Mrbgal10 gene for regulating hardness of myrica rubra fruit, product and method and application thereof

Abstract

The application provides a MrBGAL10 gene for regulating hardness of Myrica rubra fruits, a product and a method and application thereof, and determines MrBGAL10 as a key gene by screening MrBGAL family members for coding beta-Gal enzymes. The key gene and mechanism for softening of the Myrica rubra fruits are determined, the key gene regulates softening by regulating beta-Gal enzyme activity to affect pectin and hemicellulose degradation. The application has the effects of providing a basis and a means for regulating texture of the Myrica rubra fruits and improving storage and transportation resistance, can accurately regulate the softening process of the fruits, provides an innovative direction for developing related preparations, promotes development of the Myrica rubra industry, and provides a reference for researches on other fruits.

Description

Technical Field

[0001] This invention belongs to the field of plant molecular genetic engineering technology, specifically, it relates to a MrBGAL10 gene that regulates the firmness of bayberry fruit, its product, method and application. Background Technology

[0002] The Chinese bayberry (Yangmei) is a specialty fruit tree of southern China, prized for its unique flavor and rich nutritional value. However, its distinctive shape, with exposed edible segments, makes it highly susceptible to mechanical damage. Because bayberries do not ripen after harvest, they must be picked at full maturity. Previous research has shown that bayberry fruits reach their maximum firmness early in development, decreasing as they mature. Ripe bayberries are very soft and easily damaged during harvesting, sorting, packaging, and transportation, impacting their marketability and significantly limiting their sales radius. Furthermore, bayberries are primarily grown in the hilly and mountainous areas of the province, with a short ripening period coinciding with the plum rain season. This concentration of production areas and timing places higher demands on the storage and transport resistance of fresh bayberries. Therefore, understanding the physiological and molecular mechanisms of bayberry fruit softening and improving its storage and transport resistance is crucial for the development of the bayberry industry.

[0003] Fruit softening refers to the process by which a fruit changes from hard to soft during its development and ripening. Softening is often considered one of the indicators of fruit ripeness. Except for pears (which contain stone cells), the edible part of most fleshy fruits consists mainly of parenchyma cells, containing only a thin primary cell wall and rich in mesoglea between cells. The metabolic processes of the cell walls during fruit softening mainly involve the solubilization of pectin and the depolymerization of pectin and hemicellulose. Changes in cellulose content are usually not the primary cause of fruit softening in fleshy fruits. Currently, several enzymes are known to participate in the degradation of pectin and hemicellulose in fruit cell walls, including pectin methyl esterase (PME), polygalacturonase (PG), pectin lyase (PL), β-galactosidase (β-Gal), and α-L-arabinofuranase (α-AFase). Studies show that β-Gal enzymes contain the GH35 domain (PF01301) and primarily hydrolyze the non-reducing terminal β-D-galactosyl residues of lactose, glycolipids, proteoglycans, and cell wall polysaccharides containing galactosides, participating in cell wall polysaccharide metabolism. Encoded by the BGAL gene family, β-Gal enzymes play a crucial role in the softening of various fleshy fruits, including persimmons, tomatoes, apples, and peaches. However, the texture changes and key genes involved differ among different fruits. Currently, there are no reports on texture changes in bayberry fruits.

[0004] Therefore, it is urgent to analyze the physiological mechanism of softening of bayberry fruit, identify the key enzymes involved in softening, and screen key genes encoding these enzymes based on transcriptomic and quantitative analysis. This will provide a theoretical basis for regulating the texture of bayberry fruit and for controlling its storage and transportation tolerance through appropriate means. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides a MrBGAL10 gene, product, method, and application for regulating the firmness of bayberry fruit. By screening members of the MrBGAL family encoding β-Gal enzymes at the genomic level, and identifying MrBGAL10 as a key regulatory gene based on transcriptomic and quantitative analysis, overexpression and interference expression vectors were constructed and transformed into bayberries for verification. This process clearly identifies the key gene regulating the firmness of bayberry fruit and its mechanism of action, and verifies the regulatory function of these genes on fruit firmness. This invention provides a theoretical basis and technical means for regulating the texture of bayberry fruit, helps improve the storage and transportation resistance of fresh bayberry fruit, promotes the sustainable development of the bayberry industry, and also provides an important reference for research on the improvement of other fruit quality, demonstrating broad application prospects.

[0006] To achieve the above objectives, the present invention employs the following solution:

[0007] On one hand, the present invention provides a product for regulating the firmness of bayberry fruit, comprising a gene overexpression vector and / or a gene interference expression vector, wherein the gene overexpression vector has a nucleotide sequence as shown in SEQ ID NO.1, and the gene interference expression vector has a nucleotide sequence as shown in SEQ ID NO.3.

[0008] In existing technologies, the unique morphology and structure of the bayberry fruit, with its exposed edible flesh, makes it highly susceptible to mechanical damage. Furthermore, bayberries do not ripen after harvest and must be picked at full maturity. Combined with its predominantly hilly and mountainous location, short ripening period coinciding with the rainy season, and concentrated production area and time, these factors place extremely high demands on the storage and transportation resistance of fresh bayberries. However, the physiological mechanisms of bayberry fruit softening and the key factors regulating this softening have remained unclear. While softening mechanisms have been studied in some other fruits (such as tomatoes and persimmons), revealing the involvement of various enzymes in the degradation of pectin and hemicellulose in fruit cell walls, the texture change processes and key genes involved differ among different fruits and cannot be directly applied to bayberries. Therefore, a thorough analysis of the physiological mechanisms of bayberry fruit softening and the identification of key enzymes and related genes are crucial for improving the storage and transportation resistance of bayberries and developing the bayberry industry.

[0009] Therefore, in this invention, gene overexpression vectors and gene interference expression vectors with specific nucleotide sequences are constructed and first introduced into tomato fruit cells for related heterologous gene expression regulation experiments. The changes in tomato fruit firmness under different treatments are observed, and related physiological and biochemical indicators, such as changes in cell wall composition and detection of related enzyme activities, are measured and analyzed. Transient transformation and functional verification are then performed in bayberries. Through a series of in-depth experimental studies, it was found that these gene overexpression vectors and gene interference expression vectors with specific sequences can significantly affect the expression levels of key enzyme genes related to firmness in bayberry fruits, thereby regulating the metabolic processes of fruit cell walls and ultimately effectively controlling the firmness of bayberry fruits. This provides a practical technical means and theoretical basis for improving the storage and transportation resistance of bayberry fruits, and is expected to be widely applied and promoted in the bayberry industry, driving the bayberry industry towards a more efficient and sustainable development.

[0010] On the other hand, the present invention provides a method for regulating the firmness of bayberry fruit by regulating the expression of the MrBGAL10 gene, thereby regulating the firmness of bayberry fruit; the nucleotide sequence of the MrBGAL10 gene is shown in SEQ ID NO.5.

[0011] In some implementations, analysis of changes in fruit firmness and cell wall composition at three different stages of bayberry fruit development revealed that pectin, abundant in the gum layer of the cell wall, is degraded during fruit ripening, along with significant degradation of hemicellulose, which forms a cross-linked network with cellulose. This disruption of the cell wall's tight structure is likely the primary cause of the continuous softening of the bayberry fruit. β-Gal is involved in the degradation of both pectin and hemicellulose. Furthermore, measurements of enzyme activities related to cell wall degradation showed that β-Gal participates in the degradation of both pectin and hemicellulose, and the change in β-Gal enzyme activity showed the strongest correlation with changes in fruit firmness; the stronger the enzyme activity, the softer the fruit texture. This result was preliminarily validated in three bayberry varieties with different firmness levels: 'Zaojia', 'Dongkui', and 'Shuijing', indicating that β-Gal enzyme is one of the key enzymes in bayberry fruit softening.

[0012] Subsequently, based on the fact that β-Gal enzymes are encoded by the BGAL gene family, members of the MrBGAL family encoding β-Gal enzymes were screened at the genomic level, and their expression at different growth stages of bayberry fruit was analyzed. Combined with transcriptomic and quantitative analysis, MrBGAL10 and MrBGAL11 were finally identified as key genes regulating the softening of bayberry fruit. Their expression changes were closely related to fruit ripening and texture changes, providing important theoretical basis and potential regulatory targets for regulating the softening of bayberry fruit.

[0013] Furthermore, by regulating the expression of the MrBGAL10 gene, the activity of β-galactosidase was regulated, thereby controlling the firmness of bayberry fruit.

[0014] Furthermore, by increasing the expression of the MrBGAL10 gene, the activity of β-galactosidase is increased, thereby reducing the firmness of bayberry fruit; conversely, by inhibiting the expression of the MrBGAL10 gene, the activity of β-galactosidase is reduced, thereby increasing the firmness of bayberry fruit.

[0015] Furthermore, by constructing an overexpression vector for the MrBGAL10 gene and introducing it into bayberry fruit, the expression of the MrBGAL10 gene was increased; by constructing an interference expression vector for the MrBGAL10 gene and introducing it into bayberry fruit, the expression of the MrBGAL10 gene was decreased.

[0016] In some implementations, genetic engineering techniques were employed to investigate the effects of changes in MrBGAL10 and MrBGAL11 gene expression levels on the softening of bayberry fruit. Overexpression vectors containing either MrBGAL10 or MrBGAL11 genes were constructed and introduced into bayberry fruit tissues at different developmental stages using methods such as Agrobacterium-mediated transformation. A series of physiological indicators were then measured in the treated fruits. The results showed that bayberry fruits overexpressing MrBGAL10 or MrBGAL11 genes exhibited significantly increased β-galactosidase activity, a faster rate of fruit firmness reduction, and a significantly earlier fruit softening process.

[0017] These experimental findings strongly validate that the MrBGAL10 and MrBGAL11 genes can indeed affect the softening process of bayberry fruit by regulating β-galactosidase activity, providing a key theoretical basis for the precise regulation of bayberry fruit softening.

[0018] In another aspect, the present invention provides the use of a gene for preparing a reagent that regulates the pectin or hemicellulose content in bayberry fruit to regulate the softening of bayberry fruit, characterized in that the gene includes the MrBGAL10 gene, the nucleotide sequence of the MrBGAL10 gene being shown in SEQ ID NO.5.

[0019] In some embodiments, this invention constructs overexpression vectors for the MrBGAL10 and MrBGAL11 genes and introduces them into bayberry fruit tissue using Agrobacterium-mediated transformation. Culture observation and related tests revealed that regulating the expression of the MrBGAL10 and MrBGAL11 genes can effectively alter the pectin or hemicellulose content in bayberry fruits. When the MrBGAL10 and MrBGAL11 genes are overexpressed, the pectin and hemicellulose content in the fruit decreases significantly; inhibiting gene expression slows the rate of decrease. This leads to the conclusion that the MrBGAL10 and MrBGAL11 genes have a direct regulatory effect on the pectin and hemicellulose content in bayberry fruits. This discovery provides a key theoretical basis for the subsequent development of formulations targeting the improvement of bayberry fruit quality. It is expected that by artificially regulating the expression of these genes, the cell wall structure of bayberry fruits can be optimized, fruit firmness and storage and transportation resistance can be improved, which will have a positive promoting effect on the development of the bayberry industry.

[0020] Furthermore, by regulating the expression of the MrBGAL10 gene, the activity of β-galactosidase is regulated, thereby controlling the content of pectin or hemicellulose in bayberry fruit and thus regulating the softening of bayberry fruit.

[0021] Furthermore, by increasing the expression of the MrBGAL10 gene, the activity of β-galactosidase is increased, thereby reducing the content of pectin or hemicellulose in bayberry fruit and promoting the softening of bayberry fruit.

[0022] In another aspect, the present invention provides a gene overexpression vector for the preparation of a reagent that reduces the pectin or hemicellulose content in bayberry fruit, thereby reducing the firmness of bayberry fruit, wherein the gene overexpression vector has a nucleotide sequence as shown in SEQ ID NO.1.

[0023] In some implementations, the following operations were performed to develop an effective method for regulating the firmness of bayberry fruits and improving their storage and transportation resistance. MrBGAL10 and MrBGAL11 gene overexpression vectors were constructed and introduced into tomato fruit cells. Changes in tomato fruit firmness and cell wall composition were observed. Results showed that the transgenic tomato fruits had reduced firmness and decreased pectin and hemicellulose content, indicating that the overexpression vector could affect fruit cell wall metabolism. Further transient transformation was performed on bayberry fruits, and the firmness and related enzyme activities of the transformed fruits were measured. It was found that the transformed bayberry fruits had significantly reduced firmness, increased β-Gal enzyme activity, and decreased pectin and hemicellulose content. This indicates that the gene overexpression vector can also function in bayberries, enhancing β-Gal enzyme activity and promoting pectin and hemicellulose degradation by increasing the expression of MrBGAL10 and MrBGAL11 genes, thereby reducing fruit firmness. This provides a basis for developing reagents to reduce the firmness of bayberry fruits, which can be used to improve bayberry fruit quality and extend shelf life.

[0024] In another aspect, the present invention provides a gene interference expression vector for the preparation of a reagent to increase the pectin or hemicellulose content in bayberry fruit, thereby increasing the firmness of bayberry fruit, wherein the gene interference expression vector has a nucleotide sequence as shown in SEQ ID NO. 3.

[0025] In some implementations, to explore reverse strategies for regulating the firmness of bayberry fruits, interference expression vectors for the MrBGAL10 and MrBGAL11 genes were constructed. Transformation experiments were first conducted on tomato fruits, and changes in fruit firmness and related indicators were observed. The results showed that tomato fruits with interference expression of this gene exhibited increased firmness, higher pectin and hemicellulose content, and lower β-Gal enzyme activity. Subsequently, transient transformation was performed on bayberry fruits, revealing a significant increase in firmness, decreased β-Gal enzyme activity, and increased pectin and hemicellulose content. This indicates that the gene interference expression vector can effectively inhibit the expression of the MrBGAL10 and MrBGAL11 genes, reduce β-Gal enzyme activity, decrease pectin and hemicellulose degradation, and thus improve fruit firmness. This provides a possibility for preparing reagents to improve the firmness of bayberry fruits, which can help regulate fruit firmness according to needs in bayberry production, such as increasing firmness before harvesting to reduce damage.

[0026] In another aspect, the present invention provides a product for regulating the firmness of bayberry fruit, comprising a gene overexpression vector and / or a gene interference expression vector, wherein the gene overexpression vector has a nucleotide sequence as shown in SEQ ID NO.2, and the gene interference expression vector has a nucleotide sequence as shown in SEQ ID NO.4.

[0027] In another aspect, the present invention provides a method for regulating the firmness of bayberry fruit by regulating the expression of the MrBGAL11 gene, thereby regulating the firmness of bayberry fruit; the nucleotide sequence of the MrBGAL11 gene is shown in SEQ ID NO.6.

[0028] Furthermore, by regulating the expression of the MrBGAL11 gene, the activity of β-galactosidase was regulated, thereby controlling the firmness of bayberry fruit.

[0029] Furthermore, by increasing the expression of the MrBGAL11 gene, the activity of β-galactosidase is increased, thereby reducing the firmness of bayberry fruit; conversely, by inhibiting the expression of the MrBGAL11 gene, the activity of β-galactosidase is reduced, thereby increasing the firmness of bayberry fruit.

[0030] Furthermore, by constructing an overexpression vector for the MrBGAL11 gene and introducing it into bayberry fruit, the expression of the MrBGAL11 gene was increased; by constructing an interference expression vector for the MrBGAL11 gene and introducing it into bayberry fruit, the expression of the MrBGAL11 gene was decreased.

[0031] In another aspect, the present invention provides the use of a gene for preparing a reagent that regulates the pectin or hemicellulose content in bayberry fruit to regulate the softening of bayberry fruit, characterized in that the gene includes the MrBGAL11 gene, the nucleotide sequence of the MrBGAL11 gene being shown in SEQ ID NO.6.

[0032] Furthermore, by regulating the expression of the MrBGAL11 gene, the activity of β-galactosidase is regulated, thereby controlling the content of pectin or hemicellulose in bayberry fruit and thus regulating the softening of bayberry fruit.

[0033] Furthermore, by increasing the expression of the MrBGAL11 gene, the activity of β-galactosidase is increased, thereby reducing the content of pectin or hemicellulose in bayberry fruit and promoting the softening of bayberry fruit.

[0034] In another aspect, the present invention provides a gene overexpression vector for the preparation of a reagent that reduces the pectin or hemicellulose content in bayberry fruit, thereby reducing the firmness of bayberry fruit, characterized in that the gene overexpression vector has a nucleotide sequence as shown in SEQ ID NO.2.

[0035] In another aspect, the present invention provides a gene interference expression vector for the preparation of a reagent to increase the pectin or hemicellulose content in bayberry fruit, thereby increasing the firmness of bayberry fruit, characterized in that the gene interference expression vector has a nucleotide sequence as shown in SEQ ID NO.4.

[0036] The beneficial effects of this invention are as follows:

[0037] 1. This invention provides a MrBGAL10 gene, product, method, and application for regulating the firmness of bayberry fruit. It clarifies that β-Gal enzyme is one of the key enzymes in bayberry fruit softening. Simultaneously, it screens members of the MrBGAL family encoding β-Gal enzyme at the genomic level and identifies MrBGAL10 as a key gene regulating bayberry fruit softening. This provides a preliminary understanding of the molecular mechanisms related to cell wall metabolism during bayberry fruit softening; that is, the gene participates in the degradation of pectin and hemicellulose in the cell wall by regulating β-Gal enzyme activity, thereby affecting fruit softening. This provides crucial clues for a deeper understanding of the physiological and molecular mechanisms of bayberry fruit ripening and softening, filling a gap in previous research on changes in bayberry fruit texture.

[0038] 2. This invention provides a MrBGAL10 gene, product, method, and application for regulating the firmness of bayberry fruit. It clarifies that the MrBGAL10 gene has a novel use in regulating the fruit softening process and related physiological indicators. Its expression level changes are closely related to fruit ripening and texture changes, providing an important theoretical basis for further research on the function of these genes in fruit development and helping to expand our understanding of the regulatory network of plant fruit development.

[0039] 3. This invention provides a novel method for regulating the firmness of bayberry fruits, which can be achieved by regulating the expression of the MrBGAL10 gene. Overexpression of this gene can increase β-Gal enzyme activity, thereby promoting the softening of bayberry fruits; conversely, inhibiting its expression can delay fruit softening. This provides an effective technical means for the precise regulation of bayberry fruit texture, allowing for reasonable intervention in the fruit softening process according to actual needs, such as extending shelf life and improving transportation performance. It provides a practical solution for improving the storage and transportation resistance of fresh bayberries. By regulating the expression of key genes, maintaining suitable fruit firmness reduces mechanical damage during harvesting, transportation, and storage, lowers the risk of fruit decay and spoilage, effectively extends the shelf life and sales radius of bayberries, and is expected to significantly improve the economic benefits of the bayberry industry and promote its sustainable development.

[0040] 4. This invention provides a novel application of the MrBGAL10 gene in the preparation of a reagent that regulates the pectin or hemicellulose content in bayberry fruit, thereby controlling the softening of bayberry fruit. This provides an innovative direction for the development of novel fruit quality improvement agents and is expected to trigger more research and application exploration in the field of fruit quality regulation. It also lays the foundation for the subsequent development of other functionally related products and has good application prospects.

[0041] 5. This invention analyzes the softening mechanism of bayberry fruit from a physiological perspective and screens out key enzymes and their encoding genes involved in fruit softening. Through a stable tomato transformation system and a transient bayberry fruit transformation system, it is verified that MrBGAL10 positively regulates the fruit softening process by encoding β-Gal enzymes. The results of this invention provide a theoretical basis for the rational regulation of bayberry fruit softening and a scientific basis for improving the storage and transportation tolerance of fresh bayberry fruit through artificial means. Attached Figure Description

[0042] Figure 1 A shows the appearance of bayberry fruits at different growth stages (enlargement stage UR, color change stage MR, and maturity stage FR).

[0043] Figure 1 B is a graph showing the trend of hardness changes during the ripening process of bayberry fruit.

[0044] Figure 1C represents the trend of cell wall material (CWM) content changes during the ripening process of bayberry fruit.

[0045] Figure 1 D is a graph showing the trend of pectin content changes during the ripening process of bayberry fruit.

[0046] Figure 1 E is a graph showing the trend of hemicellulose content changes during the ripening process of bayberry fruit.

[0047] Figure 2 Figure A shows the changes in pectin methyl esterase (PME) activity during the ripening process of bayberry fruit.

[0048] Figure 2 Figure B shows the changes in polygalacturonase (PG) activity during the ripening process of bayberry fruit.

[0049] Figure 2 Figure C shows the changes in pectin lyase (PL) activity during the ripening process of bayberry fruit.

[0050] Figure 2 D is a graph showing the changes in β-galactosidase (β-Gal) activity during the ripening process of bayberry fruit.

[0051] Figure 3 A shows the fruit appearance of three bayberry varieties, 'Zaojia', 'Dongkui', and 'Shuijing', at different growth stages.

[0052] Figure 3 B is a comparison chart of the changes in fruit firmness at different growth stages of three bayberry varieties: 'Zaojia', 'Dongkui', and 'Shuijing'.

[0053] Figure 3 C is a comparison chart of β-Gal enzyme activity changes at different growth stages of three bayberry varieties: 'Zaojia', 'Dongkui', and 'Shuijing'.

[0054] Figure 4 This is a diagram showing the physicochemical properties of proteins from the MrBGAL gene family.

[0055] Figure 5 The FPKM changes of 11 MrBGALs genes at three different growth stages (UR, MR, FR) are shown.

[0056] Figure 6 A shows the trend of relative expression of the MrBGAL4 gene at different growth stages (UR, MR, FR) in bayberry fruit.

[0057] Figure 6 B is a graph showing the trend of relative expression of the MrBGAL10 gene at different growth stages (UR, MR, FR) of bayberry fruit.

[0058] Figure 6C represents the trend of relative expression levels of the MrBGAL11 gene at different growth stages (UR, MR, FR) in bayberry fruit.

[0059] Figure 7 A shows the relative expression trends of the MrBGAL10 gene at different growth stages (UR, MR, FR) in the fruit of three bayberry varieties: 'Zaojia', 'Dongkui', and 'Shuijing'.

[0060] Figure 7 B is a graph showing the relative expression trends of the MrBGAL11 gene at different growth stages (UR, MR, FR) in the fruit of the three bayberry varieties 'Zaojia', 'Dongkui', and 'Shuijing'.

[0061] Figure 8 A represents the process of constructing MrBGAL10 and MrBGAL11 gene overexpression vectors and overexpression transgenic tomato lines.

[0062] Figure 8 Figure B shows a comparison of the relative expression levels of the MrBGAL10 gene in transgenic tomatoes overexpressing the MrBGAL10 gene and wild-type tomatoes.

[0063] Figure 8 Figure C shows a comparison of the relative expression levels of the MrBGAL11 gene in transgenic tomatoes overexpressing the MrBGAL11 gene and wild-type tomatoes.

[0064] Figure 8 D is a comparison of the fruit firmness of transgenic tomatoes overexpressing the MrBGAL10 and MrBGAL11 genes and wild-type tomatoes.

[0065] Figure 8 E is a comparison of β-Gal enzyme activity in transgenic tomatoes overexpressing MrBGAL10 and MrBGAL11 genes and wild-type tomatoes.

[0066] Figure 9 A is a schematic diagram of the transient transformation operation of MrBGAL10 and MrBGAL11 gene overexpression vectors in bayberry fruit.

[0067] Figure 9 B is a comparison of the firmness of bayberry fruits transiently transformed by the MrBGAL10 and MrBGAL11 gene overexpression vectors with that of the control fruits.

[0068] Figure 9 C is a comparison of β-Gal enzyme activity in bayberry fruits transiently transformed with MrBGAL10 and MrBGAL11 gene overexpression vectors and control fruits.

[0069] Figure 10A 35S-MrBGAL10-YFP overexpression vector was constructed by combining the MrBGAL10 fragment with the linearized 35S-YFP vector fragment.

[0070] Figure 11 A 35S-MrBGAL11-YFP overexpression vector was constructed by combining the MrBGAL11 fragment with the 35S-YFP linearized vector fragment.

[0071] Figure 12 The pTRV2-MrBGAL10 interference vector was constructed by combining the MrBGAL10 fragment with the TRV2 linearized vector fragment.

[0072] Figure 13 The pTRV2-MrBGAL11 interference vector was constructed by combining the MrBGAL11 fragment with the TRV2 linearized vector fragment. Detailed Implementation

[0073] The present invention will be further described in detail below with reference to the embodiments. It should be noted that the embodiments described below are intended to facilitate the understanding of the present invention and do not limit it in any way.

[0074] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0075] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0076] Example 1: Study on the physiological mechanism of softening of bayberry fruit

[0077] To identify the key factors related to the softening of bayberry fruit and to provide a basis for subsequent screening of key genes, this embodiment explored the changes in physiological indicators during the softening process of bayberry fruit.

[0078] I. Plant Materials and Experimental Treatments

[0079] 1. Plant materials

[0080] Fruit samples were taken from three varieties of waxberry: the firmer variety 'Zaojia', the moderately firm variety 'Dongkui', and the softer variety 'Shuijing', at the fruit enlargement stage (UR), color change stage (MR), and ripening stage (FR), respectively.

[0081] 2. Determination of fruit firmness, cell wall components and enzyme activity in bayberry.

[0082] (1) Determination of the firmness of bayberry fruit

[0083] The firmness of bayberry fruits was determined using a Bollerfeld CT3 texture analyzer. Multiple fruits (e.g., 10) of each variety were measured repeatedly at each time period, and the average value was taken as the firmness value of the fruit for that variety at that time period.

[0084] (2) Measurement of Cell Wall Material (CWM)

[0085] The extraction method for cell wall substances was based on BASANTA MF, PONCE NMA, SALUM ML, et al. Compositional changes in cell wall polysaccharides from five sweet cherry (Prunus avium L.) cultivars during on-tree ripening[J]. Journal of agricultural and food chemistry, 2014, 62(51):12418-12427. The pectin content was determined by spectrophotometry according to the People's Republic of China Agricultural Industry Standard (NY / T 2016—2011), and the hemicellulose content was determined by hydrochloric acid hydrolysis.

[0086] (3) Enzyme activity assay

[0087] The method for determining β-galactosidase activity was referenced from Ban Qiuyan. Functional analysis of β-galactosidase genes during the ripening and softening of persimmon fruit [D]. Northwest A&F University, 2019.; BASANTA MF, PONCE NMA, SALUM ML, et al. Compositional changes in cell wall polysaccharides from five sweet cherry (Prunus avium L.) cultivars during on-tree ripening [J]. Journal of agricultural and food chemistry, 2014, 62(51): 12418-12427.

[0088] The method for determining pectin methyl esterase activity was referenced from BRUMMELL DA, LABAVITCH J M. Effect of antisense suppression of endopolygalacturonase activity on polyuronidemolecular weight in ripening tomato fruit and in fruit homogenates[J]. Plant Physiology, 1997, 115(2):717-725.; Gao Yuanyuan, Cao Hongbo, Li Dongdong, et al. Changes in cell wall-related enzyme activity during the ripening process of peaches with different flesh types[J]. Northern Horticulture, 2023, (09):15-22.

[0089] The pectin lyase activity was determined using a kit from Shanghai Enzyme-Link Biotechnology Co., Ltd. (catalog number: ml076405).

[0090] Polygalacturonase activity was measured using a kit from Shanghai Enzyme-Link Biotechnology Co., Ltd. (catalog number: ml076398).

[0091] II. Experimental Results

[0092] 1. Changes in fruit firmness

[0093] Fruit firmness is an important indicator of fruit texture. Firmness was measured in waxberry fruits collected at different growth stages: the pit hardening stage (UR), the color-changing stage (MR), and the ripening stage (FR). The results are as follows: Figure 1 A- Figure 1 As shown in B, Figure 1 A represents bayberry fruits at different growth stages. The results showed that the firmness of bayberry fruits decreased continuously with ripening, especially during the UR to MR stages, when the fruit texture deteriorated rapidly. Figure 1 B).

[0094] 2. Changes in cell wall substance content

[0095] The results of cell wall material content determination of bayberry fruits at three different growth stages showed that ( Figure 1 C- Figure 1 E) The content of cell wall substances, as well as the contents of pectin and hemicellulose, decreased significantly with fruit ripening, which was highly consistent with the trend of fruit firmness change, indicating that the softening of bayberry fruit is related to the degradation of cell wall components, especially pectin and hemicellulose.

[0096] 3. Changes in enzyme activity

[0097] Based on the close correlation between changes in firmness and pectin and hemicellulose content during the ripening process of bayberry fruit, it is speculated that cell wall degradation-related enzymes may be involved in fruit softening. Currently known enzymes involved in the degradation of pectin and hemicellulose in fruit cell walls include PME, PG, PL, and β-gal. Fruits of the 'Dongkui' variety bayberry were collected at three different stages: the green fruit stage (UR), the color-changing stage (MR), and the ripening stage (FR), and the activities of PME, PG, PL, and β-gal were measured to analyze the changes in the activities of different cell wall degradation-related enzymes at these three stages. The results showed that during the ripening process of bayberry fruit, PME activity first decreased and then increased. Figure 2 A); PG activity first increases and then decreases ( Figure 2 B); PL activity continuously decreases ( Figure 2 C); β-gal activity increases continuously with fruit growth and development. Figure 2 D). Among them, the activity of β-gal enzyme was significantly negatively correlated with the change in the firmness of bayberry fruit, and the activity level of β-gal was higher than that of other hydrolases in all three stages. Therefore, it is preliminarily speculated that β-gal is involved in the softening process of bayberry fruit.

[0098] 4. Verification of the regulatory effect of β-gal enzyme activity on the texture of bayberry fruit

[0099] To preliminarily verify the regulatory effect of β-gal enzyme activity on the texture of bayberry fruit, this example collected fruit from three bayberry varieties with significant differences in fruit firmness: 'Zaojia' (ZJ), 'Dongkui' (DK), and 'Shuijing' (SJ). Fruit firmness and β-gal enzyme activity were measured at different growth stages. The results showed that... Figure 3 A- Figure 3 C) As the firmness of the three bayberry varieties decreased during ripening, the activity of β-gal enzymes continuously increased. During the color-changing and ripening stages, the β-gal activity was lowest in the firmest variety, 'Zaojia,' and highest in the softest variety, 'Shuijing,' with significant differences in activity. This indicates that the softer the fruit, the higher the β-gal activity, and the more actively it participates in cell wall modification and component degradation. This result suggests that β-gal is a key enzyme affecting changes in the texture of bayberry fruits.

[0100] In summary, the softening of bayberry fruit is closely related to cell wall components, especially the degradation of pectin and hemicellulose. β-Gal enzyme activity showed the strongest correlation with changes in fruit firmness; the stronger the enzyme activity, the softer the fruit texture. This preliminarily indicates that β-Gal is a key enzyme affecting the texture changes of bayberry fruit. This lays a solid foundation for further research using molecular biology techniques to study the key genes encoding this enzyme, elucidate its regulatory mechanism in the softening process of bayberry fruit, and further utilize genetic engineering to rationally regulate the softening of bayberry fruit and improve its storage and transportation resistance. Simultaneously, it provides important reference for subsequent research on the potential functions of other members of this gene family in the development and quality formation of bayberry fruit, contributing to a comprehensive and in-depth understanding of the physiological and molecular mechanisms of bayberry fruit ripening and softening, thereby providing scientific theoretical support and effective technical means for the sustainable development of the bayberry industry.

[0101] Example 2: Identification and Transcriptome Analysis of the MrBGAL Family Members of Myrica rubra

[0102] In this embodiment, after identifying β-Gal as the key enzyme for softening bayberry fruit, further investigation was conducted to explore the molecular mechanism regulating bayberry fruit softening. This involved screening key genes encoding this enzyme based on transcriptomic and quantitative analyses. Since β-Gal is encoded by the BGAL gene family, members of the MrBGAL family encoding β-Gal were screened at the genomic level, and their expression at different growth stages of bayberry fruit was analyzed. This preliminarily identified MrBGAL genes potentially related to fruit softening, providing a basis for subsequently determining key genes regulating bayberry fruit softening. The specific steps are as follows:

[0103] I. Identification of MrBGALs Family Members

[0104] Our research group previously used PacBio HiFi and Hi-C sequencing data to perform de novo telomere-to-telomere (T2T) assembly of the *Myrica* cultivar 'Zaojia', obtaining the most complete and highest-quality *Myrica* genome to date. Based on the high-quality (T2T) genome assembled in this invention, hmmscan was used to screen for candidate sequences containing the PF01301 domain in the high-quality *Myrica* T2T genome. This domain is a characteristic domain of β-Gal enzymes. The selected candidate sequences were compared and analyzed with known β-Gal enzyme sequences to ensure the accuracy of the screening results. The isoelectric point, molecular weight, hydrophobicity, and other physicochemical properties of the proteins encoded by the candidate sequences were queried on the online website EXPASY (https: / / www.expasy.org / ), allowing for preliminary characterization of the MrBGALs family members.

[0105] II. Transcriptome Sequencing and Analysis

[0106] Transcriptome sequencing was performed on the cultivar 'Zaojia' of waxberry. Fruit samples were collected at the fruit enlargement (UR), color change (MR), and ripening (FR) stages. Transcriptome sequencing data analysis was commissioned to Biomarker Biotechnology Co., Ltd. Fastp and HISAT2 were used to trim and align the transcriptome data with the 'Zaojia' genome sequencing results. StringTie was used to calculate the FPKM (Fragments Per Kilobase Per Million) value, which was used to statistically analyze the expression of BGAL gene family members identified in the whole waxberry genome; a higher FPKM value indicated a higher expression level. By analyzing the FPKM values ​​of MrBGAL family members in transcriptome data from different growth stages, differentially expressed members were identified, and their expression trends were observed.

[0107] III. Experimental Results

[0108] 1. MrBGALs family member identification results

[0109] Members of the gene family encoding β-gal in the *Myrica rubra* genome were screened, resulting in 15 candidate MrBGALs, which were named MrBGAL1–15 according to their chromosomal locations. The physicochemical properties of the proteins of the *Myrica rubra* β-gal gene family members were obtained and analyzed using the EXPASY online database. Specific analytical results are as follows: Figure 4 As shown in the figure. The results indicate that the amino acid lengths of MrBGALs member proteins range from 687 to 892 aa, and their molecular weights range from 76683.66 to 100233.98 Da, with MrBGAL6 being the largest and MrBGAL2 the smallest. Their isoelectric points range from 5.89 to 8.77, indicating that the β-gal gene family members contain acidic, neutral, and basic proteins. Their average hydrophilicity coefficients range from -0.385 to -0.101, suggesting that most are hydrophilic proteins.

[0110] 2. Transcriptome analysis results

[0111] To further understand the expression of MrBGALs at different growth stages of bayberry and elucidate their potential functions, this study measured and analyzed the UR, MR, and FR transcriptome data of 'Zaojia' bayberry fruit at three growth stages. It was found that 10 out of 15 MrBGALs showed differential expression. The FPKM values ​​in the transcriptome data were analyzed, and the specific results are as follows: Figure 5 As shown, from Figure 5As can be seen, the FPKM of MrBGAL1 and MrBGAL6 increased significantly during the FR period, while the FPKM of MrBGAL8 and MrBGAL14 showed a trend of first increasing and then decreasing. MrBGAL4, MrBGAL5, MrBGAL10, MrBGAL11, and MrBGAL13 showed a continuous increase, while MrBGAL9 and MrBGAL15 showed a gradual decrease. Among them, the FR of MrBGAL1, MrBGAL4, MrBGAL5, MrBGAL6, MrBGAL8, MrBGAL10, and MrBGAL11 was significantly greater than that of UR, with MrBGAL11 being the most significant, followed by MrBGAL10, MrBGAL4, and MrBGAL1. The FPKM values ​​of MrBGAL2, MrBGAL3, MrBGAL7, MrBGAL9, and MrBGAL12 were all 0 at all three time stages, suggesting that these five genes may not be expressed or have extremely low expression levels in bayberry fruit, making them difficult to detect. Based on the transcriptome results, MrBGAL10 and MrBGAL11 may play an important role in the softening of bayberry fruit texture.

[0112] In summary, through the identification of MrBGAL family members and transcriptome analysis, this embodiment preliminarily screened MrBGAL10 and MrBGAL11 as potentially key genes regulating the softening of bayberry fruit. This provides an important foundation and direction for further in-depth research on the specific functions, regulatory mechanisms, and interactions with other genes and physiological processes of these two genes during bayberry fruit softening. It contributes to further elucidating the molecular mechanisms of bayberry fruit softening and provides key gene targets and theoretical basis for the rational regulation of bayberry fruit texture and improving the storage and transportation resistance of fresh bayberry fruit.

[0113] Example 3: Quantitative analysis of MrBGALs and further validation of key genes

[0114] In the aforementioned embodiments, it has been clearly established that β-Gal is a key enzyme affecting the texture changes of bayberry fruit, and MrBGAL10 and MrBGAL11 were preliminarily screened as potentially key genes regulating bayberry fruit softening (the nucleotide sequence of the MrBGAL10 gene is shown in SEQ ID NO.5, and the nucleotide sequence of the MrBGAL11 gene is shown in SEQ ID NO.6). Therefore, in this embodiment, the relative expression levels of some screened MrBGAL genes (MrBGAL4, MrBGAL10, and MrBGAL11) were analyzed using quantitative PCR technology to determine their expression patterns during the softening process of bayberry fruit, further screening and verifying key genes regulating bayberry fruit softening, providing a more accurate basis for subsequent in-depth research on their functions. The specific steps are as follows:

[0115] Total RNA was extracted from the fruit using the CTAB method. cDNA was synthesized via reverse PCR according to the PrimeScript™ RT reagent Kit with gDNA Eraser instructions. The qRT-PCR reaction system was prepared using the Taq SYBR Green qPCR Premix kit instructions. Quantitative reactions were performed on a Roche LighterCycler 96 real-time PCR instrument, with each stage of the reaction repeated in triplicate. (The last sentence appears to be incomplete and possibly refers to a different method.) -△△Ct Relative expression levels were calculated using the method, and significance analysis of differences was performed using SPSS Statistics 21 software.

[0116] Specific results are as follows Figure 6 A- Figure 6 As shown in C, from Figure 6 A- Figure 6 As shown in section C, this embodiment quantitatively analyzed the relative expression levels of the three genes MrBGAL4, MrBGAL10, and MrBGAL11. The results showed that MrBGAL10 and MrBGAL11 had the highest relative expression levels, and the trend of gene expression changes in the three growth stages was consistent with the trend of FPKM changes in the transcriptome data. The relative expression level of the MrBGAL10 gene gradually increased with fruit ripening, which is consistent with the first expression pattern of β-gal in the research background, that is, the enzyme activity gradually increased with fruit ripening. The relative expression level of the MrBGAL11 gene first increased and then slightly decreased with fruit ripening, but its FR relative expression level was the most significantly different from UR (p<0.05). Therefore, it is inferred that MrBGAL10 and MrBGAL11 may be the major genes regulating the softening of bayberry fruit.

[0117] The expression patterns of the MrBGALs gene were analyzed in waxberry varieties 'Zaojia', 'Dongkui', and 'Shuijing', which exhibited significant differences in fruit firmness, at different growth stages: the green fruit stage (UR), the color-changing stage (MR), and the ripening stage (FR). Specific results are as follows: Figure 7 A- Figure 7 As shown in B, Figure 7 A and Figure 7 In section B, from left to right, the stages are: green fruit stage (UR), color-changing stage (MR), and ripening stage (FR) of the waxberry. Figure 7 A- Figure 7As shown in B, the relative expression levels of MrBGAL10 and MrBGAL11 in the mature fruits of the three varieties of bayberry were significantly increased compared with the green fruit stage and the color-changing stage (p<0.05). In 'Shuijing', the expression level of MrBGAL10 showed a continuous upward trend with fruit ripening, while the expression level of MrBGAL11 showed a trend of first rapidly increasing and then slightly decreasing with fruit ripening; in 'Dongkui', the expression levels of both MrBGAL10 and MrBGAL11 showed a continuous upward trend with fruit ripening; in 'Zaojia', the expression level of MrBGAL10 showed a continuous upward trend with fruit ripening, while the expression level of MrBGAL11 showed a trend of first rapidly increasing and then decreasing with fruit ripening. At the same growth stage, the flesh of 'Crystal' was softer than that of 'Dongkui'. The relative expression levels of MrBGAL10 and MrBGAL11 were higher than those of 'Dongkui' during the color-changing and ripening stages, except that the difference was not significant during the green fruit stage. The flesh of 'Zaojia' was firmer than that of both 'Crystal' and 'Dongkui'. The differences in the relative expression levels of MrBGAL10 and MrBGAL11 during the color-changing and ripening stages compared to the green fruit stage were smaller than those of 'Crystal' and 'Dongkui'. Furthermore, the difference between 'Zaojia' and 'Dongkui' was smaller than that between 'Crystal' and 'Crystal'. This indicates that the softer the flesh of the bayberry fruit, the higher the relative expression levels of MrBGAL10 and MrBGAL11, and the higher the β-gal activity.

[0118] Therefore, this embodiment, through quantitative analysis, further verifies the important roles of MrBGAL10 and MrBGAL11 in the softening process of bayberry fruit, and their expression patterns are closely related to fruit ripening and texture changes. These two genes may participate in the degradation of pectin and hemicellulose in the cell wall of bayberry fruit by regulating the activity of β-Gal enzymes, thereby affecting the fruit softening process. This embodiment provides important experimental evidence for further in-depth research on the function and regulatory mechanism of MrBGAL10 and MrBGAL11 genes, as well as their application in improving the quality of bayberry fruit. It helps to further reveal the molecular mechanism of bayberry fruit softening and provides theoretical support and technical guidance for the development of the bayberry industry.

[0119] Example 4: Functional verification of MrBGAL10 and MrBGAL11 in tomatoes and their effect on fruit softening.

[0120] In Example 3, it was further verified that MrBGAL10 and MrBGAL11 are key genes regulating the softening of bayberry fruit, and their expression patterns are closely related to fruit ripening and texture changes. In the existing technology, due to the inability to perform efficient gene transformation and stable genetic manipulation in bayberry, and the lack of a mature research system to accurately analyze gene function, this embodiment further studies the function of these two genes in tomato in order to explore the function of MrBGAL10 and MrBGAL11 genes in the fruit softening process. By constructing overexpression vectors and interference expression vectors and performing stable transformation in tomatoes, the changes in relevant indicators of transgenic tomato fruits were analyzed to clarify the specific mechanism of action of these two genes in regulating fruit softening, providing more reference for the study of bayberry fruit softening mechanism, and also providing a theoretical basis for the improvement of other fruit quality. The specific process is as follows (in this embodiment, the nucleotide sequence of the MrBGAL10 gene overexpression vector is shown in SEQ ID NO.1, the nucleotide sequence of the MrBGAL11 gene overexpression vector is shown in SEQ ID NO.2, the nucleotide sequence of the MrBGAL10 gene interference expression vector is shown in SEQ ID NO.3, and the nucleotide sequence of the MrBGAL11 gene interference expression vector is shown in SEQ ID NO.4):

[0121] I. Construction of overexpression vectors and tomato transgenic

[0122] 1. Construction of overexpression vectors

[0123] like Figure 10 and Figure 11 As shown, MrBGAL10 and MrBGAL11 gene overexpression vectors were constructed using homologous recombination: The 35S-YFP vector plasmid was extracted and linearized by double digestion with restriction endonucleases KpnI and Xbal. A reaction system was established using the CloneExpress-II One Step Cloning Kit to ligate the MrBGAL10 and MrBGAL11 fragments with the linearized vector fragments, obtaining the recombinant products 35S-MrBGAL10-YFP and 35S-MrBGAL11-YFP. These were transformed into competent *E. coli* cells DH5α, plated on LB solid medium (50 mg / L Kan), and incubated upside down at 37°C for 12–16 h. Single colonies were then picked for amplification and PCR detection. Amplified products with matching bands were sent to Zhejiang Youkang Biotechnology Co., Ltd. for sequencing. Positive plasmids with correct sequencing results were then transformed into *Agrobacterium tumefaciens* strain GV3101, and stored at -80°C with an equal volume of 50% sterile glycerol for later use.

[0124] 'MicroTom' tomato seeds were sterilized and sown on 1 / 2 MS medium. Genetic transformation was carried out using the leaf disc method. When the seedling roots grew to 2-3 cm, they were transferred to the substrate, and a small portion of leaves were extracted for PCR testing to ensure that the target gene was effectively expressed in the transgenic tomato. Seeds harvested from the T0 generation were sown again and further screened using RT-qPCR to obtain homozygous T2 generation plants, which were used to determine fruit-related indicators, including fruit firmness, β-Gal enzyme activity, and the relative expression levels of MrBGAL10 and MrBGAL11 genes.

[0125] The fruit firmness was determined as follows: When the transgenic tomato fruits reached maturity, their firmness was measured simultaneously with that of wild-type tomato fruits. The differences in fruit firmness between transgenic and wild-type tomatoes were compared, and the effects of the MrBGAL10 and MrBGAL11 genes on fruit firmness were analyzed. The specific measurement procedure is as described in Example 1.

[0126] The β-Gal enzyme activity was determined as follows: mature transgenic tomato fruit tissue was taken, and the enzyme activity was determined according to the β-Gal enzyme activity assay kit in Example 1. Wild-type tomato fruit was used as a control to compare the differences in enzyme activity between the two, and to determine the regulatory role of MrBGAL10 and MrBGAL11 genes on β-Gal enzyme activity.

[0127] 2. Experimental Results: Verification of Transgenic Function in Tomatoes and Measurement Results of Fruit-Related Indicators

[0128] To verify the functions of MrBGAL10 and MrBGAL11 during fruit ripening, this embodiment constructed overexpression vectors for these two genes respectively during the above process, and obtained overexpression transgenic tomato lines through a stable tomato transformation system. Figure 8 A). Under suitable growing conditions, the firmness of transgenic tomato fruits was measured to be significantly lower than that of wild-type fruits. Figure 8 D), and the firmness of 35S::MrBGAL10 tomato fruit was lower than that of 35S::MrBGAL11 tomato fruit. Subsequently, the activity of β-gal in tomato fruit was measured, and the results showed that ( Figure 8 E) The β-gal activity in transgenic tomato fruits was significantly higher than that in wild-type tomato fruits, and the β-gal activity in tomatoes overexpressing MrBGAL10 was higher than that in tomatoes overexpressing MrBGAL11, consistent with the results of firmness testing. RT-qPCR results also showed that... Figure 8 B. Figure 8C) Overexpression of MrBGAL10 and MrBGAL11 significantly increased the relative expression levels of MrBGAL10 and MrBGAL11 in tomato fruits (P<0.05 compared to wild type), further demonstrating that the target genes were effectively expressed in transgenic tomatoes. The results indicate that overexpression of the MrBGAL10 and MrBGAL11 genes in tomatoes can accelerate the softening process of tomato fruits.

[0129] II. Construction of Interference Expression Vectors and Transgenic Tomato

[0130] 1. Construction of interference expression vector

[0131] like Figure 12 and 13 As shown, MrBGAL10 and MrBGAL11 gene interference expression vectors were constructed using homologous recombination: the TRV2 vector plasmid was extracted and linearized by double digestion with restriction endonucleases KpnI and Xbal. A reaction system was established using the CloneExpress-II One Step Cloning Kit to ligate the MrBGAL10 and MrBGAL11 fragments with the linearized vector fragments, obtaining the recombinant products pTRV2-MrBGAL10 and pTRV2-MrBGAL11. These were transformed into competent E. coli DH5α cells, plated on LB solid medium (50 mg / L Kan), and incubated upside down at 37°C for 12–16 h. Single colonies were then picked for amplification and PCR positive detection. The amplified products with matching bands were sent to Zhejiang Youkang Biotechnology Co., Ltd. for sequencing and transformed into Agrobacterium GV3101 for identification. The same method was used to obtain T2 generation homozygous plants for the determination of fruit-related indicators, including fruit firmness and β-Gal enzyme activity.

[0132] 2. Experimental Results: Verification of Transgenic Function in Tomatoes and Measurement Results of Fruit-Related Indicators

[0133] In this embodiment, MrBGAL10 and MrBGAL11 gene interference expression vectors were constructed, and interference-expressing transgenic tomato lines were obtained through a stable tomato transformation system. Under suitable growth conditions, the firmness of the transgenic tomato fruits was significantly higher than that of the wild-type fruits, and the firmness of pTRV2-MrBGAL10 tomatoes was lower than that of pTRV2-MrBGAL11 tomatoes. Subsequently, the activity of β-gal in the tomato fruits was measured. The results showed that the β-gal activity in the transgenic tomato fruits was significantly lower than that in the wild-type tomatoes, and the β-gal activity in the MrBGAL10 interference expression tomatoes was higher than that in the MrBGAL11 overexpression tomatoes, which was consistent with the firmness measurement results. RT-qPCR results also showed that the relative expression levels of MrBGAL10 and MrBGAL11 in the interference expression tomatoes were significantly reduced (P<0.05 compared with the wild type), further proving that the target gene was effectively expressed in transgenic tomatoes. The results showed that interfering with the expression of the MrBGAL10 and MrBGAL11 genes in tomatoes could reduce the softening process of tomato fruits and significantly increase the firmness of tomato fruits.

[0134] Based on the combined results of overexpression and interference vector experiments in tomatoes, the following conclusions can be drawn: the MrBGAL10 and MrBGAL11 genes play a crucial role in regulating tomato fruit softening. Overexpression of these two genes significantly increases β-Gal enzyme activity, accelerates fruit softening, and reduces fruit firmness; conversely, interference with the expression of these genes reduces β-Gal enzyme activity, delays fruit softening, and increases fruit firmness. This further verifies the close relationship between these two genes and β-Gal enzyme activity, and their mechanism by which they influence fruit firmness and softening by regulating β-Gal enzyme activity and participating in the degradation of pectin and hemicellulose in the cell wall. Since tomatoes and bayberries share certain similarities in fruit development and softening mechanisms, the results of this study provide important reference for a deeper understanding of the functions of the MrBGAL10 and MrBGAL11 genes in bayberry fruit softening. It also provides theoretical support and practical examples for using genetic engineering techniques to regulate the quality of other fruits, contributing to the development of research related to fruit quality improvement. In practical applications, the quality of fruit can be precisely controlled by regulating the expression of these key genes according to different needs of the fruit (such as extending shelf life or promoting ripening and softening).

[0135] Example 5: Transient conversion and functional verification of MrBGAL10 and MrBGAL11 in Myrica rubra

[0136] In Example 4, due to the inability to perform efficient gene transformation and stable genetic manipulation in bayberry, and the lack of a mature research system to accurately analyze gene function, relevant functional verification was conducted in tomato. To further investigate the functions of the MrBGAL10 and MrBGAL11 genes in the fruit softening process in bayberry, this example employed transient transformation technology. The constructed overexpression vector and interference expression vector were introduced into bayberry fruits, respectively. Changes in fruit firmness and β-Gal enzyme activity after transformation were analyzed, thus more directly clarifying the roles of these two genes in regulating bayberry fruit softening. This provides more direct evidence for the study of bayberry fruit softening mechanisms and offers a reference for subsequent bayberry fruit quality improvement. The specific experimental procedure is as follows:

[0137] 1. Instantaneous transformation of bayberries

[0138] (1) Overexpression vector

[0139] Agrobacterium culture containing the constructed 35S::MrBGAL10 and 35S::MrBGAL11 overexpression vectors was cultured to OD. 600 When the OD value was 1.0, the bacterial cells were collected by centrifugation at 5000 rpm for 15 min. The bacterial cells were resuspended by injecting an equal volume of buffer (10 mM MgCl2, 10 mM MES pH=5.7, 150 μMAS) and the OD value was measured to be 0.8-1.0. The prepared bacterial solution was allowed to stand at room temperature for 4 h. The waxberry fruit in the enlargement stage (UR) was immersed in the bacterial solution, vacuum filtered for 30 min, removed and dried, and placed in the dark at room temperature for 12 h. After standing under normal conditions for 36 h, the fruit firmness and enzyme activity index were measured (the measurement process is as described in Examples 1 and 4).

[0140] (2) Interference expression vector

[0141] Similarly, Agrobacterium tumefaciens culture containing the constructed pTRV2-MrBGAL10 and pTRV2-MrBGAL11 interference expression vectors was cultured to OD200. 600 When the concentration is 1.0, the fruit is suspended in a resuspension after centrifugation. The fruit in the UR stage of bayberry expansion is immersed in the bacterial solution, vacuumed for 30 minutes, then removed and the surface moisture is dried. The fruit is then placed in the dark at room temperature for 12 hours. After standing under normal conditions for 36 hours, the fruit firmness and enzyme activity index are measured (the measurement process is as described in Examples 1 and 4).

[0142] 2. Experimental Results: Verification of the Instantaneous Transformation of Waxberry

[0143] (1) Overexpression vector

[0144] The constructed 35S::MrBGAL10 and 35S::MrBGAL11 overexpression vectors, and 35S::empty as a control, were used for transient transformation in green bayberry fruits via vacuum filtration. Figure 9 A). The results show that ( Figure 9 B), the firmness of bayberry fruits transformed with 35S::MrBGAL10 and 35S::MrBGAL11 was significantly lower than that of the control group. Enzyme activity assay results ( Figure 9 C) also showed that after transformation with 35S::MrBGAL10 and 35S::MrBGAL11, the β-gal activity in the bayberry fruit was significantly higher than that in the control group fruit, which was consistent with the results of the firmness test.

[0145] Meanwhile, although there was no significant difference in firmness and β-gal activity between the waxberry fruits transformed by 35S::MrBGAL10 and 35S::MrBGAL11, the firmness of the waxberry fruits transformed by 35S::MrBGAL10 was lower than that transformed by 35S::MrBGAL11, and the β-gal activity of the waxberry fruits transformed by 35S::MrBGAL10 was higher than that transformed by 35S::MrBGAL11. Therefore, based on the results of Example 4, it can be concluded that both MrBGAL10 and MrBGAL11 genes play important roles in regulating the softening process of waxberry fruits, but there may be some differences in their functions. The MrBGAL10 gene may have a relatively stronger effect in promoting the increase of β-gal activity and fruit softening, while the effect of the MrBGAL11 gene is slightly weaker than that of the MrBGAL10 gene. This may suggest that the MrBGAL10 gene occupies a more critical regulatory node in the molecular regulatory network of bayberry fruit softening, or that its regulation of β-gal activity is more direct and efficient, thus having a greater impact on fruit firmness. Further research into the molecular mechanisms of this functional difference will contribute to a deeper understanding of the regulatory process of bayberry fruit softening, providing a more precise theoretical basis for the accurate regulation of bayberry fruit quality. In practical applications, if a more significant acceleration of bayberry fruit softening is desired, the MrBGAL10 gene should be prioritized for manipulation; if a certain degree of fruit softening regulation is required while maintaining relatively high fruit firmness, the MrBGAL11 gene may be a more suitable choice. This provides a valuable reference for subsequent use of genetic engineering techniques to improve bayberry fruit quality, helping to select appropriate genes for targeted regulation based on different production needs, such as extending the harvest period or promoting post-ripening softening, thereby improving the marketability and economic benefits of bayberry fruit.

[0146] (2) Interference expression vector

[0147] The constructed interference expression vectors pTRV2-MrBGAL10 and pTRV2-MrBGAL11, along with pTRV2-empty as a control, were used for transient transformation of unripe bayberry fruits via vacuum filtration. The results showed that the firmness of bayberry fruits transformed with pTRV2-MrBGAL10 and pTRV2-MrBGAL11 was significantly higher than that of the control group. Enzyme activity assays also indicated that the β-gal activity in bayberry fruits transformed with pTRV2-MrBGAL10 and pTRV2-MrBGAL11 was significantly lower than that in the control group, consistent with the firmness measurement results.

[0148] The above results demonstrate that the MrBGAL10 and MrBGAL11 genes have a clear function in regulating the softening of bayberry fruit, and the results of overexpression and interference expression experiments corroborate each other. Overexpression of both genes reduced fruit firmness and increased β-gal activity, with MrBGAL10 showing a relatively stronger effect; interference expression of both genes increased fruit firmness and decreased β-gal activity. This further confirms their mechanism of influencing fruit softening by regulating β-gal activity, and also indicates that they are positive regulators in the softening process of bayberry fruit. In bayberry fruit, regulating the expression of these two genes can effectively alter the softening process and quality characteristics. This study not only provides direct experimental evidence for a deeper understanding of the softening mechanism of bayberry fruit, but also provides a practical and feasible technical approach for subsequent precise regulation of bayberry fruit quality using genetic engineering techniques. For example, in actual production, to extend the shelf life of bayberries or improve their transport performance, a strategy of interfering with the expression of these two genes could be considered; while to promote the ripening and softening of bayberry fruit to meet specific processing or consumption needs, overexpression of these two genes could be attempted. Furthermore, the results of this study provide an important reference example for studying the softening regulation mechanisms of other similar fruits, contributing to the advancement of research in the entire field of fruit quality regulation and playing a significant role in improving the economic value and industrial competitiveness of fruits.

[0149] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

[0150] sequence list

[0151] SEQ ID NO.1 (nucleotide sequence of the MrBGAL10 gene overexpression vector):

[0152]

[0153] SEQ ID NO.2 (nucleotide sequence of the MrBGAL11 gene overexpression vector):

[0154]

[0155] SEQ ID NO.3 (nucleotide sequence of the MrBGAL10 gene interference expression vector):

[0156]

[0157] SEQ ID NO.4 (nucleotide sequence of the MrBGAL11 gene interference expression vector):

[0158]

[0159] SEQ ID NO.5 (nucleotide sequence of the MrBGAL10 gene):

[0160]

[0161] SEQ ID NO.6 (nucleotide sequence of the MrBGAL11 gene):

[0162]

Claims

1. A product for regulating the firmness of bayberry fruits, characterized in that, The invention includes gene overexpression vectors and / or gene interference expression vectors, wherein the gene overexpression vector has a nucleotide sequence as shown in SEQ ID NO.1 and the gene interference expression vector has a nucleotide sequence as shown in SEQ ID NO.

3.

2. A method for regulating the firmness of bayberry fruit, characterized in that, The firmness of bayberry fruit is regulated by controlling the expression of the MrBGAL10 gene; the nucleotide sequence of the MrBGAL10 gene is shown in SEQ ID NO.

5.

3. The method as described in claim 2, characterized in that, By regulating the expression of the MrBGAL10 gene, the activity of β-galactosidase is regulated, thereby controlling the firmness of bayberry fruit.

4. The method as described in claim 3, characterized in that, By increasing the expression of the MrBGAL10 gene, the activity of β-galactosidase is increased, thereby reducing the firmness of bayberry fruit; conversely, by inhibiting the expression of the MrBGAL10 gene, the activity of β-galactosidase is decreased, thereby increasing the firmness of bayberry fruit.

5. The method as described in claim 4, characterized in that, By constructing an overexpression vector for the MrBGAL10 gene and introducing it into bayberry fruit, the expression of the MrBGAL10 gene was increased; by constructing an interference expression vector for the MrBGAL10 gene and introducing it into bayberry fruit, the expression of the MrBGAL10 gene was decreased.

6. The use of a gene in preparing a reagent to regulate the softening of bayberry fruit by controlling the pectin or hemicellulose content in the fruit, characterized in that, The gene includes the MrBGAL10 gene, the nucleotide sequence of which is shown in SEQ ID NO.

5.

7. The use as described in claim 6, characterized in that, By regulating the expression of the MrBGAL10 gene, the activity of β-galactosidase is regulated, and the content of pectin or hemicellulose in bayberry fruit is controlled, thereby regulating the softening of bayberry fruit.

8. The use as described in claim 7, characterized in that, By increasing the expression of the MrBGAL10 gene and enhancing β-galactosidase activity, the pectin or hemicellulose content in bayberry fruit can be reduced, thereby promoting the softening of bayberry fruit.

9. The use of a gene overexpression vector in preparing a reagent to reduce the pectin or hemicellulose content in bayberry fruit, thereby reducing the firmness of bayberry fruit, characterized in that, The gene overexpression vector has a nucleotide sequence as shown in SEQ ID NO.

1.

10. The use of a gene interference expression vector in preparing a reagent to increase the pectin or hemicellulose content in bayberry fruit, thereby increasing the firmness of bayberry fruit, characterized in that, The gene interference expression vector has a nucleotide sequence as shown in SEQ ID NO.3.

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