A dandelion TmbHLH93 gene, its expressed protein, and its applications

Abstract

This invention discloses a dandelion TmbHLH93 gene, its expressed protein, and its applications, belonging to the field of plant genetic engineering technology. The nucleotide sequence of the TmbHLH93 gene is shown in SEQ ID NO.1, and the amino acid sequence of its encoded protein is shown in SEQ ID NO.2. This invention cloned the TmbHLH93 gene from dandelion, constructed its plant overexpression vector, and transformed dandelion to obtain transgenic plants. Experiments showed that overexpression of the TmbHLH93 gene significantly upregulated the expression of key enzyme genes related to the chicoric acid synthesis pathway and increased the accumulation of chicoric acid in the leaves of transgenic plants. This invention provides a key gene and technical foundation for cultivating new dandelion varieties with high chicoric acid content through transcription factor engineering.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, and more specifically, relates to a dandelion TmbHLH93 gene, its expressed protein, and its applications. Background Technology

[0002] Dandelion is a perennial herb belonging to the genus Taraxacum in the family Asteraceae, possessing various pharmacological activities. Its rich polyphenolic compounds have anti-inflammatory and anti-tumor effects, and it has been listed as a plant with both medicinal and edible properties, widely used in food and pharmaceutical fields. The Chinese Pharmacopoeia specifies chicoric acid as the representative functional component of dandelion. The entire process of chicoric acid formation in dandelion involves the following branch synthesis from the phenylalanine synthesis pathway. Phenylalanine is catalyzed by enzymes such as phenylalanine ammonia-lyase (PAL), cinnamic acid 4-hydroxylase (C4H), 4-coumarate-CoA ligase (4CL), and shikimic acid / quinine hydroxycinnamoyltransferase (HCT) to form caffeoyl-CoA. Subsequently, BAHD family member hydroxycinnamoyl-CoA quinone hydroxycinnamoyltransferase (HQT) catalyzes the formation of chlorogenic acid from caffeoyl-CoA; and tartrate hydroxycinnamoyltransferase (HTT) catalyzes the formation of caffeoyl-CoA from caffeoyl tartaric acid. Afterwards, chicoric acid synthase (CAS), a member of the SCPL IA subfamily, uses chlorogenic acid as an acyl donor and tartaric acid as an acyl acceptor to catalyze the synthesis of chicoric acid. Although the entire process of dandelion chicoric acid synthesis and the enzymes required in the catalytic synthesis pathway are relatively well understood, studies on the transcriptional regulation of chicoric acid synthase are rarely reported.

[0003] Plant transcription factors can precisely regulate the initiation and efficiency of gene transcription by recognizing cis-acting elements on target gene promoters and binding to specific sequences, thereby regulating functions such as cell differentiation, tissue and organ development, metabolic networks, and responses to plant hormones and environmental factors. bHLH transcription factors play a crucial role in regulating plant growth and development as well as plant secondary metabolic biology. Currently, bHLH family transcription factors have been identified in model plants, but functional studies of bHLH transcription factors that specifically regulate taraxacin synthesis in dandelion and their expressed proteins have not yet been reported. Summary of the Invention

[0004] To address the aforementioned problems in existing technologies, the technical problem this invention aims to solve is to provide the dandelion TmbHLH93 gene. Another technical problem this invention aims to solve is to provide the expression protein of the dandelion TmbHLH93 gene. A further technical problem this invention aims to solve is to provide an application of the dandelion TmbHLH93 gene for regulating chicoric acid synthesis in plants.

[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0006] A dandelion TmbHLH93 gene, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0007] The protein encoded by the dandelion TmbHLH93 gene has the amino acid sequence shown in SEQ ID NO.2.

[0008] A biological material, said biological material being an expression cassette, recombinant vector, recombinant bacteria, or recombinant cell containing the dandelion TmbHLH93 gene.

[0009] The application of the dandelion TmbHLH93 gene or the biomaterials described therein in increasing the chicoric acid content of dandelion.

[0010] A method to promote the increase of chicoric acid content in dandelion involves overexpressing the dandelion TmbHLH93 gene in dandelion.

[0011] In some embodiments, the method includes the following steps:

[0012] S1: Construct the overexpression vector of the dandelion TmbHLH93 gene;

[0013] S2: Transform the constructed expression vector into dandelion cells or tissues;

[0014] S3: Transgenic dandelions with increased chicoric acid content were obtained through breeding and screening.

[0015] In some embodiments, the overexpression vector is a plant expression vector.

[0016] In some embodiments, the transformation is mediated by Agrobacterium.

[0017] The application of the dandelion TmbHLH93 gene or the biological material described herein in promoting the expression of key enzyme genes in the chicoric acid synthesis pathway, wherein the key enzyme genes promoting the chicoric acid synthesis pathway are selected from at least one of the following genes: phenylalanine ammonia-lyase, cinnamic acid 4-hydroxylase, hydroxycinnamoyl-CoA quinine hydroxycinnamoyltransferase, and tartrate hydroxycinnamoyltransferase.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0019] 1) This invention constructs a plant expression vector of the dandelion TmbHLH93 gene and transforms it into dandelion. After overexpressing the TmbHLH93 gene, the transgenic dandelion significantly increases the expression of key enzyme genes TmPAL3, TmC4H1, TmHQT1 and TmHTT1 in the chicoric acid synthase pathway, elucidating its regulatory mechanism at the transcriptional level.

[0020] 2) This invention constructs a protein expression vector for the dandelion TmbHLH93 gene, transforms it into competent E. coli BL21 cells, and obtains the His-tagged TmbHLH93 protein after induction and purification. Gel migration arrest experiments demonstrate that the TmbHLH93 protein can bind to the CCGCCG cis-acting element with a biotin-labeled probe. This directly reveals the molecular mechanism by which dandelion TmbHLH93 acts as a transcription factor to regulate downstream target genes at the molecular interaction level.

[0021] 3) This invention, through genetic transformation technology, obtained dandelion plants overexpressing TmbHLH93, whose leaves contained significantly higher levels of chicoric acid than the wild-type control. This demonstrates that by regulating a single key transcription factor, the accumulation of the target metabolite (chicoric acid) can be effectively increased. This provides key genes and core technologies for the targeted improvement of dandelion varieties and the cultivation of new industrial raw material varieties with high chicoric acid yields through molecular breeding, and has excellent prospects for industrial application. Attached Figure Description

[0022] Figure 1 Images show the RNA extraction and TmbHLH93 gene CDS clone electrophoresis detection. In A, the RNA electrophoresis image is shown (M: DNA Marker; lane 1: root; lane 2: leaf; lane 3: flower); and in B, the TmbHLH93 gene CDS clone electrophoresis detection image is shown (M: DNA Marker; lane 1: full-length TmbHLH93 gene CDS sequence).

[0023] Figure 2 The diagram shows the RNA level identification of TmbHLH93 transgenic dandelion positive lines. A represents PCR identification of the GFP gene tag in the transgenic overexpressing plants; B represents PCR identification of the Kan gene tag in the transgenic overexpressing plants (M: DNA Marker; Lane 1: empty vector EV; Lane 2: TmbHLH93-OE1; Lane 3: TmbHLH93-OE2; Lane 4: TmbHLH93-OE4; Lane 5: TmbHLH93-OE7); C represents the TmbHLH93 gene expression level in different positive lines.

[0024] Figure 3 The graph shows the changes in chlorogenic acid, monocaffeoyl tartaric acid, and chicoric acid content in the TmbHLH93 overexpressing dandelion strains OE1 / 3 / 4 / 7; where A is a statistical comparison of chicoric acid content between the empty vector and the overexpressing strains; and B is a liquid chromatography detection graph of chlorogenic acid and chicoric acid in the empty vector and the overexpressing strains.

[0025] Figure 4 Figure 1 shows the results of the expression level analysis of TmPAL3, TmC4H1, TmHQT1 and TmHTT1, which are related to the chicoric acid synthesis pathway in dandelion strains.

[0026] Figure 5 The images show the combined results of TmbHLH93 protein expression and gel retardation assays. In this image, A is an SDS-PAGE plot (M: 10-250 kDa protein marker; 1: flow-through buffer; 2: flow-through buffer; 3: 50 mM imidazole elution buffer; 4: 100 mM imidazole elution buffer; 5: 250 mM imidazole elution buffer); and B is a plot showing the retardation bands generated by the binding of TmbHLH93 protein to the promoter of TmPAL3, a key enzyme gene in the chicoric acid synthesis pathway. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of this invention clearer, the invention is further described below with reference to specific embodiments. Unless otherwise specified, the technical means used in the following embodiments are all conventional means well known to those skilled in the art. The plant material used in this invention is Taraxacum mongolicum Hand.-Mazz., a plant of the genus Taraxacum in the family Asteraceae, cultivated in the nursery of the Jiangsu Provincial Institute of Botany, Chinese Academy of Sciences, Xuanwu District, Nanjing City, Jiangsu Province.

[0028] Example 1

[0029] (1) Total RNA extraction and cDNA acquisition

[0030] Total RNA was extracted from different tissues (roots, leaves, and flowers) of dandelion using a plant tissue total RNA extraction kit (Shanghai Sangon Biotech Co., Ltd.). Figure 1 As shown in Figure A. Using extracted total RNA from leaves as a template, cDNA was synthesized using a cDNA one-strand synthesis kit (genomic de-generated) (Beijing TransGen Biotech Co., Ltd.).

[0031] (2) Amplification of the CDS sequence of the dandelion TmbHLH93 gene

[0032] Based on the transcriptome sequence of the dandelion TmbHLH93 gene, the following primers were designed:

[0033] Forward: 5'- ATGATGCAACAACCAAGCGG-3' (SEQ ID NO.3);

[0034] Reverse: 5'-TTACAAACATCTGCCCCCATAAC-3' (SEQ ID NO. 4).

[0035] The CDS sequence of the TmbHLH93 gene was obtained by PCR. The PCR amplification electrophoresis image is shown below. Figure 1 As shown in B.

[0036] The final sequencing yielded the CDS sequence of the TmbHLH93 gene, as shown in SEQ ID NO.1, which is 957 bp in length and encodes 318 amino acids, as shown in SEQ ID NO.2.

[0037] Example 2

[0038] 1. Construction of the dandelion TmbHLH93 gene overexpression vector

[0039] Based on the CDS sequence of the TmbHLH93 gene and the multiple cloning site of the overexpression vector pHellsgate8-GFP (XhoI was selected as the restriction site), amplification primers were designed, and the primer sequences are shown below:

[0040] Forward: 5'-TTTGGAGAGGACACGCTCGAGATGATGCAACAACCAAGCGG-3' (SEQ ID NO.5);

[0041] Reverse: 5'-GCTCACCATGAATTCCTCGAGCAAACATCTGCCCCATAAC-3' (SEQ ID NO. 6).

[0042] The target fragment of the TmbHLH93 gene was amplified by PCR. The pHellsgate8-GFP vector was digested with XhoI at 37 ℃ for 2 h. The PCR and digestion products were recovered using a gel extraction kit (Shanghai Sangon Biotech Co., Ltd.) to obtain the target fragment and the linearized vector fragment. The target fragment was ligated into the pHellsgate8-GFP linear vector using the ClonExpress II One Step Cloning Kit (Nanjing Novizan Biotechnology Co., Ltd.) to construct a 35S::TmbHLH93 overexpression vector. The ligation system is as follows:

[0043] 2 μL of 5× CE II buffer, approximately 200 ng of linearized vector, approximately 100 ng of insert fragment, 1 μL of Exnase II, and ddH2O to a final volume of 10 μL were added. The mixture was incubated at 37°C for 30 min and then immediately transferred to ice. After the *E. coli* DH5α competent cells thawed on ice, the recombinant product was added to the competent cells and mixed thoroughly. The cells were incubated on ice for 30 min, followed by heat shock at 42°C for 45 sec, and then cooled on ice for 5 min. 500 μL of LB medium was added, and the cells were incubated at 37°C with shaking for 1 h. The bacterial culture was then spread onto plates containing 100 mg / L spec+ and incubated upside down at 37°C for 12–16 h. Single colonies were validated using colony PCR. Positive colonies were picked and inoculated into LB liquid medium containing 100 mg / L spec+ and cultured at 200 rpm and 37°C for 24 h with shaking. Plasmids were extracted and digested for verification. After successful verification, sequencing was performed. The correctly sequenced vector was the 35S::TmbHLH93 overexpression vector.

[0044] 2. Obtaining transgenic dandelion lines by root severance method

[0045] Take 6-month-old (non-sterile) wild-type Mongolian dandelion seedlings cultivated at the base, leaving 2-3 tender leaves, remove soil, wash with clean water, and set aside. Transform the constructed 35S::TmbHLH93 recombinant plasmid into K599 Agrobacterium rhizogenes using a freeze-thaw method, using 100 mL of solution containing 50 mg•L... -1 spec+ and 50 mg •L -1 Rifampicin LB liquid medium was cultured on a shaker at 28°C until the bacterial culture reached OD. 600 Centrifuge the bacterial culture at 0.6-0.8 rpm for 5000 rpm, discard the supernatant, and resuspend the culture in an equal volume of MS salt containing AS. Cut dandelion roots at a 45° angle to 2-3 cm and soak them in the above-mentioned Agrobacterium K599 containing recombinant plasmids for 15 min. Wipe dry with filter paper and place on moist vermiculite. Cover with a filter membrane (with holes punched for ventilation) for 1-2 weeks until callus or fibrous roots form, then transplant to seedling trays (soil:vermiculite = 3:1). After 4-6 weeks of routine management, use a portable fluorescent protein excitation light source LUYOR-3415RG to perform preliminary fluorescence identification of the formed fibrous roots. If identification is successful, cut the fibrous roots (above 0.5 cm) and place them on moist vermiculite to continue growing until new leaves and complete plants form (4-6 weeks).

[0046] 3. Screening and identification of positive plants

[0047] The preliminarily screened complete plants were further subjected to molecular identification. Wild-type dandelion was used as a negative control, and β-Actin was used as an internal reference gene. The expression level of TmbHLH93 in transgenic dandelion was analyzed by qRT-PCR. The results are as follows: Figure 2 As shown, Figure 2 Figure A shows the PCR identification results of the GFP gene tag in the transgenic overexpressing plant, using cDNA as a template and GFP-F and GFP-R as primers. Figure 2 Figure B shows the PCR identification results of the Kan gene tag in the transgenic overexpressing plant, which was amplified using cDNA as a template and Kan-F and Kan-R as primers. Figure 2 The results showed that the expression levels of the overexpression lines OE3 and OE4 (TmbHLH93-OE3 and TmbHLH93-OE4) were more than 10 times that of the control, and all overexpression lines were used for subsequent content determination.

[0048] 4. Analysis of the effects of TmbHLH93 gene overexpression on the content and gene expression levels of taraxacin and its precursor compounds in taraxacin.

[0049] The TmbHLH93 overexpression lines OE1 / 3 / 4 / 7 were selected for the determination of chlorogenic acid, monocaffeoyl tartaric acid, and chicoric acid. An Agilent 1100 high-performance liquid chromatography (HPLC) system was used, employing a Phenomenex Gemoni 5 u C18 110A column (5 μm × 4.6 mm × 250 mm), a flow rate of 1.0 mL / min, an absorption wavelength of 254 nm (UV detector), and a column temperature of 40 °C. Mobile phase A consisted of 0.1% formic acid aqueous solution, and mobile phase B was methanol. The separation gradient mobile phase B was as follows: 13%–20%, 0–7 min; 7–18 min, 20%–30%; 18–28 min, 30%–41%; 28–35 min, 41%–45%; 45%–13%, 35–40 min. Three biological replicates were taken for each sample and compared with the standard. The peak times were compared. The results showed that the contents of chlorogenic acid, monocaffeoyl tartaric acid and chicoric acid in the overexpressing plants were significantly increased compared with the empty vector control line. Figure 3 This indicates that overexpression of TmbHLH93 can promote the accumulation of chlorogenic acid, monocaffeoyl tartaric acid and chicoric acid, demonstrating the regulatory role of TmbHLH93 in the biosynthesis of chlorogenic acid, monocaffeoyl tartaric acid and chicoric acid.

[0050] In the TmbHLH93 overexpression line, the relative expression levels of four candidate chicoric acid biosynthesis-related enzyme genes were analyzed by qRT-PCR. The results showed that ( Figure 4In the overexpression lines, the expression levels of chicoric acid biosynthetic enzyme genes—phenylalanine ammonia-lyase (TmPAL3), cinnamic acid 4-hydroxylase (TmC4H1), hydroxycinnamoyl-CoA quinone hydroxycinnamoyltransferase (TmHQT1), and tartrate hydroxycinnamoyltransferase (TmHTT1)—were significantly increased compared to the empty vector control lines. This indicates that overexpression of TmbHLH93 may participate in the regulation of chicoric acid synthesis by modulating chicoric acid synthase genes.

[0051] Example 3

[0052] The TmbHLH93 gene sequence was constructed into a His-tagged pCold(EcoRI) vector using homologous recombination to obtain the His-TmbHLH93 recombinant vector. This recombinant vector was transformed into competent E. coli BL21(DE3) cells, and positive clones were screened after sequencing. Positive clones were picked and cultured overnight at 37°C in 20 mL of LB broth containing 100 mg / L Amp+. The culture was then transferred to a 1000 mL Erlenmeyer flask, and 400 mL of LB (Amp+) broth was added. The culture was then incubated for another 4 mL and the culture was continued at 37°C until OD was reached. 600 =0.4-0.6, take 2 mL as the pre-induction sample; add 400 μL 0.2M IPTG to the bacterial culture and incubate at 16 ℃ 100 rpm for 18-20 h, take 2 mL as the post-induction sample; centrifuge at 8000 rpm, 4 ℃ for 10 min to collect the bacterial culture; resuspend the bacterial cells in 80 mL PBS buffer, centrifuge at 8000 rpm, 4 ℃ for 10 min, and discard the supernatant; continue to resuspend the bacterial cells in 80 mL PBS buffer, add 100 mM PMSF (working concentration is 1 mM), and vortex to mix; sonicate the bacterial cells (power 35%, 45 min) until clear; centrifuge at 12000 rpm, 4 ℃ for 20 min, and discard the precipitate. Add the supernatant solution to a Ni-NTA purification column (pre-equilibrated with two column volumes of 10 mM imidazole buffer), adjust the flow rate, and collect the flow-through. Add two column volumes of 10 mM imidazole buffer to the chromatography column to remove contaminating proteins. Elute sequentially with imidazole buffer (PBS as the stock solution) at concentrations of 20 mM, 50 mM, 70 mM, 100 mM, and 250 mM, adding 14 mL of each concentration. Collect the eluent in a 2 mL centrifuge tube. Analyze the protein before and after purification using SDS-PAGE. Figure 5 (A)

[0053] The purified His-TmbHLH93 protein was used to perform a gel migration assay to detect the interaction between TmbHLH93 and proTmPAL3. The results showed that the probe could bind to His-TmbHLH93 protein and produce a corresponding blocking band, indicating that TmbHLH93 can bind to proTmPAL3 in vitro. Figure 5 (B) This confirms at the molecular level that the upregulation of proTmPAL3, a key gene for chicoric acid synthesis in transgenic lines, is directly regulated by the increased level of TmbHLH93 protein.

[0054] The above description is illustrative only and not restrictive of the present invention. Those skilled in the art will understand that many modifications, variations or equivalents can be made without departing from the spirit and scope defined by the appended claims, and all such modifications, variations or equivalents will fall within the protection scope of the present invention.

Claims

1. A dandelion TmbHLH93 gene, characterized in that, Its nucleotide sequence is shown in SEQ ID NO.

1.

2. The protein encoded by the dandelion TmbHLH93 gene as described in claim 1, characterized in that, Its amino acid sequence is shown in SEQ ID NO.

2.

3. A biomaterial, characterized in that, The biological material is an expression cassette, recombinant vector, recombinant bacteria, or recombinant cell containing the dandelion TmbHLH93 gene as described in claim 1.

4. The application of the dandelion TmbHLH93 gene of claim 1 or the biomaterial of claim 3 in increasing the chicoric acid content of dandelion, characterized in that, The application of increasing the chicoric acid content in dandelion involves overexpressing the dandelion TmbHLH93 gene in dandelion.

5. A method for increasing the chicoric acid content in dandelion, characterized in that, Overexpression of the dandelion TmbHLH93 gene as described in claim 1 in dandelion.

6. The method according to claim 5, characterized in that, Includes the following steps: S1: Construct an overexpression vector for the dandelion TmbHLH93 gene as described in claim 1; S2: Transform the constructed expression vector into dandelion cells or tissues; S3: Transgenic dandelions with increased chicoric acid content were obtained through breeding and screening.

7. The method according to claim 6, characterized in that, The overexpression vector is a plant expression vector.

8. The method according to claim 6, characterized in that, The transformation was mediated by Agrobacterium.

9. The application of the dandelion TmbHLH93 gene of claim 1 or the biomaterial of claim 3 in promoting the expression of key enzyme genes in the chicoric acid synthesis pathway, characterized in that, The key enzyme gene promoting the chicoric acid synthesis pathway is selected from at least one of the following genes: phenylalanine ammonia-lyase, cinnamic acid 4-hydroxylase, hydroxycinnamoyl-CoA quinone hydroxycinnamoyltransferase, and tartrate hydroxycinnamoyltransferase. The application of the key enzyme gene promoting the chicoric acid synthesis pathway in expression is to overexpress the dandelion TmbHLH93 gene in dandelion.

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