CsERF.E3 gene and application thereof in regulating fruit ripening

Abstract

The application discloses a CsERF.E3 gene and application thereof in regulating fruit ripening. The gene belongs to an AP2 / ERF family member, the CDS sequence of which is shown as SEQ ID NO. 1, the CDS sequence length is 762 bp, and 253 amino acids are encoded, and the amino acid encoded is shown as SEQ ID NO. 2. A primer is designed to amplify the gene CsERF.E3 on Fengjie navel orange, the gene is introduced into tomato and shanhuyan by using an agrobacterium-mediated genetic transformation method, and the obtained transgenic plants are verified by phenotype observation statistics and gene expression analysis, and it is shown that the CsERF.E3 gene has the function of promoting fruit ripening, and provides a new gene resource for molecular design breeding of fruit ripening.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of plant genetic engineering technology, and particularly relates to a gene CsERF.E3 related to citrus fruit ripening and application of the gene in regulating fruit ripening. BACKGROUND

[0002] Citrus is the world's largest fruit and one of the world's major trade agricultural products. China's citrus industry is developing rapidly, and currently still focuses on fresh consumption. To further expand the citrus market, it is necessary to increase the variety of citrus and improve fruit quality, thereby driving farmers' income and boosting the economy. At the same time, while China's citrus industry is thriving, it also faces some problems: the ripening period of varieties is relatively concentrated, and although many early and late maturing varieties have been introduced, they still cannot achieve year-round supply. In order to further expand the market for China's citrus industry, it is important to explore ripening regulation genes during the development and ripening of citrus, which is of great significance for the quality and ripening regulation of citrus.

[0003] Fruit ripening is regulated by multiple factors, including environmental factors such as light, temperature, and altitude, as well as the internal gene regulatory network, which also plays an important role in ripening. Since the rise of molecular biology, the mechanism of crop ripening has been repeatedly studied and deciphered. Among them, transcription factors participate in the regulation of fruit development and ripening by binding to target genes. According to the different transcriptional regulatory domains, the direction of regulation in the regulatory pathway is also different.

[0004] Studies have shown that transcription factors regulate various metabolic pathways during the development and ripening of various fruits, thereby affecting fruit ripening. During the ripening of citrus fruit, CsMYB77 regulates abscisic acid (ABA) signaling by inhibiting the expression of downstream gene SINAT4, while activating the expression of downstream gene PIN5, thereby regulating auxin signaling and delaying fruit ripening (Zhang et al., 2023). Overexpression of MaNAP1 and MaMADS1 transcription factors can upregulate banana cell wall-related genes and promote pericarp softening, while MaNAP1-MaMADS1 forms a cascade reaction for banana pericarp softening and finger drop, providing potential targets for enhancing the texture and shelf life of bananas (Li et al., 2025). The tomato transcription factor SlNAP1 positively regulates fruit ripening by directly activating gibberellin degradation genes and interacting with SlGID1, further affecting fruit ripening (Li et al., 2024). The transcription factor Sl ERF.D6 negatively regulates the expression of gene GAME12, thereby promoting tomato fruit ripening (Mao Mengdi, 2022). As can be seen, the study of transcription factors in horticultural crops has been very rich, and a series of transcription factors related to fruit development and ripening have been discovered.

[0005] The AP2 / ERF transcription factor has a binding domain composed of about 60 amino acids, which can directly bind to the target gene promoter (Okamuro et al., 1997). In Arabidopsis, the AP2 / ERF family is preliminarily divided into four subfamilies: dehydration response element binding protein (DREB), ethylene response element binding protein (ERF), AP2 and RAV (Feng et al., 2005). All members of the AP2 / ERF family contain at least one DNA binding domain, called the AP2 domain (Magnani et al., 2004). AP2 / ERF family transcription factors have regulatory effects in hormone response, sugar acid synthesis, carotenoid accumulation and other pathways. For example, CsERF110, as an essential activator in citrus, participates in ABA-induced carotenoid accumulation, activates the CsERF53 promoter, forms a CsERF110-CsERF53 transcriptional regulatory module, and thus positively regulates the accumulation of carotenoids in citrus (Sun et al., 2024). The AP2 family transcription factor FaABI4 affects anthocyanin accumulation by affecting the expression levels of FaCHS and FaUFGT genes, and can also affect fruit hardness and sucrose accumulation, ultimately regulating the development and maturation process of strawberry fruit (Lu et al., 2016). During apple development, ERF4 promotes fruit ripening by inhibiting the interaction between the transcription factor MYC2 and the core protein (Hu et al., 2022).

[0006] For a long time, citrus breeding has mainly relied on bud mutation and hybrid breeding. The long childhood of citrus greatly limits the efficiency of conventional breeding methods, so molecular biology breeding is an important means of future citrus breeding. SUMMARY

[0007] One of the purposes of the present application is to provide a CsERF.E3 gene, which encodes a nucleic acid sequence as shown in SEQ ID NO. 1.

[0008] Preferably, the subcellular localization of the CsERF.E3 gene is in the nucleus, and it has no transcriptional activation function.

[0009] The second purpose of the present application is to provide a protein encoded by the CsERF.E3 gene as described above, and the amino acid sequence thereof is as shown in SEQ ID NO. 2.

[0010] The third purpose of the present application is to provide an expression vector of the CsERF.E3 gene as described above.

[0011] Preferably, the present application uses the Gateway system to construct a binary expression vector, which comprises a gateway vector pDONR221 and an overexpression terminal vector pK7WG2D.

[0012] The fourth object of the present application is to provide a host bacterium of the CsERF.E3 gene.

[0013] The fifth object of the present application is to provide a primer pair for amplifying the CsERF.E3 gene, characterized in that the nucleic acid sequences thereof are shown in SEQ ID NO. 3 and 4.

[0014] The sixth object of the present application is to provide an application of the CsERF.E3 gene, the protein, the expression vector, the host bacterium or the primer pair in any one of (1) to (5) below:

[0015] (1) preparation of a product for reducing the number of days for color breaking of fruits;

[0016] (2) preparation of a product for increasing the speed of color breaking of fruits;

[0017] (3) preparation of a product for increasing the content of carotenoids in fruits;

[0018] (4) preparation of a product for reducing the content of chlorophyll in fruits;

[0019] (5) preparation of a product for promoting ripening of fruits.

[0020] Preferably, the fruits are fruits with high carotenoid content after ripening, such as citrus or tomatoes.

[0021] The seventh object of the present application is to provide a method for promoting ripening of fruits by overexpressing the CsERF.E3 gene in plants.

[0022] The eighth object of the present application is to provide an expression vector for silencing the CsERF.E3 gene.

[0023] The ninth object of the present application is to provide a host bacterium for silencing the CsERF.E3 gene.

[0024] The tenth object of the present application is to provide an application of the expression vector for silencing the CsERF.E3 gene or the host bacterium for silencing the CsERF.E3 gene in any one of (1) to (5) below:

[0025] (1) preparation of a product for increasing the number of days for color breaking of fruits;

[0026] (2) preparation of a product for reducing the speed of color breaking of fruits;

[0027] (3) preparation of a product for reducing the content of carotenoids in fruits;

[0028] (4) preparation of a product for increasing the content of chlorophyll in fruits;

[0029] (5) preparation of a product for delaying ripening of fruits.

[0030] Preferably, the fruit is a fruit with high carotenoid content after ripening, such as citrus or tomato.

[0031] Beneficial effects:

[0032] The application utilizes gene cloning technology to isolate and clone a citrus fruit ripening gene CsERF.E3 from Fengjie navel orange. On this basis, CsERF.E3 is constructed into a vector and genetically transformed in tomato and Citrus aurantium, verifying the function of CsERF.E3 in the fruit ripening process. The above-mentioned gene is transformed into tomato and Citrus aurantium by Agrobacterium-mediated genetic transformation, and the transgenic plants and fruits are verified by phenotype observation statistics and gene expression analysis. The results show that the fruit of the CsERF.E3 gene overexpressed tomato strain breaks color obviously earlier than the wild type fruit, and the average time of the fruit of the T2 generation overexpressed tomato strain is 35 days, which is about 6 days earlier than the wild type. The transient overexpression of CsERF.E3 gene in Citrus aurantium starts to change color about 6 days after injection, which is about 2 days earlier than the control group, and the silencing group starts to change color about 14 days, which is about 4 days later than the control group. The above results confirm that it has the function of significantly promoting fruit ripening, provides excellent gene resources for molecular design breeding of citrus ripening period, and has important research significance and application value for analyzing the molecular mechanism of citrus fruit ripening regulation and the development of citrus industry. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 It is a flowchart of the cloning, isolation and function verification of CsERF.E3 gene in the application.

[0034] Figure 2 It is the subcellular localization result of CsERF.E3 gene in Example 2 of the application.

[0035] Figure 3 It is the transcriptional activation activity identification result of CsERF.E3 gene in Example 3 of the application.

[0036] Figure 4 It is the DNA positive identification gel map of tomato transgenic positive seedlings in Example 4 of the application.

[0037] Figure 5 It is the quantitative detection result of CsERF.E3 gene of tomato transgenic positive seedlings in Example 4 of the application.

[0038] Figure 6 It is the fruit color change and fruit breaking color day statistics result of tomato fruit overexpressing CsERF.E3 gene in Example 4 of the application.

[0039] Figure 7The results of ethylene release amount, ABA content and carotenoid content changes of the tomato fruits overexpressing CsERF.E3 gene in Example 4 of the present application at each period are shown in Table 1.

[0040] Figure 8 The expression amount of CsERF.E3 gene in the transformed fruits of Citrus aurantium in Example 5 of the present application is shown in Table 2. In Table 2, PK7 represents pK7WG2D vector transformed Citrus aurantium, TRV represents pTRV2 vector transformed Citrus aurantium, PK7-CsERF.E3 represents CsERF.E3 gene overexpression strain, and TRV-CsERF.E3 represents CsERF.E3 gene silencing strain (the same below).

[0041] Figure 9 The phenotype observation of CsERF.E3 gene in the transiently transformed Citrus aurantium in Example 5 of the present application is shown in Table 3.

[0042] Figure 10 The comparison of ABA content, carotenoid content and chlorophyll content of the fruits of the transiently transformed Citrus aurantium and the control group in Example 5 of the present application is shown in Table 4. DETAILED DESCRIPTION

[0043] The present application will be further described below in combination with specific examples.

[0044] Example 1: Cloning of full-length cDNA of CsERF.E3 gene for citrus fruit development and maturation

[0045] The CDS sequence of the present application was obtained from the sweet orange genome database (http: / / citrus.hzau.edu.cn / orange / ), and primers were designed in the 5' non-coding region and 3' non-coding region of the sequence.

[0046] CsERF.E3-F1: 5'-AAAAAGCAGGCTCCATGTGTGGCGGTGCA CTTAT-3';

[0047] CsERF.E3-R1: 5'-AGAAAGCTGGGTTCTAACACAAGAACTGA TTGCTGTTC-3'.

[0048] Then, the wild type Fengjie navel orange cDNA was used as a template, and a high-fidelity enzyme was used for amplification reaction, and the kit was Phanta Max Super-Fidelity DNA Polymerase.

[0049] Take the wild type Fengjie navel orange leaves frozen at -80°C, use Novagen RNA reagent (FastPure Plant Total RNA Isolation kit) to extract RNA, and the specific RNA extraction method is as follows:

[0050] 1) Take an appropriate amount of plant tissue ground with liquid nitrogen and immediately add 600 μL Buffer EL or 600 μL Buffer PSL, vortex vigorously for 30 sec, mix the sample and lysis solution evenly, centrifuge at 12000 rpm (13400 x g) for 5 min, and immediately perform the following operation;

[0051] 2) Take about 500 μL supernatant to FastPure gDNA-Filter Columns III (FastPure gDNA-Filter Columns III has been placed in the collection tube), centrifuge at 12000 rpm (13400 x g) for 30 sec, discard the FastPure gDNA-Filter Columns III, and collect the filtrate;

[0052] 3) Add 0.5 times the volume of anhydrous ethanol (about 250 μL, adjust according to the actual situation of the supernatant) to the collection tube, shake and mix for 15 sec;

[0053] 4) Transfer the above mixture to FastPure RNA Columns V (FastPure RNA Columns V has been placed in the collection tube), centrifuge at 12000 rpm (13400 x g) for 30 sec, and discard the filtrate;

[0054] 5) Add 700 μL Buffer RWA to FastPure RNA Columns V, centrifuge at 12000 rpm (13400 x g) for 30 sec, and discard the filtrate;

[0055] 6) Add 500 μL Buffer RWB (check whether 48 mL of anhydrous ethanol has been added before use) to FastPure RNA Columns V, centrifuge at 12000 rpm (13400 x g) for 30 sec, and discard the filtrate;

[0056] 7) Repeat step 6);

[0057] 8) Place FastPure RNA Columns V back into the collection tube, centrifuge at 12000 rpm (13400 x g) for 2 min;

[0058] 9) Transfer the FastPure RNA Columns V to new RNase-free Collection Tubes 1.5 mL centrifuge tubes, and add 30-100 μL RNase-free ddH2O to the center of the membrane of the adsorption column, and centrifuge at 12000 rpm (13400 x g) for 1 min;

[0059] 10) The extracted RNA can be directly used for downstream experiments or stored at -20℃.

[0060] The cDNA was synthesized using the reverse transcription kit Hiscript III RT SuperMix for qPCR.

[0061] The synthesis method was performed according to the instructions. The obtained cDNA was used for PCR amplification of the CsERF.E3 gene. The PCR amplification was performed using CsERF.E3-F1 and CsERF.E3-R1 designed above as primers.

[0062] The detailed steps of PCR amplification are as follows: pre-denaturation at 95℃ for 3 min; denaturation at 95℃ for 15 s, annealing at 58℃ for 15 s, extension at 72℃ for 1 min, 35 cycles, and extension at 72℃ for 5 min after the completion of the cycles.

[0063] After the amplification, a single band of PCR product was generated, which was purified and recovered by Omega gel recovery kit after 1% agarose gel electrophoresis. The purified product was ligated with the entry vector pDOR221, and the total reaction system volume was 10 μL. After incubation at room temperature for 5 min, the E. coli competent cell DH5α was transformed.

[0064] Then, the bacterial liquid PCR positive identification was performed using the target gene sequence primers (i.e. CsERF.E3-F1 and CsERF.E3-R1), and the company was sequenced. Finally, the single clone bacterial liquid with correct sequence was selected according to the sequencing results, the plasmid was extracted using Kang Weishiji CWO500M, and then was ligated with the final vector pK7WG2D. The ligation product was transformed into Agrobacterium GV3101.

[0065] The plasmid contains the CDS sequence of the gene CsERF.E3, the nucleotide sequence of which is shown as SEQ ID NO. 1, and the amino acid sequence of the encoded protein is shown as SEQ ID NO. 2.

[0066] The CDS sequence of CsERF.E3 gene is 762 bp in length, which includes a coding reading frame that can encode 253 amino acids, and the isoelectric point is 8.32. After MEGAX analysis (https: / / www.megasoftware.net) of the sequence and the evolutionary relationship of the AP2 / ERF family in Arabidopsis thaliana, it was found that it belongs to the AP2 subfamily, and according to the arrangement order of CsAP2 subfamily on the chromosome, it is named as CsERF.E3.

[0067] Example 2: Subcellular localization of CsERF.E3 gene

[0068] The PRI101-GFP vector was selected to construct the localization vector of CsERF.E3 gene, the primers were designed according to the gene sequence, and the SaII / KPnI enzyme cutting sites were added in the forward and reverse direction primers according to the multiple cloning site of PRI101-GFP vector, the CDS without stop codon was fused to the PRI101-GFP vector to construct the fusion protein CsERF.E3:GFP, and the recombinant plasmid bacterial liquid was identified by PCR. The recombinant plasmid and empty plasmid were respectively transferred into Agrobacterium strain GV3101, and then transferred into the lower epidermis cells of Nicotiana benthamiana for transient expression.

[0069] The Agrobacterium infection of tobacco epidermis was carried out as follows:

[0070] 1) Bacterial activation: take the Agrobacterium GV3101 liquid containing recombinant plasmid and empty vector PRI101-GFP plasmid stored at-80℃, streak on LB solid medium (containing 25 mg / L Kana), and culture at 28℃ for 2 days to activate the bacteria;

[0071] 2) Small shaking of bacterial liquid: pick single colony in 5 mL LB liquid medium, shake at 28℃ and 220 r / min for 24 h;

[0072] 3) Large shaking of bacterial liquid: on the experimental day, take 300 μL of small shaking bacterial liquid and add it to a conical flask containing 30 mL of fresh liquid LB, shake at 28℃ and 220 r / min for about 10 h, until the OD value of the bacterial liquid is 0.7; 600

[0073] 4) Bacterial collection: after large shaking, the bacterial liquid was loaded into a 50 mL sterile centrifuge tube and centrifuged at 4000 r / min for 5 min, and the liquid was removed to make it flow out;

[0074] 5) Bacterial washing: add 10 mL of washing liquid (10 mM MES, 10 mM MgCl2, prepared fresh) to the bacteria, suspend thoroughly, centrifuge at 4000 r / min for 5 min, discard the liquid, add 5 mL of washing liquid and wash again, and then dissolve in 3 mL of washing liquid;

[0075] ​6) OD of the suspended bacterial solution 600 Value determination: the bacterial solution was diluted 20 times (for example: 150 μL of the bacterial solution + 2850 μL of the washing solution), and the OD of the bacterial solution:P19 was determined 600 The ratio was 0.7:0.5, 8 μL of acetyl-syringone (50 mg / mL) was added to 8 mL of the washing solution, and the mixture was mixed and incubated at 30°C for 3 h;

[0076] 7) Injection of tobacco leaves: 3 tobacco plants with the same growth and no disease were selected, and 2 leaves of each plant were injected. The back of the leaves was selected for injection (the back of the leaves was easier to inject because of the more stomata);

[0077] 8) Incubation and observation: after 24 h, fluorescence observation was performed by laser confocal.

[0078] The results are shown in Table 1. Figure 2 As shown in Table 1 (fluorescence detection results), the fluorescence of the fusion protein after recombination of CsERF.E3:GFP only appeared in the nucleus, while the fluorescence of the empty vector was distributed in the whole cell tissue. The results show that the CsERF.E3 gene is located in the nucleus and is a nuclear localization protein.

[0079] Example 3: Transcriptional activation analysis of the CsERF.E3 gene

[0080] Transcriptional activation activity is a basic feature of transcription factors. The applicant used the pGBKT7 vector to construct a recombinant, to verify whether CsERF.E3 has transcriptional activation activity.

[0081] According to the sequence of the gene, specific primers were designed, and enzyme digestion sites of EcoRI / SmaI were added to the forward and reverse primers of the pGBKT7 vector sequence information, respectively. The full-length CDS region of the gene was amplified and connected to the pGBKT7 vector to construct a recombinant plasmid, to obtain a fusion expression vector pGBKT7-CsERF.E3.

[0082] After sequencing to confirm that the sequence is correct, the fusion expression vector and the empty vector (pGBKT7) were respectively transferred into the same yeast strain AH109 (purchased from Kangwei Century). Finally, the positive identification of the bacterial solution was uniformly coated on the four incomplete solid culture media of SD / -Trp, SD / -Trp-His, SD / -Trp-His-Ade and SD / -Trp-His-Ade-Leu, respectively, and cultured on different deletion media to detect the survival of the transformants.

[0083] The results are shown in Table 2. Figure 3 As shown in Table 2, the yeast cells transformed with the empty vector could only grow on the deletion medium SD / -Trp, and the yeast transformed with the recombinant plasmid pGBKT7-CsERF.E3 could not grow on the incomplete medium.

[0084] The result shows that the transcription factor CsERF.E3 cannot bind with GAL4-BD, and cannot activate the transcription of the downstream reporter gene, so that the yeast cannot grow normally on the incomplete medium, that is, CsERF.E3 has no transcription activation function.

[0085] Example 4: Overexpression of gene CsERF.E3 in tomato

[0086] 1. Construction of plant transformation overexpression vector

[0087] A binary expression vector was constructed using the Gateway system, and the wild type Chongjiang navel orange cDNA was used as a template, and the primers were designed as follows:

[0088] F1: 5'-AAAAAGCAGGCTCCATGTGTGGCGGTGCACTTAT-3';

[0089] R1: 5'-AGAAAGCTGGGTTCTAACACAAGAACTGATTGCTGT TC-3'.

[0090] The synthesized cDNA by reverse transcription was used for PCR amplification of CsERF.E3 gene, and a single band of PCR product was produced after amplification, and after 1% agarose gel electrophoresis, the gel product obtained by amplification was purified and recovered by Omega gel recovery kit, and the product was subjected to BP reaction with the entry vector pDOR221, and then transformed into DH5α E. coli.

[0091] The next day, after picking single colonies for positive identification, the positive single colonies were selected in LB liquid medium containing kanamycin (Kan) resistance, positive clone detection and sample sequencing. After the sequencing result is correct, the ligation product is transformed into E. coli competent cells DH5α, and the positive strain is extracted by Kang Weishiji CWO500M plasmid, and the overexpression vector pK7WG2D-CsERF.E3 is constructed.

[0092] 2. Genetic transformation of tomato

[0093] Tomato is a good material for gene function verification in scientific research due to its short growth cycle and easy transformation. Mic-Tom is a dwarf plant of tomato, which is smaller than ordinary tomato, easy to plant and manage at high density. The steps of Agrobacterium-mediated genetic transformation of tomato are as follows:

[0094] 2.1 Inoculation

[0095] 1) Select seeds with full grains, soak in tap water for 1-2h;

[0096] 2) Sterilize the seeds with 75% ethanol for 1min (constant stirring);

[0097] 3) 50% 84 disinfectant solution for 15 min;

[0098] 4) 3 times of sterile water washing, sterile filter paper to dry the surface moisture of the seeds, evenly sowed on S1 medium;

[0099] 5) 16h light / 8h dark culture room for about a week.

[0100] 2.2 Cotyledon excision and Agrobacterium infection

[0101] 1) Strain activation and propagation: Agrobacterium containing the overexpression vector pK7WG2D-CsERF.E3 was streaked on LB (SPE resistant) solid medium and cultured at 28°C for 48h; after activation, the bacterial cells were re-applied to new LB (SPE resistant) solid medium and cultured at 28°C for 24h;

[0102] 2) 6-10 days after sowing, the tomato seedlings grew two cotyledons, and the leaf tips and the junctions of the leaves and petioles were excised with a sterile scalpel, and the middle sections were cut into two halves (about 0.4 cm long). The middle sections were placed on S2 co-culture medium with the back of the leaf facing up and cultured in the dark at 25±2°C for 24h;

[0103] 3) The propagated Agrobacterium was scraped with a sterile scalpel and resuspended in 50mL of Agrobacterium suspension, which was cultured at 28°C on a shaker for about 30min to an OD 600 0.2-0.6, and 50mg / L AS (acetyl-syringone) was added for standby;

[0104] 4) A sterile empty triangular flask was taken, and the cotyledons pre-cultured in the medium were placed in it, and the prepared Agrobacterium suspension was poured in and shaken for 5min for infection;

[0105] 5) The suspension was poured out, the surface moisture of the cotyledons was dried with sterile filter paper, and the cotyledons were re-placed on S2 co-culture medium (with the back of the leaf facing up) and cultured in the dark at 25±2°C for 2d.

[0106] 2.3 Selection culture

[0107] The leaves were transferred to S3 selection medium, and the cotyledons pre-cultured for 48h were inoculated on S3 selection medium with the front facing up, so that the incision was in full contact with the medium, and cultured under illumination at 25±2°C.

[0108] 2.4 Rooting culture

[0109] After about 7 days of culture, white callus grew out of the edges of the tomato leaves, and after 12 days, it was transferred to S3 subculture medium, and after 10-14 days, bud points differentiated from the callus, and after about 30 days, the buds grew into growth points and were transferred to S4 rooting medium (at this time, positive identification can be made), and after about 7 days, roots could be formed.

[0110] Table 1. Culture medium used for tomato seedling transformation

[0111]

[0112]

[0113] 3. Screening and identification of transgenic tomato seedlings with positive results

[0114] Transgenic tomato plants with the CsERF.E3 gene were obtained using the above method. Genomic DNA was extracted from the plant leaves. Forward and reverse primers were designed based on the 35S sequence on the vector and the gene sequence for amplification to verify whether the exogenous target gene had been inserted into the genome of the transformed material.

[0115] 3.1 DNA extraction from tomato leaves

[0116] 1) Preparation of DNA buffer (1L): 100mL of 1M Tris-HCl (pH 8.0), 100mL of 0.5M EDTA (pH 8.0), 300mL of 5M NaCl, and 500mL of H2O.

[0117] Among them, 1M Tris-HCl (pH 8.0): Weigh 121g of Tris-Base, add 800mL of water and stir on a magnetic stirrer until fully dissolved. Then add concentrated hydrochloric acid to adjust the pH to 8.0, add water to make up to 1000mL, sterilize and store at room temperature.

[0118] 0.5M EDTA (pH 8.0): Weigh 187g of EDTA-2Na salt and add it to about 800mL of water. While stirring on a magnetic stirrer, add solid NaOH. When both EDTA and NaOH are completely dissolved and the solution becomes clear, the pH will be around 8.0. Adjust the pH slightly with pH paper. After sterilization, store at room temperature.

[0119] 5M NaCl: Weigh 300g of NaCl, add about 800mL of distilled water and stir thoroughly on a magnetic stirrer. After about 5 minutes, stop stirring, let stand for 2-3 minutes, pour off the supernatant, add another 100mL of distilled water and repeat the previous steps until the NaCl is fully dissolved. Then, bring the volume to 1000mL, sterilize, and store at room temperature.

[0120] Preparation of phenol:chloroform:isoamyl alcohol (25:24:1): Mix water-saturated phenol, chloroform and isoamyl alcohol in a volume ratio of 25:24:1 and store in a brown bottle.

[0121] Preparation of 70% anhydrous ethanol: Mix anhydrous ethanol and water in a volume ratio of 7:3 and set aside.

[0122] The specific extraction steps are as follows:

[0123] 1) Take (0.64N x 1%) g PVP, (0.64N x 2%) g CTAB, 0.64 x N mL DNA buffer to prepare 0.64 x N mL CTAB buffer solution into a 10 mL centrifuge tube, dissolve in a 65°C water bath (N is the sample number);

[0124] 2) Take about 0.1 g of sample into a 1.5 mL centrifuge tube, add liquid nitrogen and grind;

[0125] 3) Add 100 μL of β-mercaptoethanol (1%-4%) to the CTAB buffer solution;

[0126] 4) Add 640 μL of the above CTAB buffer mixture to each sample, shake up and down (or place on a vortex shaker and mix well);

[0127] 5) 65°C water bath for 60-90 min;

[0128] 6) Add 700 μL of phenol: chloroform: isopropyl alcohol (25:24:1), shake up and down for about 5 min, then centrifuge at 13000 rpm for 8 min at 20°C;

[0129] 7) Take 500 μL of supernatant (yellow gun head), add 60 μL of 5M NaCl and 1 mL of pre-cooled anhydrous ethanol, mix well by inverting up and down, and place in a -20°C ice water bath for 30 min;

[0130] 8) Centrifuge at 13000 rpm for 6 min at 4°C;

[0131] 9) Discard the supernatant, add 1 mL of 70% anhydrous ethanol, and stand at -20°C for 2 h;

[0132] 10) Centrifuge at 10000 rpm for 5 min at 4°C;

[0133] 11) Discard the supernatant and dry on a clean bench (not too dry);

[0134] 12) Add 100 μL of TE and 1.5 μL of 10 μM RNase enzyme to each centrifuge tube (operate on a clean bench);

[0135] 13) 37°C water bath overnight.

[0136] 3.2 DNA positive identification

[0137] The positive plants were identified with specific primer 35S and gene reverse primer. In the selected transgenic lines, if a transgenic line can amplify a fragment of the expected size, it is a positive transgenic line. Finally, 8 positive plants were verified (as shown in Table 1). Figure 5

[0138] 35S: 5'-GACGCACAATCCCACTAT-3';

[0139] CsERF.E3-R1: 5'-AGAAAGCTGGGTTCTAACACAAGAACTGATTGCTGTTC-3'.

[0140] 4. Overexpression analysis of tomato transgenic positive seedlings

[0141] The RNA of the transgenic positive seedlings (denoted as #1, #2, … #8 in turn) that survived transplanting was extracted and reverse transcribed to synthesize cDNA (the RNA extraction method was the same as in Example 1), and the cDNA obtained by reverse transcription was diluted 5 times with ddH2O as a template, and the quantitative primer was designed using an online website.

[0142] The CsERF.E3 quantitative primer was:

[0143] CsERF.E3-qPCR-F: 5'-CACCCTAAGCAACCTCTCAAA-3';

[0144] CsERF.E3-qPCR-R: 5'-GCTCAAACTCGTCGTCTACTG-3'.

[0145] The tomato Actin gene was used as an internal reference gene, and the primers thereof were:

[0146] SlActin-F: 5'-GTCCTCTTCCAGCCATCCAT-3';

[0147] SlActin-R: 5'-ACCACTGAGCACAATGTTACCG-3'.

[0148] The expression amount of the gene CsERF.E3 was identified by qRT PCR method, and it was determined that the expression amount of CsERF.E3 gene in the positive transgenic tomato was relatively high (as shown in Table 2). Figure 5

[0149] 5. Fruit phenotype observation and physiological index determination of tomato transgenic positive seedlings

[0150] ​​Fruit skin color change means the beginning of maturity, which is often used to determine the starting time of fruit maturity. The phenotype of wild type (WT), and T2 generation of CsERF.E3 overexpression tomato was observed, and the time required from full bloom to the beginning of fruit color was analyzed, that is, the number of days of color breaking.

[0151] The results are as follows Figure 6 It is shown that the fruit color breaking of CsERF.E3 overexpression lines is earlier than that of wild type (WT), and the fruit of wild type begins to color at about 41 DAF (Days after flowering), and the fruit of CsERF.E3 overexpression lines begins to color at about 35 DAF. The results are as follows Figure 6 It is shown that the fruit color breaking of CsERF.E3 overexpression lines is earlier than that of wild type (WT), and the fruit of wild type begins to color at about 41 DAF (Days after flowering), and the fruit of CsERF.E3 overexpression lines begins to color at about 35 DAF. The results are as follows

[0152] The ABA content peak of tomato fruit overexpressing CsERF.E3 gene is at 35 days, and WT continues to rise until 51 days. The total amount of carotenoids continues to rise, and the content of tomato fruit overexpressing CsERF.E3 gene significantly increases from 35 days.

[0153] Example 5: Overexpression and silencing of CsERF.E3 gene in Citrus grandis

[0154] 1. VIGS silencing vector construction

[0155] The vectors used are pTRV1 and pTRV2. According to the characteristics of pTRV2 vector, EcoRI and SmaI enzymes are selected as forward and reverse enzyme digestion sites, and the primer is designed with the first 200 bp sequence of CsERF.E3 gene CDS as the template. The wild type Fengjie navel orange cDNA is used as the template, and the primer is designed as follows:

[0156] F2: 5'-GTGAGTAAGGTTACCGAATTCATGTGTGGCGGTGCTTAT-3';

[0157] R2: 5'-TGCTCGACGACAAGACCCGGGATTGCTTGAATCTTCAGCCTTC-3'.

[0158] After PCR amplification, the Omega gel recovery kit is used to purify and recover the gel product obtained by amplification, and then the linearized pTRV2 vector after double enzyme digestion is homologously recombined with the gel recovery product. After homologous recombination, E. coli DH5α is transformed, and then the company is sequenced. The correct bacterial liquid plasmid is extracted, the successfully constructed silencing vector is named pTRV2-CsERF.E3, and finally the Agrobacterium GV3101 is transformed. The positive verification correct bacterial liquid is placed in glycerol, and stored in -80℃ refrigerator.

[0159] 2. Instant transformation of kumquat

[0160] Agrobacterium culture containing the overexpression vector pK7WG2D-CsERF.E3 and the empty vector pK7WG2D was activated on LB (SPE) solid medium for 2 days. Agrobacterium culture containing the silencing vector pTRV2-CsERF.E3 and the empty vector pTRV2 was activated on LB (Kan) fixation medium for 2 days, followed by a second activation for 1 day, for subsequent genetic transformation.

[0161] The detailed steps of Agrobacterium-mediated transient transformation of kumquat are as follows:

[0162] 1) For fruit injection, after washing once with suspension buffer, mix the target bacterial solution with the P19 helper plasmid bacterial solution (P19 is a gene silencing repressor that can prevent post-transcriptional gene silencing in transgenic citrus fruits and promote high-level expression of target proteins), and adjust the OD of the mixed bacterial solution. 600 = Approximately 0.5.

[0163] 2) Inject 0.1–0.15 mL of infection solution into each fruit. The injection process should be slow and even, and the extent to which the infection solution reaches the subcutaneous layer of the fruit should be visible to the naked eye. Aspirate any excess Agrobacterium and take photographs. Photograph the fruit's condition every two days. After 14 days of infection, the CsERF.E3 gene expression level, ABA content, carotenoid content, and chlorophyll content will be measured.

[0164] Buffer formulation (10 mL): glucose (0.05 g), MES (500 mM, 1 mL), Na3PO4 (20 mM, 1 mL), acetylsyleugenol (1 M, 1 μL) and H2O (to 10 mL).

[0165] 1M Acetyleugenol (AS): Dissolve 0.0392g AS in 0.2mL DMSO, aliquot into 10μL portions and store at -20℃.

[0166] 20mM Na3PO4: Dissolve 0.17g of anhydrous Na3PO4 in 50mL of water and store at 4℃.

[0167] 500mM MES: Dissolve 4.88g of MES in 50mL of water and store at 4℃.

[0168] 3. The expression level of the gene CsERF.E3 in the transformed fruit of *Citrus aurantiacus* was identified using qRT-PCR. For example... Figure 8 As shown, the expression level in the superphenotype of Kumquat fruit was significantly higher than that in the PK7 control, and the expression level in the silent line fruit was significantly lower than that in the TRV empty vector. This indicates that the CsERF.E3 gene was successfully transiently transformed into the fruit.

[0169] 4. Phenotype observation of transgenic Poncirus fruit

[0170] The fruit color changes of the empty control group, CsERF.E3 gene overexpression lines and silencing lines were counted on the injection day and every two days. The results are shown in Table 2. Figure 9 It is shown that the fruit color breaking speed of the overexpression lines is faster than that of the wild type, and the fruit color breaking speed of the silencing lines is slower than that of the wild type.

[0171] After the carotenoid content was counted, it is shown in Table 3 that the carotenoid content of the silencing lines is significantly lower than that of the empty control, and the chlorophyll content is contrary; the carotenoid content of the overexpression lines is significantly higher than that of the wild type, and the chlorophyll content is contrary. Figure 10 It is shown that the carotenoid content of the silencing lines is significantly lower than that of the empty control, and the chlorophyll content is contrary; the carotenoid content of the overexpression lines is significantly higher than that of the wild type, and the chlorophyll content is contrary.

[0172] The above merely describes the preferred specific embodiments of the present application, but the protection scope of the present application is not limited thereto, and any person skilled in the art can make equivalent replacements or changes according to the technical solution and the inventive concept of the present application within the technical range disclosed by the present application, which should be covered within the protection scope of the present application.

Claims

1. CsERF.E3 Gene or CsERF.E3 Gene-encoded proteins or those containing CsERF.E3 Gene expression vectors or containing CsERF.E3 The host bacteria of the gene are used in any of the following (1)-(5): (1) Prepare products that reduce the number of days it takes for fruit to lose its color; (2) Prepare products that improve the rate of fruit color change; (3) Prepare products that increase the carotenoid content in fruits; (4) Prepare products that reduce the chlorophyll content in fruits; (5) Prepare products that promote fruit ripening; The CsERF.E3 Gene or CsERF.E3 Gene-encoded proteins or those containing CsERF.E3 Gene expression vectors or containing CsERF.E3 The host bacteria of the gene make the said CsERF.E3 Genes are overexpressed in plants; The fruit is a tomato or citrus; The CsERF.E3 The nucleotide sequence of the gene is shown in SEQ ID NO.1; CsERF.E3 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.

2.

2. A method for promoting fruit ripening, characterized in that, Overexpression in plants CsERF.E3 Gene; The fruit is a tomato or citrus; The CsERF.E3 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

3. Contains silence CsERF.E3 Gene expression vectors or those containing silencing agents CsERF.E3 The host bacteria of the gene are used in any of the following (1)-(5): (1) Prepare products that increase the number of days for fruit to break color; (2) Prepare products that reduce the rate of fruit discoloration; (3) Prepare products that reduce the carotenoid content in fruits; (4) Prepare products that increase the chlorophyll content in fruits; (5) Prepare products that delay fruit ripening; The fruit is either a tomato or a citrus fruit.

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