Transcription factor FanPRE1.B1 for regulating and controlling softening of strawberry fruits and application of transcription factor FanPRE1.B1

Abstract

The invention provides a transcription factor FanPRE-1. B1 for regulating and controlling softening of strawberry fruits and application of the transcription factor FanPRE-1. B1, and relates to the technical field of agricultural biology. The nucleotide sequence of the transcription factor FanPRE1. B1 is as shown in SEQ ID NO: 1, and the quality of picked fruits can be improved by inhibiting the expression of the FanPRE1. B1; a downstream gene regulated and controlled by FanPRE1.B1 is identified, and the downstream gene is a pectin lyase gene; the FanPL7a1. A1, the FanPL7a1. A1, the FanPL7a1. B1, the FanPL7b1. A1, the FanPL7b1. B1, the FanPL7b1. C1 and the FanPL7b1. D1 are specifically included, and the FanPL7a1. A1, the FanPL7b1. B1, the FanPL7b1. C1, the FanPL7b1. D1 and the The expression level of any one of the genes can be reduced by inhibiting the expression of FanPRE1.B1, so that the degradation of pectin is inhibited, and the softening of fruits is delayed. The invention provides a new thought for delaying softening of strawberry fruits and improving the quality of picked fruits.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of agricultural biotechnology, and in particular to a transcription factor FanPRE1.B1 for regulating softening of strawberry fruit and application thereof. BACKGROUND

[0002] The rot loss caused by postharvest softening of fruit is a core challenge faced by the horticultural industry, and its essence is the depolymerization of cell wall structure and loss of hardness mediated by enzymatic degradation of pectin polysaccharide. Strawberry (Fragaria × ananassa) is a typical non-climacteric fruit, and its postharvest hardness decreases rapidly, and the weight loss rate and rot rate increase significantly during storage. The shelf life of fresh fruit is usually less than a week, and the loss rate is as high as 20-30%. Studies have shown that pectate lyase (PL) plays a dominant role in the softening process: the enzyme efficiently cuts the alpha-1, 4-glycosidic bond of the pectin backbone through beta-elimination reaction, promotes the conversion of covalently bound pectin (CSP) to water-soluble pectin (WSP), and directly destroys the integrity of the cell wall; its depolymerization efficiency is significantly higher than that of polygalacturonase (PG) which relies on hydrolysis. Existing studies have preliminarily confirmed the regulatory effect of PL gene family members (such as wild strawberry FvPLA and grape VvPL15) on the softening of non-climacteric fruit, however, in octoploid cultivated strawberry, the upstream transcription factor directly regulating the expression of PL gene has not been identified, resulting in the inability to precisely intervene in the softening process through the transcriptional regulatory network. Functional redundancy is an important evolutionary strategy for organisms to respond to genetic and environmental disturbances, and plays a core regulatory role in the process of fruit softening. Current studies have shown that multiple members of the PL family such as wild strawberry FvePL1 / 4 / 7 are expressed at the same time during the maturation period. Due to the existence of subgenomic specificity differentiation of PL genes, it is difficult to effectively delay softening by regulating a single gene. SUMMARY

[0003] In view of the deficiencies in the prior art, the present application provides a transcription factor FanPRE1.B1 for regulating softening of strawberry fruit and application thereof, which can effectively alleviate the softening of postharvest strawberries by inhibiting the expression of the transcription factor FanPRE1.B1, thereby improving the storage time and shelf life of strawberries.

[0004] To achieve the above object, the present application is implemented by the following technical solutions: A transcription factor FanPRE1.B1 for regulating softening of strawberry fruit, the nucleotide sequence of the transcription factor FanPRE1.B1 is shown in SEQ ID NO: 1.

[0005] The transcription factor FanPRE1.B1 is applied to improve the postharvest quality of strawberry fruits, and the application mode is to improve the postharvest quality of strawberry fruits by inhibiting the expression or activity of the transcription factor FanPRE1.B1.

[0006] Preferably, the improvement of the postharvest quality of strawberry fruits is at least one of the following: increased fruit firmness, reduced fruit weight loss rate during storage, reduced fruit gray mold disease incidence, increased fruit cell wall matrix content, and reduced fruit water-soluble pectin content.

[0007] A pectin lyase gene regulated by the transcription factor FanPRE1.B1, the pectin lyase gene is at least one of the following genes: (1) FanPL6a1.A1, the nucleotide sequence is shown as SEQ ID NO: 2; (2) FanPL7a1.A1, the nucleotide sequence is shown as SEQ ID NO: 3; (3) FanPL7a1.B1, the nucleotide sequence is shown as SEQ ID NO: 4; (4) FanPL7b1.A1, the nucleotide sequence is shown as SEQ ID NO: 5; (5) FanPL7b1.B1, the nucleotide sequence is shown as SEQ ID NO: 6; (6) FanPL7b1.C1, the nucleotide sequence is shown as SEQ ID NO: 7; (7) FanPL7b1.D1, the nucleotide sequence is shown as SEQ ID NO: 8.

[0008] A method for delaying the softening of strawberry fruits is to inhibit the expression of FanPRE1.B1 to reduce the expression level of FanPL6a1.A1, FanPL7a1.A1, FanPL7a1.B1, FanPL7b1.A1, FanPL7b1.B1, FanPL7b1.C1, and FanPL7b1.D1, to inhibit pectin degradation, thereby delaying fruit softening.

[0009] The present application provides a transcription factor FanPRE1.B1 for regulating the softening of strawberry fruits and its application, which has the following advantages compared with the prior art: The application is verified by strawberry transient transformation test that inhibiting the expression of FanPRE1.B1 can improve the postharvest quality of fruits. The downstream gene regulated by FanPRE1.B1 is identified for the first time, and the downstream gene is pectin lyase gene; specifically including: FanPL6a1.A1, FanPL7a1.A1, FanPL7a1.B1, FanPL7b1.A1, FanPL7b1.B1, FanPL7b1.C1, FanPL7b1.D1; inhibiting the expression of FanPRE1.B1 can reduce the expression level of any one of the above genes, thereby inhibiting pectin degradation and delaying fruit softening. The application provides a new idea for delaying strawberry fruit softening and improving postharvest quality of fruits. BRIEF DESCRIPTION OF DRAWINGS

[0010] Figure 1 The figure is a schematic diagram of transcriptome analysis of strawberry FanPRE1.B1 gene fruit development related in the embodiment of the application; Figure 2 The figure is a schematic diagram of the influence of transcription factor FanPRE1.B1 on fruit softening; wherein a is the relative expression pattern of FanPRE1.B1 gene in fruit development period of cultivated strawberry varieties 'Montery', 'SanAndreas', 'Benihoppe', 'Kaolino', 'SnowPrincess' and 'Xiaobai'; the abscissa represents different stages of fruit development; b is the relative expression level of transiently expressed FanPRE1.B1 in 'Montery' fruit; WT represents wild type, OE represents overexpression, and RNAi represents silencing; c is the maturation of 'Montery' white pulp period fruit after transiently expressing FanPRE1.B1 for 1 to 11 days; d is the number statistics of fruits in white fruit period (White), turning period (Turning) and red fruit period (Red) after transient expression; the red fruit period fruit is measured (e) hardness (f) cell wall content (CWM) (g) PL activity (h-j) pectin component WSP (water-soluble pectin), CSP (covalently bound pectin), ISP (ionically bound pectin) content; each treatment includes at least 20 biological replicates, and each replicate includes at least 1 strawberry fruit; different letters represent significant differences, and Tukey multiple comparison test is used after variance analysis (p <0.05); the error bar represents standard error; Figure 3The performance of transiently regulating FanPRE1.B1 to affect the postharvest storability of 'Monterey' fruit in the embodiments of the present application; (a) typical phenotype, (b) weight loss rate, (c) malondialdehyde (MDA) content and (d) ion permeability of 'Monterey' strawberry red-fleshed fruit with transiently regulated FanPRE1.B1 expression stored for 0-5 days after harvest; (e) typical phenotype, (f) diseased fruit rate, (g) malondialdehyde (MDA) content and (h) ion permeability of 'Monterey' strawberry red-fleshed fruit with transiently regulated FanPRE1.B1 expression sprayed with Botrytis cinerea for 0-5 days; Tukey's multiple comparison test (p < 0.05) was used after variance analysis; the error bar represents the standard error; Figure 4 The PL gene family of Rosaceae plants is identified in the embodiments of the present application; Fig. a is a phylogenetic tree of Arabidopsis and Rosaceae PL genes; b is a small cluster distribution of Rosaceae species and Arabidopsis PL family members; Figure 5 The protein similarity matrix analysis of the FanPL gene family of cultivated strawberry in the embodiments of the present application; Figure 6 The expression heat map of the FanPL gene family in the strawberry development-related RNA-seq data set in the present application; the heat map data is the average of biological replicates; the upper part of the transcriptome heat map is identified as the strawberry variety name, the lower part is identified as the fruit development period, the left corresponds to the gene name, and the right corresponds to the small cluster name; the font mark red is the FanPL gene members and small clusters that are conservedly expressed in the fruit; the color gradient in the heat map corresponds to the RPKM value, indicating the expression level of the FanPL gene in different varieties and development stages; Figure 7 The correlation analysis of the FanPL gene and the fruit firmness and pectin degradation of 'Monterey' strawberry; Fig. a is a typical picture of 'Monterey' strawberry fruit during the entire development stage; b-g are dynamic detection during fruit development, in which b is fruit firmness, c is CWM content, d is WSP content, e is CSP content, f is PL activity, and g is the relative expression level of FanPL6a1, FanPL7a1 and FanPL7b1 genes; Figure 8 The influence analysis of transiently regulating the expression of three FanPL gene clusters on the fruit firmness and pectin content of 'Monterey' strawberry; Fig. a is the fruit firmness of strawberry; b-d are the CWM, WSP and CSP contents in the fruit, respectively; Figure 9 The relative expression amount of the three small clusters of FanPL6a1, FanPL7a1 and FanPL7b1 in WT, FanPRE1.B1 silenced (RNAi) and overexpressed (OE) full red stage fruit. DETAILED DESCRIPTION

[0011] In order to make the purposes, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below in conjunction with the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the protection scope of the present application.

[0012] Embodiment 1 Identifying the key transcription factor FanPRE1.B1 in the process of strawberry fruit softening 1. Transcriptome mining of the key transcription factor FanPRE1.B1 involved in strawberry fruit softening In order to identify the key transcription factor regulating strawberry fruit softening, the public transcriptome data was obtained from the NCBI database and integrated into 27 fruit development stage transcriptome data sets of 14 different strawberry varieties: Kaolino, Benihope, Snow Princess, FenYu, Monterey, San Andreas, Tokun, Toyonoka, Neinongxiang, W02, XiaoBai, Sunngberry, Kingsberry and Camrosa (Figure 1). The upper part of the picture identifies the strawberry varieties, and the lower part identifies the fruit development period.

[0013] The analysis results show that the bHLH family transcription factor gene FanPRE1.B1 presents a significant and conservative up-regulated expression pattern after the fruit large green fruit stage (BG), and the expression dynamics are highly consistent with the fruit softening process (Figure 1). The results show that FanPRE1.B1 is a key transcription factor involved in strawberry fruit ripening, and the nucleotide sequence of FanPRE1.B1 is shown as SEQ ID NO: 1.

[0014] Embodiment 2 Verification of the regulatory effect of the transcription factor FanPRE1.B1 on strawberry fruit softening, cell wall metabolism, pectin lyase activity and postharvest quality 1. Creating strawberry fruit samples with artificial regulation of FanPRE1.B1 expression The FanPRE1.B1 gene was overexpressed in the day-neutral strawberry variety Monterey by constructing an overexpression vector. The cDNA was amplified using Primer STAR GXL DNA polymerase (TaKaRa, Maebashi, Japan) in the coding region of FanPRE1.B1 and cloned into pDNOR221, and then inserted into the binary vector pK7WGF2 by LR reaction (Gateway) technology.

[0015] At the same time, the FanPRE1.B1 gene expression was silenced in the short-day strawberry variety Redcrown by constructing an RNAi vector. The partial coding sequence of FanPRE1.B1 was subcloned into pDNOR221, and then inserted into the binary vector pB7GWIWG2 by LR reaction (Gateway) technology. The correctly fused construct was transferred to Agrobacterium strain GV3101 by freeze-thaw method, and the Agrobacterium GV3101 strain was cultured at 28°C until the OD600 reached 0.8. Then, 500 μL of Agrobacterium bacterial solution was injected into each fruit at the white fruit stage from the fruit stalk.

[0016] The gene expression of FanPRE1.B1 was measured, and the total RNA of strawberry fruit was extracted using Easy Pure Plant RNA Kit (TransGen Biotech, Beijing, China) according to the manufacturer's instructions, and the qualified RNA was selected by gel electrophoresis detection. After treating the residual DNA contamination in the sample with DNase I, 2 ul of total RNA was reverse transcribed into cDNA by PrimeScript RT Reagent Kit (Perfect Real Time; Takara, Dalian, China).

[0017] PCR was performed using LightCycler 480II system (Roche, Germany). The entire antisense system was a mixture of 20 ul, including 0.2 μL cDNA sample, 1 μL primer, 10 μL 2×SYBR Green Master Mix and 4.8 μL water. The program was set as follows: 95 ℃ pre-culture for 5 minutes, 95 ℃ denaturation for 3 seconds, 60 ℃ annealing for 10 seconds, and 72 ℃ extension for 30 seconds; 35 cycles were performed. RPT6A and RPN5A were used as internal reference genes (Chen et al., 2021). The amplification efficiency of the primers during RT-qPCR was calculated using Lin-RegPCR method. The relative expression of genes in each sample was calculated using 2-ΔΔCT method.

[0018] The primers involved in this example are described in Table 1: Table 1 The experimental results show that, compared with the control group, the RNAi silencing treatment makes the expression of FanPRE1.B1 in mature fruits significantly reduced by 67.92% ± 9.85% (p < 0.001), and the overexpression (OE) treatment makes the expression level significantly increased by 3.31 ± 1.41 times (p < 0.001) Figure 2 a, b).

[0019] The phenotype observation finds that the gene expression level and the fruit ripening rate have a significant dose effect: compared with the control group, the silencing group delays the color change starting time by 3.2 ± 0.5 days (p < 0.01), and the OE group advances the color change period by 2.8 ± 0.3 days (p < 0.05) Figure 2 c, d), which indicates that FanPRE1.B1 positively regulates the fruit ripening process.

[0020] 2, verify the influence of FanPRE1.B1 on fruit hardness and cell wall metabolism: The texture analyzer is used to measure the hardness of strawberry fruits, and the instrument setting parameters are as follows: load probe P / 36R, test site is the equatorial region of the fruit, compression deformation amount is 50%, test speed before test is 3.00 mm / s, test speed during test is 1.0 mm / s, return speed after test is 5 mm / s, trigger force is 0.049 N. The probe height is calibrated before test, and the return height is set according to the size of different varieties of strawberry fruits. The determination method of cell wall matrix (CWM) in the application is as follows: the strawberry fruits are ground in liquid nitrogen, 1.0 g of sample is added into 10 mL of 80% (V / V) ethanol solution and mixed by vortex, heated in water bath for 10 min, cooled and centrifuged at room temperature for 10 min at 12000 rpm, the supernatant is discarded, and the precipitate is extracted. Then 10 mL of 80% ethanol solution is added, and the above step is repeated after centrifugation to remove the supernatant and extract the precipitate. Finally, 10 mL of acetone solution is added for washing, and the precipitate is collected after repeating the centrifugation and treating in a 40℃ oven for 24 h to obtain the cell wall matrix CWM.

[0021] The fruit hardness determination shows that, compared with the control group, the RNAi silencing treatment makes the hardness of mature fruits significantly increased by 15.7% ± 4.3% (p < 0.001), and the overexpression (OE) treatment causes the hardness to significantly decrease by 21.5% ± 5.9% (p < 0.001) Figure 2 e).

[0022] To verify the effect of FanPRE1.B1 on fruit cell wall metabolism, the determination of cell wall matrix showed that the change of CWM content was consistent with the hardness. Compared with the control group, the CWM content of the RNAi group was significantly increased, and the OE group was significantly reduced (p < 0.001) Figure 2 f). The above results show that inhibiting the expression of FanPRE1.B1 can effectively increase the fruit hardness and maintain the stability of the cell wall structure.

[0023] 3. Verify the effect of FanPRE1.B1 on pectin lyase (PL) activity and pectin components: According to the Pectin Lyase Activity Assay Kit (Beijing Boxbio Science & Technology Co., Ltd.) instruction, the pectin lyase activity detection was carried out. The generation amount of 4, 5-unsaturated oligogalacturonide was detected by ultraviolet spectrophotometer at 232 nm. According to the following formula: PL enzyme activity (U / g) = (ΔA × V2× 10 9 ) / (ε × d × V1 × W × T).

[0024] Note: ΔA = sample OD232 - control OD232; V1: the volume of crude enzyme solution added in the reaction system, 0.1 mL; V2: the total volume of the reaction, 1 mL; ε: the molar extinction coefficient of unsaturated galacturonide, 5200 L / mol / cm; d: the light path of 1 mL quartz cuvette, 1 cm; Cpr: sample protein concentration, mg / mL; W: sample mass; T: reaction time, 30 min.

[0025] Through the detection of PL enzyme activity, it was found that the RNAi silencing treatment made the PL activity significantly reduced from 4.09 ± 0.67 U / mg of the control group to 2.46 ± 0.43 U / mg (p < 0.001), while the overexpression (OE) treatment was significantly improved to 6.67 ± 0.94 U / mg (p < 0.001) Figure 2 g).

[0026] To verify the effect of FanPRE1.B1 on pectin components, 50 mmol / L sodium acetate buffer (pH 6.5) was added to the oven-dried CWM, shaken for 6 h, centrifuged for 25 min, and the supernatant (i.e. water-soluble pectin (WSP)) was taken. After centrifugation, 30 ml of 50 mmol / L ethylenediaminetetraacetic acid (CDTA) solution (dissolved in pH 6.5 sodium acetate solution) was added to the precipitate, shaken for 6 h, and the supernatant (i.e. ionically bound pectin (ISP)) was taken. Then 50 mmol / L sodium carbonate (containing 2 mmol / L CDTA) was added to the precipitate after centrifugation and shaken for 6 h, and the supernatant (i.e. covalently bound pectin (CSP)) was taken. Finally, the pectin content was determined by the carbazole colorimetric method. 1 mL of pectin solution was mixed with 6 mL of concentrated sulfuric acid, immediately cooled with ice water, then boiled for 10 min, cooled again, and then 0.25 mL of ethanol-carbazole solution was added. After incubation for 30 min, the absorbance was measured at 530 nm. Three biological replicates were set for each experiment. The results are expressed in mg / g FW.

[0027] By analyzing the pectin components, it was found that overexpression (OE) treatment resulted in a significant increase in water-soluble pectin (WSP) content by 37.5% ± 6.2% (p < 0.001) and a significant decrease in covalently bound pectin (CSP) content by 28.3% ± 4.8% (p < 0.001) compared to the control group. Figure 2 h-j).

[0028] In summary, the above results show that FanPRE1.B1 promotes pectin polysaccharide depolymerization by enhancing PL enzyme activity, thereby disrupting cell wall integrity and driving fruit softening. Therefore, inhibiting FanPRE1.B1 expression can effectively increase fruit firmness and delay fruit softening.

[0029] Example 3: To evaluate the effect of transcription factor FanPRE1.B1 on postharvest storage and disease resistance (gray mold) of strawberry fruits, the present invention conducted postharvest storage experiments and pathogen infection resistance experiments on 'Montrey' strawberry full-red stage fruits with artificially regulated FanPRE1.B1 expression obtained in Example 2. The following steps were included: 1. Evaluate the effect of transcription factor FanPRE1.B1 on postharvest storage of fruits: Postharvest storage experiments were conducted on 'Montrey' strawberry full-red stage fruits with artificially regulated FanPRE1.B1 expression obtained in Example 2 for 5 days, with the temperature controlled at 20 ± 2°C and the humidity controlled at 60-70%. After 5 days, the weight loss rate, malondialdehyde (MDA) content, and ion permeability of the strawberry fruits were measured. The measurement methods are as follows: 1) Determination of fruit weight loss rate: the present application adopts weighing method for determination, and the formula is as follows: weight loss rate (%) = (weight before storage - mass on the day of storage period) / mass before storage x 100%.

[0030] 2) Determination of fruit malondialdehyde (MDA) content: 1.0 g of strawberry fruit sample is weighed and grinded in 5 mL of 10% trichloroacetic acid solution (TCA). After centrifugation for 20 min (4°C, 12000 rpm), 2 mL of supernatant is taken, 2 mL of 0.67% thiobarbituric acid solution (TBA) is added, the test tube is placed in boiling water, and after 20 min, it is placed in ice for accelerated cooling. The absorbance value is determined at 450 nm, 532 nm and 600 nm. The calculation formula is as follows: MDA (μmol g-1) = 6.45 (OD532- OD600) - 0.56 x OD450.

[0031] 3) Determination of fruit ion permeability: the present application determines ion leakage rate by relative conductivity. First, the strawberry is cut into cubes of about 5 mm and immersed in 15 mL of deionized water, and a test tube containing the same volume of deionized water is used as a control. The test tube is shaken on a shaker at room temperature for 1 h (20 rpm), and then the initial conductivity of the sample (C1) and the control (CK1) is measured. The test tube is boiled for 10 min and cooled to room temperature, and then the second conductivity (C2, CK2) is measured. The ion leakage rate is represented by the relative conductivity (C), and the following formula is used for calculation: C (%) = (C1- CK1) / (C2- CK2) x 100%.

[0032] The experimental results are shown in Figure 3 a-d, compared with the control group: 1) The fruit weight loss rate of FanPRE1.B1-RNAi is significantly lower than that of the control group; 2) The malondialdehyde (MDA) content of FanPRE1.B1-RNAi fruit is significantly lower than that of the control group, indicating that the degree of membrane lipid peroxidation is reduced; 3) The ion permeability of FanPRE1.B1-RNAi fruit is significantly lower than that of the control group, indicating that the cell membrane integrity is better maintained; 4) The fruit weight loss rate of FanPRE1.B1-OE is significantly higher than that of the control group; 5) The malondialdehyde (MDA) content of FanPRE1.B1-OE fruit is significantly higher than that of the control group, indicating that the degree of membrane lipid peroxidation is increased; 6) The ion permeability of FanPRE1.B1-OE fruit is significantly higher than that of the control group, indicating that the cell membrane integrity is maintained worse.

[0033] The above results prove that the inhibition of FanPRE1.B1 expression can effectively improve the postharvest storage quality of strawberry fruit.

[0034] 2. Assessing the effect of transcription factor FanPRE1.B1 on fruit resistance (gray mold) to disease: The present application artificially regulates the expression of FanPRE1.B1 in the Monterey full red stage fruit obtained in Example 2, and sprays Botrytis cinerea to perform pathogen infection experiments. Five days after spraying the pathogen, the diseased fruit rate, malondialdehyde (MDA) content and ion permeability of the strawberry fruit are measured, respectively. The measurement method is as follows: 1) Measurement of disease rate: the formula is as follows, disease rate (%) = number of diseased fruits / total number of fruits x 100%.

[0035] 2) The detection method of malondialdehyde (MDA) content and ion permeability is the same as that in Example 4.1 The experimental results are shown in Figure 3 f-h, compared with the control group: 1) The disease rate of FanPRE1.B1-RNAi fruit is significantly lower than that of the control group; 2) The malondialdehyde (MDA) content of FanPRE1.B1-RNAi fruit is significantly lower than that of the control group; 3) The ion permeability of FanPRE1.B1-RNAi fruit is significantly lower than that of the control group; 4) The disease rate of FanPRE1.B1-OE fruit is significantly higher than that of the control group; 5) The malondialdehyde (MDA) content of FanPRE1.B1-OE fruit is significantly higher than that of the control group; 6) The ion permeability of FanPRE1.B1-OE fruit is significantly higher than that of the control group.

[0036] The above results prove that the resistance of strawberry postharvest fruit to the main disease (gray mold) can be significantly enhanced by inhibiting the expression of FanPRE1.B1.

[0037] Example 4: Clarify the molecular mechanism of transcription factor FanPRE1.B1 driving strawberry fruit softening by regulating the expression of downstream pectin lyase gene.

[0038] In order to analyze the molecular mechanism of FanPRE1.B1 regulating fruit softening, especially its relationship with downstream pectin lyase (PL) gene, the following researches are carried out in this embodiment, including the following steps: 1. Identification of FanPL gene family Based on 696 PL protein sequences of 32 species of Rosaceae, the present application uses MAFFT software to reorder all identified gene families, and uses IQ-TREE2 software to construct a phylogenetic tree by maximum likelihood method (ML). The phylogenetic tree of PL genes of Arabidopsis thaliana and Rosaceae plants is as follows: Figure 4a, Multiple sequence alignment of PL protein sequences was performed using MAFFT software, and a maximum likelihood phylogenetic tree was constructed using IQ-TREE2 with 1000 bootstrap resampling, and the numbers represent bootstrap values, with a scale of 20 units being 100 bases. The PL genes of Rosaceae were reorganized into 7 subfamilies and 31 clusters. The five-star symbol on the phylogenetic tree (left) represents the whole genome duplication (WGD) event, and the bubble chart on the right shows the distribution of PL gene family members in different species, with the bubble size corresponding to the number of members in the cluster Figure 4 b); this grouping obtained the similarity matrix support of the subgroup clustering Figure 5 , Protein similarity analysis of cultivated strawberry PL genes was performed based on Clustal Omega software. The Similiarity value represents protein similarity, with higher values corresponding to higher similarity and redder color.

[0039] 2, Transcriptome analysis of FanPL gene family The present application obtains public transcriptome data from the NCBI database and integrates it into a cultivated strawberry fruit development transcriptome dataset Figure 6 Through analysis of the transcriptome data, the present application screens three FanPL gene clusters with high expression specificity during the ripening period of strawberry fruit: FanPL6a1, FanPL7a1, and FanPL7b1, which contain a total of 7 member genes: FanPL6a1.A1, FanPL7a1.A1, FanPL7a1.B1, FanPL7b1.A1, FanPL7b1.B1, FanPL7b1.C1, and FanPL7b1.D1. The nucleotide sequences of the 7 member genes are shown in SEQ ID NO: 2 to SEQ ID NO: 8, respectively.

[0040] 3, Analysis of the correlation between candidate FanPL genes and strawberry fruit hardness Dynamic analysis during fruit development (index detection method same as in Example 2) found that the expression of these FanPL genes peaked during the turning color period to the red fruit period, showing significant negative correlation and high synchronicity with physiological softening indicators such as fruit hardness decrease, CWM content reduction, WSP content increase, and PL enzyme activity improvement Figure 7 ); the development period of strawberry includes small green period (SG, 8DAF), large green period (BG, 14DAF), fade green period (DG, 18DAF), white fruit period (White, 22DAF), turning color period (T, 25DAF), half red period (PR, 28DAF), full red period (R, 31DAF), post-mature I period (OR, 33DAF), and post-mature II period (OR, 35DAF); each treatment includes at least 20 biological replicates, and each replicate includes at least 1 strawberry fruit.

[0041] 4. Functional verification of FanPL genes By transient overexpression technology (the method is the same as that in Example 2), the expression levels of FanPL6a1, FanPL7a1, and FanPL7b1 were increased, respectively, which resulted in a significant decrease in fruit hardness, a decrease in CWM content, an increase in WSP accumulation, and accelerated CSP degradation (Example 3). Figure 8 On the contrary, RNAi interference treatment designed in the public sequence region can significantly increase fruit hardness and CWM content, and inhibit WSP accumulation and CSP degradation (Example 4). Figure 8

[0042] The above results show that the seven FanPL genes in claim 4: FanPL6a1.A1, FanPL7a1.A1, FanPL7a1.B1, FanPL7b1.A1, FanPL7b1.B1, FanPL7b1.C1, and FanPL7b1.D1: synergistically participate in the regulation of the strawberry fruit softening process.

[0043] The primers involved are described in Table 2: Table 2 5. Clarification of the transcriptional regulation of FanPRE1.B1 on downstream FanPL genes qRT-PCR was used to detect the expression of downstream FanPL genes (the specific detection method is described in Example 2). The results showed that in the fruits silenced by FanPRE1.B1-RNAi, the expression of the seven FanPL genes in claim 4 was significantly decreased by 71%-76%. On the contrary, in the fruits overexpressed by FanPRE1.B1-OE, the expression of these genes was significantly increased by 2.15-4.69 times (p<0.01) (Example 5). Figure 9 This indicates that the transcription factor FanPRE1.B1 positively regulates the expression of downstream key FanPL genes.

[0044] The primers involved are described in Table 3: Table 3 In summary, the above results show that the transcription factor FanPRE1.B1 enhances PL enzyme activity by positively regulating the expression of downstream pectin lyase gene clusters FanPL6a1, FanPL7a1, and FanPL7b1 (including the seven member genes in claim 4), thereby promoting pectin degradation, and ultimately leading to strawberry fruit softening (Example 6). Figure 9 ​). Therefore, inhibiting the expression of FanPRE1.B1 can effectively reduce the expression level of these PL genes, inhibit pectin degradation, thereby delaying fruit softening, improving fruit hardness and postharvest quality.

[0045] The above examples are only used to illustrate the technical solutions of the present application, but not to limit it; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that the technical solutions recorded in the foregoing examples can be modified, or some technical features can be replaced by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A transcription factor FanPRE1.B1 that regulates strawberry fruit softening, characterized by: The nucleotide sequence of the transcription factor FanPRE1.B1 is shown in SEQ ID NO:

1.

2. Use of the transcription factor FanPRE1.B1 as claimed in claim 1 in improving the postharvest quality of strawberry fruit.

3. The use according to claim 2, characterized in that: The application method is to improve the post-harvest quality of strawberry fruit by inhibiting the expression or activity of the transcription factor FanPRE1.B1.

4. The use according to claim 2, characterized in that The improvement of the post-harvest quality of strawberry fruit is at least one of increasing the firmness of the fruit, reducing the weight loss rate of the fruit during storage, reducing the gray mold infection rate of the fruit, increasing the cell wall matrix content of the fruit, and reducing the water-soluble pectin content of the fruit.

5. A pectin lyase gene regulated by the transcription factor FanPRE1.B1, characterized in that: The pectin lyase gene is at least one of the following genes: (1) FanPL6a1.A1, the nucleotide sequence of which is shown in SEQ ID NO: 2; (2) FanPL7a1.A1, the nucleotide sequence of which is shown in SEQ ID NO: 3; (3) FanPL7a1.B1, the nucleotide sequence of which is shown in SEQ ID NO:4; (4) FanPL7b1.A1, the nucleotide sequence of which is shown in SEQ ID NO:5; (5) FanPL7b1.B1, the nucleotide sequence of which is shown in SEQ ID NO:6; (6) FanPL7b1.C1, the nucleotide sequence of which is shown in SEQ ID NO:7; (7) FanPL7b1.D1, the nucleotide sequence is shown in SEQ ID NO:

8.

6. A method for delaying the softening of strawberry fruit, characterized in that: The method is to inhibit the expression of FanPRE1.B1 to reduce the expression levels of FanPL6a1.A1, FanPL7a1.A1, FanPL7a1.B1, FanPL7b1.A1, FanPL7b1.B1, FanPL7b1.C1, and FanPL7b1.D1, thereby inhibiting pectin degradation and delaying fruit softening.

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