Uses of SmDREB1D in increasing anthocyanin content in plants

Abstract

This invention discloses the use of SmDREB1D in increasing anthocyanin content in plants, belonging to the field of biotechnology. This invention is the first to experimentally demonstrate that the protein SmDREB1D and its encoding gene provided by this invention can increase the anthocyanin content of plants: compared with the eggplant variety Africa eggplant, the total anthocyanin content in the stems and floral organs of T1 generation eggplants transgenic with the SmDREB1D gene is significantly increased. Therefore, the protein SmDREB1D and its encoding gene have important theoretical significance and practical value in regulating anthocyanin synthesis in plants.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to the use of SmDREB1D in increasing the anthocyanin content of plants. Background Technology

[0002] Anthocyanins are naturally occurring water-soluble pigments. Due to their antioxidant activity, they not only serve as a protective agent for plants against biotic and abiotic stresses, but also offer medicinal and health benefits in humans, particularly in combating diabetes, cancer, inflammation, bacteria, obesity, and preventing cardiovascular diseases. The higher the anthocyanin content of a plant, the greater its cultivation and economic value.

[0003] Eggplant (Solanum melongena L.) is one of the few purple vegetables, and its purple skin contains a large amount of anthocyanins. The main types of anthocyanins in purple eggplant skin are delphinidin-3-(p-coumaroylrhamnoside)-5-glucoside and delphinidin-3-rhamnoside. Only a few eggplant varieties and some wild species contain delphinidin-3-glucoside and petunidin-3-(p-coumaroylrhamnoside)-5-glucoside. In addition, some eggplant varieties are also rich in small amounts of delphinidin-3-caffeoylrhamnoside-5-glucoside (Azuma et al., 2008).

[0004] Genes influencing anthocyanin biosynthesis are divided into structural genes and regulatory genes. Structural genes directly encode the catalytic enzymes involved in anthocyanin biosynthesis, determining the final type of anthocyanin synthesized; these include PAL, CHS, CHI, DFR, and ANS. Regulatory genes encode transcription factors that bind to the promoters of structural genes, regulating their expression and intensity, thereby promoting or inhibiting their expression. Currently, transcription factors with relatively clear understanding in model plants include MYB, bHLH, and WD40. Compared to model plants such as Arabidopsis thaliana, research on anthocyanin biosynthesis in eggplant is significantly lagging ("Research Progress on Eggplant Anthocyanins," Molecular Plant Breeding, 2018). Therefore, screening and cloning key genes in eggplant anthocyanin biosynthesis can lay the foundation for genetic improvement of eggplant and the creation of new eggplant germplasm with high anthocyanin content. Summary of the Invention

[0005] In view of the above-mentioned prior art, the purpose of this invention is to provide the use of SmDREB1D in increasing the anthocyanin content of plants. This invention is the first to discover that overexpression of the SmDREB1D gene in eggplant can increase the anthocyanin content in the stems and floral organs of eggplant, which has significant application value.

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

[0007] In a first aspect, the present invention provides the use of the SmDREB1D gene as a positive regulatory gene in the following (1) or (2):

[0008] (1) Increase the anthocyanin content of plants;

[0009] (2) Cultivate plant varieties with increased anthocyanin content;

[0010] The SmDREB1D gene is a nucleic acid molecule as shown in i) or ii) below:

[0011] i) The nucleotide sequence is the nucleic acid molecule shown in SEQ ID NO.1;

[0012] ii) Nucleic acid molecules other than those in i) that encode the amino acid sequence shown in SEQ ID NO.2.

[0013] In a second aspect, the present invention provides the application of the protein encoded by the SmDREB1D gene as a positive regulator in increasing the anthocyanin content of plants.

[0014] Furthermore, the amino acid sequence of the protein encoded by the SmDREB1D gene is shown in SEQ ID NO.2.

[0015] In the above applications, the eggplant SmDREB1D gene or eggplant transcription factor SmDREB1D is used as the target. By overexpressing the eggplant SmDREB1D gene or increasing the expression level or activity of the eggplant transcription factor SmDREB1D, the anthocyanin content in eggplant can be positively regulated, thereby improving the quality and application value of eggplant.

[0016] A third aspect of the present invention provides the use of a recombinant expression vector or engineered bacteria containing the SmDREB1D gene in the following (1) or (2):

[0017] (1) Increase the anthocyanin content of plants;

[0018] (2) Cultivate plant varieties with increased anthocyanin content.

[0019] A fourth aspect of the present invention provides a method for increasing the anthocyanin content in eggplant, comprising the step of overexpressing the SmDREB1D gene in eggplant.

[0020] The above methods can induce SmDREB1D gene overexpression through the following pathways:

[0021] Exogenous transfer of the SmDREB1D gene;

[0022] Alternatively, upregulate the expression of the SmDREB1D gene in the eggplant genome.

[0023] A fifth aspect of the present invention provides a method for cultivating eggplant varieties with high anthocyanin content, comprising the following steps:

[0024] The SmDREB1D gene was transferred into wild-type eggplant plants, resulting in overexpression of the SmDREB1D gene in the wild-type eggplant plants, thus obtaining transgenic eggplant plants.

[0025] In the above method, the anthocyanin content of the transgenic eggplant plant is higher than that of the wild-type eggplant plant.

[0026] The methods described above for transferring the SmDREB1D gene into wild-type eggplant plants include: polyethylene glycol method, Agrobacterium-mediated method, or gene gun bombardment method.

[0027] The beneficial effects of this invention are:

[0028] This invention is the first to experimentally demonstrate that the protein SmDREB1D and its encoding gene provided by this invention can increase the anthocyanin content of plants: compared with the eggplant variety Africa eggplant, the total anthocyanin content in the stems and floral organs of the T1 generation eggplant transgenic with the SmDREB1D gene was significantly increased. Therefore, the protein SmDREB1D and its encoding gene have important theoretical significance and practical value in regulating anthocyanin synthesis in plants. This invention has significant applications and market prospects in the agricultural field. Attached Figure Description

[0029] Figure 1 To identify the transcriptional level of T1 generation transgenic eggplant containing the SmDREB1D gene.

[0030] Figure 2 Phenotypic identification of T1 generation eggplant transgenic with the SmDREB1D gene.

[0031] Figure 3 The results show the total anthocyanin content of T1 generation eggplant transgenic with the SmDREB1D gene. Detailed Implementation

[0032] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0033] As mentioned earlier, the higher the anthocyanin content in a plant, the greater its cultivation and economic value. Eggplant is one of the few purple vegetables, and its purple skin contains a large amount of anthocyanins, making it a promising candidate for various applications. However, compared to model plants such as Arabidopsis thaliana, research on the biosynthesis process of anthocyanins in eggplant is significantly lagging behind.

[0034] In view of this, this invention conducts an in-depth study on the biosynthesis of anthocyanins in eggplant. The AP2 / ERF transcription factor, named for its AP2 / ERF domain consisting of 60-70 amino acids, is a superfamily of transcription factors with numerous members. The AP2 / ERF family includes the DREB / CBF subfamily and the ERF subfamily. Current research on members of the DREB / CBF subfamily mainly focuses on plant resistance to abiotic stresses.

[0035] SmDREB1D is a member of the DREB subfamily of AP2 / ERF transcription factors. The nucleotide sequence of the SmDREB1D gene is shown in SEQ ID NO.1, as follows:

[0036] ATGAATAATAACTTGATGTCATGTTTGTCAGATTCTAATACGATAAACTTGGAGGAAGCATATGGAGGAGTATTCCCATTAGCTTCGAGTCAACCAAAGAAACGTGCTGGAAGAAAGAAGTTCAAGGAAACTCGCCACCCAATTTATAGGGGAGTGAGGAGAAGGAATAAT AACAAGTGGGTTTGCGAAGTACGTGAGCCTAGTCAACAAAAAAGAATATGGTTAGGGACTTATCCTACTCCAGAAATGGCGGCTCGAGCTCATGATGTAGCTGCATTAGCACTTAGAGGTAATCTAGCCACTTTAAATTTCGCAGACTCTCATTGGCGATTACCAATGCCGG TATCTAAGGACCCTAAAGACATACGTCAGGCGGCTATAAAAGCAGCTAAAGCATTTTGTCAGAATACTGAATTAGTTGGGGTTGATTCCAATAGTATTGAAGAAGTAAAGTACCAAGAGGTTGATGTTGCGAGGGCTAATAGTGGAAGTAGTAGTGATTTTGGCGCGAAGG AATTGAATATGGACATGGAGAACATTTTATGTTGTAATTCAGGAGAAGATAATGAGATATTGGAGATGGAAGGATGGCGAGAAAAGATGGCAGAGGGGCTTCTGTTTTCACCAACTCCGCGGTTAAGTAGTTGTTTCAGTTGGGATAACATGGAGATTAGTGACATTGAGGT GTCTTTGTGGAGTTATAATATTTAA.

[0037] The amino acid sequence of the protein SmDREB1D encoded by the SmDREB1D gene is shown in SEQ ID NO.2, as follows:

[0038] MNNNLMSCLSDSNTINLEEAYGGVFPLASSQPKKRAGRKKFKETRHPIYRGVRRRNNNKWVCEVREEPSQQKRIWLGTYPTPEMAARAHDVAALALRGNLATLNFADSHWRLPMPVSKD PKDIRQAAIKAAKAFCQNTELVGVDSNSIEEVKYQEVDVARANSGSSSDFGAKELNMDMENILCCNSGEDNEILEMEGWREKMAEGLLFSPTPRLSSCFSWDNMEISDIEVSLWSYNI.

[0039] The function of the SmDREB1D gene and its encoded protein is currently unclear. This invention is the first to explore the function of this gene. Through transgenic experiments, this invention demonstrates that overexpression of the SmDREB1D gene in eggplant significantly increases the anthocyanin content. Therefore, the SmDREB1D gene is involved in anthocyanin synthesis in eggplant and has significant application value, hence this invention.

[0040] To enable those skilled in the art to better understand the technical solutions of this application, the technical solutions of this application will be described in detail below with reference to specific embodiments. The experimental materials used in the embodiments of this invention are all conventional experimental materials in the art and can be purchased through commercial channels.

[0041] Example 1: Cloning of the eggplant SmDREB1D gene

[0042] 1. Plant materials:

[0043] The transgenic recipient material used in this invention is African red tomato, whose anthocyanins mainly accumulate in the stem epidermis and calyx.

[0044] 2. RNA extraction and reverse transcription:

[0045] Total RNA was extracted from eggplant using the MiniBEST Universal RNA Extraction Kit (TaKaRa) following the standard instruction manual. It was then reverse transcribed into cDNA (PrimeScript). TM RT Master Mix (Bao Ri Medical Biotechnology Co., Ltd.)

[0046] 3. Cloning verification of SmDREB1D:

[0047] Based on the gene number SMEL4.1_08g001300.1.01 in the eggplant genome (https: / / solgenomics.net / organism / Solanum_melongena / genome) V4.1, primers were designed as follows:

[0048] F: 5′-TCTCTCTCTCAAGCTTATGAATAATAACTTGATGTCATGTTTGTC-3′; (SEQ ID NO.3)

[0049] R: 5′-TGCAGCTCGAGGATCCTTAAATATTATAACTCCACAAAGACACCTC-3′. (SEQ ID NO.4)

[0050] The PCR amplification product was ligated into the GFP-tagged plant expression vector pHB (described in the literature "MicroRNA171c-targeted SCL6-II, SCL6-III, and SCL6-IV genes regulate shootbranching in Arabidopsis, doi.org / 10.1093 / mp / ssq042"), transformed into Escherichia coli Trans1-T1 (purchased from Beijing TransGen Biotech Co., Ltd.), and sent to Sangon Biotech (Qingdao) Co., Ltd. for sequencing. Plasmids were extracted from the bacterial cultures with correct sequencing results and transformed into Agrobacterium LBA4404 (purchased from Shanghai Weidi Biotechnology Co., Ltd.). After PCR verification, the samples were stored at -80℃, thus successfully constructing the SmDREB1D-GFP vector.

[0051] Example 2: Obtaining transgenic eggplant plants by Agrobacterium infection

[0052] Eggplant seeds of the 'African Red Eggplant' variety were sown in a clean bench and cultured in the dark until germination. After germination, the seeds were transferred to light and cultured for approximately 7 days until the cotyledons expanded. The cotyledons were then cut and laid flat on a pre-medium. Agrobacterium LBA4404 containing the SmDREB1D-GFP vector was shaken overnight. The next day, the cells were centrifuged at 3000g for 10 minutes. The supernatant was discarded in a clean bench. The Agrobacterium precipitate adhering to the bottom of the centrifuge tube was dissolved in MS liquid medium to prepare an Agrobacterium suspension. An appropriate amount was added to 20 ml of MS liquid medium to achieve an infection concentration (OD value) of approximately 0.3. Using sterile forceps, the cotyledonary explants on the pre-medium were gently immersed in the Agrobacterium suspension for 15–20 minutes, shaking every 5 minutes. After infection, the cotyledons were removed by forceps, taking care not to damage them. The surface of the explants was blotted dry with sterile paper, and the cotyledons were then inoculated onto a co-medium and co-cultured in the dark for 2 days. The infected cotyledons were placed on a selection medium for growth. The medium was changed approximately every 14 days, and regenerated plants were obtained through callus differentiation in about 4 months.

[0053] Pre-medium: MS medium + 30 mg / L sucrose -1 +Zephyll 2.0 mg·L -1 +7g / L agar powder -1 The pH value is 5.8.

[0054] Co-culture medium: MS + 30 mg / L sucrose -1 +Zephyll 2.0 mg·L -1 + Acetyleugenone 100 mmol·L -1 +7g / L agar powder -1 The pH value is 5.8.

[0055] Selection medium: MS medium + 30 mg / L sucrose -1 +Zephyll 2.0 mg·L -1 +Carbenicillin 300 mg / L -1 + Hygromycin 50 mg / L -1 +7g / L agar powder -1 The pH value is 5.8.

[0056] Example 3: Validation of SmDREB1D transgenic eggplant plants

[0057] Real-time quantitative PCR was used to detect the expression of the SmDREB1D gene in regenerated plants. The specific steps are as follows:

[0058] ① Total RNA was extracted from eggplant using the MiniBEST Universal RNA Extraction Kit (TaKaRa) following the standard instruction manual. It was then reverse transcribed into cDNA (PrimeScript).TM RT Master Mix (Bao Ri Medical Biotechnology Co., Ltd.)

[0059] ②Real-time quantitative PCR was used to detect the relative expression level of the SmDREB1D gene (with the SmActin7 gene as an internal reference primer).

[0060] Design quantitative primers using Primer Express 3.0.1 software:

[0061] The specific primers for SmDREB1D gene quantification analysis are:

[0062] qRT-SmDREB1D-F: 5′-AAGTGGGTTTGCGAAGTACG-3′; (SEQ ID NO.5)

[0063] qRT-SmDREB1D-R: 5′-TCCTTAGATACCGGCATTGG-3′. (SEQ ID NO.6)

[0064] The internal reference gene is SmActin7, and the specific primers for its quantitative analysis are:

[0065] qRT-ACTIN-F: 5′-GTCGGAATGGGACAGAAGGATG-3′; (SEQ ID NO.7)

[0066] qRT-ACTIN-R: 5′-GTGCCTCAGTCAGGAGAACAGGGT-3′. (SEQ ID NO.8)

[0067] Test results are shown Figure 1 The expression level of SmDREB1D in regenerated plants was significantly higher than that in wild-type plants.

[0068] Example 4: Detection of anthocyanin content in SmDREB1D transgenic eggplant plants

[0069] After the plants to be tested enter the flowering and fruit-setting stage, observe the plant phenotype. For example... Figure 2 As shown, the purple color of the stem epidermis and floral organs of the transgenic eggplant plants is significantly deepened.

[0070] The anthocyanin content in the stems and floral organs of wild-type and SmDREB1D transgenic eggplant plants during their full-blown flowering period was determined as follows: A certain mass of sample was weighed and placed in a 1.5 mL centrifuge tube. Steel beads were added, and the tubes were frozen in liquid nitrogen. The samples were then thoroughly ground using a high-throughput grinder. 300 μL of 1% hydrochloric acid in methanol (V / V) was added to each centrifuge tube, and the tubes were incubated overnight at 4°C in the dark. Then, 200 μL of water and 500 μL of chloroform were added, and the tubes were centrifuged at 14000 rpm for 5 minutes. 400 μL of the supernatant was collected, and 400 μL of 60% methanol in 1% HCl (1% hydrochloric acid in methanol:water = 6:4 (V / V)) was added to each centrifuge tube. The absorbance at 530 nm and 657 nm was read using a spectrophotometer, and the anthocyanin content was quantified based on the difference in absorbance.

[0071] The results showed that the anthocyanin content in the stem epidermis and floral organs of transgenic eggplant plants was significantly increased. Figure 3 The above results demonstrate that overexpression of the SmDREB1D gene can significantly increase the total anthocyanin content of eggplant.

[0072] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. SmDREB1D Use of the gene as a positive regulatory gene in (1) or (2) below: (1) Increase the anthocyanin content in the stem epidermis and floral organs of African red eggplant plants; (2) Cultivate plant varieties with increased anthocyanin content; The SmDREB1D Genes are nucleic acid molecules as shown in i) or ii) below: i) The nucleotide sequence is the nucleic acid molecule shown in SEQ ID NO.1; ii) Nucleic acid molecules other than those in i) encoding the amino acid sequence shown in SEQ ID NO. 2; The plant species is African red eggplant.

2. SmDREB1D The application of a gene-encoded protein as a positive regulator in increasing anthocyanin content in the stem epidermis and floral organs of African red eggplant plants is characterized by, The SmDREB1D The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.

2.

3. Contains SmDREB1D The application of recombinant gene expression vectors or engineered bacteria in the following (1) or (2): (1) Increase the anthocyanin content in the stem epidermis and floral organs of African red eggplant plants; (2) Cultivate plant varieties with increased anthocyanin content; The SmDREB1D Genes are nucleic acid molecules as shown in i) or ii) below: i) The nucleotide sequence is the nucleic acid molecule shown in SEQ ID NO.1; ii) Nucleic acid molecules other than those in i) encoding the amino acid sequence shown in SEQ ID NO. 2; The plant species is African red eggplant.

4. A method for increasing the anthocyanin content in the stem epidermis and floral organs of African red eggplant plants, characterized in that, include: In eggplant SmDREB1D The steps involved in gene overexpression; The SmDREB1D Genes are nucleic acid molecules as shown in i) or ii) below: i) The nucleotide sequence is the nucleic acid molecule shown in SEQ ID NO.1; ii) Nucleic acid molecules other than those in i) that encode the amino acid sequence shown in SEQ ID NO.

2.

5. The method according to claim 4, characterized in that, Through the following means SmDREB1D Gene overexpression: Exogenous transfer SmDREB1D Gene; Or, upregulate the eggplant genome SmDREB1D Gene expression.

6. A method for cultivating eggplant varieties with high anthocyanin content, characterized in that, Includes the following steps: Will SmDREB1D Genes were transferred into wild-type eggplant plants, resulting in wild-type eggplant plants... SmDREB1D Gene overexpression was used to obtain transgenic eggplant plants; The SmDREB1D Genes are nucleic acid molecules as shown in i) or ii) below: i) The nucleotide sequence is the nucleic acid molecule shown in SEQ ID NO.1; ii) Nucleic acid molecules other than those in i) encoding the amino acid sequence shown in SEQ ID NO. 2; The wild-type eggplant plant mentioned is the African Red Eggplant.

7. The method according to claim 6, characterized in that, The anthocyanin content of the transgenic eggplant plants was higher than that of the wild-type eggplant plants.

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