Eggplant bHLH family transcription factor SmbHLH93 protein and coding gene thereof

Abstract

The invention discloses an eggplant bHLH family transcription factor SmbHLH93 protein and a coding gene thereof, the gene participates in regulating eggplant anthocyanin synthesis, the SmbHLH93 gene is overexpressed in wild-type arabidopsis thaliana, so that arabidopsis thaliana anthocyanin synthesis can be promoted, and a foundation is laid for improving plant quality by utilizing a genetic engineering technology in the future. A theoretical basis is provided for obtaining medicines or foods with high anthocyanin, and the method has a great application value.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, relates to key enzymes in an eggplant light signal pathway and coding genes thereof, and specifically relates to an eggplant bHLH family transcription factor SmbHLH93 protein and a coding gene and application thereof. Background Art

[0002] Anthocyanidins, also known as flower pigments, are the most important component of flavonoid compounds. They are mainly present in fruits and vegetables in the form of water-soluble pigments. They have the functions of reducing the risk of cardiovascular diseases, anti-oxidation, and anti-mutagenicity. Eggplant contains a large amount of anthocyanidins, which is very beneficial to human health. This makes the study of the anthocyanidin synthesis mechanism in eggplant an important topic for improving the nutritional value of crops and developing functional foods.

[0003] The synthesis of anthocyanins requires basic helix-loop-helix transcription factors, i.e., bHLH transcription factors. bHLH transcription factors contain a highly conserved basic helix-loop-helix (bHLH) domain and are commonly found in various eukaryotic organisms. These transcription factors participate in epidermal differentiation, respond to environmental stresses, or regulate secondary metabolism in plants. They bind to specific DNA sequences to activate or inhibit the expression of downstream target genes, thereby regulating a series of complex biological processes. In particular, the bHLH transcription factor family plays an indispensable role in the biosynthesis of anthocyanins, ensuring the precise regulation of the anthocyanin synthesis pathway.

[0004] The bHLH transcription factor family plays an important role in the biosynthesis of anthocyanins in eggplant. However, the current research on bHLH is mainly focused on a few model plants, such as Arabidopsis thaliana, but the verification of bHLH genes and their functions in non-model crops, especially economic crops such as eggplant, is relatively scarce. The main reason for this phenomenon is that the bHLH gene family is very large and has many branches, making it difficult to systematically screen and analyze its functions using traditional methods. Summary of the invention

[0005] The invention provides an eggplant bHLH family transcription factor SmbHLH93 protein and a coding gene thereof.

[0006] In a first aspect, the present invention provides an eggplant bHLH family transcription factor SmbHLH93 protein, comprising the amino acid sequence shown in SEQ ID No.2.

[0007] In a second aspect, the present invention provides a nucleic acid sequence of a gene encoding eggplant bHLH family transcription factor SmbHLH93 protein.

[0008] Furthermore, the cDNA sequence of the gene includes the base sequence shown at positions 1 to 1185 shown in SEQ ID NO.1.

[0009] Furthermore, the base sequences of the primer pair for amplifying the gene of the eggplant bHLH family transcription factor SmbHLH93 protein are shown in SEQ ID NO.3 and SEQ ID NO.4.

[0010] Furthermore, the base sequences of the primer pairs used for fluorescence quantitative PCR analysis of the gene encoding the eggplant bHLH family transcription factor SmbHLH93 protein are shown in SEQ ID NO.5 and SEQ ID NO.6.

[0011] Preferably, the eggplant SmbHLH93-related nucleotide full-length sequence or a fragment thereof can be obtained by PCR amplification, recombination or artificial synthesis.

[0012] In a third aspect, the present invention provides an application of the gene encoding the eggplant bHLH family transcription factor SmbHLH93 protein in promoting the synthesis of plant anthocyanins.

[0013] Furthermore, the application includes overexpressing a gene encoding the eggplant bHLH family transcription factor SmbHLH93 protein in plants to promote the synthesis of anthocyanins.

[0014] Furthermore, the plant is Arabidopsis thaliana.

[0015] In a fourth aspect, the present invention provides a method for cultivating transgenic plants with high anthocyanin content, wherein Agrobacterium containing the gene infects Arabidopsis inflorescence, cultured, and obtains infected T0 generation Arabidopsis seeds; the T0 generation Arabidopsis seeds are subcultured, and transgenic Arabidopsis plants with high anthocyanin content are screened.

[0016] Compared with the prior art, the present invention has the following beneficial effects: The present invention overexpresses the SmbHLH93 gene in wild-type Arabidopsis thaliana, which can promote the synthesis of anthocyanins in Arabidopsis thaliana, provides a method for improving plant quality by using genetic engineering technology, and obtains medicines or foods with high antioxidant properties, which has great application value.

[0017] The present invention discloses the regulatory mechanism of bHLH genes in the anthocyanin synthesis of eggplant, and the biosynthesis of anthocyanins in eggplant may be promoted by up-regulating the expression of anthocyanin synthesis structural genes (AtCHS, AtANS, AtDFR, AtF3H). BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings: Figure 1 The expression of the eggplant SmbHLH93 gene in different tissues in Example 2 of the present invention; Figure 2 The phenotypic changes of leaves of transgenic plants obtained from wild-type seeds and T2 generation seeds in Example 3 of the present invention; Figure 3 The changes in anthocyanin synthesis in leaves of transgenic plants obtained from wild-type seeds and T2 generation seeds in Example 3 of the present invention; Figure 4 The changes in the main structural genes for anthocyanin synthesis in the transgenic Arabidopsis thaliana plants obtained from the wild-type seeds and T2 generation seeds in Example 3 of the present invention are shown. DETAILED DESCRIPTION

[0019] The present invention is described in detail below in conjunction with specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, several variations and improvements may be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0020] The experimental methods in the following examples where specific conditions are not specified are generally performed under conventional conditions, such as those described in Sambrook et al. Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer.

[0021] Example 1: Cloning of Eggplant SmbHLH93 Gene

[0022] S1. Acquisition of plant materials; The plant material used in this experiment is the excellent eggplant germplasm resource 'Lanshan Hexianqie'. The experimental material was cultivated in an artificial plastic greenhouse at the Nongcuiyuan Base of Anhui Agricultural University. The seedlings were raised under natural conditions, grew and matured, and the leaves of the eggplant were collected for RNA extraction; S2. RNA extraction; Total RNA was extracted using the TRIzol method (TRIzol was purchased from Sangon Biotech (Shanghai) Co., Ltd.). The integrity of RNA was identified by formaldehyde denaturing gel electrophoresis, and then the purity and concentration of RNA were determined on a spectrophotometer (Thermo Scientific NANODROP 1000 Spectrophotometer); S3, gene cloning; Primer design and PCR amplification: The extracted RNA was reverse transcribed (Prime Script II 1st Strand cDNASynthesis Kit: Takara Biotech (Dalian) Co., Ltd.), using the first-strand cDNA as the template; PCR amplification was performed using primers, and the specific primers were designed as follows: F1 (SEQ ID NO.3): 5′-ATGAATCTTCAGGAATTTGGATCTG -3′ R1 (SEQ ID NO.4): 5′-TCAACACTTGGGGTGATGGATTGGAT-3′ After the expected length was obtained by amplification, the fragment was recovered and connected to the Blunt Simple Vector (Beijing Quanshijin Biotechnology Co., Ltd.), and transformed into Escherichia coli DH5α; S4, sequence information analysis; The positive clones were screened by colony PCR, and the SmbHLH93 gene was obtained, which was sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing, and the cDNA sequence SEQ ID NO.1 was obtained. The full-length CDS open reading frame sequence of the SmbHLH93 gene was 1185 bp; S5, amino acid sequence derivation; Eggplant SmbHLH93 The amino acid sequence of SEQ ID NO. 2 has 395 amino acid residues, a molecular weight of 43.65 KDa, and a theoretical isoelectric point (pI) of 7.008. S6, conserved domain analysis; The conserved domain analysis of SmbHLH93 by NCBI's Conserved Domain revealed that SmbHLH93 has a typical bHLH domain, indicating that SmbHLH93 belongs to the SmbHLH family gene.

[0023] Example 2: Expression of eggplant SmbHLH93 gene in different tissues

[0024] S1. Acquisition of plant materials; During the fruit maturity period, eggplant roots, stems, leaves, flowers, sepals, and peels were collected, wrapped in aluminum foil, and immediately immersed in liquid nitrogen, and then transferred to a -80°C ultra-low temperature refrigerator for storage. S2. RNA extraction; Total RNA was extracted using the TRIzol method (TRIzol was purchased from Sangon Biotech (Shanghai) Co., Ltd.). The integrity was tested using ordinary agarose gel electrophoresis (gel concentration 1.2%; 0.5×TBE electrophoresis buffer; 150 v, 15 min). The maximum rRNA brightness in the electrophoresis band should be 1.5-2.0 times the brightness of the second rRNA, otherwise it indicates the degradation of the rRNA sample. RNA with better purity, A 260 / A 280 and A 260 / A 230 It is about 2.0. Use a spectrophotometer to measure the OD value and calculate the RNA content; S3, cDNA acquisition; Using 500 ng of total RNA as template, the TaKaRa PrimeScript TM RT reagentKit Perfect Real Time Kit operating instructions for reverse transcription to obtain cDNA for use; S4, design specific primers; Design specific primers for real-time fluorescence quantitative PCR analysis to determine the expression of genes in different tissues. Based on the obtained eggplant SmbHLH93 gene sequence, primer design software was used to design specific primers for quantitative analysis of the SmbHLH93 gene in real-time PCR. The specific primers were designed as follows: SmbHLH93-F (SEQ ID NO.5): 5′-CGGACTCTGTCGCTGCTAAAT-3′ SmbHLH93-R (SEQ ID NO.6): 5′-TAGGCCACAAACTGGGACTTG-3′ The internal reference gene is Actin (GU984779.1), and its primers are: SmACTIN-F (SEQ ID NO.7): 5′-GTCGGAATGGGACAGAAGGATG-3′ SmACTIN-R (SEQ ID NO.8): 5′-GTGCTCAGTCAGGAGAACAGGGT-3′ S5. Prepare standard curves of target gene and internal reference gene; Use EASY Dilution (provided by the kit) to dilute the standard cDNA solution in a gradient manner, and then use the diluted cDNA solution as a template to perform real-time PCR amplification with specific primers of the target gene and the internal reference gene. After the reaction is completed, draw a melting curve and a standard curve. Analyze the melting curve to determine whether the melting curves of the target gene and the internal reference gene have a single peak, so as to determine whether a single PCR amplification product can be obtained using the primers. Determine the appropriate dilution multiple of the template cDNA through the standard curve; S6, real-time fluorescence quantitative analysis of target genes in the sample to be tested; The first strand of the synthesized cDNA was used as a template, and specific primers of the target gene and the internal reference gene were used for amplification and fluorescence quantitative analysis. The real-time PCR reaction was performed on the FTC-3000 real-time quantitative instrument, and the reaction system was 20 µL. The reaction used a three-step method, denaturation at 95°C for 1 min, followed by 40 cycles: 95°C for 30s; 58°C for 30s; 72°C for 45s. After each amplification, a melting curve was performed to verify whether the amplified product was specifically produced; S7, using 2 -△△Ct Relative quantitative analysis was performed by the method; The results showed that the SmbHLH93 gene was expressed in the root, stem, leaf, flower, sepal, and pericarp of eggplant, with the highest expression in the leaf, followed by the flower, and lower expression in the stem and root, indicating that the SmbHLH93 gene was highly expressed in the site where anthocyanins are synthesized ( Figure 1 ).

[0025] Example 3: Functional verification of eggplant SmbHLH93 gene transgenic Arabidopsis

[0026] S1. Material preparation; The gene is the SmbHLH93 gene obtained in Example 1, the Arabidopsis material is the wild type (Columbia) plant, and the transformation vector used is PHB. Agrobacterium containing SmbHLH93-PHB was cultured to OD 0.8-2.0, centrifuged at 6000rpm for 5min, the supernatant was discarded, the bacterial precipitate was resuspended with MS solution, and the OD was adjusted. 600 0.8~1.2; S2, plant transformation; Infect Arabidopsis inflorescence for 10-60 seconds, incubate in the dark for 12 hours, then culture normally and collect mature seeds; S3, seed screening; The collected T0 transgenic seeds were spread on 1 / 2 MS plates containing 50 mg / L hygromycin resistance, vernalized at 4°C for 3 days, cultured in a light incubator for 6-10 days, and strong seedlings with long roots and true leaves were selected and transplanted into soil pots for culture; the collected T1 generation seeds were continued to be spread on 1 / 2 MS plates containing 50 mg / L hygromycin resistance, and the strains with a resistant: non-resistant ratio of 3:1 were selected to collect T2 generation seeds; the survival rate of T2 generation on 50 mg / L hygromycin resistance plates was 100%, which was considered to be pure and resistant plants; S4, expression level determination; Real-time PCR was used to detect the expression level of SmbHLH93 in the obtained strains. The Arabidopsis Actin (NM_179953) primers used were as follows: SEQ ID NO.9: 5′-GTCTGGATTGGAGGGTC-3′ SEQ ID NO.10: 5′-TGAGAAATGGTCGGAAA-3′ The positive transformants obtained by screening were cultured together with the wild type under 8h darkness / 16h light; Leaves of plants obtained from wild-type seeds and T2 generation seeds are shown in Figure 2. Figure 2 As shown, WT is wild Arabidopsis, i.e. Arabidopsis without SmbHLH9393 gene inoculation; SmbHLH93-OE2, SmbHLH93-OE3, SmbHLH93-OE4 are all Arabidopsis plants obtained after T2 generation seed cultivation; S5, anthocyanin content determination; The anthocyanin content was then determined. Figure 3 As shown, the SmbHLH93-OE transgenic line accumulated more anthocyanins in its leaves compared with the wild type; the anthocyanin contents of WT, SmbHLH93-OE2, SmbHLH93-OE3, and SmbHLH93-OE4 were 0.023 mg / g, 0.078 mg / g, 0.067 mg / g, and 0.065 mg / g, respectively.

[0027] Through Arabidopsis genetic transformation experiments, it was found that overexpression of SmbHLH13 can promote the accumulation of anthocyanins, which indicates that the SmbHLH93 gene has the function of promoting anthocyanin synthesis; that is, the quality of Arabidopsis was improved through genetic engineering, and Arabidopsis transgenic plants with anthocyanin content were obtained; S6, structural gene expression analysis; Further analysis of its gene structure revealed that the SmbHLH13 gene may promote the biosynthesis of anthocyanins by upregulating the expression of anthocyanin biosynthesis structural genes (AtCHS, AtANS, AtDFR, AtF3H). Figure 4).

[0028] Sequence Listing NO.1 SmbHLH93 (Smechr0602178) gene cDNA sequence: Amino acid sequence of the No. 2 SmbHLH93 gene: MNLQEFGSAFDPIDIVSSWKISQRQEAATKLAADSVAAKSGTGSIGNGTKSKIGSSSSSSSSFPPQNNYMPYFGNNQVPVCGLMADLNYIGLSSKTKSPDCLLSTTNTSDTDSSVELDGAILSEDSRSFWNVNANNAVSSGESPIDVLKSKPIYANGNNNVHYSVNELDQTVSYNNLITSSEAKRYNIDKRSHNDLLQSDFSNRDYGFQLILENDQPMSKKSRSDYKLPSSSNINFQQACSSECSIVEPNSEAIAQMKEMIYCAAAFRPVNSVMEEVMVEKPKRKNVRISMDPQTAAARRRREKISERIRILQKLVPGGTYMDTASMLDEAANYLKFLRTQVNTLEAFGFKIDPIIINNNSFTTISSIPLINYPFPMQPHFPLQNFNPIHHPKC*

Claims

1. An eggplant bHLH family transcription factor SmbHLH93 protein, characterized in that: The amino acid sequence of the protein is shown in SEQ ID NO.

2.

2. A gene encoding the eggplant bHLH family transcription factor SmbHLH93 protein according to claim 1, characterized in that: The nucleic acid sequence of the gene is shown in SEQ ID NO.

1.

3. Use of the gene encoding the eggplant bHLH family transcription factor SmbHLH93 protein according to claim 2 in promoting plant anthocyanin synthesis.

4. The use according to claim 3, characterized in that: The gene encoding the eggplant bHLH family transcription factor SmbHLH93 protein was overexpressed in plants to promote anthocyanin synthesis.

5. The use according to claim 4, characterized in that: The plant is Arabidopsis thaliana.

6. A method for cultivating a transgenic plant with a high anthocyanin content, characterized in that: Agrobacterium containing the gene of claim 2 infects Arabidopsis inflorescence, cultures it, and obtains infected T0 generation Arabidopsis seeds; subcultures the T0 generation Arabidopsis seeds, and screens out transgenic Arabidopsis plants with high anthocyanin content.

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