Gene TsMYB75 for regulating and controlling leaf color of cedrela sinensis and application of gene TsMYB75
By overexpressing the gene TsMYB75 that regulates the leaf color of toon sinensis, activating the genes of the anthocyanin synthesis pathway, changing the leaf color of toon sinensis and increasing the anthocyanin content, the problem of color regulation of toon sinensis buds was solved, the color quality of toon sinensis buds was improved, and the regulation mechanism was understood.
Patent Information
- Application Number
- CN202510851955.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-24
- Publication Date
- 2025-10-10
AI Technical Summary
The existing technology lacks effective means to regulate the color of toon buds, which affects their sensory selection and taste.
By screening and overexpressing the gene TsMYB75 that regulates the leaf color of Toona sinensis, the promoter of the anthocyanin synthesis pathway gene is directly activated, the expression level of Toona sinensis leaf color is changed, the green leaves turn red and the anthocyanin content is increased.
It has achieved effective regulation of the color of toon leaves, improved the color quality of toon buds, provided new technical ideas for cultivating high-quality toon buds, and deeply understood the color regulation mechanism of toon buds.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the field of plant molecular biology, and particularly relates to a gene TsMYB75 for regulating leaf color of Toona sinensis and application thereof. BACKGROUND
[0002] Toona sinensis belongs to the family Meliaceae and the genus Toona, and its tender bud, also known as Toona sinensis head or Toona sinensis bud, has various nutritional components such as protein, mineral and vitamin, has the effects of clearing heat and detoxifying, invigorating stomach and regulating qi, dispelling wind and dampness, and has a unique pungent smell, which is deeply loved by people. However, there is a great difference in leaf color of Toona sinensis bud, which affects the sensory selection and taste. The main component of red Toona sinensis bud is a water-soluble natural pigment anthocyanin, which is mainly distributed in the form of glycoside in leaf cells.
[0003] At present, there is a lack of regulation research and new germplasm for color of Toona sinensis bud. SUMMARY
[0004] The present application aims to provide a gene TsMYB75 for regulating leaf color of Toona sinensis and application thereof.
[0005] Based on previous gene sequencing research, the present application accurately screens a key regulatory factor TsMYB75 for regulating leaf color of Toona sinensis, the nucleotide sequence of the gene is shown as SEQ ID NO: 1, the gene is located in the nucleus as a MYB transcription factor, the expression level and anthocyanin are closely related, overexpression of the gene can directly change the leaf color of Toona sinensis from green to red, and further determination shows that the gene can change the expression level of the whole anthocyanin synthesis and metabolism pathway gene of Toona sinensis. The gene can be used for changing the leaf color of Toona sinensis, and can also be used as an auxiliary breeding marker gene screening, which has important significance for germplasm resource identification.
[0006] In order to achieve the object of the present application, in the first aspect, the present application provides a gene TsMYB75 for regulating leaf color of Toona sinensis, which is a gene encoding the following protein (a) or (b): (a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2; or (b) a protein derived from (a) by substitution, deletion or addition of one or more amino acids and having equivalent function.
[0007] Further, the gene TsMYB75 is: i) a nucleotide sequence shown in SEQ ID NO: 1; ii) a nucleotide sequence shown in SEQ ID NO: 1 by substitution, deletion and / or addition of one or more nucleotides and expressing the same functional protein; iii) a nucleotide sequence that hybridizes to the sequence set forth in SEQ ID NO: 1 under stringent conditions and expresses the same functional protein, the stringent conditions being hybridization in 0.1 x SSPE or 0.1 x SSC containing 0.1% SDS at 65°C, and washing the membrane with the same solution; or iv) a nucleotide sequence that has more than 90% homology to the nucleotide sequence of i), ii) or iii) and expresses the same functional protein.
[0008] In a second aspect, the present application provides a biological material containing the gene TsMYB75, which includes but is not limited to recombinant DNA, expression cassette, transposon, plasmid vector, viral vector or engineered bacteria.
[0009] In a third aspect, the present application provides the use of the gene TsMYB75 or the biological material containing the gene in regulating the leaf color and anthocyanin content of Toona sinensis.
[0010] In a fourth aspect, the present application provides a method for changing the leaf color and increasing the anthocyanin content of Toona sinensis, which comprises overexpressing the gene TsMYB75 in Toona sinensis, making the green leaf of Toona sinensis red, increasing the expression level of key genes in the anthocyanin synthesis pathway and increasing the anthocyanin content.
[0011] The overexpression mode can be selected from the following 1) to 5), or optional combinations: 1) by introducing a plasmid containing the gene; 2) by increasing the copy number of the gene on the chromosome of Toona sinensis; 3) by changing the promoter sequence of the gene on the chromosome of Toona sinensis; 4) by operably linking a strong promoter to the gene; 5) by introducing an enhancer.
[0012] Further, the expression vector containing the gene TsMYB75 is transformed into Agrobacterium, and the gene is introduced into Toona sinensis by Agrobacterium-mediated transformation method, so as to overexpress the gene TsMYB75 in Toona sinensis.
[0013] Further, the key genes in the anthocyanin synthesis pathway include PAL, 4CL, C4H-1, C4H-2 and DFR.
[0014] In a fifth aspect, the present application provides the use of the transgenic Toona sinensis obtained according to the method in plant breeding.
[0015] Further, the breeding method includes but is not limited to transgenesis, crossbreeding, backcrossing, selfing or vegetative reproduction.
[0016] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects: This invention reveals for the first time the function of the TsMYB75 gene, and its expression level is directly related to the color of toon buds. By controlling the expression of TsMYB75, the color of toon buds can be directly changed, providing new technical ideas for cultivating toon buds with high-quality color, and at the same time playing an important role in further understanding the color regulation mechanism of toon buds. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] Figure 1 The evolution and functional localization analysis of TsMYB75 gene in the preferred embodiment of the present invention is shown in Figure A. The evolution analysis of TsMYB75 homologous genes and the subcellular functional localization analysis of TsMYB75 are shown in Figure B.
[0018] Figure 2 This figure demonstrates the regulatory relationship between TsMYB75 and toona sinensis leaf color gene expression in a preferred embodiment of the present invention. A represents a toona sinensis leaf, B represents the anthocyanin content in the corresponding leaf, and C represents the TsMYB75 gene expression level in the corresponding leaf. Y1-Y10 represent 10 toona sinensis leaves, ranging from small to mature.
[0019] Figure 3 This is a map of the SK expression vector.
[0020] Figure 4 This is the LUC vector map.
[0021] Figure 5 This is an identification of the effect of TsMYB75 on the promoters of the anthocyanin synthesis pathway genes PAL (A), 4CL (B), C4H-1 (C), C4H-2 (D), and DFR (E) in the preferred embodiment of the present invention.
[0022] Figure 6 This is the map of the pCambia1304-MCS35S vector.
[0023] Figure 7 This is an analysis of TsMYB75-overexpressed toona sinensis leaves in a preferred embodiment of the present invention. A: Overexpression of the TsMYB75 gene can turn green toona sinensis leaves red and significantly increase the anthocyanin content. B: Overexpression of the TsMYB75 gene can increase the expression levels of key genes in the anthocyanin synthesis pathway. Among them, EV represents the control treatment experiment, MYB75OX represents the TsMYB75 gene overexpression experiment; EV1 and EV2 represent control 1 and control 2 treatment experiments, respectively, and MYB75OE-1, MYB75OE-2, and MYB75OE-3 represent TsMYB75 gene overexpression 1-3 treatment experiments, respectively. DETAILED DESCRIPTION
[0024] The application aims to provide a key gene for changing color of Toona sinensis leaves (a key gene for directly regulating color of Toona sinensis sprouts), and provide a new technical idea for cultivating high-quality color Toona sinensis sprouts.
[0025] The application adopts the following technical scheme: (1) The application provides a Toona sinensis sprout color regulation gene TsMYB75, the coding region nucleotide sequence of which is shown as SEQ ID NO:1, which is an Arabidopsis thaliana MYB75 / PAP1 homologous gene.
[0026] (2) The application is verified by analysis, and the TsMYB75 gene coding protein is located in the cell nucleus, and is significantly related to Toona sinensis sprout color and anthocyanin content.
[0027] (3) The application is verified by analysis, and the TsMYB75 gene directly activates the promoter of an anthocyanin synthesis pathway gene and regulates its metabolic flow.
[0028] (4) The application is verified by experiments, and overexpression of the TsMYB75 gene can make green Toona sinensis leaves red, change the color of Toona sinensis, and can be applied in molecular breeding of Toona sinensis sprout color quality and creation of red sprout new germplasm.
[0029] Researches show that the TsMYB75 gene in Toona sinensis directly activates the promoter of an anthocyanin synthesis pathway gene, regulates its metabolic flow, and overexpression can make green Toona sinensis leaves red.
[0030] The following examples are used to illustrate the application, but are not used to limit the scope of the application. If not specifically indicated, the technical means used in the examples are conventional means familiar to those skilled in the art, and the raw materials used are commercially available goods.
[0031] Example 1 Cloning of TsMYB75 gene 1. Total mRNA of Toona sinensis is extracted by a general RNA extraction kit and is reversely transcribed into cDNA; upstream and downstream primers are designed starting from the start codon and the stop codon of TsMYB75 for amplification, the upstream primer PF is 5'-ATGACGGGCTCTTTGGC-3', and the downstream primer PR is 5'-TTAAATCAAAGTATTTTGATCTGC-3'. The PCR amplification conditions are as follows: 95℃ for 2 min, 35 cycles of 98℃ pre-denaturation for 10 s, 50℃ annealing for 30 s, 68℃ extension for 1 min, 72℃ final extension for 5 min, and the company is sequenced, the base sequence of the TsMYB75 gene is shown as SEQ ID NO:1, and the amino acid sequence of the coding protein is shown as SEQ ID NO:2.
[0032] 2. Sequence alignment and evolutionary analysis with other plants, which is MYB75 / PAP1 homologous gene and located in the nucleus Figure 1 ).
[0033] The TsMYB75 gene provided by the application is accurately presented by DNA sequence, but the gene sequences of different Chinese toon materials may have several base differences. Therefore, without deviating from the spirit of the application, all belong to the scope claimed by the application.
[0034] Example 2 Verification of the expression regulation relationship between TsMYB75 and Chinese toon leaf color gene 1. The young tissues of Chinese toon are red, and the synthesis of anthocyanin decreases and the content of chlorophyll increases when mature, so that the leaves are red. The regulation relationship between TsMYB75 and Chinese toon leaf color was analyzed by collecting leaves at different development stages, and the total RNA and anthocyanin content of different leaves were extracted by using a kit Figure 2 A).
[0035] 1g of leaves was weighed, and the content of anthocyanin was detected by using an anthocyanin content detection kit, and the absorbance values at 620nm and 650nm wavelengths of the extract were measured, and the anthocyanin content was calculated by using the Greey formula Figure 2 B).
[0036] After total RNA extraction, cDNA was reverse transcribed, and quantitative PCR detection was performed by using a common quantitative kit. The upstream primer PF of TsMYB75 quantitative detection is 5'-AGAAGGTGAAGACATCGCAAATC-3', and the downstream primer PR is 5'-CCAAAGCTCCAAGACAAAAGTG-3'. After the reaction, the was used to calculate the relative expression amount Figure 2 C).
[0037] 2. The results show that the expression level of TsMYB75 is consistent with the trend of leaf color and anthocyanin content, indicating that TsMYB75 is a positive expression regulation gene of Chinese toon leaf color.
[0038] Example 3 TsMYB75 directly regulates and activates the promoter of anthocyanin synthesis metabolic pathway gene 1. Based on the Chinese toon genome, the promoter sequences of key genes in the anthocyanin synthesis pathway were retrieved, and it was found that the promoter sequences of anthocyanin synthesis pathway genes (PAL, 4CL, C4H-1, C4H-2, DFR) contain TsMYB75 binding regulation elements, and the promoters containing the regulation elements are cloned.
[0039] 2. Domestic ordinary kit was used for dual luciferase experiment, and TsMYB75 was constructed into SK expression vector (purchased from Hainan Ni Xing Biological Technology Co., Ltd., and the plasmid map is shown inFigure 3 ), the anthocyanin synthesis pathway gene promoter was constructed into a LUC vector (purchased from Hainan Ni Xing Biotechnology Co., Ltd., and the plasmid map is shown in Figure 4 ), the bacteria liquid containing the SK vector and the bacteria liquid containing the LUC vector were mixed at a volume ratio of 1:1, and the mixed bacteria liquid was injected into tobacco leaves growing for 4-5 weeks.
[0040] 3. After normal light culture for 4 days, the fluorescence intensity of the tobacco leaves co-injected with TsMYB75-SK and LUC carrying the PAL promoter, LUC carrying the 4CL promoter, LUC carrying the C4H-1 promoter, LUC carrying the C4H-2 promoter, and LUC carrying the DFR promoter was higher than that of the control treatment of TsMYB75-SK and empty LUC (P<0.05). Figure 5 ).
[0041] Example 4: Overexpression of TsMYB75 promotes the leaf color of Chinese toon to turn red 1. The cloned TsMYB75 gene was constructed into a pCambia1304-MCS35S overexpression vector (purchased from Hainan Ni Xing Biotechnology Co., Ltd., and the plasmid map is shown in Figure 6 ), and the transformed GV3101 Agrobacterium competent cells were cultured at a rotation speed of 180-220 rpm and 28°C until the OD 600 was about 0.6.
[0042] 2. The seedling material was obtained from cultured Chinese toon tissue culture seedlings, which were subcultured in MS + 0.5-2 mg / L 6-BA (6-benzylaminopurine) + 0.1-1.0 mg / L IBA (indolebutyric acid) for about 1 month until the seedlings were 3-5 cm, and the Chinese toon seedlings with consistent growth were mixed with Agrobacterium for vacuum infection for 30 min, the excess water was absorbed with filter paper, and the seedlings were cultured under normal light for 4-5 days for phenotype observation.
[0043] 3. The phenotype observation results showed that overexpression of the TsMYB75 gene could make the green Chinese toon leaves turn red and significantly increase the anthocyanin content (Fig. Figure 7 A), and the expression level of key genes in the anthocyanin synthesis pathway (Fig. Figure 7 B) was increased.
[0044] The present application first found that the Chinese toon TsMYB75 gene has a similar function of promoting anthocyanin, and first improves the color quality of Chinese toon sprouts, which plays an important role in cultivating high-quality Chinese toon sprouts.
[0045] While the application has been described in detail and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that various modifications or changes can be made therein without departing from the spirit and scope thereof. Accordingly, it is intended that all such modifications and changes be included within the scope of the application as claimed.
Claims
1. A gene TsMYB75 that regulates toona sinensis leaf color, characterized in that: These are genes encoding proteins (a) or (b) as follows: (a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2; or (b) A protein derived from (a) with equivalent function, wherein one or more amino acids are substituted, deleted or added to the sequence shown in SEQ ID NO:
2.
2. The gene according to claim 1, characterized in that It is: i) the nucleotide sequence shown in SEQ ID NO: 1; ii) a nucleotide sequence in which one or more nucleotides are substituted, deleted, and / or added to the nucleotide sequence shown in SEQ ID NO: 1 and the nucleotide sequence expresses a protein with the same function; iii) a nucleotide sequence that hybridizes to the sequence shown in SEQ ID NO: 1 under stringent conditions, wherein the hybridization is carried out in 0.1× SSPE containing 0.1% SDS or in 0.1× SSC containing 0.1% SDS at 65°C, and the membrane is washed with the solution; or iv) a nucleotide sequence that has more than 90% homology with the nucleotide sequence of i), ii) or iii) and expresses a protein with the same function.
3. The biological material containing the gene according to claim 1 or 2, characterized in that: The biological material is recombinant DNA, expression cassette, transposon, plasmid vector, virus vector or engineered bacteria.
4. Use of the gene according to claim 1 or 2 or the biomaterial according to claim 3 in regulating the leaf color and anthocyanin content of Toona sinensis.
5. A method for changing the leaf color of Toona sinensis and increasing the anthocyanin content, characterized in that: The method comprises: overexpressing the gene according to claim 1 or 2 in toona sinensis, so that green toona sinensis leaves turn red, the expression level of key genes in the anthocyanin synthesis pathway increases, and the anthocyanin content is improved.
6. The method according to claim 5, characterized in that The overexpression method is selected from the following 1) to 5), or an optional combination: 1) by introducing a plasmid carrying the gene; 2) by increasing the copy number of the gene on the toon chromosome; 3) by changing the promoter sequence of the gene on the chromosome of Toona sinensis; 4) by operably linking a strong promoter to the gene; 5) By introducing enhancers.
7. The method according to claim 5, characterized in that The expression vector containing the gene is transformed into Agrobacterium, and the gene is transferred into Toona sinensis by an Agrobacterium-mediated transformation method, so that the gene is overexpressed in Toona sinensis.
8. The method according to any one of claims 5 to 7, characterized in that: The key genes in the anthocyanin synthesis pathway include PAL, 4CL, C4H-1, C4H-2, and DFR.
9. Use of the transgenic Toona sinensis obtained according to the method according to any one of claims 5 to 8 in plant breeding.
10. The use according to claim 9, characterized in that Breeding methods include transgenics, hybridization, backcrossing, selfing or asexual reproduction.