Function and application of transcription factor PsJUB1.1 in rain resistance and crack resistance of plum fruits

Abstract

The invention belongs to the field of plant genetic engineering, and particularly relates to application of an NAC transcription factor PsJUB1.1 in rain resistance and crack resistance of plum fruits. Two NAC transcriptional regulation factors which participate in regulation of fruit rain splitting and have large protein sequence difference are screened by utilizing peel transcriptome data in a Wushan crisp plum fruit rain splitting process. The PsJUB1.1 specifically responds to rainfall and cracking signals in the fruit rain cracking occurrence process, and expression is up-regulated. The PsJUB1.1 is positioned in a cell nucleus and has transcriptional self-activation activity. The PsJUB1.1 provided by the invention actively participates in rain-resistant and crack-resistant regulation and control of mature fruits. In the rain crack resisting process of the fruits, the induced expression PsJUB1.1 enhances the rain resistance and crack resistance of the fruits by influencing the arrangement of epicarp cells and cuticle deposition. The research result expands the functional cognition of the plant JUB1 gene, and has potential application value in the aspect of screening and cultivating new rain-resistant and crack-resistant strains.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of plant genetic engineering, and particularly relates to application of a NAC transcription factor PsJUB1.1 in rain resistance and crack resistance of plum fruit. BACKGROUND

[0002] In recent years, new varieties such as 'Wushan Crispy Plum', 'Yubei Wai-Mouth Plum', 'Wanzhou Powder-Da Crispy Plum' and 'Late Frost Crispy Plum' have been selected in Chongqing. These varieties are favored by the market and consumers due to their moderate sweet and sour taste and excellent quality (Fang et al., 2020; Xiong et al., 2020). At the same time, thanks to the excellent natural ecological conditions in Chongqing, the plum industry has become a characteristic pillar industry that effectively connects income increase and the promotion of rural comprehensive revitalization in the Three Gorges Reservoir area. However, the maturation of plum fruit in the reservoir area coincides with the period of rain and heat, and in recent years, the extreme climate has become increasingly severe. The serious pre-harvest fruit rain cracking problem has become a bottleneck restricting the sustainable development of the plum industry in Chongqing (Zhou et al., 2024).

[0003] NAC class protein JUB1 plays an important role in regulating plant development and stress resistance. Overexpression of NAC transcription factor AtJUB1 can improve the drought tolerance of tomato and Arabidopsis and delay the aging of Arabidopsis (Wu et al., 2012; Ebrahimian et al., 2017; Thirumalaikumar et al., 2018; Zhang et al., 2022). Banana JUB1, namely MaNAC42, can improve the drought tolerance and salt tolerance of banana (Taka et al., 2017). AtJUB1 directly inhibits the expression of AtGA3ox1 and AtDWF4 genes involved in the synthesis of plant hormones GA and BR, resulting in delayed flowering, dwarfing and male sterility in Arabidopsis, and improving the resistance of Arabidopsis plants (Shahnejat-Bushehri et al., 2016). The role of AtJUB1 in tomato is conservative, and it can regulate the expression of SlDWF4 and SlDELLA genes related to the synthesis of GA and BR. Studies have shown that overexpression of AtJUB1 in wild-type tomato (S. lycopersicum cv. Moneymaker) can cause delayed flowering and color change in tomato, smaller leaves, and dwarfing of tomato plants (Shahnejat-Bushehri et al., 2017).

[0004] At present, the existence of JUB1 in plum and the role of JUB1 in the process of rain resistance and crack resistance of plum fruit are not clear. SUMMARY

[0005] The present application obtains the transcriptional regulation genes differentially expressed in the rain cracking process of 'Wushan Crisp Plum' fruit by analyzing the pericarp transcriptome data in the rain cracking process of 'Wushan Crisp Plum' fruit. Among the 7 transcriptional regulation factors screened, two NAC transcriptional regulation factors, namely PsJUB1.1 and PsJUB1.2, are contained. The whole genome identification result of plum JUB1 shows that there are 3 JUB1s in the plum genome, and the 3 identified JUB1s have great differences in protein sequences. PsJUB1.1 and PsJUB1.2 can be continuously expressed up-regulated in response to rain and cracking signals, while PsJUB1.3 there is no significant difference in the expression in the fruit rain cracking process. In view of PsJUB1.1 and PsJUB1.2 the great differences in protein sequences, it is speculated that the two NAC transcriptional regulation factors perform different functions in the regulation of fruit rain cracking. The present application studies the gene characteristics of PsJUB1.1 and the functions thereof in the regulation of fruit rain cracking, and lays a theoretical and gene resource foundation for plum fruit rain resistance and cracking resistance molecular breeding.

[0006] Specifically, the present application aims to provide the following aspects: 1) In a first aspect, the present application provides 7 transcriptional regulation factors specifically responding to rain and cracking signals in the rain cracking process of plum fruit, and provides PsJUB1s expression analysis of response to fruit rain cracking; 2) In a second aspect, the present application provides a characteristic analysis of a NAC transcriptional regulation factor PsJUB1.1 specifically responding to rain and cracking signals, and the characteristics include PsJUB1.1 expression change in plum fruit rain cracking, PsJUB1.1 tissue expression characteristics of subcellular localization and transcriptional self-activation activity of PsJUB1.1; PsJUB1.1 3) In a third aspect, the present application provides a functional analysis of a NAC transcriptional regulation factor PsJUB1.1 specifically responding to rain and cracking signals, and the functional analysis includes the influence of on the rain resistance and cracking resistance of plum fruit and the influence on the pericarp structure; PsJUB1.1 4) In a fourth aspect, the present application provides a method for improving the cracking resistance of tomato fruit, which uses tomato 'Micro-Tom' as a stable transformation material, uses promoter E8 to mediate the specific expression of mature tomato fruit, and stably overexpresses , which significantly improves the rain resistance and cracking resistance of tomato fruit.

[0007] The beneficial effects of the present application include: 1) The present application provides 3 plum JUB1 protein sequences with great differences, only PsJUB1.1 and PsJUB1.2It can respond to rainfall and cracking signals and participate in the regulation of fruit rain cracking. PsJUB1.1 is located in the cell nucleus and has transcriptional autoactivation activity.

[0008] 2. The PsJUB1.1 provided by this invention actively participates in the regulation of rain resistance and cracking resistance in mature fruits. During the process of fruit resisting rain cracking, the induced expression of… PsJUB1.1 By influencing the arrangement of exocarp cells and cuticle deposition, the study enhances the fruit's resistance to rain and cracking. The findings expand our understanding of plant... JUB1 Understanding gene function has potential applications in screening and breeding new rain-resistant and crack-resistant strains. Attached Figure Description

[0009] Figure 1 The results show the screening of key genes regulating rain-induced fruit splitting in plum; where a represents differentially expressed transcriptional regulatory genes during the rain-induced fruit splitting process of 'Wushan Crisp Plum'; b is the phylogenetic tree of JUB1, where At represents Arabidopsis thaliana, Sl represents tomato, Musa represents banana, and Ps represents plum; c is protein sequence alignment; d is the similarity matrix of plum JUB1; e is... PsJUB1s Transcriptome data during the fruit rain-cracking process.

[0010] Figure 2 The results of the characterization analysis of the transcriptional regulator PsJUB1.1; where a is... PsJUB1.1 Expression changes in plum fruit cracking during rain; b is... PsJUB1.1 c represents the tissue expression characteristics of PsJUB1.1; d represents the subcellular localization of PsJUB1.1; and d represents the transcriptional autoactivation activity analysis of PsJUB1.1.

[0011] Figure 3 The results of functional analysis of PsJUB1.1 in rain resistance and crack prevention of plum fruit are shown; where 'a' represents transient overexpression in plum pericarp. PsJUB1.1 Quantitative analysis was performed afterward; b represents transient overexpression in plum pericarp. PsJUB1.1 Fruits treated with rain-induced fruit cracking on days 0 and 4, where DAT represents the number of days after the rain-induced fruit cracking simulation treatment, and cracked areas are circled in white; c represents transient overexpression of plum pericarp. PsJUB1.1 Changes in the fruit cracking index during the post-rain cracking simulation treatment; d represents the transient overexpression of plum pericarp. PsJUB1.1 Subsequent optical microscopy observations showed that paraffin sections of plum pericarp were stained with toluidine blue O, with pericarp transfected with no vector serving as a control; e represents transient overexpression in plum pericarp. PsJUB1.1 Post-pericarp thickness analysis; f represents transient overexpression of plum pericarp. PsJUB1.1 Analysis of keratinization degree of postepidermal cells; g represents transient overexpression in plum pericarp. PsJUB1.1 Post-stratum corneum infiltration thickness analysis; h represents the transient silencing time of plum pericarp. PsJUB1.1 Quantitative analysis following; i represents transient silencing of plum pericarp. PsJUB1.1Fruits subjected to rain-induced cracking simulation on days 0 and 4, where DAT represents the number of days after the simulation, and cracked areas are circled in white; j represents the momentary silencing of plum pericarp. PsJUB1.1 Changes in the fruit cracking index during the post-rain cracking simulation treatment; k represents the instantaneous silencing of plum pericarp. PsJUB1.1 Subsequent optical microscopic observations included staining plum pericarp paraffin sections with toluidine blue O, using untreated pericarp as a control; l represents transient silencing of plum pericarp. PsJUB1.1 Post-pericarp thickness analysis; m represents the instantaneous silencing of the plum pericarp. PsJUB1.1 Analysis of the degree of keratinization of posterior epidermal cells; n represents the transient silencing of plum pericarp. PsJUB1.1 Analysis of the thickness of the posterior stratum corneum.

[0012] Figure 4 This refers to the application of PsJUB1.1 in the rain resistance and crack prevention of tomato fruits; where a represents the PCR detection of generation T0. PsJUB1.1 -E8 genetically modified tomatoes PsJUB1.1 The expression; b is PsJUB1.1 -E8 genetically modified tomato strains PsJUB1.1 The expression level and relative expression level were compared with wild-type tomato WT as the control; c was... PsJUB1.1 -Microscopic observation of the exocarp of E8 transgenic tomatoes and wild-type tomatoes. Paraffin sections of T2 generation tomatoes at the green-ripe stage and 7 days after color change were stained with toluidine blue O, with wild-type tomatoes serving as a control; d represents... PsJUB1.1 -Analysis of pericarp thickness in E8 transgenic tomatoes and wild tomatoes at two developmental stages: MG and BR7; e represents... PsJUB1.1 Analysis of the degree of keratinization in epidermal cells of E8 transgenic tomatoes and wild tomatoes at two growth stages (MG and BR7); f is PsJUB1.1 -Analysis of cuticle penetration thickness at two stages: MG and BR7 in E8 transgenic tomatoes and wild-type tomatoes; g is PsJUB1.1 - The degree of toluidine blue O staining in E8 transgenic tomatoes and wild-type tomatoes, with a staining time of 24 hours; h represents the change in fruit cracking index during the simulated rain cracking treatment; i represents the fruit cracking index on days 0 and 5 of the simulated rain cracking treatment. PsJUB1.1 -E8 genetically modified tomato fruit, DAT indicates the number of days after the fruit was subjected to rain cracking simulation treatment, and the cracked areas are circled in white.

[0013] Figure 5 A technical roadmap for the function and application of transcription factor PsJUB1.1 in rain resistance and crack prevention of plum fruit. Detailed Implementation

[0014] The following detailed description of various example embodiments of the application should not be considered to be limiting of the application, but rather a description of certain example aspects, features and embodiments of the application. Unless otherwise defined, technical terms used in the examples have the same meaning as commonly understood by one of ordinary skill in the art.

[0015] Plant materials used in the examples: The 5-year-old self-rooted Prunus simonii seedlings used in the study were obtained from the Gujia Village of Quzi Township, Wushan County, Chongqing. The pericarp samples at different stages of rain-induced cracking were obtained by artificial rainfall treatment of P. simonii in the field. “CK” represents the pericarp without water spraying treatment, “WCK” represents the pericarp without cracking after water spraying, “WSCK” represents the pericarp on the opposite side of the slightly cracked region after water spraying, “WSC” represents the pericarp with slight cracking after water spraying, “WMCK” represents the pericarp on the opposite side of the moderately cracked region after water spraying, and “WMC” represents the pericarp with moderate cracking after water spraying (Zhou et al., 2024). The Prunus mira fruits used for transient expression were obtained from the Chuanfei Strawberry Orchard in Chongqing. The tomato variety used for genetic transformation was ‘Micro-Tom’ (Solanum lycopersicum) (Li et al., 2019), and the Nicotiana benthamiana used for subcellular localization was N. benthamiana (Li et al., 2019). Solanum lycopersicum Nicotiana benthamiana

[0016] Strains and vectors used in the examples: The E. coli strain used for constructing the vectors was DH5α, and the Agrobacterium strain was GV3101. Both the competent cells were prepared in the laboratory. The Cambia-2302 vector was used for subcellular localization experiments, and OsGhd7-mCherry was used as the nuclear localization marker. The Y2H Gold yeast strain was used for the transcription self-activation experiment, and the vectors were pGADT7 and pGBKT7. The overexpression vector for transient transformation was pNmGFP, and the silencing vector was pNmGFP-RNAi. The E8 vector based on the modification of Cambia-2300 was used for tomato genetic transformation.

[0017] The primer sequence information designed in the examples of the application is shown in Table 1.

[0018] Table 1 Primer sequence information Figure 5 The technical route map showing the function and application of the transcription factor PsJUB1.1 in rain-resistant cracking of plum fruits is shown. The application will be described in detail below in conjunction with Figure 5 and the examples.

[0019] Example 1 Screening of key genes for regulating rain-induced cracking of plum fruits ​​1. Screening of transcriptional regulatory genes differentially expressed in the rain cracking process of plum fruit: Among the transcriptome sequencing results of the fruit peel in the rain cracking process of ‘Wushan Crispy Plum’, the peel of the fruit that did not crack after being sprayed with water and the peel of the fruit that did not undergo water spraying, the peel of the fruit that cracked slightly after being sprayed with water and the peel of the fruit that cracked slightly on the opposite side of the sprayed area, the peel of the fruit that cracked moderately after being sprayed with water and the peel of the fruit that cracked moderately on the opposite side of the sprayed area (“WCK vs. CK”, “WSC vs. WSCK” and “WMC vs. WMCK”), the differentially expressed genes with a fold change greater than 2 or less than 0.5 were obtained, which may be involved in the regulation of rain cracking of plum fruit (Zhou et al., 2024). According to the functional annotation of the differential genes, it was found that among the 157 differentially expressed genes obtained, only 7 transcriptional regulatory factors existed, and among the 7 transcriptional regulatory factors, 2 NAC transcriptional regulatory factors, namely PsJUB1.1 and PsJUB1.2, were included (Zhou et al., 2024). Figure 1 a). JUB1 plays an important role in regulating plant development and stress resistance, so it is speculated that PsJUB1.1 and PsJUB1.2 are involved in the regulation of fruit rain cracking.

[0020] 2. Genome-wide identification and expression analysis of plum: JUB1 The protein sequences of Arabidopsis thaliana, Solanum lycopersicum and Musa JUB1 were obtained from the NCBI database (https: / / www.ncbi.nlm.nih.gov). Using the Blast tool of TBtools, the protein sequence of Arabidopsis thaliana JUB1 was used as a reference to align the ‘Wushan Crispy Plum’ genome (https: / / www.rosaceae.org / Analysis / 16031258), and the protein sequence information of 5 genes with high similarity was obtained. The MEGA software (Version 11) was used to draw the phylogenetic tree, and then the ITOL online website (https: / / itol.embl.de) was used to complete the beautification of the phylogenetic tree. Phylogenetic analysis showed that Chr5.1702 and Chr4.888 had high homology with SlJUB1 and AtJUB1, Chr4.1200 had high homology with Musa JUB1, and Chr5.2435 and Chr1.2695 did not cluster with JUB1 in existing research (Zhou et al., 2024). Figure 1b). The protein sequences in the phylogenetic analysis were analyzed visually using the DNAMAN software (Version 9). Sequence analysis was performed using JUB1 in Arabidopsis thaliana, Solanum lycopersicum and Musa acuminate (Wu et al., 2012; Thirumalaikumar et al., 2017; Yan et al., 2021), and the N-terminal of the candidate protein sequence has the conserved A, B, C, D, E domains, and the C domain has a nuclear localization signal NLS, belonging to the NAC transcription factor. Among them, Chr5.2435 and Chr1.2695 have obvious differences in the TRD region sequence between JUB1 proteins, while Chr5.1702, Chr4.888 and Chr4.1200 have smaller differences in the TRD region sequence of JUB1 Figure 1 c). Based on the above analysis, it can be concluded that there are three JUB1s in the plum genome, namely Chr5.1702, Chr4.888 and Chr4.1200. According to the protein sequence similarity, the three genes are named PsJUB1.1 (PsJUB1.1), PsJUB1.2 (PsJUB1.2) and PsJUB1.3 (PsJUB1.3) respectively. Protein sequence similarity analysis shows that the protein sequence similarity of PsJUB1.1 and PsJUB1.2 is 55.52%, the protein sequence similarity of PsJUB1.1 and PsJUB1.3 is 52.70%, and the protein sequence similarity of PsJUB1.2 and PsJUB1.3 is 50.34%. The larger sequence difference indicates that the three PsJUB1s may have obvious functional differentiation Figure 1 d). PsJUB1s The analysis of the transcriptome sequencing results in the fruit rain cracking process shows that PsJUB1.1 and PsJUB1.2 can continuously respond to rain and cracking signals to express up-regulation, and PsJUB1.3 does not respond to rain and cracking signals Figure 1 e). The characteristics and the role of PsJUB1.1 in plum fruit rain cracking are further studied in the present application.

[0021] Example 2 Characteristic analysis of transcriptional regulator PsJUB1.1 1、 PsJUB1.1Expression changes in plum fruit during rain cracking: RNA was extracted from samples using the RNAprep Pure Polysaccharide-Polyphenol Plant Total RNA Extraction Kit (DP441, TIANGEN), and then reversed using the PrimeScript™ RT reagent Kit with gDNA Eraser (RR047A, Takara). RT-qPCR analysis was performed using the Novo Start® SYBR qPCR Super Mix plus kit (E096-01A, novoprotein). The usage method was followed according to the manufacturer's instructions. RT-qPCR validation showed that as the degree of fruit rain cracking increased, PsJUB1.1 The level of expression gradually improved ( Figure 2 a). This indicates that PsJUB1.1 may play an important regulatory role in plum fruit rain cracking.

[0022] 2. PsJUB1.1 Tissue expression characteristics analysis: Using the roots, stems, leaves, flowers, young fruits, mature pericarps and pulp of 'Wushan Crisp Plum', we analyzed... PsJUB1.1 The tissue expression characteristics. Quantitative results showed that... PsJUB1.1 High expression levels were observed in roots, leaves, and flowers. Figure 2 b). However, in both young and mature fruits, PsJUB1.1 The expression levels of these substances were all very low, and the maturation period was also very short. PsJUB1.1 The expression level in the peel is higher than that in the pulp. This indicates that... PsJUB1.1 It may not be related to fruit development, but rather to a key gene in the specific response of the mature fruit peel to rainwater-regulated cracking.

[0023] 3. Subcellular localization of PsJUB1.1: based on correct sequencing... PsJUB1.1 Using the T-plasmid as a template, a gene fragment with homologous arms was amplified, cloned and ligated into a double-digested vector using a one-step method, and a GFP fluorescent tag vector was constructed. PsJUB1.1 -GFP. The one-step cloning reagent was the ClonExpress II One Step Cloning Kit (C112-02, Vazyme), and the method was as per the manufacturer's instructions. The empty GFP vector and... PsJUB1.1-GFP transformed Agrobacterium, the obtained resuspended bacteria were mixed with OsGhd7-mCherry resuspended bacteria located in the nucleus, and then injected into N. benthamiana leaves (Luo et al., 2024). After 24 hours of dark culture and 24 hours of light culture, the subcellular localization of PsJUB1.1 was observed by laser confocal microscopy imaging. The OsGhd7-mCherry with red fluorescence was used as a nuclear localization marker. The tobacco leaves transformed with GFP empty vector had green fluorescence in the nucleus and cell membrane, and the tobacco leaves transformed with PsJUB1.1 GFP had green fluorescence coinciding with the red fluorescence of the nuclear marker, indicating that PsJUB1.1 was located in the nucleus (Fig. 2c). Figure 2 c).

[0024] 4. Analysis of the transcriptional self-activation activity of PsJUB1.1: To study the transcriptional self-activation activity of PsJUB1.1, the coding sequence of PsJUB1.1 was amplified by PCR and connected to the pGBKT7 vector. PsJUB1.1 The transcriptional self-activation activity of PsJUB1.1 was analyzed using the yeast two-hybrid system. After the successful construction of the pGBKT7- PsJUB1.1 vector, the pGADT7 empty vector was co-transformed into yeast (Y2H-Gold). The positive transformants were selected using the selective medium SD / -Leu / -Trp, and then 10 randomly selected transformant colonies were inoculated into the auxotrophic medium SD / -Leu / -Trp / -His / -Ade, and the growth of the yeast was observed. The yeast in each group grew well in the two-lacking medium (SD / -Leu / -Trp), and the yeast that was transformed with the pGADT7 empty vector and the pGBKT7- PsJUB1.1 grew well in the four-lacking medium (SD / -Leu / -Trp / -His / -Ade) (Fig. 2d). Figure 2 d). The results showed that the protein encoded by PsJUB1.1 had transcriptional self-activation activity. Since the A subdomain of the N-terminal of the NAC transcription factor can participate in the formation of functional dimers, and the C-terminal transcriptional regulatory domain has the ability to bind other proteins (Han et al., 2023). Subsequently, the gene fragment encoding the N-terminal 178 amino acids of PsJUB1.1 containing the NAC domain and the gene fragment encoding the C-terminal 179-302 aa containing the TRD domain were constructed into pGBKT7- PsJUB1.1 -N and pGBKT7- PsJUB1.1 -C vectors, and the yeast was transformed. The results showed that the NAC domain of PsJUB1.1 had no self-activation activity, while the TRD domain had self-activation activity (Fig. 2d). Figure 2 d).

[0025] Example 3 Functional analysis of PsJUB1.1 in rain resistance and crack resistance of plum fruit 1. Transient overexpressionPsJUB1.1 Effects of overexpression vectors on plum fruit resistance to rain cracking PsJUB1.1 After transformation of Agrobacterium with GFP, Agrobacterium resuspensions were prepared separately from the empty GFP vector. Transient expression was performed on immature green plum fruits in the pericarp region. One to two pericarp regions near the equator were injected into each fruit, with 1 mL of bacterial suspension injected around each region. The injection sites were marked with a black marker. After transient overexpression, the green plums were bagged to prevent rain interference. Bagged fruits were harvested 7 days later for subsequent analysis. Fluorescence in the plum pericarp region around the injection sites was detected using a Chemi Dog Ultra multi-functional imaging system. Fluorescent pericarp samples were frozen, with three biological replicates per treatment, each replicate including 6-10 fruits. The fluorescence in the transiently expressed samples was quantitatively detected. PsJUB1.1 The expression level was compared with that of fruit peel samples transformed with empty GFP vector as a control. Transient transformation PsJUB1.1 -GFP ( PsJUB1.1 -OE) in plum peel PsJUB1.1 The expression level of GFP was significantly higher than that of plum peel transformed with empty GFP (EV). PsJUB1.1 expression level ( p <0.05), indicating PsJUB1.1 Successful overexpression was achieved in plum pericarp, with each treatment containing 3 biological replicates denoted as 1-3. Figure 3 a).

[0026] After transient expression, plum fruits were placed in containers of the same size, and an equal amount of ultrapure water was added to cover the fruits. Seven to ten fruits were grouped together, with three replicates. The changes in the cracking grade of the injected area were recorded every 24 hours, with the ultrapure water being replaced during each instance. The experiment was conducted at room temperature (25℃). The evaluation index for cracking grade was based on the jujube cracking disease grading system (Yuan Zan et al., 2013). Based on the number, length, and area of ​​cracks on the fruit surface, cracked fruits were divided into six grades from 0 to 5, where grade 0 indicates no cracks and grade 5 indicates that the cracked area exceeds half the fruit surface area. The calculation method for the cracking index was the same as that for jujube cracking index. The specific calculation method is: Cracking Index = ∑i0(i×L) / (a×n), where i = cracking grade, L = number of cracked fruits at each cracking grade, a = most severe cracking grade, and n = total number of fruits surveyed.

[0027] Four days after the fruit was subjected to rain-induced cracking simulation treatment, varying degrees of cracking were observed in the pericarp area of ​​the plums subjected to transient expression. Figure 3 b). Transient overexpression PsJUB1.1 The average fruit cracking index was lower than the control 1 to 4 days after the fruit rain cracking simulation treatment, indicating that overexpression... PsJUB1.1 It can delay the occurrence of plum fruit cracking to some extent.Figure 3 c)。

[0028] 2、Transient overexpression of P sJUB1.1 Effects on the structure of plum epicarp: After transient expression, the plum epicarp with a length and width of about 1 cm was taken with a sterile scalpel, paraffin sections were made and observed by toluidine blue O staining. The specific steps are as follows: (1) The cut plum epicarp tissue block was fixed with 50% FAA buffer for 24-48 hours; (2) The sample was sequentially placed in 30%, 50%, 70%, and 90% ethanol for 1 hour each time. Then it was dehydrated with anhydrous ethanol twice for 1 hour each time; (3) After taking out the sample, it was immersed in a mixture of 50% paraffin and 50% xylene overnight, and then immersed in pure wax for 3 times, 1 hour each time; (4) The embedded wax block was sectioned using a microtome, and the cut sections were baked in a 42°C incubator for 48 hours; (5) The sections were deparaffinized using pure xylene for 10 minutes each time; (6) The sections were sequentially placed in 90%, 70%, 50%, and 30% ethanol for 15 minutes each time, and finally placed in distilled water for 20 minutes; (7) The sections were stained with 0.5% toluidine blue O dye for 10 seconds; (8) The sections were sequentially placed in 30%, 50%, 70%, and 90% ethanol for 15 minutes each time, and then treated with pure xylene twice for 15 minutes each time; (9) The sections were sealed with glycerol gelatin sealing agent; (10) The sections were observed and photographed under an optical microscope (OM).

[0029] Each treatment observed 3 fruits, and each fruit observed 2-3 visual fields. Transient overexpression PsJUB1.1 Compared with the plum epicarp with empty vector, the number of cell layers in the epicarp region increased significantly, and the cutin layer of the epicarp appeared different degrees of thickening Figure 3 d). Statistical analysis of the thickness of the epicarp, the degree of cutinization of the epidermal cells, and the degree of cutin layer invagination in the transient expression epicarp region showed that the thickness of the epicarp after overexpression PsJUB1.1 p <0.01, Figure 3 e), the degree of cutinization of the epidermal cells was significantly higher than that of the control group p <0.01, Figure 3 f), and the average value of the degree of cutin layer invagination was higher than that of the control group Figure 3 g). ​

[0030] 3. Momentary silence PsJUB1.1 The impact of rain-induced cracking resistance on plum fruit: Constructing a silent carrier PsJUB1.1 -RNAi, using an empty RNAi vector as a control, was used to transform Agrobacterium and prepare a resuspension for use on unripe green plums growing on trees. PsJUB1.1 Transient silencing. The injection method is the same as that for transient overexpression. Rainwater is removed through bagging to prevent transient silencing. PsJUB1.1 Interference from the fruit of the green plum. Fruits were harvested 7 days later for subsequent analysis. Quantitative detection of [unclear - possibly referring to a specific substance or component] in transiently silent samples. PsJUB1.1 The expression level was compared with that of pericarp samples transformed with empty vector RNAi as a control. Transient transformation PsJUB1.1 -RNAi in plum pericarp PsJUB1.1 The expression level was significantly lower in plum pericarp transformed with empty vector RNAi than in other samples. PsJUB1.1 expression level ( p <0.05), indicating successful silencing in plum peel. PsJUB1.1 The expression ( Figure 3 h).

[0031] Instant silence PsJUB1.1 The fruit cracking simulation treatment method was the same as that used for overexpression. Four days later, varying degrees of fruit cracking appeared in the pericarp area of ​​the plum after transient silencing. Figure 3 i) Momentary silence PsJUB1.1 The average fruit cracking index was higher than the control 1 to 3 days after the fruit rain cracking simulation treatment, and significantly higher than the average fruit cracking index of transient empty vector RNAi expression 4 days after the fruit rain cracking simulation treatment. p <0.01), indicating silence. PsJUB1.1 It can accelerate the occurrence of plum fruit cracking during rain. Figure 3 j).

[0032] 4. Instantaneous Silence P sJUB1.1 Effects on plum pericarp structure: Plum pericarps with transient silencing regions, approximately 1 cm in length and width, were harvested using a sterile scalpel. Paraffin sections were prepared and stained with toluidine blue O for observation. The method was the same as for transient overexpression samples. Three fruits were observed for each treatment, with 2-3 fields of view observed for each fruit. Transient silencing PsJUB1.1 Compared with the plum pericarps transferred to empty plants, the cells in the exocarp region were significantly larger, the number of layers decreased, and the pericarp cuticle was thinner. Figure 3 k). Statistical analysis of the pericarp thickness, epidermal cell keratinization degree, and cuticle invagination degree in the transient expression region showed that silencing... PsJUB1.1 The thickness of the pericarp was significantly reduced afterward. p <0.01, Figure 3 l), the degree of epidermal cell keratinization and stratum corneum invagination were significantly lower than those in the control group (p <0.01, Figure 3 m, 3n).

[0033] Example 4: Application of PsJUB1.1 in rain resistance and crack prevention of tomato fruits 1. PsJUB1.1 -E8 transgenic tomato acquisition and positive identification: The genetic transformation method was based on previous research (Hong et al., 2019). 'Micro-Tom' seeds were sterilized with 20% NaClO solution for 15 minutes, then washed 3-5 times with sterile water. They were sown in 1 / 2 MS solid medium and cultured in the dark for 3-4 days until germination. After germination, they were cultured in a tissue culture room with 16 hours of light and 8 hours of darkness daily until the cotyledons unfolded. The cotyledons were cut, ensuring cut ends, and placed in pre-medium medium. After 1 day in the dark, they were cultured in the light for 2-3 days.

[0034] Build PsJUB1.1 After sequencing and alignment confirmed, the E8 vector was used to extract plasmids, which were then transformed into Agrobacterium to prepare a resuspension. This resuspension was then used to infect pre-cultured tomato cotyledons. The infected cotyledons were transferred to an induction medium and cultured in the dark for 2-3 days. Then, the cotyledons were placed face up in a differentiation medium and cultured under light. Subsequently, the sprouted parts were transferred to a subculture medium. When the resistant shoots reached 3-4 cm in height, they were transferred to a rooting culture to induce rooting. The rooted tomatoes were then transplanted into nutrient soil and cultured in a greenhouse.

[0035] Extraction was performed using the Biospin All-Purpose Plant Genomic DNA Extraction Kit (BSC13S1B, Bioer). PsJUB1.1 DNA from E8 transgenic tomato fruits. The DNA of transgenic tomatoes was amplified using E8 vector primers (Table 1), and the target band was detected. Wild-type tomatoes were used as a negative control. PsJUB1.1 - The E8 plasmid was used as a positive control. PCR detection was performed on the selected tomatoes using universal primers for the E8 vector. All transgenic tomatoes amplified the plasmid, matching the positive control. PsJUB1.1 The presence of bands of the same size as the E8 vector plasmid, while the negative control wild-type tomato did not amplify any bands, indicates that... PsJUB1.1 The -E8 vector was successfully transferred into 7 tomato plants, achieving... PsJUB1.1 overexpression ( Figure 4 a).

[0036] After rooting and transplanting PsJUB1.1 RNA from E8 transgenic tomato fruits was quantitatively analyzed to detect... PsJUB1.1 The expression level of [the substance / molecule]. Quantitative results showed that the fruits of 7 transgenic tomato plants [expressed the substance / molecule]. PsJUB1.1 The relative expression levels of both were significantly higher than those of wild type ( p <0.05, Figure 4 b). Replace T0PsJUB1.1 -E8 transgenic tomato seeds were sown on a sowing medium containing kanamycin, and offspring that could be stably inherited were screened for research. PsJUB1.1 Gene function.

[0037] 2. PsJUB1.1 Effects on the structure of the tomato pericarp: Wild-type tomatoes at two stages, namely the green ripening stage (MG) and 7 days after color change (BR7) and the T1 generation, were examined using a sterile scalpel. PsJUB1.1 -E8 transgenic tomatoes had their pericarps near the equator cut into pieces approximately 1 cm in length and width. After being embedded in paraffin, the pieces were sectioned and stained with toluidine blue O for observation, using the same method as for plum fruits. This was done at both the MG and BR7 stages. PsJUB1.1 -E8 1#、 PsJUB1.1 -E8 3# and PsJUB1.1 -E8 4# genetically modified tomatoes showed an increase in the number of cell layers and cuticle in the exocarp compared to wild-type tomatoes. Figure 4 c). Statistical analysis of the exocarp thickness, epidermal cell keratinization degree, and cuticle invagination degree in the tomato pericarp region during the two periods showed that... PsJUB1.1 -E8 1#、 PsJUB1.1 -E8 3# and PsJUB1.1 -E8 4# transgenic tomatoes showed significantly increased pericarp thickness and epidermal cell keratinization at both growth stages compared to wild-type tomatoes. p <0.05, Figure 4 d, 4e). PsJUB1.1 -E8 4# strain showed no significant difference in cuticle invagination between the MG stage and wild-type tomatoes, but the degree of invagination was significantly higher at the BR7 stage. PsJUB1.1 -E8 3# and PsJUB1.1 -E8 4# genetically modified tomatoes showed significantly greater cuticle invagination at both stages compared to wild-type tomatoes. p <0.05, Figure 4 f).

[0038] 3. PsJUB1.1 Effects on the osmotic water absorption capacity of tomato fruits: Wild-type tomatoes and PsJUB1.1 Fruits from the MG and BR7 stages of the E8 transgenic tomatoes were immersed in a 2.5% toluidine blue O solution. After 24 hours, the fruits were removed, rinsed with ultrapure water, and the staining was observed and photographed (Liu et al., 2024). Eight fruits were immersed at each stage, with three replicates. Forty-six hours after staining treatment, numerous blue spots appeared on the surface of the MG and BR7 wild-type tomatoes. PsJUB1.1 -E8 1#、 PsJUB1.1 -E8 3# and PsJUB1.1The number of blue spots on the surface of tomato fruits of the E8 4# strain was significantly reduced compared to the wild type (P < 0.05) Figure 4 g). The results showed that, PsJUB1.1 The thickening of the exocarp and cuticle of the E8 transgenic tomato helped the tomato fruit resist osmotic water absorption.

[0039] 4、 PsJUB1.1 Analysis of the rain cracking resistance of E8 transgenic tomato fruits: The rain cracking simulation treatment of transgenic tomato fruits was similar to that of plum fruits. Red-ripe tomato fruits were harvested, 10 tomato fruits were used as a group, 3 replicates were set for each group, and the tomato fruits were soaked in containers of the same size using an equal amount of ultrapure water. The cracking of tomato fruits after 0-5 days of soaking was counted, and the cracking index was calculated. The statistical standards and calculation methods were the same as those for the rain cracking simulation treatment of plum fruits. After 1 day of treatment, the tomato fruits showed slight cracking. After 5 days, the number and degree of cracking of transgenic tomato fruits were significantly lower than those of the wild type (P < 0.05) Figure 4 i). The 3rd to 5th days after the rain cracking simulation treatment of the fruits JUB1.1 The E8 1#, PsJUB1.1 The E8 3# and PsJUB1.1 The cracking index of tomato fruits of the E8 4# strain was significantly lower than that of the wild type tomato (P < 0.05) p <0.05, Figure 4 h). The results showed that overexpression of E8 can improve the resistance of tomato fruits to cracking caused by rain. PsJUB1.1

[0040] Although the present application has been described in detail above with specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present application. Therefore, these modifications or improvements made on the basis of not deviating from the spirit of the present application, all belong to the scope of protection claimed by the present application.​

Claims

1. Application of NAC transcription factor PsJUB1.1 in rain resistance and crack resistance of plant fruits, characterized in that, The genes are introduced into the genome of a plant and overexpressed in the transgenic plant. PsJUB1.1 The genes are introduced into the genome of a plant and overexpressed in the transgenic plant.

2. Use according to claim 1, wherein Transgenic plants overexpressing PsJUB1.1 Afterwards, the cell layers of the outer pericarp region increased significantly, and the cuticle of the pericarp thickened.

3. Use according to claim 1 or 2, characterized in that, The plant is plum or tomato.

4. Use according to claim 1 or 2, wherein the compound is ###0002### The gene is introduced into the genome by Agrobacterium-mediated method.

5. A method of improving rain resistance and cracking resistance of a fruit of a plant, characterized by, The genes are introduced into the genome of a plant and overexpressed in the transgenic plant. PsJUB1.1 The genes are introduced into the genome of a plant and overexpressed in the transgenic plant.

6. The method of claim 4, wherein, The plant is plum or tomato.

7. Use according to claim 4 or 5, wherein the compound is ###0002### The gene is introduced into the genome by Agrobacterium-mediated method.

8. The method of claim 5, wherein, The plant is tomato, and compared with wild type tomato, the number of cell layers of the exocarp of transgenic tomato is significantly increased, and the cuticle of the exocarp is obviously thickened at two periods of green-ripe period and 7 days after turning color.

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