Ramie MYB transcription factor and application thereof

Abstract

The invention relates to the technical field of genetic engineering and molecular breeding, in particular to a ramie MYB transcription factor and application thereof. The MYB transcription factor is BnMYB6, the amino acid sequence of the MYB transcription factor is as shown in SEQ ID NO.1, and the gene sequence for coding the amino acid of the MYB transcription factor is as shown in SEQ ID NO.2. The transcription factor protein BnMYB6 can specifically and negatively regulate the biosynthesis of plant lignin, and has application value in the aspects of inhibiting the synthesis of plant lignin, promoting the growth and development of a root system and maintaining related traits such as the activity of the root system under drought stress.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and molecular breeding technology, and in particular to a ramie MYB transcription factor and its application. Background Technology

[0002] Lignin is a major component of plant secondary cell walls, playing a crucial role in plant mechanical support, water transport, and stress resistance. However, in certain applications, excessively high lignin content can be detrimental. For example, in fiber crops (such as ramie and flax), high lignin content can make fibers coarse, hard, and brittle, affecting fiber strength, color, and elasticity; in forage crops (such as alfalfa and forage corn), high lignin content can severely reduce feed digestibility and nutritional value; and in fruit and vegetable crops, lignification can affect palatability and edibility. Therefore, effectively reducing the lignin content in specific plant tissues or at specific growth stages has significant economic and ecological value.

[0003] Currently, strategies for reducing lignin content in plants mainly include traditional breeding and genetic engineering. Genetic engineering often achieves this by inhibiting or knocking out key enzyme genes (such as PAL, C4H, 4CL, CCR, CAD, etc.) in the lignin synthesis pathway. However, directly inhibiting structural genes often leads to severe lignin synthesis obstruction, causing serious negative phenotypes such as malformed plant growth and development, dwarfism, reduced fertility, and even death. Furthermore, it easily leads to abnormal accumulation of other phenolic substances due to metabolic compensation effects. Therefore, finding gene resources that can reduce lignin content while maintaining or even enhancing plant growth (especially root development) has become a significant challenge in this field.

[0004] Transcription factors, as upstream regulatory elements, can regulate the entire metabolic pathway in a more precise and coordinated manner. Using negatively regulating transcription factors to inhibit lignin synthesis, compared to directly knocking out key enzyme genes involved in lignin synthesis, may achieve a "fine-tuning" of lignin content, reducing lignin levels while minimizing negative impacts on normal plant growth and development. Currently, the number of reported negatively regulating lignin synthesis MYB transcription factors (such as EgMYB1 and TaMYB4) is limited, and their inhibitory effects, action spectrum, and conservation across different species require further exploration and application.

[0005] Therefore, discovering new and efficient transcription factors that negatively regulate lignin synthesis from specific plants (such as ramie, which has high-quality fiber and relatively low lignin content) is of great theoretical and applied value for developing new lignin improvement technologies and cultivating high-quality crop varieties with low lignin content. Summary of the Invention

[0006] In view of this, the present invention proposes a novel negative regulatory transcription factor protein BnMYB6 derived from ramie and its encoding gene. This transcription factor can specifically inhibit the expression of key genes in the lignin synthesis pathway, thereby effectively reducing the lignin content of plants. At the same time, it also has a significant regulatory effect on plant phenotype, especially root length and root activity under drought stress, and has great application value for breeding high-quality crop varieties.

[0007] The technical solution of this invention is implemented as follows: In a first aspect, the present invention provides a ramie MYB transcription factor, wherein the MYB transcription factor is BnMYB6, and its amino acid sequence is shown in SEQ ID NO.1.

[0008] The sequence of SEQ ID NO.1 is as follows: MGRSPCCEKAHTNKGAWTKDEDQRLIDYIRVHGEGCWRSLPKAAGLLRCGKSCRLRWINYLRPDLKRGNFTEEEDELIIKLHGLLGNKWSLIAGRLPGRTDNEIKNYWNTHIKRKLISRGLDPQTHRPLNDAAPPPPPPAAAAAKLHASARLDFRNASPAPAPAD KRNNLSAPNTNTKLAIHPKAETIVFDEAARSNCTSSGTTTEEEDQRHDNIAHQHAHHRHVMTSDLHVAVNLELSIGLAPFQSRDQAHDSAAATRLSFANSAESKPLRVGNGVNSNGNNCNNYGQLFGGVGVCLCCQLGHQSSTELCRNCQASNAFFRFHNLPLNS.

[0009] Amino acid sequences that have been substituted, deleted, or have one or more amino acids added to the SEQ ID NO.1 sequence, i.e., have more than 95% homology with the SEQ ID NO.1 sequence and have the same regulatory effect as SEQ ID NO.1, are also within the scope of protection of this invention.

[0010] Secondly, a gene for a ramie MYB transcription factor is provided, which encodes the ramie MYB transcription factor as described above.

[0011] Based on the above technical solutions, preferably, the nucleotide sequence of the gene is shown in SEQ ID NO.2.

[0012] The sequence of SEQ ID NO.2 is as follows: ATGGGAAGGTCTCCTTGCTGCGAGAAAGCTCACACCAACAAAGGCGCTTGGACCAAAGACGAAGATCAACGCCTCATCGACTACATCCGCGTTCACGGTGAGGGCTGCTGGCGCTCCCTCCCCAAGGCCGCAGGGTTACTTAGATGCGGCAAGAGTTGCAGGCTGAGGTGGATAAACTACCTCCGACCCGACCTCAAGCGAGGGAATTTCACCGAAGAGGAAGACGAATTGATCATCAAGCTTCACGGCTTACTCGGAAATAAATGGTCACTGATAGCGGGAAGATTGCCGGGAAGAACTGACAACGAGATAAAAAACTACTGGAACACGCACATCAAGCGGAAACTCATAAGCCGCGGCCTCGACCCTCAGACGCACCGCCCCCTAAACGACGCCGCACCGCCGCCGCCACCACCCGCCGCCGCCGCCGCTAAATTACACGCCTCGGCCCGCTTGGACTTCAGAAACGCCTCCCCCGCGCCCGCGCCCGCCGATAAGCGCAACAACTTATCGGCACCCAATACCAATACCAAGCTTGCGATACACCCGAAAGCGGAAACCATAGTGTTCGACGAAGCCGCCAGGTCGAACTGCACGAGCAGCGGAACGACAACCGAGGAGGAGGACCAGCGACACGATAATATTGCCCACCAGCACGCTCACCACCGCCACGTGATGACCAGCGACCTCCACGTGGCGGTCAACCTGGAGCTCTCCATCGGCCTCGCGCCGTTCCAGTCACGTGACCAAGCCCACGACTCGGCCGCCGCGACTCGGCTGTCGTTCGCCAACTCGGCCGAGTCGAAACCGCTCCGAGTCGGTAACGGCGTGAATAGTAATGGCAATAACTGTAATAACTACGGTCAGTTGTTTGGTGGCGTTGGCGTGTGCCTGTGTTGCCAGTTGGGGCACCAAAGCAGCACCGAGTTGTGCAGGAATTGCCAAGCCTCAAATGCCTTCTTCAGATTTCACAATTTGCCTCTTAATTCATAG。

[0013] Thirdly, a recombinant expression vector is provided, which contains the gene of the ramie MYB transcription factor as described above.

[0014] Fourthly, a host cell is provided that contains the ramie MYB transcription factor as described above, the gene of the ramie MYB transcription factor as described above, or the recombinant expression vector as described above.

[0015] Fifthly, the application of the ramie MYB transcription factor as described above, the gene of the ramie MYB transcription factor as described above, the recombinant expression vector as described above, or the host cell as described above in regulating plant lignin biosynthesis, expression of lignin synthesis-related genes, plant lignin deposition, and / or plant phenotype is provided.

[0016] Based on the above technical solutions, preferably, the regulation of plant lignin biosynthesis includes: negative regulation of plant lignin biosynthesis.

[0017] Based on the above technical solutions, preferably, the regulation of lignin synthesis-related gene expression includes: negative regulation of lignin biosynthesis-related gene expression; the lignin biosynthesis-related genes include one or more of AtPAL2, AtCAD4, AtCAD6, AtCCoAOMT1, At4CL5, or AtCOMT.

[0018] Based on the above technical solutions, preferably, the regulation of plant lignin deposition includes: negative regulation of plant lignin deposition.

[0019] Based on the above technical solutions, preferably, the regulation of plant phenotype includes: positively regulating plant root length or root activity under drought stress conditions.

[0020] The present invention also provides a method for reducing the lignin content of plants or improving plant-related qualities, the method comprising: introducing a polynucleotide encoding the BnMYB6 transcription factor into a target plant or plant cell, and overexpressing or heterologously expressing it, thereby negatively regulating the expression of lignin synthesis-related genes and reducing the lignin content of the plant; more preferably, the introduction and expression of the polynucleotide are achieved through Agrobacterium-mediated genetic transformation technology.

[0021] The ramie MYB transcription factor of the present invention has the following advantages over the prior art: 1. This invention provides a novel negative regulatory transcription factor protein BnMYB6 derived from ramie and its encoding gene. This transcription factor can specifically regulate plant lignin biosynthesis, lignin synthesis-related gene expression, plant lignin deposition, and plant phenotype.

[0022] 2. Compared with wild-type plants, the transgenic plants of this invention exhibit the following: significantly downregulated transcription levels of key genes in the lignin synthesis pathway (AtPAL2, AtCAD4, AtCAD6, AtCCoAOMT1, At4CL5, AtCOMT); significantly higher lignin staining intensity in transgenic seedlings and mature leaves compared to wild-type plants; significantly longer root systems in transgenic seedlings compared to wild-type control plants, demonstrating enhanced nutrient absorption potential; and significantly higher root vigor in transgenic seedlings under simulated drought stress compared to wild-type control plants, demonstrating the potential to maintain root vigor under drought stress. This has significant application value in the breeding of new varieties of plants such as Arabidopsis thaliana and ramie. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 This is a PCR electrophoresis image of the transcription factor BnMYB6 gene clone of the present invention. M: Marker, lane 1: BnMYB6 gene amplification result; Figure 2 The diagram shows the predicted conserved domains and conserved amino acid analysis of the transcription factor BnMYB6 protein of this invention. (A) is the SMART online analysis diagram of the conserved domains of BnMYB6, and (B) is the amino acid sequence alignment result of the R2R3 domain of BnMYB6 and its homologous protein. Figure 3 The diagram shows the transcriptional activation activity of the transcription factor BnMYB6 of this invention. Diagram (A) shows the transcriptional activity of the complete BnMYB6 and its VP16 fusion construct in a yeast system. Diagram (B) shows the different domains of BnMYB6 (N-terminus and C-terminus) interacting with pGBKT7 and pGBKT7-, respectively. VP16 A graph showing the transcriptional activity analysis of the fused yeast cells; Figure 4 This is a diagram showing the expression levels of BnMYB6 and related lignin synthesis genes under different stem bark lignin deposition conditions according to the present invention. Figure (A) shows the low lignin stem, stem cross section and stem bark fiber lignin staining diagram; Figure (B) shows the high lignin stem, stem cross section and stem bark fiber lignin staining diagram; and Figure (C) shows the expression patterns of BnMYB6 and key genes for ramie lignin synthesis in high and low lignin stem bark. Figure 5The images show the PCR identification of transgenic Arabidopsis plants and the detection of BnMYB6 gene overexpression in this invention. In the images, (A) represents the PCR detection, (B) represents the RT-PCR detection, and (C) represents the RT-qPCR detection. Figure 6 The above are phenotypic comparison diagrams of transgenic Arabidopsis thaliana plants and wild-type plants of the present invention. In the diagram (A), the growth status of wild-type and transgenic seedlings is compared. In the diagram (B), the lignin histochemical staining diagram of Arabidopsis thaliana seedlings is shown. In the diagram (C), the root length analysis diagram of transgenic Arabidopsis thaliana plants and wild-type plants is shown. In the diagrams (D) and (E), the growth status of mature wild-type and transgenic plants after transplanting is compared. Figure 7 This is a graph showing the expression levels of key genes for lignin synthesis in the transgenic plants of this invention. Figure 8 This is a graph showing the root activity of the transgenic plants and wild-type plants of this invention under drought stress. Detailed Implementation

[0025] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0026] Example 1: Cloning and sequence analysis of the transcription factor BnMYB6 gene of the present invention 1.1 Extraction of ramie RNA and synthesis of cDNA Young leaves of ramie variety Huazhu 5 were selected as material, and total RNA (TIANGEN, DP441) was extracted using the RNAprep Pure Polysaccharide and Polyphenol Total RNA Extraction Kit. cDNA was obtained by reverse transcription using the TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix kit (Beijing TransGen Biotech Co., Ltd., AT311-03). Specific procedures were strictly followed according to the product instructions.

[0027] 1.2 Cloning and sequencing of the BnMYB6 gene Using the CDS sequence of ramie transcription factor BnMYB6 as a reference, specific primers BnMYB6-F and BnMYB6-R were designed. The specific primer sequences are as follows: BnMYB6-F: 5'-ATGGGAAGGTCTCCTTGCT-3' (SEQ ID NO. 3); BnMYB6-R, 5'-CTATGAATTAAGAGGCAAATTGTG-3' (SEQ ID NO. 4).

[0028] Using the cDNA obtained in step 1.1 as a template, the BnMYB6 gene fragment was amplified. The reaction system for amplification with the high-fidelity enzyme PrimeSTARMax DNA Polymerase is shown in Table 1 (25 μL system): Table 1

[0029] The reaction conditions are shown in Table 2: Table 2

[0030] The reaction solution was analyzed by 1.5% TAE agarose gel electrophoresis. The electrophoresis results were as follows: Figure 1 The results showed that the target fragment obtained by PCR amplification was approximately 1000 bp in size, consistent with the size of the BnMYB6 gene. The correctly identified PCR product was sent to Wuhan Tianyi Huayu Gene Technology Co., Ltd. for sequencing, and the gene DNA sequence is shown in SEQ ID NO.2 of the sequence listing.

[0031] 1.3 Amino acid sequence analysis of BnMYB6 in Huazhu 5 ramie The BnMYB6 amino acid sequence was submitted to the SMART online analysis tool for conserved protein domain analysis (http: / / smart.embl-heidelberg.de / ). The results showed ( Figure 2 (Figure A) shows that the BnMYB6 gene of ramie var. chinensis contains two conserved domains. Multiple sequence alignment of the amino acid sequence was performed using ClustalX software, and the results showed that (…). Figure 2 (Figure B) shows that BnMYB6 possesses a highly conserved R2R3 domain at its N-terminus, and a C1 repressive motif was also identified at its C-terminus. These results suggest that BnMYB6 may be an R2R3 MYB transcriptional repressor.

[0032] Example 2: Analysis of BnMYB6 transcriptional activation activity in ramie Huazhu No. 5 2.1 Enzyme digestion and recovery of pGBKT7 and pGBKT7-VP16 vectors The gene cloning primer adapter sequences contain EcoRI and BamHI restriction sites. The pGBKT7 vector was double-digested with the restriction endonucleases EcoRI and BamHI. The double digestion system (20 μl) is shown in Table 3.

[0033] Table 3

[0034] The reaction conditions are shown in Table 4: Table 4

[0035] The gene cloning primer adapter sequence contains a BamHI restriction site, and the pGBKT7-VP16 vector was digested with the restriction endonuclease BamHI. The single digestion system (20 μl) is shown in Table 5:

[0036] Table 5

[0037] The reaction conditions are the same as Table 4.

[0038] The two fragments, one long and one short, after double enzyme digestion were separated by agarose gel electrophoresis, and PCR was performed using the Wizard® SV Gel and PCR Clean-Up System (Promega, A9281). The long fragment was then cut and recovered from the gel. The specific steps were strictly followed according to the product instructions.

[0039] 2.2 Construction of full-length and truncated BnMYB6 vectors containing restriction enzyme sites Based on the domain characteristics of BnMYB6, it was truncated into two fragments: BnMYB6N (containing the N-terminus and R2R3 DNA-binding domains) and BnMYB6C (containing the C1 repressor motif and C-terminal sequence). Adapter primers were designed based on the BnMYB6 gene sequence amplified in step 1.7. The designed primer sequences are shown in Table 6.

[0040] Table 6

[0041] Using the cDNA obtained in step 1.1 as a template, the reaction system and conditions for amplification with the high-fidelity enzyme PrimeSTAR Max DNA Polymerase were the same as in step 1.2. PCR products were purified using the Wizard® SV Gel and PCR Clean-Up System (Promega, A9281) nucleic acid purification kit, with specific steps strictly following the product instructions.

[0042] The gene fragment obtained in step 2.2 was ligated to the linearized vector obtained in step 2.1 via homologous recombination using the ClonExpress II One Step Cloning Kit (Vazyme, C112). The specific steps were strictly performed according to the product instructions. The fragment was introduced into *E. coli* DH5α competent cells using the heat shock method, and positive clones were screened on kanamycin plates and sent to Wuhan Tianyi Huayu Gene Technology Co., Ltd. for sequencing.

[0043] 2.3 Analysis of transcriptional activation activity The negative control pGBKT7, the positive control pGBKT7-VP16, and the recombinant plasmids pGBKT7-BnMYB6N, pGBKT7-BnMYB6C, pGBKT7-BnMYB6, pGBKT7-VP16-BnMYB6, pGBKT7-VP16-BnMYB6N, and pGBKT7-VP16-BnMYB6C obtained in step 2.5 were transformed into yeast AH109 competent cells (Coolaber, CC300) using the PEG / LiAC method. The specific steps were strictly performed in accordance with the product instructions.

[0044] Positive yeast colonies were picked and agaricated onto SD / -Trp and SD / -Ade-His-Trp plates, and incubated in the dark for 3-5 days. Results showed (e.g.) Figure 3 As shown in the figure, all transformants, including the negative control, grew well on SD / -Trp plates; on SD / -Ade-His-Trp plates, only pGBKT7-VP16-BnMYB6 and pGBKT7-VP16-BnMYB6C could resume yeast growth, but their colony-forming ability was weaker than that of the positive control. This indicates that BnMYB6 has transcriptional repressive activity, and its N-terminal and R2R3 domains are essential for exerting this transcriptional repressive function.

[0045] Example 3: Expression level analysis of BnMYB6 gene in ramie stem bark with high and low lignin deposition Ramie stems with high and low lignin deposition were selected and fixed in FAA fixative (70% ethanol: glacial acetic acid: formaldehyde = 90:5:5) for 24 h. After gradient dehydration with ethanol, clearing with xylene, and paraffin embedding, transverse or longitudinal sections (8-12 μm) were cut using a rotary microtome, spread and fixed at 40℃. Dewaxing with xylene and rehydration with gradient ethanol were performed. Lignin histochemical staining was performed using the Wiesner staining method: soaking in 2% phloroglucinol solution for 10 min, reacting with 18% hydrochloric acid for 5 min, and observing and photographing under a microscope. Results are as follows: Figure 4 As shown ( Figure 4Figures (A) and (B) show that, compared with ramie stem bark with high lignin deposition, ramie stem bark with low lignin deposition is lighter in color and has lower lignin staining intensity.

[0046] Total RNA was extracted from ramie stem bark with high and low lignin deposition, and cDNA was obtained by reverse transcription, following the same steps as in 1.1.

[0047] Using the obtained cDNA as a template, quantitative real-time PCR was performed using ChamQ Universal SYBR qPCR Master Mix (Vazyme, Q711-02), primers for the BnMYB6 gene, and primers for the lignin synthesis gene. The Bne1Fα gene was used as an internal reference gene. Primer sequences are shown in Table 7.

[0048] Table 7

[0049] The reaction system (10 μL) is shown in Table 8: Table 8

[0050] The reaction conditions are shown in Table 9: Table 9

[0051] Figure 4 Figure (C) shows that, compared with ramie stem bark with high lignin deposition, the expression levels of lignin synthesis-related genes BnPAL, BnCCR1, BnCCoAOMT, BnCOMT1, and BnCAD were significantly decreased in ramie stem bark with low lignin deposition, but the expression level of the BnMYB6 gene was significantly increased. These results suggest that BnMYB6 may negatively regulate lignin biosynthesis.

[0052] Example 4: Construction of plant overexpression vectors 4.1 Double digestion and purification of the overexpression vector pCAMBIA2300 The gene cloning primer adapter sequences contain BamHI and XbaI restriction enzyme sites. The experiment used double digestion with the restriction endonucleases BamHI and XbaI. The double digestion system (20 μl) is shown in Table 10:

[0053] Table 10

[0054] The reaction conditions are shown in Table 11: Table 11

[0055] Long segments are cut and recycled (same as step 2.1).

[0056] 4.2 Construction of BnMYB6 overexpression vector containing restriction enzyme sites Based on the BnMYB6 gene sequence amplified in Example 1, adapter primers were designed. The upstream primer contained a BamHI restriction site, and the downstream primer contained an XbaHI restriction site. The primer sequences are as follows: OE-BnMYB6-F: 5'-CTTGGAATTCCTGCTGGATCCATGGGAAGGTCTCCTTGCT-3' (SEQ ID NO.31), OE-BnMYB6-R: 5'-CTCTTCGAACCCGGGTCTAGACTATGAATTAAGAGGCAAATTGTGA-3' (SEQ ID NO.32).

[0057] Using the cDNA obtained in step 1.1 as a template, the reaction system and conditions for amplification with the high-fidelity enzyme PrimeSTAR Max DNA Polymerase were the same as in step 1.2. PCR products were purified using the Wizard® SV Gel and PCR Clean-Up System (Promega, A9281) nucleic acid purification kit, with specific steps strictly following the product instructions.

[0058] The gene fragment obtained in step 4.2 was ligated to the linearized vector obtained in step 4.1 via homologous recombination using the ClonExpress II One Step Cloning Kit (Vazyme, C112). The specific steps were strictly performed according to the product instructions. The fragment was introduced into *E. coli* DH5α competent cells using the heat shock method, and positive clones were screened on kanamycin plates and sent to Wuhan Tianyi Huayu Gene Technology Co., Ltd. for sequencing.

[0059] Example 5: Obtaining and Identifying Transgenic Arabidopsis 5.1 Obtaining transgenic Arabidopsis The recombinant vector pCAMBIA2300-35S::BnMYB6 obtained in step 4.2 was transformed into Agrobacterium GV3101 competent cells, following the product instructions. Wild-type Arabidopsis inflorescences were immersed in Agrobacterium GV3101 bacterial culture containing the pCAMBIA2300-35S::BnMYB6 recombinant vector using the inflorescence immersion method, gently agitated for 20-30 seconds, and cultured under normal conditions until seed maturity. The transformed Arabidopsis seeds were then harvested. Mature Arabidopsis seeds were screened on MS / Kana (Kana 100 mg / L) solid medium. Seedlings showing normal growth and green color were considered positive in the initial screening. T1 generation seeds were harvested after the positive plants matured. After two generations of screening, a high proportion of positive plants was achieved, gradually obtaining homozygous T3 generation lines.

[0060] 5.2 PCR identification of transgenic Arabidopsis thaliana T3 generation transgenic Arabidopsis homozygotes and wild-type Arabidopsis leaves were selected as materials. The genome was extracted using the Ezup column-based plant genomic DNA extraction kit (Shanghai Sangon Biotech, B518261-0100). The specific steps were strictly followed according to the product instructions.

[0061] Genomic DNA and pCAMBIA2300-35S::BnMYB6 plasmid DNA were diluted to 100 ng / μl and used as templates for gene amplification. The target gene was amplified using p2300-F / p2300-R primers. The primer sequences are as follows: p2300-F: 5'-GGCAAGCTTGGAATTCCTGCT-3' (SEQ ID NO.33), p2300-R: 5'-CTCTTCGAACCCGGGTCTAGA-3' (SEQ ID NO.34).

[0062] The reaction system (25 μl) is shown in Table 12: Table 12

[0063] The reaction conditions are shown in Table 13: Table 13

[0064] The reaction solution was analyzed using 1.5% TAE agarose gel electrophoresis. Electrophoresis results (e.g.) Figure 5 Figure (A) shows that, except for the wild-type control, all eight independent transgenic lines tested showed amplification of the target gene-specific band, confirming the acquisition of positive transgenic lines.

[0065] 5.3 Semi-quantitative PCR identification of transgenic Arabidopsis thaliana T3 generation transgenic Arabidopsis homozygotes and wild-type Arabidopsis leaves were selected as materials. Total RNA was extracted using the Trizol method, and cDNA was obtained by reverse transcription, following the same steps as in 1.1. The obtained cDNA was used as a template, and semi-quantitative PCR was performed using qBnMYB6F / qBnMYB6R primers. The reaction system and conditions were the same as in step 5.2.

[0066] The reaction solution was analyzed using 1.5% TAE agarose gel electrophoresis. Electrophoresis results (e.g.) Figure 5 Figure (B) shows that, except for the wild-type control, all eight independent transgenic lines tested showed amplification of the target gene-specific band, further confirming the acquisition of positive transgenic lines.

[0067] 5.4 Detection of BnMYB6 gene expression level To detect the expression of the target gene BnMYB6 integrated into the genome in transgenic Arabidopsis lines, the cDNA obtained in step 5.3 was used as a template. Real-time quantitative PCR was performed using ChamQ Universal SYBR qPCR Master Mix (Vazyme, Q711-02) and primers qBnMYB6F / qBnMYB6R, with the reaction system and conditions identical to those in Example 3. AtActin was used as an internal reference gene. The primer sequences were designed as follows: qAtActin-F: 5'-GCCATCCAAGCTGTTCTCTC-3' (SEQ ID NO. 35), qAtActin-R: 5'-CAGTAAGGTCACGTCCAGCA-3' (SEQ ID NO. 36).

[0068] The results show (e.g.) Figure 5 (Figure C): The expression level of BnMYB6 in transgenic Arabidopsis lines was significantly higher than that in wild type, indicating that the BnMYB6 gene was successfully overexpressed in Arabidopsis.

[0069] Example 6: Phenotypic and Lignin Analysis of Transgenic Arabidopsis 6.1 Phenotype of transgenic Arabidopsis thaliana Plump T3 generation transgenic Arabidopsis homozygous lines and wild-type Arabidopsis seeds were selected as materials. Transgenic Arabidopsis seeds were sown on 1 / 2 MS medium supplemented with 100 mg / L kanamycin and cultured for 5 days. Wild-type Arabidopsis seeds were sown on 1 / 2 MS medium and cultured for 5 days. Seedlings were then transferred to 1 / 2 MS medium for vertical culture for 3 days. Photographs were taken, root lengths were measured, and statistical analysis was performed. Results showed (e.g.) Figure 6 Figures (A) and (C) show that the root length of transgenic Arabidopsis thaliana was significantly longer than that of wild-type Arabidopsis thaliana. This indicates that BnMYB6 overexpression can promote root elongation in Arabidopsis thaliana seedlings.

[0070] Simultaneously, plump T3 generation transgenic Arabidopsis homozygous lines and wild-type Arabidopsis seeds were selected as materials, cultured for 10 days, and then transplanted into nutrient soil for continued growth. Seedling phenotypes were observed after 3 weeks, and the results showed (e.g.) Figure 6 Figures (D) and (E) show that there was no significant difference in seedling morphology between transgenic and wild-type Arabidopsis thaliana. This indicates that BnMYB6 overexpression does not affect the seedling morphology of Arabidopsis thaliana.

[0071] 6.2 Lignin staining in transgenic Arabidopsis thaliana Lignin histochemical staining was performed using the Wiesner staining method, following the same procedure as in Example 3. Results showed (e.g.) Figure 6 (Figure B) shows that the lignin staining of transgenic Arabidopsis seedlings was significantly weaker than that of wild-type, indicating that the BnMYB6 gene has a negative regulatory effect on lignin deposition in Arabidopsis seedlings.

[0072] 6.3 Expression of lignin synthesis-related genes in transgenic Arabidopsis thaliana Using the cDNA obtained in step 5.3 as a template, quantitative real-time PCR was performed using ChamQ Universal SYBR qPCR Master Mix (Vazyme, Q711-02). The AtActin gene was used as an internal reference gene. Primer sequences are shown in Table 14.

[0073] Table 14

[0074] The reaction system and conditions are the same as in Example 3. The results show (e.g.) Figure 7 Compared to wild-type Arabidopsis, the expression levels of key lignin synthesis genes AtPAL2, AtCAD4, AtCAD6, AtCCoAOMT1, At4CL5, and AtCOMT were significantly downregulated in transgenic plants. These results indicate that overexpression of BnMYB6 inhibits the expression of genes related to lignin biosynthesis.

[0075] Example 7: Overexpression of BnMYB6 enhances plant tolerance to salt and drought stress; root length and activity assays. 7.1 Construction of the Root Vigor Standard Curve (1) Preparation of 0.1 mol / L phosphate buffered PBS (pH=7.0-7.2): Mix approximately 61 mL of solution A (0.2 mol / L Na2HPO4) with 39 mL of solution B (0.2 mol / L NaH2PO4), adjust the pH to 7.0-7.2 using a pH meter, and dilute with distilled water to a total volume of 200 mL to obtain 0.1 mol / L PBS. Store at 4℃ for later use.

[0076] (2) Preparation of TTC working solution (0.4%, w / v): Weigh 0.4 g of TTC powder, dissolve it in the above-mentioned 0.1 mol / L PBS and make up to 100 mL in a brown volumetric flask. This is a stock solution, stored at 4°C protected from light, and is valid for one week.

[0077] (3) Preparation of TTF stock solution: Take 500 μL of 0.4% TTC (requires 2 mg TTC powder) and add 2 mL of 0.1 M PBS. Then add excess Na2S2O4 (a small amount) to ensure that the TTC is completely reduced. Add 5 mL of ethyl acetate and extract for 1 min to ensure that the inorganic phase is colorless. Transfer the organic phase to a new 10 mL centrifuge tube, and make up to 10 mL with ethyl acetate. Store at 4 °C. MTTC: 334.39 g / mol, MTTF: 340.37 g / mol; mTTF = 2.0·(340.37 / 334.39) = 2.036 mg; cstock = 2.036 / 10 = 0.2036 mg / mL = 203.6 μg / mL.

[0078] (4) Dilute TTF in a gradient and bring the volume to 1 mL with ethyl acetate.

[0079] (5) Absorbance measurement: Pipette 200 μL of TTF standard dilution into each well of a 96-well plate and measure the absorbance at 485 nm. Record this value as A. 标准 Pipette 200 μL of ethyl acetate into a 96-well plate and measure the absorbance at 485 nm, denoted as A. 空白 ; Calculate ΔA 标准 =A 标准 -A 空白 Note: Each concentration standard tube and blank tube only needs to be measured 1-2 times.

[0080] (6) Establishment of the standard curve: The concentrations of 40 μg / mL, 30 μg / mL, 20 μg / mL, 10 μg / mL, 5 μg / mL, and 2.5 μg / mL LTTF standard dilutions were plotted on the x-axis, and the corresponding ΔA was plotted on the y-axis. 标准 Using the ordinate (y), a standard curve is plotted, yielding the linear regression equation y = kx + b (R²). 2 ≥0.99).

[0081] 7.2 Determination of root length and activity in plants overexpressing BnMYB6 to withstand salt and drought stress Plump T3 generation transgenic Arabidopsis homozygous lines and wild-type Arabidopsis seeds were selected as materials. Transgenic Arabidopsis seeds were sown on 1 / 2 MS medium supplemented with 100 mg / L kanamycin and cultured for 7 days. Wild-type Arabidopsis seeds were sown on 1 / 2 MS medium and cultured for 7 days. After that, the seedlings were transferred to drought stress (1 / 2 MS + 200 mM mannitol) medium and cultured vertically for 10 days. Root vigor was then measured.

[0082] The steps are as follows: (1) Samples: Take the root system of seedlings, three seedlings as a group, weigh the root weight, put them into 10mL centrifuge tubes, 5 replicates per group, and mark them. (2) Reaction: Add 5.0 mL of 0.1 M PBS and 5.0 mL of 0.4% TTC working solution to each tube, place them in an incubator at 37 ℃ and incubate in the dark for 3-4 h; (3) Termination: Add 1.0 mL of 1 M H2SO4 to each tube; (4) Pour out the liquid and remove the root system; take a picture of part of the root system with a stereomicroscope, and dry the rest with filter paper; (5) Place the roots into a 2mL centrifuge tube, freeze-mill, add ethyl acetate after milling, and bring the volume to 2mL. Centrifuge at 4000rpm for 10min, take the supernatant, and measure the absorbance at 485nm wavelength;

[0083] According to the calculation formula: Root activity [μg TTF / (g·h)] = (X·V SE ) / (W·T); Note: X: Theoretical TTF concentration derived from the standard curve; V SE : Total volume of ethyl acetate used to make up to volume in the reaction system; W: Mass of the sample to be tested added to the reaction system; T: Reaction time at 37℃ in the dark.

[0084] The results are as follows Figure 8 As shown, under drought stress (200 mM mannitol), the root activity of transgenic Arabidopsis thaliana was significantly higher than that of wild-type Arabidopsis thaliana. This indicates that BnMYB6 overexpression positively regulates root activity in Arabidopsis thaliana under drought stress.

[0085] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A ramie MYB transcription factor, characterized in that: The MYB transcription factor is BnMYB6, and its amino acid sequence is shown in SEQ ID NO.

1.

2. A gene for a ramie MYB transcription factor, characterized in that: Encoding the ramie MYB transcription factor as described in claim 1.

3. The ramie MYB transcription factor gene as described in claim 2, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO.

2.

4. A recombinant expression vector, characterized in that: A gene containing the ramie MYB transcription factor as described in claim 3.

5. A host cell, characterized in that: The gene containing the ramie MYB transcription factor of claim 1, the ramie MYB transcription factor of claim 2 or 3, or the recombinant expression vector of claim 4.

6. The application of the ramie MYB transcription factor as described in claim 1, the gene of the ramie MYB transcription factor as described in claim 2 or 3, the recombinant expression vector as described in claim 4, or the host cell as described in claim 5 in regulating plant lignin biosynthesis, expression of lignin synthesis-related genes, plant lignin deposition, and / or plant phenotype.

7. The application as described in claim 6, characterized in that, The regulation of plant lignin biosynthesis includes: negative regulation of plant lignin biosynthesis.

8. The application as described in claim 6, characterized in that, The regulation of lignin synthesis-related gene expression includes: negative regulation of lignin biosynthesis-related gene expression; the lignin biosynthesis-related genes include one or more of AtPAL2, AtCAD4, AtCAD6, AtCCoAOMT1, At4CL5, or AtCOMT.

9. The application as described in claim 6, characterized in that, The regulation of plant lignin deposition includes: negative regulation of plant lignin deposition.

10. The application as described in claim 6, characterized in that, The regulation of plant phenotypes includes: positively regulating plant root length or root activity under drought stress.

Images