Eggplant bHLH family transcription factor SmbHLH93 protein and its encoding gene

Abstract

The present invention discloses an eggplant bHLH family transcription factor SmbHLH93 protein and its encoding gene. This gene is involved in regulating the synthesis of anthocyanins in eggplant. Overexpressing the SmbHLH93 gene in wild-type Arabidopsis can promote the synthesis of anthocyanins in Arabidopsis, providing a theoretical basis for improving plant quality using genetic engineering techniques in the future and obtaining drugs or foods with high anthocyanins, and having great application value.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and relates to key enzymes and their encoding genes in the light signal pathway of eggplant, specifically to an eggplant bHLH family transcription factor SmbHLH93 protein, its encoding gene and applications. Background Art

[0002] Anthocyanin, also known as anthocyanidin, is the most important component of flavonoids, mainly existing in fruits and vegetables in the form of water-soluble pigments, and has functions such as reducing the risk of cardiovascular diseases, antioxidation, and antimutagenesis. Eggplant contains a large amount of anthocyanin, which is very beneficial to human health, making the study of the anthocyanin synthesis mechanism in eggplant an important topic for improving crop nutritional value and developing functional foods.

[0003] The synthesis of anthocyanin requires basic helix-loop-helix transcription factors, namely bHLH transcription factors. The bHLH transcription factor contains a highly conserved basic helix-loop-helix (bHLH) domain and is ubiquitous in various eukaryotes. Such transcription factors are involved in epidermal differentiation, response to environmental stress, or regulation of 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. Especially in the process of anthocyanin biosynthesis, the bHLH transcription factor family plays an indispensable role, ensuring the precise regulation of the anthocyanin synthesis pathway.

[0004] In the process of anthocyanin biosynthesis in eggplant, the bHLH transcription factor family plays an important role. However, current research on bHLH mainly focuses on a few model plants such as Arabidopsis thaliana, but there is relatively little research on bHLH genes and their functional verification in non-model crops, especially economic crops such as eggplant. 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 present invention provides an eggplant bHLH family transcription factor SmbHLH93 protein and its encoding gene.

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

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

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

[0009] Furthermore, the nucleotide 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 nucleotide sequences of the primer pair 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 full-length sequence or fragment of the eggplant SmbHLH93-related nucleotide can be obtained by PCR amplification, recombination or artificial synthesis methods.

[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 anthocyanin synthesis in plants.

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

[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. Agrobacterium containing the gene infects the inflorescence of Arabidopsis thaliana, and after culturing, the infected T0 generation Arabidopsis thaliana seeds are obtained; the T0 generation Arabidopsis thaliana seeds are subcultured, and transgenic Arabidopsis thaliana plants with high anthocyanin content are screened.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] Overexpressing the SmbHLH93 gene in wild-type Arabidopsis thaliana in the present invention can promote anthocyanin synthesis in Arabidopsis thaliana, providing a method for improving plant quality using genetic engineering technology to obtain drugs or foods with high antioxidant properties, which has great application value.

[0018] The present invention reveals the regulatory mechanism of the bHLH gene in anthocyanin synthesis in eggplant, and may promote anthocyanin biosynthesis in eggplant by upregulating the expression levels of anthocyanin synthesis structural genes (AtCHS, AtANS, AtDFR, AtF3H). Brief Description of the Drawings

[0019] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments read in conjunction with the accompanying drawings:

[0020] Figure 1 It shows the expression of the eggplant SmbHLH93 gene in different tissues in Example 2 of the present invention;

[0021] Figure 2 It shows the phenotypic changes of the leaves of transgenic plants obtained from wild-type seeds and T2-generation seeds in Example 3 of the present invention;

[0022] Figure 3 It shows the changes in anthocyanin synthesis in the leaves of transgenic plants obtained from wild-type seeds and T2-generation seeds in Example 3 of the present invention;

[0023] Figure 4 It shows the changes in the main structural genes of Arabidopsis anthocyanin synthesis in the leaves of transgenic plants obtained from wild-type seeds and T2-generation seeds in Example 3 of the present invention. Detailed Embodiments

[0024] The present invention will be described in detail below with reference to specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be noted that those of ordinary skill in the art can make several modifications and improvements without departing from the concept of the present invention. These all fall within the protection scope of the present invention.

[0025] For the experimental methods without specific conditions noted in the following embodiments, they are usually carried out under conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer.

[0026] Example 1: Cloning of Eggplant SmbHLH93 Gene

[0027] S1. Obtaining plant materials;

[0028] The plant material used in this experiment is the excellent germplasm resource of eggplant 'Lanshan Hexianqie'. The experimental materials were cultivated in an artificial plastic greenhouse in the Nongcuiyuan Base of Anhui Agricultural University. Seedlings were raised, grown, and fruited under natural conditions, and the leaves of eggplants were collected for RNA extraction;

[0029] S2. RNA extraction;

[0030] Total RNA was extracted using the TRIzol method (TRIzol was purchased from Sangon Biotech (Shanghai) Co., Ltd.). The integrity of the RNA was identified by formaldehyde denaturing gel electrophoresis, and then the purity and concentration of the RNA were measured on a spectrophotometer (Thermo Scientific NANODROP 1000 Spectrophotometer);

[0031] S3. Gene cloning;

[0032] Primer design and PCR amplification: The extracted RNA was reverse transcribed (Prime Script II 1st Strand cDNA Synthesis Kit: Takara Bio Inc. (Dalian)), using the first-strand cDNA as a template;

[0033] PCR amplification was carried out using the primers. The specific primer design was as follows:

[0034] F1 (SEQ ID NO.3): 5′- ATGAATCTTCAGGAATTTGGATCTG -3′

[0035] R1 (SEQ ID NO.4): 5′- TCAACACTTGGGGTGATGGATTGGAT -3′

[0036] After amplification to obtain the expected length, it was recovered and ligated to the Blunt Simple Vector (TransGen Biotech Co., Ltd., Beijing) vector, and then transformed into Escherichia coli DH5α;

[0037] S4. Sequence information analysis;

[0038] Colony PCR was used to screen for positive clones to obtain the SmbHLH93 gene, which was sent to Sangon Biotech Co., Ltd. in Shanghai 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;

[0039] S5. Amino acid sequence derivation;

[0040] Based on the CDS open reading frame sequence, the SmbHLH93 amino acid sequence of eggplant was deduced, with a total of 395 amino acid residues, a molecular weight of 43.65 KDa, and a theoretical isoelectric point (pI) of 7.008. The detailed sequence is shown in the sequence of SEQ ID NO.2;

[0041] S6. Conserved domain analysis;

[0042] Analysis of the conserved domain of SmbHLH93 through the Conserved Domain of NCBI found that SmbHLH93 has a typical bHLH domain, indicating that SmbHLH93 belongs to the SmbHLH family of genes.

[0043] Example 2: Expression of Eggplant SmbHLH93 Gene in Different Tissues

[0044] S1. Obtaining plant materials;

[0045] At the fruit ripening stage, collect eggplant roots, stems, leaves, flowers, sepals, and pericarp respectively. Wrap the samples with aluminum foil and immediately put them into liquid nitrogen, and then transfer them to a -80 °C ultra-low temperature refrigerator for storage for later use;

[0046] S2. RNA extraction;

[0047] Total RNA was extracted using the TRIzol method (TRIzol was purchased from Sangon Biotech (Shanghai) Co., Ltd.). Integrity was detected by ordinary agarose gel electrophoresis (gel concentration 1.2%; 0.5×TBE electrophoresis buffer; 150 v, 15 min). The brightness of the largest rRNA in the electrophoresis band should be 1.5 - 2.0 times that of the second rRNA brightness, otherwise it indicates degradation of the rRNA sample. For RNA with better purity, the A 260 / A 280 and A 260 / A 230 is about 2.0 or so. Measure the OD value with a spectrophotometer and calculate the RNA content;

[0048] S3. Obtaining cDNA;

[0049] Using 500 ng of total RNA as a template, reverse transcription was carried out according to the operation instructions of the TaKaRa PrimeScript TM RT reagentKit Perfect Real Time kit to obtain cDNA for later use;

[0050] S4. Designing specific primers;

[0051] Design specific primers for real-time fluorescence quantitative PCR analysis to determine the expression levels of genes in different tissues. According to the obtained eggplant SmbHLH93 gene sequence, use primer design software to design specific primers for quantitative analysis of the SmbHLH93 gene in Real-time PCR. The specific primers are designed as follows:

[0052] SmbHLH93-F (SEQ ID NO.5): 5′- CGGACTCTGTCGCTGCTAAAT -3′

[0053] SmbHLH93-R (SEQ ID NO.6): 5'-TAGGCCACAAACTGGGACTTG-3'

[0054] The reference gene is Actin (GU984779.1), and its primers are:

[0055] SmACTIN-F (SEQ ID NO.7): 5'-GTCGGAATGGGACAGAAGGATG-3'

[0056] SmACTIN-R (SEQ ID NO.8): 5'-GTGCCTCAGTCAGGAGAACAGGGT-3'

[0057] S5. Prepare the standard curves of the target gene and the reference gene;

[0058] Use EASY Dilution (provided by the kit) to perform gradient dilution on the standard cDNA solution. Then, using the diluted cDNA solution as the template respectively, perform Real-time PCR amplification with the specific primers of the target gene and the reference gene. After the reaction, draw the melting curve and the standard curve. Analyze the melting curve to determine whether the melting curves of the target gene and the reference gene obtain a single peak, so as to judge whether a single PCR amplification product can be obtained using these primers. Determine the appropriate dilution factor of the template cDNA through the standard curve;

[0059] S6. Real-time fluorescence quantitative analysis of the target gene in the test samples;

[0060] Using the first-strand cDNA synthesized as the template, perform fluorescence quantitative analysis by amplifying with the specific primers of the target gene and the reference gene respectively. The Real-time PCR reaction is carried out on an FTC-3000 real-time quantitative instrument, and the reaction system is 20 µL. The reaction adopts the three-step method: denaturation at 95°C for 1 min, followed by 40 cycles: 95°C for 30 s; 58°C for 30 s; 72°C for 45 s. After each amplification is completed, a melting curve is made to check whether the amplification product is specifically generated;

[0061] S7. Use the 2 -△△Ct method for relative quantitative analysis;

[0062] The results show that the SmbHLH93 gene is expressed in the roots, stems, leaves, flowers, sepals, and pericarp of eggplants. Among them, the expression level in leaves is the highest, followed by flowers, while the expression levels in stems and roots are relatively low, indicating that the SmbHLH93 gene has a relatively high expression level in the parts where anthocyanins are synthesized ( Figure 1 ).

[0063] Example 3: Functional Verification of Eggplant SmbHLH93 Gene Transformed into Arabidopsis thaliana

[0064] S1. Material preparation;

[0065] The gene is the SmbHLH93 gene obtained in Example 1. The Arabidopsis material is a wild-type (Columbia) plant, and the transformation vector used is PHB. The Agrobacterium containing SmbHLH93-PHB was cultured to an OD of 0.8 - 2.0, centrifuged at 6000 rpm for 5 min, the supernatant was discarded, and the bacterial cell pellet was resuspended with MS solution to adjust the OD 600 to 0.8 - 1.2;

[0066] S2. Plant transformation;

[0067] The inflorescence of Arabidopsis was infected for 10 - 60 sec, cultured in the dark for 12 h and then normally cultured, and the mature seeds were collected;

[0068] S3. Seed screening;

[0069] The collected T0 generation transgenic seeds were spread on a 1 / 2 MS plate containing 50 mg / L hygromycin resistance, vernalized at 4°C for 3 d, cultured in a light incubator for 6 - 10 d, and the robust seedlings with long root length and true leaves were selected and transplanted into a soil pot for cultivation; the collected T1 generation seeds were continued to be spread on a 1 / 2 MS plate containing 50 mg / L hygromycin resistance, and the lines with a resistance:non-resistance ratio of 3:1 were selected to collect T2 generation seeds; the lines with a 100% survival rate on a 50 mg / L hygromycin resistance plate in the T2 generation were considered to have screened out homozygous resistant plants;

[0070] S4. Expression level determination;

[0071] For the obtained lines, the expression level of SmbHLH93 was detected by Real-time PCR. The primers for Arabidopsis Actin (NM_179953) used were as follows:

[0072] SEQ ID NO.9: 5′-GTCTGGATTGGAGGGTC-3′

[0073] SEQ ID NO.10: 5′-TGAGAAATGGTCGGAAA-3′

[0074] The selected positive transformants and the wild type were cultured together under 8 h darkness / 16 h light;

[0075] The leaves of the plants obtained from the wild-type seeds and the T2 generation seeds were as Figure 2 shown, where WT is wild Arabidopsis, that is, Arabidopsis not inoculated with the SmbHLH9393 gene; SmbHLH93-OE2, SmbHLH93-OE3, and SmbHLH93-OE4 are all Arabidopsis plants obtained after culturing the T2 generation seeds;

[0076] S5, Determination of anthocyanin content;

[0077] Subsequently, its anthocyanin content was determined. As Figure 3 shown, compared with the wild type, the SmbHLH93-OE transgenic lines had more anthocyanin accumulation in the leaves; 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.

[0078] Through Arabidopsis thaliana genetic transformation experiments, it was found that overexpression of SmbHLH13 could promote anthocyanin accumulation, indicating that the SmbHLH93 gene has the function of promoting anthocyanin synthesis; that is, the quality of Arabidopsis thaliana was improved through genetic engineering, and transgenic Arabidopsis thaliana plants with anthocyanin content were obtained;

[0079] S6, Analysis of structural gene expression;

[0080] Further analysis of its gene structure revealed that the SmbHLH13 gene might promote anthocyanin biosynthesis by upregulating the expression levels of anthocyanin synthesis structural genes (AtCHS, AtANS, AtDFR, AtF3H) ( Figure 4 ).

[0081] Sequence Listing

[0082] NO.1 cDNA sequence of SmbHLH93 (Smechr0602178) gene:

[0083]

[0084] Amino acid sequence of the No. 2 SmbHLH93 gene:

[0085] 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 as claimed in claim 1, characterized in that, The nucleic acid sequence of the gene is shown in SEQ ID NO.

1.

3. Use of a gene encoding the eggplant bHLH family transcription factor SmbHLH93 protein as described in claim 2 in promoting anthocyanin synthesis in plants, characterized in that, Overexpress the gene encoding the eggplant bHLH family transcription factor SmbHLH93 protein in plants to promote the synthesis of anthocyanins; the plant is Arabidopsis thaliana.

4. A method for cultivating a transgenic plant with a high anthocyanin content, characterized in that, Agrobacterium containing the gene recited in claim 2 infects the inflorescence of Arabidopsis thaliana, and after culturing, the infected T0 generation Arabidopsis thaliana seeds are obtained; the T0 generation Arabidopsis thaliana seeds are subcultured, and transgenic Arabidopsis thaliana plants with high anthocyanin content are screened.

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