SmERF1B gene cloned in eggplant and application thereof

Abstract

The invention relates to a SmERF1B gene cloned from eggplant and application of the SmERF1B gene in regulating and controlling the anthocyanin content of eggplant peel. The maximum reading frame sequence of the SmERF1B gene is represented by SEQ ID NO.1, and the amino acid sequence coded by the SmERF1B gene is represented by SEQ ID NO.2. The eggplant is transformed by adopting an agrobacterium tumefaciens-mediated method, and the result proves that compared with a non-transformed plant, the anthocyanin content in the peel of the transgenic eggplant is obviously increased, so that the color of the peel is darkened. The method is helpful for further understanding the eggplant peel coloring mechanism, so that the eggplant peel coloring can be regulated and controlled by utilizing a molecular means, and a new eggplant variety with high anthocyanin content is bred.

Description

Technical Field

[0001] This invention relates to a SmERF1B gene cloned from eggplant and its application in regulating the anthocyanin content of eggplant pericarp. Background Technology

[0002] Eggplant (Solanum melongena L.), a vegetable belonging to the genus Solanum in the family Solanaceae, originated in India, with China being its second place of origin. my country's eggplant production is 38.32 million tons, accounting for 64.6% of the world's total production (FAO, 2025). Due to differences in the types and contents of anthocyanins, eggplant fruits come in various colors, including purplish-red and purplish-black (purple eggplant), white (white eggplant), and green (green eggplant). Purple eggplant has the highest anthocyanin content and is one of the few purple vegetables. Anthocyanins have antioxidant, anti-inflammatory, anti-aging, and cardiovascular disease prevention effects (Wang et al., 2022; Lee et al., 2023; Li et al., 2023). Therefore, breeding purple eggplants with high anthocyanin content has become one of the breeding goals for eggplant.

[0003] ERF (ethylene responsive factor) transcription factors are a subfamily of the AP2 / ERFBP transcription factor family. ERF transcription factors all contain four functional domains: a DNA-binding domain, a transcriptional regulatory domain, a nuclear localization signal, and an oligomerization site. The DNA domain, a highly conserved functional domain consisting of approximately 60 amino acids (the AP2 / ERF domain), can specifically bind to DNA, enabling transcriptional regulation of target genes to be expressed at specific times, spaces, and in specific intensities. Therefore, investigating the regulatory role of the SmERF1B gene in eggplant on anthocyanin content in the pericarp is of great significance for breeding new eggplant varieties with high anthocyanin content. Summary of the Invention

[0004] In view of the above problems, according to an objective of the present invention, a SmERF1B gene cloned from eggplant pericarp is provided, the largest reading frame (ORF) sequence of which is represented by SEQ ID NO:1.

[0005] According to a specific embodiment of the present invention, the ORF of the SmERF1B gene is 696 bp in size, encoding 232 amino acids, and has a highly conserved AP2 / ERF domain (e.g., Figure 1 As shown in the figure, this indicates that SmERF1B belongs to the ERF transcription factor family.

[0006] Furthermore, the amino acid sequence encoded by the SmERF1B gene is represented by SEQ ID NO:2.

[0007] According to another specific embodiment of the present invention, the SmERF1B gene is expressed in the pericarp of the fruit at different developmental stages. Specifically, using 18S rRNA as an internal reference gene, real-time fluorescence quantitative expression analysis showed that the SmERF1B gene was expressed in the pericarp of the fruit at different developmental stages under different temperature treatments, with the highest expression level observed at 25℃ for 20 days. Specifically, the expression level of the SmERF1B gene at 35℃ was significantly lower than that at 25℃ (e.g., ...). Figure 2 (As shown).

[0008] According to another aspect of the present invention, the application of the SmERF1B gene in regulating the anthocyanin content of eggplant pericarp is also provided. The application includes forward ligation of the SmERF1B gene to a plant expression vector (e.g., pCAMBIA1302) to obtain a recombinant plasmid, such as pCAMBIA1302-SmERF1B. The recombinant plasmid (e.g., pCAMBIA1302-SmERF1B) is then transformed into an Agrobacterium strain (e.g., GV3101). After transformation of eggplant using Agrobacterium-mediated transformation, the anthocyanin content in the eggplant pericarp increases significantly, for example, by 150-200%, or for example, 180%, compared to the anthocyanin content of non-transformed plants.

[0009] According to another aspect of the present invention, a vector for the above-mentioned SmERF1B gene is provided.

[0010] According to another aspect of the present invention, a protein encoded by the above-mentioned SmERF1B gene is provided, the amino acid sequence of which is represented by SEQ ID NO:2.

[0011] The SmERF1B gene of this invention can increase the anthocyanin content of eggplant peel, resulting in a darker peel color. This helps to further understand the coloring mechanism of eggplant peel, thus enabling the use of molecular methods to regulate eggplant peel coloring and breed new eggplant varieties with high anthocyanin content. Attached Figure Description

[0012] Figure 1 This indicates the amino acid sequence alignment of the ERF homologous gene.

[0013] Figure 2 The expression of SmERF1B at different developmental stages of fruit under different temperature treatments is shown.

[0014] Figure 3 The PCR detection results of the SmERF1B transgenic plants are shown.

[0015] Figure 4 The results of anthocyanin content determination in SmERF1B transgenic plants are shown. Detailed Implementation

[0016] The present invention will be further described in detail below with reference to specific embodiments. These embodiments are only used to illustrate the present invention and do not limit the scope of the present invention.

[0017] The ERF1B gene provided by this invention was cloned from eggplant pericarp and named SmERF1B gene. The largest ORF sequence is represented by SEQ ID NO:1 (Sequence 1).

[0018] The protein encoded by the SmERF1B gene is named SmERF1B protein, and its amino acid sequence is represented by SEQ ID NO:2 (Sequence 2).

[0019] I. Cloning and Sequence Analysis of the SmERF1B Gene

[0020] The cloning method and sequence analysis of the SmERF1B gene described above include the following steps:

[0021] 1. RNA extraction

[0022] Using the pericarp of the high-generation inbred line 30# of purple-red eggplant as material, total RNA was extracted from the eggplant pericarp using the Beijing Huayueyang RNA Extraction Kit. The specific method is as follows:

[0023] (1) Grind the eggplant peel into powder in a mortar containing liquid nitrogen. Take 50-100 mg of sample into a centrifuge tube, add 1 mL of cell lysis buffer, and shake well to mix.

[0024] (2) Add 300 μL of protein removal solution and 200 μL of chloroform to a centrifuge tube, and vortex for 1 min to mix thoroughly. At this time, the solution is a uniform emulsion.

[0025] (3) Let stand at room temperature for 2 min, then centrifuge at 12000 rpm at room temperature for 10 min, and transfer the supernatant to another clean centrifuge tube. The supernatant should not exceed 700 μL.

[0026] (4) Add an equal volume of rinsing solution to the supernatant, mix thoroughly by inverting, transfer the mixture to a centrifugal adsorption column, centrifuge at 12000 rpm for 1 min at room temperature, and discard the breakthrough liquid.

[0027] (5) Add 500 μL of washing buffer, centrifuge at 12000 rpm for 1 min at room temperature, and discard the breakthrough liquid; repeat this step, and then centrifuge at 12000 rpm for 1 min at room temperature, and discard the breakthrough liquid;

[0028] (6) Add 6 μL of RNase-free DNaseI to 44 μL of DNase buffer (thawed on ice), mix well, add to the center of the membrane of the centrifugal adsorption column, and incubate at 37°C for 20–30 min.

[0029] (7) Add 500 μL of enzyme removal solution directly to the centrifugal adsorption column, invert and mix several times, centrifuge at 12000 rpm for 1 min at room temperature, and discard the breakthrough liquid; repeat this step once.

[0030] (8) Centrifuge at 12,000 rpm for 2 min at room temperature to remove residual ethanol;

[0031] (9) Transfer the centrifugal adsorption column to an RNase-free tube, add 50 μL of RNA elution buffer, and incubate at room temperature for 3 min;

[0032] (10) Centrifuge at 12,000 rpm for 1 min at room temperature. The sample in the centrifuge tube is the RNA sample.

[0033] (11) Take 2 μL of RNA for electrophoresis to check its integrity.

[0034] 2. Reverse transcription

[0035] Reverse transcription was performed using a reverse transcription kit from Shanghai Toyobo Biotechnology Co., Ltd. to obtain the first strand of cDNA, which was used as a template for subsequent PCR amplification.

[0036] 3. Primer design

[0037] Primers for full-length SmERF1B cDNA amplification were designed based on eggplant genome data (http: / / eggplant.kazusa.or.jp / ):

[0038] ERF1B-F:5'-AGCTATTTAGCGACAGATTA-3'

[0039] ERF1B-R:5'-TCAGTATTTTCAACACGAGA-3'

[0040] The reaction system consisted of 12.5 μL PCR buffer, 8.4 μL sterile deionized water, 0.1 μL Taq enzyme, 1 μL each of forward and reverse primers (10 μM), and 2 μL cDNA template. The PCR amplification conditions were: denaturation at 94℃ for 3 min; denaturation at 94℃ for 0.5 min, annealing at 48℃ for 0.5 min, extension at 72℃ for 2 min, for 35 cycles; and extension at 72℃ for 10 min.

[0041] 4. The PCR reaction solution was sent to Qingke Biotechnology Co., Ltd. for sequencing, and the results confirmed that the obtained product was the full-length eggplant SmERF1B cDNA. Its full length is 889bp, the largest ORF is 696bp (SEQ ID NO:1), and it encodes 232 amino acids (SEQ ID NO:2).

[0042] 5. Sequence Analysis

[0043] Homologous sequence searches and alignments on NCBI revealed that SmERF1B shares a conserved AP2 / ERF domain with other homologous sequences. Figure 1 This indicates that SmERF1B belongs to the ERF family of transcription factors, and these structures are closely related to their protein functions.

[0044] II. Construction of plant expression vectors containing the SmERF1B gene

[0045] PCR reaction system: The total reaction volume of 25 μL contains 2 μL of template first-strand cDNA, 1 μL each of forward and reverse primers (10 μmol / L), 0.1 μL of Taq DNA polymerase (5 U / μL), 12.5 μL of 2×Mix buffer, and the volume is made up with sterile distilled water.

[0046] The PCR reaction conditions were: 94℃ for 3 min; 94℃ for 30 sec, 48℃ for 30 sec, 72℃ for 2 min, 35 cycles; extension at 72℃ for 10 min. The amplified ERF1B was ligated to pMD19-T and named pMD19-SmERF1B. After transformation into DH5α competent cells, transformants were selected using a blue-white screening method. White transformants were used for PCR detection, and positive transformants were sent to the company for sequencing. The sequencing results confirmed that the obtained gene was the SmERF1B gene containing the coding region and restriction enzyme sites.

[0047] 2. Construction of plant expression vectors

[0048] The target gene fragment was excised from the recombinant plasmid pMD19-SmERF1B containing the target gene using restriction endonucleases BglII and SpeI. Simultaneously, the pCAMBIA1302 vector was digested with both BglII and SpeI. After separation by agarose gel electrophoresis, the target gene fragment and the large fragment of the digested pCAMBIA1302 vector were recovered using a gel recovery kit from Tiangen Biotech Co., Ltd. SmERF1B was inserted forward into the pCAMBIA1302 vector using T4 DNA ligase. The ligation product was transformed into *E. coli* DH5α competent cells, and the plant expression vector pCAMBIA1302-SmERF1B was obtained after PCR identification.

[0049] 3. Introduction of Agrobacterium into plant expression vectors

[0050] After thawing competent Agrobacterium GV3101 cells on ice, add 1 μg pCAMBIA1302-SmERF1B plasmid DNA, mix well, incubate on ice for 30 min, place in liquid nitrogen for 1 min, and then quickly transfer to a 37℃ water bath for 5 min. After thawing, add 0.8 mL of freshly prepared LB liquid medium (without antibiotics) and incubate at 28℃ with shaking for 2–4 h. Spread 200 μL of the bacterial culture onto LB solid medium containing 20 mg / L rifampicin (Rif), incubate at 28℃ for 2–3 days, and perform PCR detection on the colonies.

[0051] 4. Transformation of eggplant with the target gene

[0052] Select a single clone of the identified positive Agrobacterium and culture it in 10 mL of LB liquid medium containing 20 mg / L Rif, shaking at 28°C and 180 rpm until the optical density (OD) is approximately 0.6. One day before transformation, transfer 1 mL of this culture to 100 mL of LB liquid medium containing 20 mg / L Rif and culture overnight with shaking. The next day, when the OD600 of the culture is between 0.8 and 1.2, it is ready for use.

[0053] Pour Agrobacterium-methyl bacterial solution into a petri dish, immerse the eggplant explants in the solution, and soak for 10 minutes, shaking occasionally. Remove the explants and co-culture them in MS medium supplemented with 0.2 mg / L 6-benzylaminopurine (6-BA) and 0.2 mg / L naphthaleneacetic acid (NAA) for 2 days. Then transfer them to an antibacterial selection medium (MS medium supplemented with 0.2 mg / L 6-BA, 0.2 mg / L NAA, and 400 mg / L termethin). After callus formation, shoots emerge, and the shoots are cut off and inoculated into rooting medium (MS medium supplemented with 30 mg / L kanamycin). After rooting, transplant them into nutrient pots.

[0054] 5. Application of the SmERF1B gene

[0055] 1) PCR detection of transformed plants

[0056] Genomic DNA was extracted from transformed plants and used as a template. PCR verification was performed using primers YZ-ERF1B-F: CTT CGC AAG ACC CTT CCTCT and YZ-ERF1B-R: GTT GGC CAC GGA ACA GGTA. The results showed that the SmERF1B gene had been integrated into the eggplant genome. Figure 3 ).

[0057] 2) Determination of anthocyanin content in transformed plants

[0058] Non-transformed plants from the same period were used as controls. The anthocyanin extraction method was based on and slightly optimized from the method described by Shao Wenting (2013) for extracting anthocyanins from eggplant peel. 4 mL of 2% HCl-C2H5OH extractant was added to each sample, and the mixture was extracted at 32℃ in the dark for 2 hours, shaking several times during the extraction. The sample was then centrifuged at 8000g for 10 minutes at 4℃, and the supernatant was collected. This extraction was repeated twice, and the supernatants were combined and diluted to 10 mL. The anthocyanin extract was diluted 10-fold with pH 1.0 and pH 4.5 buffer solutions, respectively, and stabilized for 60 minutes. The absorbance was measured at 530 nm and 700 nm, respectively. The results are as follows: Figure 4 As shown, compared with non-transformed plants, the anthocyanin content in the peel of eggplant from transformed plants was significantly higher, indicating that SmERF1B promoted the accumulation of anthocyanins in the peel, resulting in a darker peel color.

[0059] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention.

[0060]

[0061]

Claims

1. A SmERF1B gene cloned from eggplant, the maximum reading frame of which is represented by SEQ ID NO:

1.

2. The SmERF1B gene according to claim 1, which encodes 232 amino acids and has a highly conserved AP2 / ERF domain of the ERF transcription factor family.

3. The SmERF1B gene according to claim 1, wherein, The amino acid sequence encoded by the SmERF1B gene is represented by SEQ ID NO:

2.

4. The SmERF1B gene according to claim 1, wherein, The SmERF1B gene is expressed in the pericarp at different developmental stages of the fruit.

5. The application of the SmERF1B gene according to any one of claims 1 to 4 in regulating the anthocyanin content of eggplant pericarp.

6. The application according to claim 5, wherein, The applications include: The SmERF1B gene was forward-linked to a plant expression vector to obtain a recombinant plasmid. The recombinant plasmid was transformed into Agrobacterium, and the transformation of eggplant using Agrobacterium-mediated transformation increased the anthocyanin content in the eggplant peel.

7. The application according to claim 6, wherein, The plant expression vector was pCAMBIA1302, and the Agrobacterium strain was GV3101.

8. The vector of the SmERF1B gene according to any one of claims 1 to 4.

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