AtruMYB23 gene and application thereof

Abstract

This invention provides a method for regulating the synthesis of chlorogenic acid in Acer truncatum. AtruMYB23 Genes and their applications, involving the field of plant genetic engineering technology. AtruMYB23 The gene contains the nucleotide sequence shown in SEQ ID NO:1. AtruMYB23 The gene encodes a protein with the amino acid sequence shown in SEQ ID NO:2, which is used to increase chlorogenic acid content and regulate chlorogenic acid synthesis in plants such as tobacco.

Description

Technical Field

[0001] This invention provides AtruMYB23 Genes and their applications, involving the field of plant genetic engineering technology. Background Technology

[0002] Chlorogenic acids are a class of phenolic acids formed by the esterification of caffeic acid and quinic acid. In plants, chlorogenic acids participate in pathogen defense and abiotic stress responses, serving as an important component of the plant's stress resistance system. Research on chlorogenic acids in perennial woody plants is relatively limited, mainly because their metabolic pathways differ slightly among different plants. Several regulatory factors participate in the biosynthesis of chlorogenic acids in plants by regulating related catalytic enzymes. Regarding biological functions, current research on chlorogenic acids in *Acer truncatum* mainly focuses on evaluating the overall activity of leaf extracts, with limited research on monomeric chlorogenic acids. Identifying candidate key genes regulating chlorogenic acid synthesis in *Acer truncatum* leaves is of great significance for promoting the high-quality development of related industries. Summary of the Invention

[0003] This invention provides a method for regulating the synthesis of chlorogenic acid in Acer truncatum. AtruMYB23 The study of genes and their applications has solved the technical problem of the lack of research on key genes regulating chlorogenic acid in Acer truncatum in existing technologies.

[0004] This invention is implemented as follows: AtruMYB23 The gene contains the nucleotide sequence shown in SEQ ID NO:1.

[0005] As a further preferred option, this AtruMYB23 The gene encodes a protein with the amino acid sequence shown in SEQ ID NO:2.

[0006] Should AtruMYB23 Genes include at least one of the following applications: (a) increasing the content of chlorogenic acid in plants; (b) regulating the synthesis of chlorogenic acid in plants.

[0007] The beneficial effects of this invention are: AtruMYB23 Overexpression can significantly promote the accumulation of total chlorogenic acid in plant leaves. Attached Figure Description

[0008] Figure 1 yes AtruMYB23 Amplification diagram of the full-length gene; Figure 2 yes AtruMYB23 Image showing a positive result for bacterial culture. Figure 3 This is the sequence alignment diagram of the AtruMYB23 protein from Example 1; Figure 4 This is Example 1 AtruMYB23 Phylogenetic tree; Figure 5 This is a hydrophobicity diagram of the AtruMYB23 encoded protein from Example 1. The horizontal axis represents the amino acid sequence of the encoded protein, and the vertical axis represents the hydrophilicity / hydrophobicity value; "-" indicates hydrophilicity, and "+" indicates hydrophobicity. Figure 6 Example 1 shows the analysis of conserved domains of the AtruMYB23-encoded protein; Figure 7 This is Example 2 AtruMYB23 Gene tissue expression specificity chart; Figure 8 This is Example 3 AtruMYB23 Electrophoresis image of recombinant gene plasmid; Figure 9 This is Example 3 AtruMYB23 Electrophoresis diagram of Agrobacterium bacterial suspension for positive detection; Figure 10 This is a schematic diagram of the infection process of the tobacco leaf disc conversion method in Example 3; Figure 11 It is the overexpression in Example 3 AtruMYB23 Schematic diagram of phenotypic characteristics of tobacco plants; Figure 12 It is the overexpression in Example 3 AtruMYB23 Electrophoresis diagram for DNA verification in tobacco plants; Figure 13 It is the overexpression in Example 3 AtruMYB23 Semi-quantitative verification of tobacco plants; Figure 14 This is a fluorescence quantitative verification image of tobacco plants overexpressing AtruMYB23, as shown in Example 3. Figure 15 This is a graph showing the content of six types of chlorogenic acid compounds in Arabidopsis thaliana plants overexpressing AtruMYB23, as described in Example 3. Figure 16 This is a graph showing the total chlorogenic acid content in tobacco plants overexpressing AtruMYB23, as shown in Example 3. Detailed Implementation

[0009] The Acer truncatum leaves used in the examples were all collected from the Acer truncatum germplasm resource conservation nursery of the Gaoguanzhai Conservation Bank of the Shandong Provincial Forestry and Grassland Germplasm Resource Center, Zhangqiu District, Jinan City, Shandong Province. The main reagents used in this application were RNAprep Pure polysaccharide and polyphenol plant total RNA extraction kits purchased from Tiangen Biotech (Beijing) Co., Ltd. Example 1

[0010] AtruMYB23 Gene cloning and sequence information Leaves of *Acer truncatum* were collected, dried in an oven at 80℃, and then stored using silica gel. The concentration, purity, and total RNA of the RNA samples were determined using the Tiangen RNAprep Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit and Nanodrop, following the kit instructions.

[0011] Using extracted total RNA as a template, reverse transcription was performed using high-fidelity reverse transcriptase to synthesize first-strand cDNA. PCR primers were designed, with the upstream primer sequence shown in SEQ ID NO:3 and the downstream primer sequence shown in SEQ ID NO:4. Subsequently, PCR amplification was performed using high-fidelity DNA polymerase to obtain the target gene. AtruMYB23 The amplification system is shown in Table 1.

[0012] Table 1 PCR amplification system

[0013]

[0014] See Figure 1 The PCR products were then subjected to electrophoresis in a 1×TAE gel with 1.5% agarose gel. The band size in the gel electrophoresis image was observed based on the DL2000 marker. The single target band was then purified and recovered.

[0015] The amplified fragment was recovered from the gel and transformed into competent E. coli cells. Using the pEASY®-Blunt Zero Cloning Kit (Beijing TransGen Biotechnology Co., Ltd.), the vector and fragment were gently mixed in centrifuge tubes at a molar ratio of 1:7. The reaction system is shown in Table 2. The reaction was carried out at room temperature (20℃~37℃) for 5 min. After the reaction, the centrifuge tubes were placed on ice.

[0016] Table 2. Escherichia coli transformation reaction system

[0017] Finally, eight single clones were selected and tested using three primer pairs: M13F / R, M13F / target gene R, and target gene F / R. AtruMYB23 The test results are shown below. Figure 2 After being identified as a positive bacterium, sequencing was performed. AtruMYB23 The gene contains the nucleotide sequence shown in SEQ ID NO:1.

[0018] Using ExPASy ProtParam AtruMYB23 The physicochemical properties of the protein were analyzed, and subcellular localization was predicted using Euk-mPLoc 2.0. The results are shown in Table 3.

[0019] Table 3. Physicochemical properties of basic proteins

[0020] See Figure 3 , AtruMYB23 Sequence alignment with other plant MYB homologous proteins showed that its amino acid sequence was highly conserved. AtruMYB23 It is relatively stable during evolution and has the structural characteristics of typical MYB-type transcription factors.

[0021] See Figure 4 The results of the phylogenetic analysis show that AtruMYB23 First, with the golden maple ( Dipteronia sinensis The MYB homologous proteins clustered into one branch, and the branch support was high. This result indicates that... AtruMYB23 It is closely related to homologous proteins of closely related species, suggesting that they also have certain functional similarities.

[0022] Using the ProtScale online tool to AtruMYB23 The hydrophobicity of the gene-encoded protein was analyzed. (See also...) Figure 5 , AtruMYB23 Proteins exhibit hydrophilicity in their primary amino acid structure. The amino acid with the lowest hydrophobicity score is position 5, with a score of -2.511, indicating the strongest hydrophilicity at this position. Conversely, amino acid position 76 has a hydrophobicity score of 1.444, indicating the strongest hydrophobicity at this position.

[0023] To further analyze the structural characteristics of the proteins encoded by candidate genes of Acer truncatum, the NCBI Conserved Domain Database was used. AtruMYB23 Conserved domain prediction was performed on the protein sequence. (See also...) Figure 6 The AtruMYB23 protein contains a typical conserved PLN03212 domain in the amino acid region from position 9 to 112. This domain belongs to the SANT / Myb-like DNA-binding domain type and is a characteristic DNA-binding domain of the MYB transcription factor. AtruMYB23 The protein sequence is shown in SEQ ID NO:2. Example 2

[0024] Tissue-specific gene expression analysis Using the method described in Example 1, RNA was extracted from the roots, stems, leaves, flowers, and seeds of *Acer truncatum*, and cDNA was obtained through reverse transcription. qRT-PCR analysis was then performed on these different tissues. (See also...) Figure 7 , AtruMYB23Expression levels were highest in roots, with no significant difference between roots and stems; both were at relatively high levels. Root expression was approximately 15-20 times higher than in flowers and over 20 times higher than in leaves. In contrast, seed expression was lower, while flower and leaf expression was the lowest, with overall levels at extremely low levels. AtruMYB23 It is highly expressed mainly in vegetative organs such as roots and stems, indicating that its regulatory role has obvious tissue specificity, suggesting that it may act as a transcriptional regulator and participate in regulating the expression of phenylpropanone metabolism-related genes in these tissues. Example 3

[0025] overexpression AtruMYB23 Functional verification of tobacco plants The cloning plasmid and the pCAMBIA1300 expression vector plasmid were double-digested with restriction endonucleases Hind III and EcoRI. After electrophoresis to confirm that the plasmid loops were cleaved, the target gene fragment and the large vector fragment were recovered separately. Then, they were ligated using the Novavitase recombinant kit to construct the recombinant plasmid pCAMBIA1300- AtruMYB23 See also Figure 8 Sequencing results showed that the expression vector was successfully constructed and the corresponding bacterial culture plasmid was extracted.

[0026] The recombinant plasmid pCAMBIA1300- AtruMYB23 Transformed into Agrobacterium GV3101, and positive clones were detected by colony PCR. (See also...) Figure 9 Electrophoresis results showed that the amplified bands were clear, the size distribution was normal, and there was no obvious non-specific amplification, indicating that the target gene had been successfully integrated into Agrobacterium.

[0027] Agrobacterium-mediated genetic transformation was used to transform plasmids containing recombinant plasmid pCAMBIA1300- AtruMYB23 Agrobacterium tumefaciens culture of GV3101 (OD=0.5-0.6) was used to infect tobacco leaves. See also... Figure 10 Tobacco plants that tested positive were transferred to nutrient soil for further growth, resulting in resistant plants. (See also...) Figure 11 After collecting the seeds, the screening process was repeated to obtain T2 generation plants. The obtained resistant plants underwent DNA verification; tobacco plants identified as positive were used for subsequent functional verification.

[0028] To verify AtruMYB23To determine whether the gene was successfully transferred into tobacco plants, genomic PCR, semi-quantitative RT-PCR, and qRT-PCR expression analyses were performed on the obtained transgenic lines. Primers used for genomic PCR are shown in SEQ ID NO:3 and SEQ ID NO:4; primers used for qRT-PCR expression analysis are shown in SEQ ID NO:5 and SEQ ID NO:6; and primers used for semi-quantitative RT-PCR expression are shown in SEQ ID NO:7 and SEQ ID NO:8. (See also...) Figure 12 DNA electrophoresis results showed that specific bands of the expected size were amplified in transgenic lines 3-33, 3-34, 3-35, 3-36 and 3-37.

[0029] Further use of tobacco internal reference genes NtRPL25 As a control, semi-quantitative RT-PCR and qRT-PCR were used to... AtruMYB23 The transcriptional level was detected. See [reference needed]. Figure 13 and Figure 14 The results showed that, compared with the wild type, each transgenic line had... AtruMYB23 The expression levels of all genes were increased. The transgenic lines were numbered 3-33, 3-34, 3-35, 3-36, and 3-37 (the first number 3 represents the target gene). AtruMYB23 The line with the highest expression level (the second number represents different positive plant individuals within that batch) was the transformation batch, approximately 5-6 times that of WT, significantly higher than other lines.

[0030] overexpression AtruMYB23 The contents of six types of chlorogenic acid compounds in the leaves of tobacco strains (3-33, 3-34, 3-35, 3-36, 3-37) and wild-type tobacco were determined. (See also...) Figure 15 Compared to the wild type, the content of chlorogenic acid in all transgenic lines showed an overall increasing trend, but the differences between different lines were significant. Among them, line 3-35 had the highest chlorogenic acid content, significantly higher than the other lines; lines 3-33 and 3-34 also showed significant increases, while lines 3-36 and 3-37 showed relatively smaller increases. Meanwhile, neochlorogenic acid and cryptochlorogenic acid also showed an increasing trend in most transgenic lines, with line 3-35 showing the most prominent increase. Of the three isochlorogenic acids, isochlorogenic acid C had a higher overall content than isochlorogenic acid A and isochlorogenic acid B, but its variation was relatively smaller. Overall, AtruMYB23 Overexpression primarily promoted the accumulation of core components such as chlorogenic acid, neochlorogenic acid, and cryptochlorogenic acid.

[0031] Further statistical analysis was conducted on the total amounts of chlorogenic acid, neochlorogenic acid, and cryptochlorogenic acid. (See reference...) Figure 16Compared with the wild type, the total chlorogenic acid content of all transgenic lines was significantly increased, reaching 12-65 times that of the wild type, with the highest level observed in line 3-35. This indicates that... AtruMYB23 It has a significant promoting effect on the accumulation of chlorogenic acid.

[0032] The beneficial effects of this invention are: AtruMYB23 Overexpression can significantly promote the accumulation of total chlorogenic acid in plant leaves.

Claims

1. AtruMYB23 Genes, characterized by, The gene includes the nucleotide sequence shown in SEQ ID NO:

1.

2. As described in claim 1 AtruMYB23 Genes, characterized by, This gene encodes a protein with the amino acid sequence shown in SEQ ID NO:

2.

3. As described in claim 1 AtruMYB23 The application of genes is characterized by, Including at least one of the following applications: (a) Increase the content of chlorogenic acid in plants; (b) Regulate the synthesis of chlorogenic acid in plants.

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