Saffron bHLH transcription factor CsbHLH30, encoding gene and application

Abstract

This invention discloses a saffron bHLH transcription factor CsbHLH30, its encoding gene, and its applications. The transcription factor CsbHLH30 comprises a polypeptide having the amino acid sequence shown in SEQ ID NO. 2. This invention also provides a nucleic acid sequence encoding the above protein, SEQ ID NO. 1. This invention provides a theoretical basis for enhancing the antioxidant activity of saffron using genetic engineering technology and has significant application value.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering application technology, and relates to an important transcription factor in saffron that can significantly enhance antioxidant activity in response to JA signal during stress resistance. Specifically, it relates to the saffron bHLH transcription factor CsbHLH30, its encoding gene, and its applications. Background Technology

[0002] Saffron is a plant belonging to the Iridaceae family. Crocus sativus The dried stigmas of saffron (L.), also known as saffron, are native to Europe, the Mediterranean, and Central Asia. The unique natural pigments and aromatic substances in saffron make it one of the world's most expensive spices, often referred to as "red gold," and it has significant potential for development in high-end textiles, daily chemical products, and health supplements. However, saffron medicinal yields are extremely low, and its quality is easily affected by the environment, resulting in unstable yields and quality.

[0003] The bHLH (basic / helix-loop-helix) transcription factor family, named for its highly conserved basic / helix-loop-helix domain, is one of the largest transcription factor families in plants, widely distributed in both plants and animals. The bHLH transcription factor family participates in many plant physiological processes, playing a crucial role in plant growth regulation, stress response regulation, and secondary metabolism regulation, and possessing broad functions in biosynthesis, metabolism, and signal transduction. Multiple studies have confirmed that bHLH transcription factors can regulate plant stress responses through JA signaling, and transcriptome analysis has revealed that saffron... CsbHLH The gene responded to the JA signal. However, there are currently few research reports on the role of CsbHLH transcription factor in enhancing the antioxidant activity and stress resistance of saffron. Summary of the Invention

[0004] To fill the saffron CsbHLH30 This invention discloses the cloning, expression pattern, and functional analysis of the gene, as well as the blank of the saffron transcription factor CsbHLH30; CsbHLH30 The gene sequence and encoded amino acid sequence, protein subcellular localization, related gene expression and enzyme activity analysis lay the foundation for revealing the role of CsbHLH30 transcription factor in saffron stress response, elucidating the formation law of saffron medicinal material quality and cultivating high-quality saffron medicinal material.

[0005] On one hand, the present invention provides saffron bHLH transcription factor CsbHLH30, wherein the transcription factor CsbHLH30 comprises a polypeptide (protein) having the amino acid sequence shown in SEQ ID NO.2; or a protein having the characteristics of saffron bHLH transcription factor CsbHLH30, wherein the amino acid sequence shown in SEQ ID NO.2 has been substituted, deleted, or added with one or more amino acids. The saffron bHLH transcription factor CsbHLH30 can respond to JA signaling and enhance the antioxidant activity of transgenic plants.

[0006] On the other hand, the present invention provides a coding gene encoding the above-mentioned saffron bHLH transcription factor CsbHLH30. The nucleotide sequence of the above-mentioned coding gene is specifically: (a) the base sequence is as shown in positions 1 to 756 of SEQ ID NO.1; or (b) a sequence having at least 70% homology with the nucleic acid shown in positions 1 to 756 of SEQ ID NO.1.

[0007] In this invention, "isolated DNA" and "purified DNA" refer to DNA or fragments that have been isolated from sequences flanking them in their natural state, and also to DNA or fragments that have been separated from components that accompany nucleic acids in their natural state, and from proteins that accompany them in the cell.

[0008] In this invention, the gene encoding the saffron bHLH transcription factor CsbHLH30 protein refers to the nucleotide sequence encoding a polypeptide with saffron protein activity, such as the nucleotide sequence from position 1 to 756 of SEQ ID NO.1 and its degenerate sequence. This degenerate sequence refers to a sequence in which one or more codons in positions 1 to 756 of SEQ ID NO.1 are replaced by degenerate codons encoding the same amino acid. Due to codon degeneracy, a degenerate sequence with less than 70% homology to the nucleotide sequence from position 1 to 756 of SEQ ID NO.1 can also encode the sequence shown in SEQ ID NO.2. The aforementioned encoding gene can also refer to a nucleotide sequence with at least 70% homology to the nucleotide sequence shown in SEQ ID NO.1.

[0009] In this invention, saffron can be analyzed using real-time quantitative PCR. CsbHLH30 The expression pattern of gene products, i.e., analyzing saffron CsbHLH30 The presence and quantity of gene mRNA transcripts in cells.

[0010] Furthermore, according to the present invention, saffron CsbHLH30 Gene sequences and amino acid sequences can be used to screen saffron based on nucleic acid homology or expressed protein homology. CsbHLH30Gene-related homologous genes or homologous proteins.

[0011] The saffron of this invention CsbHLH30 Full-length sequences or fragments of gene-related nucleotides can typically be obtained using PCR amplification, recombination, or artificial synthesis. For PCR amplification, primers can be designed based on the nucleotide sequences disclosed in this invention, and commercially available cDNA libraries or cDNA libraries prepared using conventional methods known to those skilled in the art can be used as templates to amplify the relevant sequences. When the sequences are long, two or more PCR amplifications are often required, and then the fragments amplified from each amplification are spliced ​​together in the correct order.

[0012] Once the relevant sequence is obtained, it can be obtained in large quantities using recombination methods. This typically involves cloning it into a vector, transferring it into cells, and then isolating the relevant sequence from the proliferated host cells using conventional methods.

[0013] In addition, mutations can be introduced into the protein sequence of the present invention through chemical synthesis.

[0014] Besides being produced by recombinant methods, fragments of the protein of the present invention can also be produced by solid-phase technology through direct peptide synthesis. The individual fragments of the protein of the present invention can be chemically synthesized separately and then chemically linked to produce a full-length molecule.

[0015] This invention also provides a recombinant expression vector containing the encoding gene of the saffron bHLH transcription factor CsbHLH30. The recombinant expression vector is pCAMBIA1304- CsbHLH30 .

[0016] The present invention also provides an application of the gene encoding the above-mentioned saffron bHLH transcription factor CsbHLH30 in enhancing the antioxidant activity of saffron.

[0017] The above applications include: constructing a recombinant expression vector containing the above-mentioned transcription factor CsbHLH30 encoding gene, transforming it into a plant host, and culturing and screening to obtain transgenic plants.

[0018] Beneficial Effects: Saffron, as a rare and precious traditional Chinese medicine, has a large market demand. This invention is the first to clone the coding sequence of CsbHLH30, an important regulatory protein in saffron's stress resistance process, and analyzes it using real-time quantitative PCR. CsbHLH30 Gene expression patterns, transient expression analysis of transcription factor CsbHLH30 in tobacco leaf epidermal cells, subcellular localization, and overexpression analysis of related antioxidant activities were studied to provide a basis for future regulation using genetic engineering techniques. CsbHLH30The study of spatiotemporal gene expression provides a theoretical basis for improving the antioxidant activity, stress resistance, and breeding of new saffron varieties, and has great application value. Attached Figure Description

[0019] Figure 1 The saffron of this invention CsbHLH30 Genes and Arabidopsis AtbHLH Phylogenetic tree analysis of homologous genes;

[0020] Figure 2 Map showing the localization of saffron transcription factor CsbHLH30 in tobacco leaf epidermal cells;

[0021] Figure 3 Saffron CsbHLH30 Diagram showing differential gene expression in different plant tissues;

[0022] Figure 4 Saffron induced by MeJA CsbHLH30 Gene expression analysis diagram;

[0023] Figure 5 Saffron CsbHLH30 A graph showing the differences in antioxidant enzyme activity in genetically modified tobacco plants;

[0024] Figure 6 Saffron under drought stress CsbHLH30 Growth chart of genetically modified tobacco. Detailed Implementation

[0025] The technical solution of the present invention will be described in detail below through embodiments, but the scope of protection of the present invention is not limited to the embodiments described.

[0026] Experimental methods not specifically described in the following examples are generally performed under standard conditions, such as those described in Molecular Cloning: A Laboratory Manual (4th Edition), or as recommended in the reagent instructions.

[0027] Example 1: Saffron CsbHLH30 Cloning of genes

[0028] 1. Obtaining plant materials

[0029] Select healthy saffron bulbs and harvest the saffron flowers on the day of flowering into sterile centrifuge tubes. Quickly freeze the tubes in liquid nitrogen for the extraction of total RNA from the saffron flowers.

[0030] 2. RNA extraction

[0031] Total RNA was extracted from saffron according to the instructions of the RNAprep Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (TIANGEN). The integrity of the RNA was identified by gel electrophoresis, and the purity and concentration of the RNA were determined by spectrophotometer (Nanodrop 2000).

[0032] 3. Full-length cloning of genes

[0033] Based on the nucleic acid sequence and protein function annotation results provided by the laboratory's previous full-length transcriptome analysis, saffron was obtained CsbHLH30 Full-length gene. The extracted RNA was reverse transcribed (TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix) to obtain cDNA. Using the first-strand cDNA as a template, primers were used... CsbHLH -F(5'-ATGGTTTTTTGGCAATATGGCT-3') and ​ PCR amplification was performed using -R (5'-TCAACAAGAGGATGAGAGTGTGGAT-3'), yielding a 756 bp fragment. This fragment was recovered, ligated into the pMD19-T vector, and sent to a sequencing company for sequencing.

[0034] The secondary and tertiary structures of the saffron transcription factor CsbHLH30 protein were predicted using the online websites SOPMA (https: / / npsa-prabi.ibcp.fr / cgi-bin / npsa_automat.pl?page=npsa%20_sopma.html) and SWISS-MODEL (https: / / swissmodel.expasy.org / ). The prediction results showed that the secondary structure of the CsbHLH30 protein consisted of 44.22% α-helices, 13.35% extended strands, 3.19% β-turns, and 40.24% random coils.

[0035] Example 2, Saffron ​ Gene sequence information and homology analysis

[0036] The saffron of this invention ​ The full-length open reading frame (ORF) sequence of the gene is 756 bp, and the detailed sequence is shown in SEQ ID NO.1. Based on the ORF sequence, the amino acid sequence of the saffron transcription factor CsbHLH30 protein was deduced, consisting of 251 amino acids, with a molecular weight of 27.62 kDa and an isoelectric point (pI) of 8.77. The detailed sequence is shown in SEQ ID NO.2.

[0037] 162 AtbHLH transcription factor sequences were downloaded from the Arabidopsis protein database (https: / / www.arabidopsis.org / ) and used to construct a phylogenetic tree with the screened CsbHLH transcription factors. Phylogenetic analysis showed that the saffron transcription factor CsbHLH30 exhibited high homology with bHLH transcription factors from other known species in the phylogenetic tree, such as... ​ As shown.

[0038] Example 3: Subcellular localization analysis of saffron transcription factor CsbHLH30 in tobacco leaves

[0039] Design specific primers at the start codon and stop codon respectively. ​ -F (5'- TCGGTACCCGGGGATCCATGGTTTTTTGGCAATATGGCTTCGG-3'), ​ -R(5'-TGCTCACCATGTCGACACAAGAGGATGAGAGTGTGGATTCG-3'), and introduced on both sides of the full-length gene sequence. ​ I and ​ I. Restriction Sites. The plasmid containing the target fragment with restriction sites is then coupled with the 35S-GFP vector. ​ I and ​ I. Double enzyme digestion, recovery of the digested 35S-GFP vector and... ​ The fragments were ligated with T4 ligase at 37℃ for 1 h to carry out homologous recombination, constructing a recombinant expression vector, and the correctly identified recombinant vector was transformed into Agrobacterium EHA105.

[0040] The identified EHA105 strain was cultured in 50 mL of LB (Kana + Rif) liquid medium at 28°C and 220 rpm for 36 h until the bacterial OD reached the target value. 600 Approximately 0.5-0.6; centrifuge at 5000 rpm for 15-20 min, collect the bacterial cells; resuspend the bacterial cells using resuspension buffer, and finally calculate the OD value. 600 The concentration was 0.4; the plants were placed at room temperature for 2-3 hours; the solution was injected into tobacco leaves, and the injected tobacco plants were cultured in the dark for 12 hours, followed by a 12-hour photoperiod at 21°C for 2 days. Observations were then performed using a laser confocal microscope at an excitation wavelength of 514 nm. ​ As shown.

[0041] Example 4, Saffron ​ Spatiotemporal expression analysis of genes

[0042] 1. Material acquisition: Saffron bulbs with uniform growth were selected. On the day the saffron flowers bloomed, samples were taken from the bulbs, leaves, petals, stamens, and stigmas. The samples were then rapidly frozen in liquid nitrogen and stored at -80℃ for tissue expression analysis. Saffron stigmas treated with 100 μmol / L MeJA for different time periods were selected, rapidly frozen in liquid nitrogen, and stored at -80℃ for expression analysis in response to JA signals.

[0043] 2. RNA extraction, determination of RNA integrity, purity, and concentration, and acquisition of cDNA are described in Example 1.

[0044] 3. Design specific primers for real-time quantitative PCR analysis of gene expression levels in various tissues, based on the obtained saffron... ​ Gene sequence designed for use in real-time PCR. ​ Specific primers for gene quantification analysis, primer q ​ -F(5'-CCATCAAGGCATCGCTCTCC-3'), primer q ​ -R(5'-TGGCGAACATTTGCCGTACA-3'), internal reference gene ​ Primers are ​ F(5'-ACCATGTTTCCCGGGATTGC-3'), ​ -R(5'-TGCGGTGAACGATTGAAGGG-3').

[0045] 4. Standard curves for the target gene and internal reference gene: The standard cDNA solution was serially diluted with ddH2O. Then, using the diluted cDNA as templates, Real-time PCR amplification was performed with specific primers for the target gene and internal reference gene, respectively. Melting curves and standard curves were plotted. The melting curves were analyzed to determine whether a single peak was obtained for the target gene and internal reference gene, in order to determine whether a single PCR amplification product could be obtained using the primers. The appropriate dilution factor of the template cDNA was determined by the standard curves.

[0046] 5. Real-time quantitative analysis of the target gene in the test sample: Using the first strand of the synthesized cDNA as a template, the target gene and the internal reference gene were amplified with specific primers for real-time quantitative analysis. The Bio-Rad CFX real-time quantitative PCR reaction was performed. The reaction volume was 20 µL, and the reaction program was: 95℃ for 30s; 95℃ for 5s; 58℃ for 30s; 65℃ for 5s; 40 cycles.

[0047] 6. Use 2 -△△Ct Relative quantitative analysis was performed using the method, and the results showed that saffron ​The gene was expressed in all five sites, with the highest expression level in the stigma, followed by the leaves, stamens, and corms, and the lowest expression level in the petals. ​ As shown; saffron ​ Gene expression levels increased dramatically during MeJA treatment, more than 20-fold compared to the control group, and were a positive response factor to JA signaling. ​ As shown.

[0048] Example 5, Saffron ​ Genetic transformation in tobacco

[0049] 1. Constructing plant expression vectors

[0050] The plasmid containing the target fragment with restriction enzyme sites was coupled with the overexpression vector pCAMBIA1304. ​ I and ​ II. After double enzyme digestion, the digested vector was recovered and ligated via homologous recombination to obtain the recombinant expression vector pCAMBIA1304- ​ The recombinant expression vector was then transformed into Agrobacterium EHA105.

[0051] 2. Tobacco Conversion

[0052] The specific steps for EHA105 tobacco contamination are as follows:

[0053] ① Take 2 μL of Agrobacterium tumefaciens containing recombinant plasmids, which has been stored at low temperature, and streak it in LB (Kana 50 mg / L, Rif 25 mg / L) solid medium. Incubate at 28°C with the medium upside down for 2 days. Pick single colonies and reactivate them in sterile 1.5 mL centrifuge tubes. Add 1 mL of LB (Kana + Rif) liquid medium to the centrifuge tubes and incubate at 28°C with shaking for 24 h.

[0054] ② Take 100 μL of activated Agrobacterium bacterial culture and incubate it in 25 mL of LB (Kana + Rif) liquid medium at 28°C with shaking until OD reaches 100 μL. 600 Approximately 0.4-0.6.

[0055] ③ Centrifuge at 4℃, 6000 rpm for 6 min, and discard the supernatant.

[0056] ④ Resuspend the bacterial cells in 20 mL of MS (AS 100 μmol / L) liquid medium, centrifuge at 4℃, 6000 rpm for 6 min, and repeat the washing once.

[0057] ⑤ Resuspend the bacterial culture in MS (AS 100 μmol / L) liquid medium and adjust the OD. 600 0.5 is reserved.

[0058] ⑥ Take sterile tobacco plants that have grown for about one month, cut them into 1 cm × 1 cm pieces using a sterile scalpel, place them in MS bud culture medium, and incubate in the dark for 3 days.

[0059] ⑦ Place the tobacco leaves that have been pre-cultured for about 3 days into the resuspended bacterial solution obtained in step 5. Shake gently for about 8 minutes, then place the tobacco leaves on sterile filter paper to absorb the residual bacterial solution on the surface of the tobacco leaves. Lay the tobacco leaves flat with the back side up in the solid culture medium for MS co-culture. After culturing in the dark for 2 days, perform the first sterilization.

[0060] ⑧ Wash the leaves twice with sterile water containing Tim (300 mg / L), then wash them three times with sterile water without Tim. Blot the surface of the tobacco leaves dry on sterile filter paper, lay the leaves flat on MS bud culture medium containing Tim (300 mg / L) and Hyg (25 mg / L), and perform a second sterilization after 7 days of light culture.

[0061] ⑨ If no Agrobacterium grows, the leaves can be placed directly on MS bud culture medium, and the corresponding antibiotics can be added for 14 days of culture.

[0062] ⑩ Repeated sterilization can reduce the concentration of Tim in the culture medium. After the tobacco leaves sprout, they are placed in MS blank medium to induce rooting and harden off.

[0063] 3. Screening of transgenic positive lines

[0064] Genomic DNA and RNA were extracted to identify positive plants.

[0065] Example 6, Overexpression ​ Analysis of the activity of antioxidant enzymes in tobacco genes

[0066] For research ​ The regulation of antioxidant activity in tobacco by genes was investigated, and the differences in enzyme activity between transgenic tobacco and wild-type tobacco (WT) were analyzed by measuring SOD and CAT enzyme activities. ​ As shown.

[0067] 1. Obtaining materials: Selection ​ Genetically modified tobacco and wild-type tobacco were sampled, rapidly frozen in liquid nitrogen, and then stored at -80°C for analysis of antioxidant enzyme activity.

[0068] 2. Sample preparation: Weigh fresh tobacco leaf tissue and add 9 volumes of phosphate buffer (PBS, 0.1 mmol / L, pH 7.2-7.4) at a weight (g):volume (mL) ratio of 1:9. Grind the mixture into a homogenate in an ice bath, centrifuge at 3500 r / min for 10 min, collect the supernatant, dilute it with buffer to an appropriate concentration, and perform various physiological and biochemical tests. All samples were tested three times.

[0069] 3. Superoxide dismutase (SOD) and catalase (CAT) activity assays: The corresponding kits from Nanjing Jiancheng Bioengineering Institute were used for the assays. The specific methods were described in the kit instructions. All samples were 10% concentration tobacco leaf tissue homogenates.

[0070] 4. The results show that in ​ In transgenic tobacco overexpression, the activities of both SOD and CAT were significantly increased, such as ​ As shown, this demonstrates that the transcription factor CsbHLH30 can affect the stress resistance of tobacco by influencing its antioxidant activity.

[0071] Example 7, Overexpression ​ Analysis of drought resistance in genetically modified tobacco

[0072] 1. Take ​ Overexpressing tobacco and wild-type tobacco (WT) were planted in a culture room at 25°C with 16 hours of light. After 30 days, tobacco plants with consistent growth were selected, watering was stopped, and drought stress treatment was applied.

[0073] 2. The growth of drought-treated materials was observed, and the results showed that after 15 days of drought treatment... ​ Tobacco plants with overexpressed genes showed less leaf wilting and exhibited better growth than wild-type tobacco. ​ As shown, this demonstrates that the transcription factor CsbHLH30 can improve the drought resistance of tobacco.

[0074] Unless otherwise specified, all technologies mentioned above refer to existing technologies.

[0075] As described above, although the invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the invention itself. Various changes in form and detail may be made without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. The application of overexpression of the gene encoding the saffron bHLH transcription factor CsbHLH30 in enhancing the drought resistance of saffron, characterized in that, The amino acid sequence of saffron bHLH transcription factor CsbHLH30 is shown in Sequence 2.

2. The application of the gene encoding the saffron bHLH transcription factor CsbHLH30, as described in claim 1, in enhancing the drought resistance of saffron, characterized in that... The application includes: constructing a recombinant expression vector containing the encoding gene of the transcription factor CsbHLH30, transforming it into a plant host, and culturing and screening to obtain transgenic plants.

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