Grape c3h33 gene and application thereof

Abstract

The present application belongs to the field of genetic engineering, and relates to a grape C3H33 gene and application thereof. The present application finds that the grape C3H33 gene has the functions of high-temperature resistance, increase of anthocyanin content of fruits and promotion of fruit ripening.

Description

Technical Field

[0001] This invention relates to a grape C3H33 gene and its application, belonging to the field of genetic engineering. Background Technology

[0002] CCCH-type zinc finger proteins (C3H proteins) are a class of distinctive transcriptional regulators. Their conserved domains consist of three cysteine ​​residues and one histidine residue arranged in a specific conserved sequence pattern. By binding to zinc ions to stabilize their structure, they achieve specific binding to DNA or RNA, regulating gene expression at the transcriptional or post-transcriptional levels. CCCH proteins are widely distributed in eukaryotes. Plant genomes encode a large number of CCCH family members, including 68 in Arabidopsis thaliana, 67-73 in rice, 68 in maize, 80 in tomato, 91 in poplar, 40 in pineapple, and 81 in dragon fruit.

[0003] In regulating plant growth and development, members of the C3H family exhibit functional diversity. For example, Arabidopsis thaliana HUA1 and AtTZF4 can promote seed germination (Genes (Basel). 2025, 16 (4): 429); rice OsC3H38 exhibits salt tolerance (CN110028564A); and the poplar C3H35 gene can regulate lignin content (CN119979748A). There are 69 members of the C3H family in grapes, and the specific function of each member remains unclear.

[0004] Anthocyanin content in grapes is a key trait determining grape quality, and identifying the genes and regulatory networks that regulate anthocyanin content is crucial for genetic improvement of this trait. This invention, through transcriptomic and metabolomic analysis of grape anthocyanin content, identified the key gene VvC3H33 affecting anthocyanin content. Further research revealed that overexpression of VvC3H33 has the technical effects of improving plant heat tolerance, increasing fruit anthocyanin content, and promoting fruit ripening. Summary of the Invention

[0005] The purpose of this invention is to provide a grape C3H33 gene and its application.

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

[0007] This invention provides the application of a protein in improving plant heat tolerance and / or increasing anthocyanin content in fruits and / or promoting fruit ripening, characterized in that the amino acid sequence of the protein is as shown in SEQ ID NO. 2.

[0008] The present invention also provides the application of nucleic acid molecules in improving plant heat tolerance and / or increasing anthocyanin content in fruits and / or promoting fruit ripening, characterized in that the nucleic acid encodes the aforementioned protein.

[0009] In some implementations, the above-mentioned nucleic acid sequence is shown as either SEQ ID NO. 1 or SEQ ID NO. 3.

[0010] The present invention also provides an expression cassette for improving plant heat tolerance and / or increasing anthocyanin content in fruits and / or promoting fruit ripening, characterized in that the expression cassette contains the aforementioned nucleic acid molecules.

[0011] The present invention also provides an application of an expression vector in improving plant heat tolerance and / or increasing anthocyanin content in fruits and / or promoting fruit ripening, characterized in that the expression vector contains the above-mentioned expression cassette.

[0012] The present invention also provides the application of a host cell in improving plant heat tolerance and / or increasing anthocyanin content in fruits and / or promoting fruit ripening, characterized in that the host cell contains the above-mentioned expression cassette or the above-mentioned expression vector.

[0013] In some implementations, the host cell is an Escherichia coli cell or an Agrobacterium cell.

[0014] The present invention also provides a method for improving plant heat tolerance and / or increasing anthocyanin content in fruit and / or promoting fruit ripening, characterized in that plants are transformed with the above-mentioned expression cassette or expression vector or host cell, and plants with improved heat tolerance and / or increased anthocyanin content in fruit and / or faster fruit ripening are screened.

[0015] Compared with existing technologies, the beneficial effects of this invention are: this invention has found that overexpression of the C3H33 gene in grapes and tomatoes can improve plant heat tolerance, increase anthocyanin content in fruits, and promote fruit ripening. Attached Figure Description

[0016] Figure 1 The energy spectrum curve of the ultraviolet radiation lamp (A) and the installation diagram of the lamp (B).

[0017] Figure 2 Infrared radiation lamp energy spectrum curve (A) and lamp installation diagram (B).

[0018] Figure 3 Results of transcriptomic and metabolomic analyses of grapes under UV irradiation. A: Bar chart of co-enrichment of differentially expressed metabolites and genes; B, C: Differentially expressed gene clusters; D, E: Differentially expressed metabolite sets. The horizontal axis UV1-5 represents five biological replicates of UV irradiation treatment.

[0019] Figure 4 Results of combined transcriptomic and metabolomic analysis of grapes under infrared light irradiation. A: Co-enrichment pathway of differentially expressed genes and metabolites; B: Downregulated gene clusters; C: Upregulated gene clusters; D: Downregulated metabolite clusters; E: Upregulated metabolite clusters. The horizontal axis IR1~5 represents 5 biological replicates of infrared irradiation treatment.

[0020] Figure 5 Weighted gene co-expression network analysis. A: Gene hierarchical clustering tree, different branches of the clustering tree represent different gene modules, and different colors represent different modules; B: Association analysis diagram between modules and flavonoid and anthocyanin traits; C: Correlation between gene identity (MM) and gene significance (GS) in the Black module; D: Screening of key genes in the Black module. Trait 1: Pelargonidin 3-O-glucoside Dp-3-O-Glu; Trait 2: Chrysanthemin; Trait 3: Methyl anthocyanin 3-O-glucoside Pt-3-O-Glu; Trait 4: Dimethyl anthocyanin 3-O-glucoside Mv-3-O-Glu; Trait 5: Dimethyl anthocyanin-3-B-glucoside Mv-3-B-Glu; Trait 6: Methyl anthocyanin 3-O-(6-O-acetyl)glucoside Pn-3-acetylglc; Trait 7: Dimethyl anthocyanin 3-O-(6-O-acetyl)glucoside Mv-3-acetylglc; Trait 8: Pelargonidin 3-O-[2-O-(6-O-(E)-feruloyl-beta-D-glucopyranosyl)-beta-D-glucopyranoside]-5-O-(beta-D-glucopyranoside); Trait 9: Methyl cylindrical 3-O-(6-O-acetyl)glucoside Pt-3-acetylglc; Trait 10: Methyl anthocyanin 3-O-(6-O-p-coumaryl)glucoside Pn-3-p-coumglc; Trait 11: Cyanidin 3-(6''-p-coumarylsambubioside); Trait 12: Dimethicone cylindrical 3-O-(6-O-caffeoyl)glucoside Mv-3-caffglc; Trait 13: Proanthocyanidin B2; Trait 14: Kandelin A-1; Trait 15: Quercetin; Trait 16: Quercetin 3-O-glucoside.

[0021] Figure 6 Image of VvC3H33 overexpression vector.

[0022] Figure 7Transient overexpression of VvC3H33 in grape berries under dark conditions. A: Control; B: Transient overexpression of VvC3H33.

[0023] Figure 8 Transient overexpression of VvC3H33 in grape berries under UV irradiation. A: Control; B: Transient overexpression of VvC3H33.

[0024] Figure 9 Transient overexpression of VvC3H33 in grape fruit under infrared irradiation. A: Control; B: Transient overexpression of VvC3H33.

[0025] Figure 10 Anthocyanin content in transiently rotated grape berries. A: Anthocyanin content in transiently rotated grape berries under darkness; B: Anthocyanin content in transiently rotated grape berries under ultraviolet irradiation; C: Anthocyanin content in transiently rotated grape berries under infrared irradiation.

[0026] Figure 11 The phenotype of tomato overexpression with VvC3H33. A~H: Photos taken at 95, 96, 97, 98, 99, 100, 101 and 102 days after transplanting, respectively.

[0027] Figure 12 Phenotypic characteristics of VvC3H33 overexpressing tomato seedlings under low-temperature stress. A: Wild-type tomato seedlings before and after low-temperature treatment; B: Low-temperature treatment of overexpressing tomato seedlings before and after low-temperature treatment; C: DAB staining of wild-type leaves after low-temperature treatment; D: DAB staining of overexpressing leaves after low-temperature treatment; E: Measurement of relative conductivity of wild-type and overexpressing leaves after low-temperature treatment.

[0028] Figure 13 Phenotypic characteristics of transgenic tomato seedlings under high temperature stress. A: Wild-type tomato seedlings before and after high temperature treatment; B: High temperature in overexpressing tomato seedlings before and after high temperature treatment; C: DAB staining of wild-type leaves after high temperature treatment; D: DAB staining of overexpressing leaves after high temperature treatment; E: Measurement of relative conductivity of wild-type and overexpressing leaves after high temperature treatment. Detailed Implementation

[0029] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of this application. Unless otherwise specified, the examples are conducted under conventional experimental conditions, such as those described in Sambrook et al.'s *Molecular Cloning: A Laboratory Manual* (Sambrook J & Russell DW, 2001), or according to the conditions recommended in the manufacturer's instructions. Unless otherwise specified, the chemical reagents used in the examples are all commercially available and conventional methods well known to those skilled in the art.

[0030] Example 1: Joint analysis of transcriptomics and metabolomics to identify genes related to anthocyanin synthesis in grape fruits

[0031] Anthocyanin content in grapes is a key trait determining grape quality, and identifying genes and regulatory networks that regulate anthocyanin content is crucial for the genetic improvement of this trait. The inventors treated grape berries with ultraviolet (UVA) and infrared (IR) light, respectively, and measured the transcriptome and metabolome of the fruit samples. The aim is to identify genes related to anthocyanin synthesis in grape berries through combined transcriptomic and metabolomic analysis. Specific methods and results are as follows:

[0032] Using Cabernet Sauvignon grapes (commercial variety purchased from open sources) 70 days after flowering as experimental material, 27 bunches of grapes of similar size that had not yet turned color were collected and cultured in vitro in a constant temperature incubator (2% sucrose solution, 80% humidity, 25℃), with distilled water as a control. UVA lamps (UVA340, LONGPRO, Guangzhou, China) were installed at the top, with 9 bunches per treatment. Figure 1 Samples were collected after 3 days (20% color conversion rate), 6 days (50% color conversion rate), and 9 days (100% color conversion rate) of light treatment, as shown in Table 1. The remaining time was spent in darkness. The ultraviolet radiation received by the grape berries was measured using an ultraviolet spectroradiometer (HP350UV, Hangzhou Shuangse Intelligent Detection Instrument Co., Ltd., Hangzhou, China). The average ultraviolet radiation intensity was 1.1 W / m². 2 .

[0033] An infrared radiation lamp box (R10-470-300, Longpro, Guangzhou, China) was installed on top of another constant temperature incubator, connected to an external constant temperature control distribution box (RS400, Longpro, Guangzhou, China). The energy spectrum curve of the infrared radiation lamp and a schematic diagram of its installation are shown below. Figure 2Samples were collected after 3 days (20% color change rate), 6 days (50% color change rate), and 12 days (100% color change rate) of light treatment, as shown in Table 3-2. All other treatment times were consistent with the above. Infrared radiation received by the grape berries was measured using an infrared spectroradiometer (SM206-NIR, Xinbao Instruments, Shenzhen, China). The average infrared radiation intensity was 61 W / m². 2 .

[0034] Nine grape berries treated with UVA for 3 days and nine grape berries treated with infrared radiation for 3 days were randomly selected. After removing the seeds, the samples were sent for metabolomics analysis. Nine grape berries treated with UVA for 3 days and nine grape berries treated with infrared radiation for 3 days were also randomly selected. After peeling, the grape skins were sent for transcriptome sequencing analysis. Each treatment was repeated five times. After metabolomics and transcriptome identification, co-enrichment analysis of differentially expressed genes and metabolites revealed that the enrichment pathways of differentially expressed genes mainly included phenylpropane biosynthesis, plant hormone signal transduction, isoquinoline alkaloid biosynthesis, phenylalanine metabolism, and tyrosine metabolism. The enrichment pathways of differentially expressed metabolites mainly included amino acid biosynthesis, pyruvate metabolism, and thiamine metabolism (vitamin B1). Figure 3 A). Differential metabolites and genes were categorized using K-means for each group, and then plotted according to the categories to identify metabolites and genes with consistent trends. In gene cluster 1, 319 genes showed a consistent trend, indicating that the expression levels of genes treated with UVA+ were lower than those in the control group. Figure 3 B); In gene cluster 2, 383 genes showed a consistent trend, with UVA+ treatment showing higher gene expression levels than the control. Figure 3 C). In metabolite set 1, 52 metabolites showed a consistent trend in content, indicating that the content of metabolites treated with UVA+ was higher than that in the control ( Figure 3 D); In metabolite set 2, 86 metabolites showed a consistent trend in content, indicating that the content of metabolites treated with UVA+ was lower than that in the control ( Figure 3 E). Therefore, gene cluster 1 and metabolite cluster 2 are highly correlated, and gene cluster 2 and metabolite cluster 1 are highly correlated.

[0035] Compared with the control, nine candidate genes related to photoregulation of anthocyanin synthesis in grapes under UVA radiation were identified, including HYH, HY5, CRY, PHY, and UVR8. Thirty structural genes related to anthocyanin synthesis were identified, including F3'H, F3'5'H, LDOX, UFGT, OMT, and GST. Eleven transcription factors were identified related to anthocyanin synthesis, including MYBA1, MYBA2, MYBA3, and C3H33. Among the candidate genes for UVA-regulated anthocyanin synthesis, VvHYH (FC=4.34) showed the highest fold change. Among the anthocyanin structural genes, VvPAL (FC=4.88) showed the highest fold change. Among the transcription factors, VvMYBA1 (FC=2.28) showed the highest fold change (see Table 2 for details).

[0036]

[0037]

[0038] Gene enrichment pathways include the degradation of valine, leucine, and isoleucine; galactose metabolism; secondary metabolite synthesis; phenylpropane biosynthesis; thiamine metabolism; biosynthesis of hyoscyamine, scopolamine, and hyoscyamine alkaloids; and biosynthesis of ubiquinone and other terpenoid quinones. Metabolite enrichment pathways include the biosynthesis of valine, leucine, and isoleucine and purine metabolism. By analyzing highly co-expressed gene clusters and metabolites with highly consistent content changes among different samples, candidate genes that may be involved in anthocyanin synthesis were identified, which can be divided into three main categories: light-regulated genes, structural genes, and transcription factors. Gene set 5 contains 260 genes ( Figure 4 B), gene set 10 has 93 genes ( Figure 4 C), Metabolite set 2 contains 28 metabolites ( Figure 4 D), Metabolite set 1 contains 46 metabolites ( Figure 4 E).

[0039] Compared with the control, nine candidate genes related to photoregulation of anthocyanin synthesis in grapes under infrared radiation were identified, including HYH, HY5, CRY, PHY, and UVR8. Thirty structural genes related to anthocyanin synthesis were identified, including F3'H, F3'5'H, LDOX, UFGT, OMT, and GST. Eleven transcription factors related to anthocyanin synthesis were identified, including MYBA1, MYBA2, MYBA3, and C3H33. Among the candidate genes for anthocyanin synthesis regulated by infrared radiation, VvHY5 (FC=3.74) showed the highest fold change. Among the anthocyanin structural genes, VvF3'5'H (FC=5.38) showed the highest fold change. Among the transcription factors, VvMYBA1 (FC=2.19) showed the highest fold change. (See Table 3 for details.)

[0040]

[0041]

[0042] Further WGCNA analysis identified 11 modules. The black and dark blue modules showed a predominantly positive correlation with anthocyanin-related components, while the blue and brown modules showed a predominantly negative correlation. The black module had a cor=0.85p=2.3e-103, the dark blue module had a cor=0.45p=0.00012, the blue module had a cor=0.54p=2.6e-125, and the brown module had a cor=0.3p=0.00032. Higher cor values ​​and lower p values ​​are better, indicating that module members are more representative of the module's characteristics. Therefore, the black module was selected as the key module. This module contains the structural genes for anthocyanin synthesis: PAL (VIT_13s0019g04460), OMT (VIT_01s0010g03470, VIT_01s0010g03490, ... VIT_01s0010g03510, VIT_18s0001g09400), CHS (VIT_14s0068g00920, VIT_14s0068g00930, VIT_05s0136g 00260), LDOX (VIT_02s0025g04720), F3H (VIT_04s0023g03370), F3'5'H (VIT_06s0009g03010), CHI (VIT_13 Genes such as s0067g03820, UFGT (VIT_16s0039g02230), anthocyanin transport-related genes GST4 (VIT_04s0079g00690), transcription factors MYBA1 (VIT_02s0033g00410), ERF107 (VIT_16s0013g00890), and C3H33 (VIT_11s0016g04280) belong to this module and may play an important regulatory role in anthocyanin synthesis. The black module is significantly positively correlated with traits 2, 4, 5, and 7 (p<0.05), and extremely significantly positively correlated with traits 6, 8, and 10 (p<0.01). The genes in the black module were imported into Cytoscape and sorted directly by weight value. The top 14 genes were selected as Hub genes. The first one was VIT_11s0016g04280 (VvC3H33), which may be a key gene involved in UVA and infrared radiation response.

[0043] Example 2 Phenotypic Identification of VvC3H3 Overexpression in Grapes

[0044] To further verify the regulatory role of VvC3H33 in the anthocyanin biosynthesis pathway, a transient overexpression experiment was conducted on the coding region of the VvC3H33 gene (SEQ ID NO. 3) in the Cabernet Sauvignon grape variety to observe the effect of VvC3H33 on grape berries. The overexpression vector diagram is shown below. Figure 6 As shown, VvC3H33 is located between the EcoRV restriction sites, driven by the CaMV35S promoter, and linked to the nos terminator. This vector also carries a hygromycin resistance gene expression cassette as a selection marker during genetic transformation. The vector was transformed into Agrobacterium GV3101, cultured overnight by shaking, and after two rounds of activation, the cells were collected by centrifugation and resuspended in 10 mM MgCl2 (containing AS and MES) to OD. 600 =0.8-1.0. Pour an appropriate amount of Agrobacterium infection solution into a 10 mL centrifuge tube, immerse the treated grape stalks in the Agrobacterium infection solution, and place them in a light incubator for incubation at 25°C in the dark overnight (12 h). The next day, discard the bacterial solution, replace it with sucrose solution, and continue incubation. The light incubator is set to a constant temperature of 25±1°C and a humidity of 80%. The light cycle is shown in Table 1.

[0045] After obtaining grape berries with transient overexpression, they were simultaneously subjected to dark, ultraviolet, and infrared treatments, respectively. Results are shown below. Figure 7 , Figure 8 , Figure 9 Wild-type grapes were significantly greener, while grapes overexpressing VvC3H33 were redder and had a significantly higher color change rate. Anthocyanin content analysis showed that, under darkness, UVA, and infrared radiation, the anthocyanin content in the overexpressing grapes was significantly higher than that in the wild-type control grapes, increasing by 1.84 times, 73.68%, and 2.83 times, respectively. Figure 10 This indicates that overexpression of VvC3H33 can increase the accumulation of anthocyanins in grape berries and promote fruit ripening.

[0046] Example 3 Phenotypic identification of tomato overexpression of VvC3H3

[0047] The inventors further overexpressed VvC3H33 in tomatoes to identify its function. The overexpression vector was the same as in Example 2, and the tomato genetic transformation method was as follows:

[0048] Add 1 μL of plasmid to 50 μL of GV3101 Agrobacterium competent cells, mix thoroughly, and then transfer to an electroporation cuvette. After electroporation, add 1 mL of LB liquid medium, mix thoroughly, and then transfer to a 1.5 mL centrifuge tube. Incubate in a shaker at 30℃ and 180 rpm for 30 min. Inoculate 50 μL of the activated Agrobacterium culture onto LB solid medium and incubate in the dark at 30℃ for 48 h.

[0049] Tomato seeds were washed with sterile water for 2 minutes, disinfected with 75% alcohol for 1 minute, soaked in 5% hypochlorous acid solution for 5-8 minutes, and washed twice with sterile water for 10 minutes each time. The seeds were then inoculated onto sterile filter paper and air-dried. The disinfected seeds were sown in germination medium and cultured at 23℃ for 2 days. After the seeds sprouted white buds, they were cultured at 23℃ for 16 hours (light) / 8 hours (dark) for 4-5 days. Once the cotyledons of the germinated tomato seedlings were fully expanded, the cotyledon petioles and tips were surgically removed, leaving the middle portion cut into 2-3 segments. These segments were then inoculated onto a pre-culture medium and cultured at 23℃ for 1-2 days.

[0050] Agrobacterium was picked into the infection solution to prepare OD. 600 Inoculate the explants with a 0.2% Agrobacterium suspension for 10-15 min, then inoculate the dried explants into a co-culture medium and incubate at 23°C in the dark for 2 days. Wash the co-cultured explants twice with 1 g / L cephalosporin solution for 15 min each time, then inoculate them into recovery medium and incubate at 23°C for 16 h (light) / 8 h (dark) for 3-5 days. Inoculate the recovered callus into selection medium and incubate at 23°C for 16 h (light) / 8 h (dark) for 15-30 days. Selected callus is then inoculated into differentiation medium and incubated at 23°C for 16 h (light) / 8 h (dark) for 30-40 days. When the differentiated seedlings reach approximately 2-3 cm in height, remove them from the callus and inoculate them into rooting medium, incubating at 23°C for 16 h (light) / 8 h (dark) for 10-15 days. Finally, extract DNA, confirm positive results by PCR, and observe the phenotype of the transgenic plants.

[0051] The results showed that overexpression of the VvC3H33 gene did not significantly affect the growth of tomato plants, including root system, plant height, leaf size, and number. After fruit set, fruit thinning was used to maintain a similar number of fruits between the overexpressing and wild-type plants. The fruits of the overexpressing plants completed color change, while the fruits of the wild-type plants began to color change, with a time difference of 8 days. Stable overexpression of VvC3H33 accelerated tomato fruit color change and promoted fruit ripening. Figure 11 ).

[0052] The inventors further determined whether the VvC3H33 gene possesses stress resistance function through stress treatment. The specific treatment method and results are as follows:

[0053] 1. Low-temperature treatment. Wild-type tomato seedlings and VvC3H33 overexpressing transgenic tomato seedlings grown in culture medium were transplanted into organic matter and placed in an incubator. They were treated with normal light (white light, 8 / 16 h) at 25℃. After the growth vigor recovered, the incubator temperature was set to 15℃, and the seedling response was observed. One week later, both wild-type and overexpressing seedlings showed varying degrees of wilting and leaf drooping. DAB staining and conductivity results showed no significant difference in the response to low-temperature stress between the overexpressing and wild-type seedlings. Figure 12 This indicates that tomato seedlings overexpressing VvC3H33 are not sensitive to low temperatures.

[0054] 2. High-temperature treatment. Wild-type and transgenic seedlings grown in the culture medium were transplanted into organic matter and placed in an incubator under normal light (white light, 8 / 16 h) at a temperature of 25℃. After the growth vigor recovered, the incubator temperature was set to 35℃, and the seedling response was observed. Three days later, the overexpression seedlings showed severe wilting and drooping leaves, while the wild-type seedlings showed no obvious signs. DAB staining and conductivity results both indicated that the wild-type seedlings were subjected to more severe stress, and the difference between the two groups was significant. Figure 13 This indicates that tomato seedlings overexpressing VvC3H33 have a certain degree of tolerance to high temperatures.

[0055] The above results indicate that overexpression of the C3H33 gene in grapes and tomatoes can improve plant heat tolerance, increase anthocyanin content in fruits, and promote fruit ripening.

[0056] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention are within the scope of protection claimed by the present invention.

Claims

1. The application of protein in improving plant heat tolerance and / or increasing anthocyanin content in fruits and / or promoting fruit ripening, characterized in that, The amino acid sequence of the protein is shown in SEQ ID NO.

2.

2. The application of nucleic acid molecules in improving plant heat tolerance and / or increasing anthocyanin content in fruits and / or promoting fruit ripening, characterized in that, The nucleic acid encodes the protein of claim 1; optionally, the nucleic acid sequence is shown in either SEQ ID NO. 1 or SEQ ID NO.

3.

3. The application of expression cassettes in improving plant heat tolerance and / or increasing anthocyanin content in fruits and / or promoting fruit ripening, characterized in that, The expression cassette contains the nucleic acid molecule as described in claim 2.

4. The application of the expression vector in improving plant heat tolerance and / or increasing anthocyanin content in fruits and / or promoting fruit ripening, characterized in that, The expression vector contains the expression cassette as described in claim 3.

5. The application of host cells in improving plant heat tolerance and / or increasing anthocyanin content in fruits and / or promoting fruit ripening, characterized in that, The host cell contains the expression cassette as described in claim 3 or the expression vector as described in claim 4; optionally, the host cell is an Escherichia coli cell or an Agrobacterium cell.

6. A method for improving plant heat tolerance and / or increasing anthocyanin content in fruits and / or promoting fruit ripening, characterized in that, Plants transformed using the expression cassette of claim 3, the expression vector of claim 4, or the host cell of claim 5 were screened for plants with improved heat tolerance and / or increased anthocyanin content in fruit and / or faster fruit ripening.

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