Method for improving aroma of refrigerated Nanguo pear fruits

Abstract

The invention discloses a method for improving aroma of refrigerated Nanguo pears, which is characterized in that methyl jasmonate (MeJA) is used for treatment before refrigeration, and decomposition of ester volatile substances of the refrigerated Nanguo pears is inhibited. Meanwhile, the mechanism of the MeJA for inhibiting the aroma decomposition of the refrigerated Nanguo pears is clarified on the transcriptional regulation level, and a theoretical foundation is laid for directionally improving the aroma quality of the fruits.

Description

Technical Field

[0001] The invention relates to the technical field of fruit and vegetable storage and cultivation, and in particular to a method for improving the aroma quality of refrigerated Nanguo pear fruits and cultivating plants. Background Art

[0002] Fruit is an important reproductive organ of plants and an important source of dietary nutrition for humans. With the development of social economy, fruit quality has become the core of the healthy development and market competitiveness of the current and future fruit industry. In the past, research on fruit quality mainly focused on aspects such as fruit soluble sugar, organic acid and peel coloring, while research on fruit volatiles was relatively small. Surveys in the United States and Europe in recent years have found that the loss of fruit flavor is leading to a decline in fruit consumption.

[0003] Nanguo Pear (Pyrus ussuriensis'Nanguo Pear'.) is a local autumn pear variety in Liaoning Province. It has become a characteristic industry in Anshan, Liaoyang, Haicheng and other regions (Li Hongjun et al., 2023). Nanguo Pear fruit is famous for its strong aroma, and ester volatile substances are the main source of fruit aroma (Li et al., 2022). Nanguo Pear fruit is a typical respiratory climacteric fruit. After harvesting, the fruit can only be stored for 15-20 days at room temperature and quickly loses its commercial value (Li Xiaojing, 2023). Therefore, refrigeration is often used in production to extend the storage period of the fruit. However, long-term refrigeration of Nanguo Pear fruit is prone to the phenomenon of aroma dilution, which seriously reduces the commercial value of the fruit and affects the healthy development of the Nanguo Pear industry. Therefore, revealing the occurrence law of aroma dilution of cold-stored Nanguo Pear fruit and seeking regulatory technology to inhibit the aroma dilution of cold-stored Nanguo Pear are of great theoretical and practical significance to the sustainable development of the Nanguo Pear industry. Summary of the invention

[0004] The purpose of the invention is to provide a method for improving the aroma quality of refrigerated fruits, a key regulatory gene for the aroma of refrigerated Nanguo pear, and a method for cultivating plants.

[0005] A method for regulating the aroma of refrigerated pear fruit, wherein the method comprises up-regulating the transcription level of the MYC2 gene and / or inhibiting the acetylation level of the histones H3 and H4 of the CXE15 gene promoter. In some embodiments, the method comprises up-regulating the transcription level of the MYC2 gene; inhibiting the acetylation level of the histones H3 and H4 of the CXE15 gene promoter; and inhibiting the acetylation level of the histones H3 and H4 of the CXE15 gene promoter at the same time by up-regulating the transcription level of the MYC2 gene.

[0006] In some embodiments, the decomposition of volatile ester substances in refrigerated fruits is inhibited after any of the above treatments.

[0007] A method for improving the aroma of refrigerated fruit, wherein the fruit is treated with a methyl jasmonate solution. In some embodiments, the fruit is treated with the methyl jasmonate solution before refrigeration. In some embodiments, the fruit is soaked in the methyl jasmonate solution before refrigeration.

[0008] In some embodiments, the treatment concentration of the methyl jasmonate solution is 10-500 μM, 50-500 μM, 50-200 μM, 50-100 μM or 100-200 μM. In some specific embodiments, a small amount of anhydrous ethanol is used to dissolve and then diluted with water and constant volume to obtain the treatment concentration of the corresponding methyl jasmonate. In some specific embodiments, the treatment concentration of the methyl jasmonate is 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, 100 μM, 110 μM, 120 μM, 130 μM, 140 μM, 150 μM, 160 μM, 170 μM, 180 μM, 190 μM, 200 μM, 300 μM, 400 μM, 500 μM. In some specific embodiments, the treatment concentration of methyl jasmonate is 50 μM, 100 μM or 200 μM.

[0009] In some embodiments, the MeJA treatment significantly enhances the expression of MYC2, inhibits the acetylation levels of histones H3 and H4 in the CXE15 gene promoter, and inhibits the transcription of CXE15.

[0010] A method for cultivating plants, wherein the transcription level of the MYC2 gene of the plant is upregulated, and / or the acetylation level of the histones H3 and H4 of the CXE15 gene promoter is inhibited; the fruit of the plant has the improved characteristic of maintaining or improving the aroma after cold storage.

[0011] A method for cultivating plants, wherein the transcription level of the MYC2 gene of the plant is upregulated relative to that of the wild type, and the expression amount of MYC2 is increased.

[0012] A method for breeding a plant, wherein the acetylation levels of histones H3 and H4 of the promoter of a CXE15 gene in the plant are decreased compared with the wild type.

[0013] A method for breeding a plant, wherein the transcription level of CXE15 in the plant is reduced relative to wild type.

[0014] A method for cultivating plants, wherein the expression level of MYC2 in the plants is increased, the acetylation level of histones H3 and H4 in the promoter of the CXE15 gene is decreased, and / or the transcription level of CXE15 is decreased relative to the wild type.

[0015] In some embodiments, the method of cultivating plants comprises: regulating the gene using transgenic technology or gene editing technology. In some specific embodiments, the recipient plant is made to overexpress the exogenously introduced MYC2 gene.

[0016] In some embodiments, the expression of genes or elements that positively regulate MYC2 in the recipient plant is enhanced, and the expression of genes or elements that negatively regulate MYC2 is suppressed.

[0017] In some embodiments, CXE15 is silenced by Agrobacterium injection.

[0018] In some embodiments, CXE15 is silenced by Agrobacterium injection.

[0019] In some embodiments, the fruit comprises pear, apple, strawberry, peach, tomato, orange, tangerine, or mandarin.

[0020] In some embodiments, the pear is Nanguo pear.

[0021] In some embodiments, the MYC2 is PuMYC2.

[0022] In some embodiments, CXE15 is PuCXE15.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] First, obtaining the regulatory technology to inhibit the dilution of the aroma of refrigerated Nanguo pear fruit is of great significance to the development of the Nanguo pear industry.

[0025] Second, the mechanism by which MeJA inhibits the aroma decomposition of cold-stored Nanguo pear was elucidated at the transcriptional regulation level, laying a theoretical foundation for the targeted improvement of fruit aroma quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The effect of 50, 100, and 200 μM MeJA treatment before refrigeration on the total content of volatile esters in refrigerated Nanguo pear fruit on the 9th day of shelf life. Cold: refrigerated control group; MeJA+Cold: MeJA treatment group; 50, 100, and 200 represent MeJA concentrations of 50, 100, and 200 μM, respectively.

[0027] Figure 2 It is the content of volatile ester substances in Nanguo pear fruit during its shelf life after refrigeration. Figure 2 A, total ester content; Figure 2B-2D, characteristic esters (ethyl butyrate, ethyl hexanoate and hexyl acetate) content. RT, Cold and MeJA+Cold indicate storage at room temperature, refrigerated (0±0.5℃) for 90 days after harvest without treatment and methyl jasmonate treatment; 0d indicates the day of harvest or the day of shipment, and OTP indicates storage until the best consumption period.

[0028] Figure 3 The jasmonic acid content and PuAAT1 expression level of Nanguo pear fruit. Figure 3 A, jasmonic acid content; Figure 3 B, Expression level of PuAAT1.

[0029] Figure 4 This is a phylogenetic analysis of CXEs in Pyrus nanguoensis and other plants. Orange circles represent CXEs in Pyrus nanguoensis, and green hollow triangles represent CXEs in other plants. PuCXE1-40 represents CXEs in Pyrus nanguoensis, AtCXE1-20 represents CXEs in Arabidopsis thaliana, PpCXE1-3 represents CXEs in peach, MdCXE1 represents CXEs in apple, SlASH1, SlASH2, SlCXE1, and Lehsr203J represent CXEs in tomato, and Nthsr203J represents CXEs in tobacco.

[0030] Figure 5 It is the phylogenetic tree, gene structure, conserved motifs and markers of 7 conserved motifs of PuCXEs. Figure 5 A, Phylogenetic tree of PuCXEs; Figure 5 B, PuCXEs gene structure. The thick yellow lines represent exons, and the thin black lines represent introns; Figure 5 C, Motif structure. Motifs 1-7 are represented by boxes of different colors; Figure 5 D, Motif amino acid sequence.

[0031] Figure 6 It is the detection of cis-acting elements in the PuCXEs gene of Nanguo pear. Figure 6 A, Promoter cis-acting elements. Cis-acting elements with different functions are represented by boxes of different colors. The black line represents the promoter length of the PuCXEs gene. Figure 6 B, Statistics of cis-acting elements; Figure 6 C, Classification of cis-acting elements. The sum of cis-acting elements that respond to light, stress, development, and hormones is represented by different colored bars.

[0032] Figure 7 The tertiary structure predictions of 40 PuCXE proteins. Blue and red represent low-activity and high-activity structures, respectively.

[0033] Figure 8The VOCs changes of Nanguo pear fruits 0, 5, 10, 15 and 20 days after harvest. Figure 8 A, relative content of 7 types of volatile substances; Figure 8 B, the quantity of 7 types of volatile substances; Figure 8 C, Contents of 24 ester volatiles.

[0034] Fig. 9 It is the correlation between PuCXEs expression and volatile ester content during the ripening process of Nanguo pear fruit. Fig. 9 A, Expression of PuCXEs. Data are presented as mean ± SD of three independent biological replicates; Fig. 9 B, Correlation between the expression levels of PuCXE7, PuCXE15, PuCXE20 and PuCXE25 and the content of ethyl butyrate (P<0.05); Fig. 9 C, Correlation between the expression levels of PuCXE7, PuCXE15, PuCXE20 and PuCXE25 and the content of ethyl hexanoate (P<0.05); Fig. 9 D, Correlation between the expression levels of PuCXE7, PuCXE15, PuCXE20 and PuCXE25 and the content of hexyl acetate (P<0.05).

[0035] Fig.10 The effects of overexpression and silencing of PuCXE15 on the content of volatile esters in Nanguo pear fruit. Fig.10 A, Phenotype of Nanguo pear fruit after injection. Nanguo pear fruit was injected 135 days after anthesis (0 days after harvest) and stored at 25℃ for 15 days. The scale is 2 cm. Fig.10 B, Expression level of PuCXE15; Fig.10 C, total ester content; Fig.10 D, hexyl acetate content; Fig.10 E, content of ethyl hexanoate. ns indicates no significant difference, and asterisks indicate statistically significant differences: ** indicates extremely significant difference (P<0.01), and * indicates significant difference (P<0.05).

[0036] Fig.11 It is the subcellular localization of PuCXE15. GFP: GFP image; mCherry: mCherry image; Brightfield: bright field; Merge: the image after merging GFP image, mCherry image and bright field; PM-mCherry: mCherry marks the plasma membrane marker; NF-YA4-mCherry: mCherry marks the nuclear marker; the scale bar represents 50μm.

[0037] Fig.12 This is the expression analysis of PuCXE15.

[0038] Fig.13 is the expression level of PuMYCs during the shelf life of Nanguo pear fruit after MeJA treatment and cold storage. Fig.13 A, PuMYC2.1-3 expression level at the optimal edible period of Nanguo pear fruit after cold storage treated with MeJA; Fig.13 B, Expression level of PuMYC2 in Nanguo pear fruit during shelf life 0-12 days after cold storage treated with MeJA.

[0039] Fig.14 It is the subcellular localization of PuMYC2. GFP: GFP image; mCherry: mCherry image; Brightfield: bright field; Merge: the image after merging GFP image, mCherry image and bright field; NF-YA4-mCherry: mCherry marks nuclear marker; The scale bar represents 50μm.

[0040] Fig.15 was the analysis of the content of ester volatile substances and the expression level of PuCXE15 after transient expression of PuMYC2 in cold-stored Nanguo pear fruits. Fig.15 A, PuMYC2 expression; Fig.15 B, PuCXE15 expression level; Fig.15 C, total content of ester substances; Fig.15 D, ethyl hexanoate content; Fig.15 E, ethyl butyrate content; Fig.15 F, Hexyl acetate content.

[0041] Fig.16 Y1H assay analysis of PuMYC2 binding to the PuCXE15 promoter.

[0042] AD-Rec-P53 / P53-Promoter: positive control; AD-Empty / ProPuCXE15: negative control. +AbA: AbA (Aureobasidin A) was added to the culture medium, -AbA culture medium did not contain AbA, 150: the final concentration of AbA added was 150 ng mL -1 .

[0043] Fig.17 This is an EMSA analysis of PuMYC2 binding to the PuCXE15 promoter. Hot probe: biotin-labeled probe; Cold probe: non-biotin-labeled probe; Mutant probe: non-biotin-labeled mutant probe; ++, +++: the concentration of non-biotin-labeled probe is 200, 300 times that of biotin-labeled probe; Free probe: biotin-labeled probe that is not bound to the target protein.

[0044] Fig.18This is the regulatory analysis of PuMYC2 on PuCXE15 promoter activity.

[0045] Fig.19 This is the result of the effect of MeJA treatment on the histone acetylation level of PuCXE15 gene and its promoter in cold-stored Nanguo pear fruit. Fig.19 A, Promoter structure of PuCXE15; Fig.19 B, Acetylation levels of histones H3 and H4 on the PuActin promoter; Fig.19 C, Acetylation level of histone H3 on the PuCXE15 promoter; Fig.19 D, Acetylation level of histone H4 on the PuCXE15 promoter.

[0046] Fig. 20 This is an analysis chart of the interaction between PuMYC2 and PuHDAC15 and the expression level of PuHDAC15. Fig. 20 A, Expression level of PuHDAC15; Fig. 20 B, Yeast two-hybrid experiment verified the interaction between PuMYC2 and PuHDAC15. PuHDAC15 / PuMYC2: co-transfection of PuHDAC15-AD and PuMYC2-BD; P53 / SV40: positive control; AD / PuMYC2, PuHDAC15 / BD and AD / BD: negative control; DDO: SD solid medium lacking Leu and Trp; QDO: SD solid medium lacking Leu, Trp, His, and Ade; QDO / X / A: QDO medium with X-α-gal and AbA added. DETAILED DESCRIPTION

[0047] The present invention will be described in detail below in conjunction with examples, but the examples provided herein are only for illustrative purposes and are not intended to limit the present invention.

[0048] Example 1: Study on the aroma of Nanguo pear after refrigeration

[0049] 1 Materials and reagents

[0050] The fruits of Pyrus ussriensis Maxim were collected at the Experimental Farm of Tangjiafang Town, Qianshan District, Anshan City, Liaoning Province.

[0051] MeJA (Cat. No.: M813622, McLean, China) was dissolved in a small amount of anhydrous ethanol and then diluted with water to make up the volume.

[0052] 3-Octanol (Cat. No. 218405, Sigma-Aldrich, Germany).

[0053] 2 Experimental methods

[0054] 2.1 Cold storage of Nanguo pear

[0055] The harvested fruits were divided into five groups (60 in each group). The first group of fruits were not treated and left to ripen naturally at room temperature (20±1℃); the fruits in the second to fifth groups were soaked in 0, 50, 100, and 200μM MeJA for 15 minutes, respectively, and left to dry at room temperature. The first group of fruits were stored at room temperature for 15 days, and samples were taken every 5 days. The fruits in other groups were refrigerated at 0±0.5℃ for 90 days and then transferred to room temperature for 12 days, and samples were taken every 3 days. Each sampling was repeated 3 times, and 5 fruits were collected from each repeat, immediately frozen with liquid nitrogen, and stored at -80℃ for subsequent experiments.

[0056] 2.2 Detection of volatile ester substances

[0057] First, 1.5 g NaCl, 4 mL pear juice, and 1.5 μL 3-octanol (1.2 mg mL -1 ) was added as an internal standard to a 20 mL headspace bottle. Then, volatile compounds were extracted by headspace solid phase microextraction (HS-SPME), and the volatile content was determined by gas chromatography-mass spectrometry (GC-MS) (7890A-5975C, Agilent Technologies, USA); gas chromatography conditions refer to: Li Xiaojing, Molecular mechanism of PuWRKY74 / PuNAC37 regulating the key gene PuAAT1 for the formation of fruit aroma of 'Nanguo Pear' [D]. 2023, Shenyang Agricultural University). Finally, the types of volatiles were analyzed by searching the NIST / WILEY standard spectral library (NISTII), and the volatile content was determined by the internal standard method. Each sample was repeated at least three times, and the volatile content was expressed in μg·g -1 express.

[0058] 2.3 Determination of jasmonic acid content

[0059] Fresh pulp material was ground with liquid nitrogen and weighed 1g. After adding 4mL of 80% pre-cooled methanol and extracting at -20℃ for 12h, centrifuged at 10000rpm for 10min; the supernatant was taken in a new centrifuge tube; the remaining precipitate was added with 3mL of 80% pre-cooled methanol and extracted again in a -20℃ refrigerator for 4h; centrifuged at 10000rpm for 10min at 4℃, the supernatant was taken, and the supernatants collected twice were combined. The combined supernatant was concentrated and evaporated to dryness using a freeze vacuum dryer (Gene company ss4055) until no liquid flowed at the bottom of the centrifuge tube. The evaporation conditions were: 2000rpm, 10pa, 4℃. After adding 400μL of 80% methanol to dissolve the concentrate in the centrifuge tube and diluting to 1mL, it was filtered twice using a 0.2μm nylon filter for standby use.

[0060] The jasmonic acid content was measured using a Waters high performance liquid chromatography-mass spectrometer (ACQUITY UPLC XEVOTQD, Waters), and the separation column was a C18 column (CSH 21x50 mm 1.7μm, Waters). Chromatographic conditions refer to: Pengtao Yue et al., Jasmonate activates aCsMPK6-CsMYC2 module that regulates the expression ofβ-citraurin biosynthetic genes and fruit coloration in orange (Citrus sinensis), The Plant Cell, 2023, 35(4): 1167–1185, with at least 3 biological replicates each time.

[0061] 3 Experimental results

[0062] 3.1 Analysis of volatile ester content in cold-stored Nanguo pear fruit after MeJA treatment

[0063] Compared with the control group at room temperature (RT), the content of volatile esters in Nanguo pear fruit during the optimal taste period (OTP; in this study, OTP was the 9th day of shelf life) after refrigeration was significantly reduced ( Figure 2 However, compared with the cold storage control group (Cold), the volatile ester content in Nanguo pear fruits treated with 50, 100 and 200 μM MeJA (MeJA+Cold) was significantly increased, among which the 100 μM MeJA treatment had the best effect (see Figure 1 100 μM MeJA treatment significantly inhibited the reduction of characteristic volatile esters (ethyl butyrate, ethyl hexanoate and hexyl acetate) in Nanguo pear fruit (see Figure 2 ).

[0064] 3.2 Analysis of JA content and PuAAT1 expression in cold-stored Nanguo pear fruit after MeJA treatment

[0065] In order to reveal the formation mechanism of MeJA inhibiting the aroma dilution phenomenon of cold-stored Nanguo pear fruit, the JA content and PuAAT1 expression in the fruits of the MeJA+Cold treatment group and the Cold control group were measured. The results showed that the JA content in the fruits of the MeJA+Cold treatment group was significantly higher than that in the Cold control group, but there was no significant difference in the expression of PuAAT1 in the fruits of the MeJA+Cold treatment group and the Cold control group ( Figure 3The above results showed that MeJA treatment significantly increased the JA content in the fruit, but MeJA treatment did not affect the aroma of cold-stored Nanguo pear fruit by regulating the expression of PuAAT1.

[0066] Example 2: Identification and analysis of the PuCXEs gene in Nanguo pear

[0067] The content of volatile esters in fruits is regulated by both synthesis and decomposition pathways. In previous studies, it was found that MeJA treatment increased the content of volatile esters in cold-stored Nanguo pear fruits, but the expression of PuAAT1, a key gene for the synthesis of volatile esters, did not change significantly. Therefore, further research was conducted on PuCXEs, a key gene for the decomposition of volatile esters.

[0068] Using the amino acid sequences of CXEs in Arabidopsis, apple and peach as templates, a total of 40 PuCXEs genes were identified in the genome of Pyrus nanguoensis through BlastP local alignment, and these genes were named PuCXE1-PuCXE40 according to their locations in the genome.

[0069] The amino acid sequences of CXEs in Pyrus nanguoensis and other plants (Arabidopsis thaliana, apple, peach, tomato, and tobacco) were used to construct an unrooted phylogenetic tree using the neighbor-joining method (see Figure 4 ), which included 20 Arabidopsis AtCXE1-20 sequences, 1 apple MdCXE1 sequence, 4 tomato SlCXEs sequences, 1 tobacco Nthsr203J sequence, and 3 peach PpCXEs sequences. Phylogenetic analysis showed that the 40 Nanguo pear PuCXEs were closely homologous to CXEs in other species, indicating that CXEs are highly conserved among species (see Figure 4 ).

[0070] Phylogenetic analysis showed that PuCXEs can be divided into seven groups based on phylogenetic relationships (see Figure 5 A). In order to study the evolutionary characteristics and functional diversification of PuCXEs genes, TBtools was used to analyze the exon-intron structure of 40 PuCXEs gene sequences. The analysis results showed that 21 PuCXEs genes did not contain introns, and 11 genes contained 1 intron. The 8 PuCXEs randomly distributed in different groups were found to have >2 introns (see Figure 5B). The diversity of introns and exons showed that most PuCXEs in Nanguo pear contained one or fewer introns, and PuCXEs genes underwent different splicing events during translation, which may be related to the function of PuCXEs genes. Seven motifs were found in PuCXEs of Nanguo pear using MEME Suite software. These results show that PuCXEs with close phylogenetic relationships have similar motifs and gene structures, indicating that they may have similar biological functions.

[0071] The distribution of cis-acting elements in the PuCXEs promoter is as follows Figure 6 A. Analysis of the cis-acting elements of the PuCXEs promoter using PlantCare software revealed that 30 cis-acting elements in the PuCXEs promoter were involved in a variety of abiotic stress responses, including development (7), light signal (10), stress response (5) and plant hormone response (8) ( Figure 6 C). Among these cis-acting elements, the cis-acting elements that respond to light signals and plant hormone synthesis account for the majority. There are 8 hormone-responsive cis-acting elements, including abscisic acid response element, methyl jasmonate response element, salicylic acid response element, gibberellin response element and auxin response element ( Figure 6 C). Among them, light and hormone response elements account for the largest proportion in the promoter of PuCXEs gene ( Figure 6 B) These results suggest that PuCXEs may play a key role in the response of Pyrus nanguoensis to light and hormones.

[0072] The three-dimensional structure of PuCXEs protein is mainly composed of α-helix, β-fold and random coil ( Figure 7 ). After grouping these proteins according to their phylogenetic relationships, it was found that proteins in the same group had similar three-dimensional structures, such as PuCXE4, PuCXE5, PuCXE11, PuCXE12, and PuCXE20 in the third group; PuCXE25, PuCXE26, and PuCXE27 in the seventh group ( Figure 7 ). The protein three-dimensional structure prediction results further showed that proteins in the same group may have similar functions.

[0073] Example 3: Identification of key genes for decomposition of volatile ester substances in Nanguo pear fruit

[0074] 1. Analysis of the composition and content of volatile substances in Nanguo pear fruit during ripening

[0075] The types and contents of volatile substances in Nanguo pear fruit change with storage time. Aldehydes are the main aroma components of Nanguo pear fruit in the early stage of storage. However, as the storage time increases, the content of aldehydes in the fruit gradually decreases, while the content of esters and terpenes gradually increases, forming a strong fruity aroma ( Figure 8 A). During the fruit ripening process, a total of 69 volatile substances were detected, including 1 alcohol, 3 acids, 7 benzenes, 7 terpenes, 8 aldehydes, 24 esters and 21 other volatile substances ( Figure 8 B) As the fruit matures, the content and types of aldehyde volatile substances decrease, while the content and types of ester volatile substances increase ( Figure 8 B, C). The volatile ester substances increased from 2 on day 0 to 24 on day 15, with a significant change in quantity ( Figure 8 B). Analysis of the contents of 24 volatile ester substances revealed that ethyl butyrate, ethyl hexanoate and hexyl acetate had high contents and low aroma thresholds ( Figure 8 C), was identified as the characteristic esters of Pyrus nanguoensis fruit.

[0076] 2. Analysis of PuCXEs gene expression pattern in Nanguo pear fruit

[0077] Of the 40 PuCXEs identified in the genome of Nanguo pear, 27 were expressed during fruit ripening. Among them, PuCXE15, PuCXE25, PuCXE7, and PuCXE20 were expressed at higher levels than the other 23 PuCXEs ( Fig. 9 A). The expression level of PuCXE15 increased during storage 0-5 days and decreased during storage 5-20 days; the expression level of PuCXE25 showed the opposite trend, decreasing during storage 0-10 days and increasing during storage 10-20 days. During the period of 0-20 days after harvest, the expression levels of PuCXE7 and PuCXE20 did not change significantly compared with PuCXE15 and PuCXE25. The expression of the remaining 13 PuCXEs was not detected during the period of 0-20 days after harvest, and these genes may be involved in other metabolic processes of Nanguo pear.

[0078] Ethyl hexanoate, ethyl butyrate and hexyl acetate are characteristic ester volatile substances accumulated during the ripening of Nanguo pear fruit ( Fig. 9 C). PuCXE15, PuCXE25, PuCXE7, and PuCXE20, which have higher expression levels, are considered candidate genes for regulating the decomposition of volatile esters ( Fig. 9BD). Correlation analysis showed that the expression level of PuCXE15 was highly negatively correlated with the contents of ethyl hexanoate, hexyl acetate and ethyl butyrate, with correlation coefficients of 0.85, 0.86 and 0.92, respectively. In addition, the expression level of PuCXE7 was positively correlated with the contents of ethyl hexanoate and hexyl acetate, with correlation coefficients of 0.46 and 0.44, respectively. The expression level of PuCXE7 was moderately correlated with the content of ethyl butyrate, with a correlation coefficient of 0.62. The expression level of PuCXE20 was moderately correlated with the content of ethyl butyrate, with a correlation coefficient of 0.48, and had a low correlation with the contents of hexyl acetate and ethyl hexanoate. PuCXE15 has a high expression level and a high negative correlation with the content of ester volatile substances, so it may be a key CXE gene for the degradation of ester volatile substances in Nanguo pear fruit.

[0079] 3. Functional verification of PuCXE15

[0080] PuCXE15 (GenBank ID: OQ235138.1) was transiently expressed in postharvest Nanguo pear fruit to verify its function ( Fig.10 A). In the overexpression of PuCXE15 fruit, the expression of PuCXE15 gene was increased by about 4 times compared with the empty load fruit, and the content of total volatile esters was reduced by 50% ( Fig.10 B, C), while inhibiting the expression of PuCXE15 had the opposite effect ( Fig.10 B, C). After PuCXE15 was transiently overexpressed in the fruit, the content of hexyl acetate decreased by 48% compared with the empty load; the content of ethyl hexanoate decreased to 42%; for the transiently silenced fruit, the opposite effect was observed ( Fig.10 D, E). These results showed that increasing the expression of PuCXE15 in Nanguo pear fruit reduced the content of ester volatiles, further indicating that PuCXE15 is a key CXE gene for the degradation of ester volatiles in Nanguo pear fruit.

[0081] 4. Subcellular localization of PuCXE15

[0082] In plants, CXEs located in different subcellular locations perform different biological functions. To explore the subcellular localization of PuCXE15, PuCXE15-GFP fusion protein was heterologously expressed in tobacco leaves, using plasma membrane Maker (PM-mCherry) and nuclear Maker (NF-YA4-mCherry) as markers. The experimental results showed that PuCXE15 is located in the nucleus and plasma membrane ( Fig.11 ).

[0083] 5.MeJA treatment inhibits the expression of PuCXE15 in cold-stored Nanguo pear fruit

[0084] In order to reveal the formation mechanism of MeJA inhibiting the aroma dilution phenomenon of cold-stored Nanguo pear fruit, the expression of PuCXE15, a key gene for the decomposition of volatile esters, was further analyzed by RT-qPCR. Compared with the Cold control group, the expression of PuCXE15 in the fruits of the Cold+MeJA treatment group was inhibited ( Fig.12 The above results indicate that MeJA may inhibit the decomposition of volatile esters in cold-stored Nanguo pear fruit by inhibiting the expression of PuCXE15 gene.

[0085] Example 4: Identification of PuMYC2 from Pyrus nanguoensis

[0086] 1. Expression analysis of PuMYC2 in MeJA-treated fruits

[0087] MYC2 is the core transcription factor of the JA signaling pathway, and there are multiple MYC2 binding sites (G-box motif) 1100bp upstream of the PuCXE15 promoter. PuMYC2 was selected as a candidate transcription factor for regulating PuCXE15. Using the amino acid sequence of the MYC2 gene (Gene ID GenBank: PQ584614.1) that has been identified as playing an important regulatory role in fruit quality in species such as apple and citrus as a template, four MYC2 genes with the closest structure were found in the Nanguo pear genome by BlastP alignment, and were initially named PuMYC2.1-4. After analyzing the expression levels of these four MYC2 genes, it was found that PuMYC2.1-3 were all expressed in Nanguo pear fruits and were significantly induced by MeJA, while PuMYC2.4 was not detected in Nanguo pear fruits ( Fig.13 A). Among them, the expression of PuMYC2.2 was most significantly induced by MeJA treatment, so this gene was named PuMYC2. Further measurement of the expression of PuMYC2 found that the expression level of PuMYC2 in the fruits of the Cold+MeJA treatment group was significantly higher than that in the fruits of the Cold control group during the 0-12 days of the shelf life ( Fig.13 B). The above results indicate that PuMYC2 may play an important role in the process of MeJA inhibiting the decomposition of volatile esters in cold-stored Nanguo pear fruit.

[0088] 2. Subcellular localization of PuMYC2

[0089] In order to explore the subcellular location of PuMYC2 in cells, the fusion recombinant protein PuMYC2-GFP was heterologously expressed in Nicotiana benthamiana leaves, and NF-YA4-mCherry was used as a nuclear localization marker. Fig.14It can be seen that the green fluorescence of PuMYC2-GFP completely overlaps with the red fluorescence of NF-YA4-mCherry, indicating that PuMYC2 is located in the cell nucleus, which is consistent with the spatial characteristics of transcription factors.

[0090] 3. Functional verification of PuMYC2

[0091] The role of PuMYC2 in the formation of volatile esters in cold-stored Nanguo pear fruits was investigated through Agrobacterium-mediated transient transformation experiments. PuMYC2 was transiently expressed in Nanguo pear fruits that had been cold-stored for 90 days. Compared with the empty vector (pRI), the expression level of PuMYC2 in Nanguo pear fruits overexpressing PuMYC2 increased by about one-fold, the expression level of PuCXE15 decreased by 70%, and the total content of volatile esters and the content of three characteristic esters increased significantly ( Fig.15 AF). In contrast, silencing PuMYC2 in Nanguo pear fruit had the opposite effect ( Fig.15 AF). After treatment with MeJA, compared with the untreated fruit, the expression of PuMYC2 in the PuMYC2-silenced Nanguo pear fruit decreased by more than 50%, the expression of PuCXE15 increased by about 6 times, and the total content of ester volatile substances and the content of three characteristic ester volatile substances were significantly reduced, while the effect was the opposite in the fruit overexpressing PuMYC2 ( Fig.15 AF). The above results further indicated that PuMYC2 promoted the accumulation of volatile esters in Nanguo pear fruit after cold storage by inhibiting the expression of PuCXE15.

[0092] Example 5: Analysis of the regulation of PuMYC2 on PuCXE15

[0093] 1PuMYC2 binds to the PuCXE15 promoter

[0094] In order to further verify the role of PuMYC2 in the metabolism of volatile esters in Nanguo pear fruit, a yeast one-hybrid assay (Y1H) was performed and it was found that PuMYC2 could bind to the promoter of PuCXE15 but not to the promoter of PuAAT1 ( Fig.16 At the same time, the PuMYC2 protein with GST tag (PuMYC2-GST) was purified in Escherichia coli, and the PuCXE15 promoter fragment containing G-box motif and 5'-end biotin label was designed as a labeled probe for gel retardation assay (EMSA). GST protein does not bind to the biotin labeled probe and serves as a negative control ( Fig.17 , Lane 1). The experimental results showed that PuMYC2-GST binds to the biotin-labeled probe containing the G-box motif ( Fig.17, Lane 2); adding 200-fold or 300-fold non-biotinylated probes containing the G-box motif can compete with the biotinylated probe containing the G-box motif for binding to PuMYC2-GST, and the binding band disappears ( Fig.17 , Lanes 3 and 4). The addition of 300 times the probe containing two bases of the mutant G-box motif without biotin labeling could not compete with the biotin-labeled probe containing the G-box motif for binding to PuMYC2-GST ( Fig.17 , Lane 5). The above results indicate that PuMYC2 specifically binds to the G-box motif on the PuCXE15 promoter.

[0095] 2PuMYC2 regulates the transcriptional activity of the PuCXE15 promoter

[0096] To study the regulation mode of PuMYC2 on PuCXE15, GUS experiments were conducted in Nicotiana benthamiana leaves. Pro35S:PuMYC2 and ProPuCXE15:GUS were infected into tobacco leaves by Agrobacterium, and the control group (Control) was kept in the dark for 48 hours, the cold treatment group (Cold) was kept in the dark for 12 hours at 12℃ in the dark after 36 hours, the MeJA treatment group (MeJA) was kept in the dark for 44 hours after 10mM MeJA was injected into the leaves, and then kept in the dark for 4 hours, and the MeJA and cold stress co-treatment group (MeJA+Cold) was kept in the dark for 36 hours, then kept in the dark for 8 hours at 12℃, and then kept in the dark for 4 hours after 10mM MeJA was injected into the leaves. When Pro35S:PuMYC2 was co-transformed with ProCXE15:GUS, the activity of the PuCXE15 promoter was significantly inhibited in the Control group; this inhibitory effect was enhanced in the Cold or MeJA treatment group; and the inhibitory effect was further enhanced in the MeJA+Cold treatment group ( Fig.18 The above results indicate that PuMYC2 has an inhibitory effect on the promoter activity of PuCXE15, and this inhibitory effect is enhanced by cold treatment and MeJA treatment.

[0097] Example 6: Effect of MeJA treatment on the histone acetylation level of PuCXE15 promoter in cold-stored Nanguo pear fruit

[0098] 1MeJA treatment reduces histone acetylation level of PuCXE15 promoter

[0099] Histone acetylation plays an important role in gene expression. It changes chromatin conformation through the antagonistic effects of HAT and HDAC, thereby regulating gene expression. In the study of banana and Nanguo pear, it was found that the level of promoter histone acetylation is a key regulatory factor affecting the expression of genes related to fruit quality and chilling injury. In the ChIP-PCR experiment, histone H3 and H4 acetylation antibodies were used to detect the histone acetylation level of the PuCXE15 promoter in the fruits of the Cold+MeJA treatment group and the Cold control group. First, the acetylation levels of histone H3 and H4 of the internal reference gene PuActin in Nanguo pear fruit were detected, and no difference was found ( Fig.19 B), so PuActin was used as an internal reference. The first 1000bp of the PuCXE15 promoter was divided into 6 segments (S1-S6). It was found that the acetylation levels of histone H3 in the S2, S5 and S6 segments of the PuCXE15 promoter and the acetylation levels of histone H4 in the S1, S2, S5 and S6 segments of the fruits of the Cold+MeJA treatment group were significantly lower than those of the fruits of the Cold control group ( Fig.19 C, D). This indicates that the reduction of the expression of PuCXE15 gene by MeJA in cold-stored Nanguo pear fruit is related to the increase of its histone acetylation level.

[0100] 2PuMYC2 interacts with PuHDAC15 protein

[0101] In order to explore the reason why MeJA treatment reduces the acetylation level of PuCXE15 promoter, this study conducted transcriptome sequencing on the fruits of the Cold+MeJA treatment group and the Cold control group, and discovered a histone deacetylation gene PuHDAC15 whose expression was significantly induced by MeJA treatment. RT-qPCR detection found that compared with the fruits of the Cold control group, the expression level of PuHDAC15 gene in the Cold+MeJA treatment group was significantly increased at 3, 6, 9, and 12 days after cold storage ( Fig. 20 A). Histone deacetylases usually do not have the ability to bind to DNA directly, and need to interact with proteins with DNA binding ability such as transcription factors to approach chromatin to perform their functions. In order to explore whether the effect of PuMYC2 in inhibiting PuCXE15 transcription is related to the level of histone acetylation, yeast two-hybrid assay (Y2H) was used to study whether PuMYC2 and PuHDAC15 interact. The experimental results showed that PuMYC2 and PuHDAC15 interact ( Fig. 20 B). The above results indicate that the transcription factor PuMYC2 can recruit PuHDAC15 to the promoter of PuCXE15 by interacting with PuHDAC15, thereby reducing the acetylation level of histones in the promoter of PuCXE15, inhibiting the transcription of PuCXE15, and increasing the accumulation of ester aroma substances.

[0102] The above results show that the endogenous jasmonic acid content of Nanguo pear fruits treated with MeJA before refrigeration increased significantly during the shelf life after refrigeration, and the expression level of PuMYC2 in the fruit increased significantly. PuMYC2 inhibits the transcription of PuCXE15, and at the same time recruits PuHDAC15 to the promoter of PuCXE15 by interacting with PuHDAC15, reducing the histone acetylation level of the PuCXE15 promoter, further inhibiting the transcription of PuCXE15, and increasing the accumulation of volatile esters in refrigerated Nanguo pear fruits.

[0103] This specific embodiment is merely an explanation of the present invention and is not a limitation of the present invention. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed. However, as long as they are within the scope of the claims of the present invention, they are protected by the patent law.

Claims

1. A method for regulating the aroma of refrigerated pear fruit, characterized in that: The decomposition of volatile ester substances in cold-stored fruits was inhibited by upregulating the transcription level of the MYC2 gene and / or inhibiting the acetylation level of histones H3 and H4 in the promoter of the CXE15 gene.

2. The method according to claim 1, characterized in that The refrigeration condition is 0±0.5°C for 90 days.

3. The method according to claim 2, characterized in that Before refrigerating the fruit, soak it in a 10-500 μM methyl jasmonate solution; use a small amount of anhydrous ethanol to dissolve the methyl jasmonate and then dilute it with water to the corresponding treatment concentration.

4. The method according to claim 3, characterized in that The treatment concentration of methyl jasmonate is 100 μM.

5. The method according to claim 4, characterized in that The pear is Nanguo pear.

6. The method according to claim 5, characterized in that The CXE15 is PuCXE15, and the MYC2 is PuMYC2; the PuCXE15 gene sequence is shown in OQ235138.1, and the PuMYC2 gene sequence is shown in PQ584614.

1.

7. A method for growing plants, characterized in that: The transcription level of the MYC2 gene of the plant is upregulated by transgenic technology or gene editing technology, and / or the acetylation level of histones H3 and H4 of the CXE15 gene promoter is inhibited; the fruit of the plant has the improved characteristic of maintaining or improving aroma after refrigeration.

8. The method according to claim 7, characterized in that The fruit is selected from apples, peaches, tomatoes and pears.

9. The method according to claim 8, characterized in that The pears include Nanguo pear.

10. The method according to claim 9, characterized in that The MYC2 is PuMYC2, and the CXE15 is PuCXE15.

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