment and linearization vector, yielding the pART-CAM-his-FaWRKR21 recombinant plasmid (see schematic diagram of plasmid construction). Figure 1 As shown in (A), the recombinant plasmid was transformed into Escherichia coli DH5α, plated on LB solid medium (50 mg / L Kana) and cultured at 37°C, and then single-clone screening was performed.

[0032] Positive monoclonal colonies containing recombinant plasmids were screened and sent for sequencing. Strains with correct sequencing were cultured to the logarithmic growth phase, mixed with sterile glycerol, and frozen at -80°C.

[0033] The pART-CAM-his-FaWRKR21 recombinant plasmid (plasmid image shown) was used. Figure 1(B) Agrobacterium competent cells GV3101 were transformed and cultured at 28°C on LB solid medium containing three antibiotics (50 mg / L Kana; 40 mg / L Gen; 20 mg / L Rif) for screening of positive monoclonal bacteria for subsequent experiments.

[0034] 4. Agrobacterium-mediated transient overexpression of FaWRKY21 in strawberry infection

[0035] Agrobacterium GV3101 containing the recombinant plasmid pART-CAM-his-FaWRKY21 and its empty vector plasmid pART-CAM-his was cultured in liquid LB medium containing triple antibodies (50 mg / L Kana; 40 mg / L Gen; 20 mg / L Rif) at 28°C and 220 rpm overnight. The cells were collected by centrifugation at 5000 rcf for 10 min, and the cells were resuspended in freshly prepared infection solution (containing 10 mM MgCl2, 10 mM MES pH 5.6, and 200 μL MAS). The OD value of the infection solution was adjusted to 0.8-1.0, and the cells were allowed to stand at room temperature in the dark for 1 h before use. Using 'Hongyan' strawberry fruits at the ginkgo stage as experimental material, 1 mL of infection solution was drawn up with a sterile syringe and slowly injected into the strawberry stem until water droplets ooze from the skin. After drying, the fruits were incubated in the dark at 25℃ and 80-90% humidity. The injection and inoculation times of FaWRKY21 for Agrobacterium and Botrytis cinerea were recorded in [reference needed]. Figure 2 (A)

[0036] 5. q-PCR detection of FaWRKY21 gene overexpression efficiency

[0037] On days 2 and 4 after injection, pulp from strawberry fruits near the equatorial region overexpressing FaWRKY21 was collected, frozen in liquid nitrogen, and ground into powder under liquid nitrogen. Total RNA was extracted and cDNA was synthesized, following the same method as described in section 2 above. The q-PCR primers for the FaWRKY21 gene were designed as follows:

[0038] The nucleotide sequence of the forward primer for q-PCR of the FaWRKY21 gene is shown in SEQ ID NO.5: 5'-GAGCTGGGACAACGCTTACT-3'; the nucleotide sequence of the reverse primer for q-PCR of the FaWRKY21 gene is shown in SEQ ID NO.6: 5'-CTTCCCGCCAAATCAGACCT-3'.

[0039] The reverse-transcribed cDNA was diluted 10-fold and used as a template. Strawberry FaActin was selected as the internal reference gene. After q-PCR amplification, 2 -ΔΔCtThe relative expression of FaWRKY21 was calculated, with three technical replicates for each sample.

[0040] The results are as follows Figure 2 As shown in Figure (B), compared with the control group injected with empty vector Agrobacterium, the expression level of the FaWRKY21 gene in strawberry fruits injected with pART-CAM-his-FaWRKY21 Agrobacterium was significantly upregulated, and the expression level at 4 days was more than 160 times that of the control group, indicating that the transient overexpression system was successfully constructed.

[0041] Example 2: Identification of disease resistance in strawberry fruits overexpressing FaWRKY21.

[0042] This invention discovered that FaWRKY21 plays an important role in regulating plant disease resistance. Therefore, the following experiments were conducted to observe the phenotype, disease incidence, and lesion diameter of strawberries overexpressing FaWRKY21 after inoculation with Botrytis cinerea.

[0043] Strawberry fruits that underwent transient transformation in Example 1 (FaWRKY21 overexpression group and empty vector control group) were used as experimental materials. The transiently transformed strawberry fruits were artificially punctured at the equator using sterile nails (3×3×3 mm), and 10 μL of 1×10⁻⁶ granules were inoculated at the wound site. 5 concentration per mL B. cinerea Spore suspension. At 24, 48, 72, 84, and 96 hours after inoculation, the phenotype, incidence rate, and diameter of lesions on the fruit were observed and photographed to record the disease.

[0044] like Figure 2 As shown in (C)-(E), 60 h after inoculation with *Botrytis cinerea*, the disease incidence in strawberry fruits overexpressing FaWRKY21 was significantly lower than that in the control group. Furthermore, the incidence rate and lesion diameter of FaWRKY21-overexpressing strawberry fruits were also significantly lower than those in the control group. These results fully demonstrate that overexpression of the FaWRKY21 gene can effectively activate the defense response of strawberry fruits, significantly inhibit the infection and spread of *Botrytis cinerea*, thereby improving the postharvest disease resistance of strawberry fruits. This provides direct evidence for using this gene to breed disease-resistant varieties or develop postharvest treatment technologies.

[0045] Example 3: FaWRKY21 overexpression in strawberry fruit increases the content of the endogenous hormone jasmonic acid (JA) and the expression of key genes in the JA pathway.

[0046] This invention discovered that FaWRKY21 overexpression can increase the endogenous JA content in strawberry fruit and affect the expression of disease resistance genes.

[0047] In Example 1, strawberry fruit samples that underwent transient transformation were used as experimental materials. The JA content in strawberry fruit was determined using a plant jasmonic acid (JA) enzyme-linked immunosorbent assay kit (SBJ-P239) produced by Nanjing Senbeiga Biotechnology Co., Ltd. 0.2 g of strawberry fruit powder, which had been ground under liquid nitrogen, was weighed, dissolved in 1.8 mL of PBS buffer, vortexed, centrifuged at 3000×g for 20 min, and the supernatant was collected. First, add 40 μL of sample diluent to each well of the enzyme-labeled plate, followed by 10 μL of sample supernatant. Incubate at 37°C for 30 min, then discard the liquid, shake off excess water, and fill each well with a 30-fold diluted wash buffer. Let stand for 30 s, then discard the wash buffer. Repeat this process 5 times. After shaking off excess water, add 50 μL of enzyme-labeled reagent, incubate at 37°C for 30 min, then discard the liquid, shake off excess water, and fill each well with a 30-fold diluted wash buffer. Let stand for 30 s, then discard the wash buffer. Repeat this process 5 times. After shaking off excess water, add 50 μL of chromogenic reagent A, followed by 50 μL of chromogenic reagent B. Shake to mix, and incubate at 37°C in the dark for 15 min. Add 50 μL of stop solution to terminate the reaction. At this point, the solution in the well will change from blue to yellow. Within 15 min after adding the stop solution, measure the absorbance (OD value) of each well sequentially at 450 nm using an enzyme-labeled plate reader. The OD value of the sample is converted into the actual concentration of the sample based on the standard curve, with the unit being pmol / L.

[0048] The cDNA from the transiently transformed strawberry fruit in Example 1 was diluted 10-fold as a template, and strawberry FaActin was selected as an internal reference gene. After q-PCR amplification, 2 -ΔΔCt Methods for calculating key genes in the JA pathway ( FaOPR3 Ⅱ , FaJMT , FaAOS-C The expression of ) was analyzed, and 5 technical replicates were set for each sample.

[0049] design JA The q-PCR primers for synthesizing the key gene are as follows:

[0050] FaOPR3Ⅱ The nucleotide sequence of the forward primer for q-PCR of the gene is shown in SEQ ID NO.7: 5'-CGCCAATGACGAGGCTTAAC-3';

[0051] FaOPR3Ⅱ The nucleotide sequence of the reverse primer for q-PCR of the gene is shown in SEQ ID NO.8: 5'-ACCATAGGCAGCGTAACGAG-3';

[0052] FaJMTThe nucleotide sequence of the forward primer for q-PCR of the gene is shown in SEQ ID NO.9: F: 5'-TGGGTTGCTCATCTGGACCT-3';

[0053] FaJMT The nucleotide sequence of the reverse primer for q-PCR of the gene is shown in SEQ ID NO.10: 5'-TCTAAGCTCCGTTACTGGCG-3';

[0054] FaAOS-C The nucleotide sequence of the forward primer for q-PCR of the gene is shown in SEQ ID NO.11: 5'-CAATGTCCCTCCGACCTTCC-3';

[0055] FaAOS-C The nucleotide sequence of the reverse primer for q-PCR of the gene is shown in SEQ ID NO.12: 5'-CATGTCGAAGAGGTGCGAGA-3'.

[0056] like Figure 3 As shown, the JA content in strawberry fruits of the control group showed a gradually decreasing trend. FaWRKY21 The JA content in the -OE strawberry fruits was significantly higher than that in the control group, showing a trend of first increasing and then slowly decreasing, but its content remained higher than that on day 0. Meanwhile, as... Figure 4 (A) Figure 4 (B) and Figure 4 As shown in (C), compared with the control group, FaWRKY21 -OE strawberry fruit FaOPR3Ⅱ , FaJMT , FaAOS-C The relative expression levels of all genes were significantly induced, and FaOPR3Ⅱ, FaJMT, and FaAOS-C are key genes in the JA synthesis pathway. Therefore, FaWRKY21 may affect the postharvest resistance of strawberry fruit to gray mold by participating in the JA pathway.

[0057] 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 a strawberry transcription factor FaWRKY21 in improving postharvest resistance to gray mold in strawberries, characterized in that, The application is to improve the postharvest resistance of strawberries to gray mold by overexpressing the strawberry transcription factor FaWRKY21 gene; the nucleotide sequence of the FaWRKY21 gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.

2.

2. A method for improving postharvest resistance to gray mold in strawberries, characterized in that, This is achieved by increasing the expression level of the FaWRKY21 gene in strawberry cells; the nucleotide sequence of the FaWRKY21 gene is shown in SEQ ID NO.

1.

3. The method according to claim 2, characterized in that, The method includes the step of treating harvested strawberry fruits with transient expression technology to transiently overexpress the FaWRKY21 gene.

4. The method according to claim 3, characterized in that, The transient expression technology is Agrobacterium-mediated transient transformation.

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