Camellia sinensis transcription factor csmyb5 gene and application thereof

Abstract

The present application relates to the technical field of plant genetic engineering, and discloses a tea tree transcription factor CsMYB5 gene and application thereof. Firstly, the present application studies the trait performance of leaves of green varieties and low-temperature-sensitive albino varieties in the temperature change process through joint analysis of physiological and biochemical data and transcriptome data, mines key regulatory transcription factors, and constructs a transcription factor regulation network. Further, the present application performs function verification on the key regulatory factors, and performs expression analysis and phenotype identification on the instantaneously silenced / overexpressed materials. The key regulatory transcription factor MYB5 provided by the present application is helpful for in-depth research on the low-temperature response of leaf color whitening and the temperature rise response of leaf color greening of low-temperature-sensitive albino tea trees at the transcription level, provides support for further understanding of the leaf color variation mechanism of albino tea trees in the new shoot development stage in spring, and helps to improve the field management measures of albino tea varieties, so that the quality can be better stabilized.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, and more specifically, to tea plant transcription factors. CsMYB5 Genes and their applications. Background Technology

[0002] tea tree( Camellia sinensis (L.) O. Kuntze originated in China, with a vast planting area and abundant germplasm resources. Through long-term evolution and artificial breeding, tea tree leaf color has varied, resulting in the successful cultivation of tea tree germplasm resources with a variety of unique leaf colors, including purplish-black, purple, orange, red, yellow, white, green, and multi-colored varieties. Among them, albino tea has albino buds and leaves rich in amino acids and relatively low in tea polyphenols, making it an ideal raw material for producing high-quality green tea. Albino tea trees are divided into ecologically sensitive and ecologically insensitive types. Ecologically sensitive types include those sensitive to temperature, light, and photothermal. Temperature-sensitive tea trees are typically sensitive to low temperatures; during the spring shoot development stage, when the environment is below a certain temperature, the leaves exhibit albino characteristics, turning green as the temperature rises. The temperature threshold varies among different varieties, typically between 20℃ and 25℃.

[0003] Currently, research on the formation mechanism of temperature-sensitive albino tea trees is quite extensive. Numerous varieties have been studied, and as perennial plants, 'Baiye No. 1', 'Xiaoxueya', 'Qiannianxue', 'Ruixue No. 2', and 'Jingbai No. 2' all exhibit stable, reversible albino or yellowing traits annually. Among these, 'Baiye No. 1' has the longest research history and the most reported findings. Studies often select materials with different leaf colors at different developmental stages in the field or at the same developmental stage, or conduct indoor temperature-controlled treatments to study the material's response under different temperature conditions. In terms of research methods and directions, cytology, metabolomics, transcriptomics, and proteomics have been widely applied. The chloroplast genomes of several albino varieties have been successfully analyzed, revealing a leaf color regulatory network composed of DNA methylation, transcriptional regulation, post-transcriptional modification, protein translation, protein modification, and competitive endogenous RNA regulation. Most studies focus on chloroplast development and pigment metabolism pathways. Abnormal expression of genes regulating early chloroplast development, abnormal expression and accumulation of thylakoid membrane proteins, blocked pigment synthesis pathways, and enhanced pigment degradation pathways can all lead to leaf leukoplakia. Many genes associated with the leukoplakia phenotype have been identified. However, existing research primarily focuses on phenotypic expression under different leaf color states or temperature conditions, and there are no reports on gene response regulation and leaf color phenotype formation during temperature changes (heating / cooling). Therefore, it is necessary to identify and recognize new genes that play an important role in temperature response and leaf color regulation in low-temperature sensitive leukoplakia varieties, and to elucidate their functions and regulatory pathways.

[0004] Thylakoid membrane proteins are composed of many components that work together to complete photosynthesis; the absence of any part will affect structural stability and functional performance. Among them, the light-harvesting pigment-protein complex directly binds to chlorophyll, fixing free pigments to prevent degradation and simultaneously transferring light energy. LHCB has been extensively studied in many species and its important role in leaf green phenotype formation has been verified in tobacco, Arabidopsis thaliana, kiwifruit, and other species. The expression level of CsLHCII is closely related to chlorophyll biosynthesis and may also play a role by forming a complex with CssHSP and CsSGR proteins. In many related studies on tea plants, the distinction between these proteins remains unclear. LHCB Differences exist among members of the gene family, and existing research on transcriptional regulation mechanisms has relatively little direct link to temperature. Summary of the Invention

[0005] The purpose of this invention is to provide tea plant transcription factors. CsMYB5 Genes and their applications.

[0006] To achieve the objectives of this invention, in a first aspect, this invention provides a tea plant transcription factor. CsMYB5 A gene that encodes either (a) or (b) a protein: (a) A protein consisting of the amino acid sequence shown in SEQ ID NO:2; or (b) Proteins derived from (a) with the sequence shown in SEQ ID NO:2 substituted, deleted or added with one or more amino acids and having the same function.

[0007] The tea tree transcription factor CsMYB5 The nucleotide sequence of the gene is as follows: i) The nucleotide sequence shown in SEQ ID NO:1; or, ii) A nucleotide sequence with one or more nucleotides substituted, deleted, and / or added to the nucleotide sequence shown in SEQ ID NO:1, and having the same function; or, iii) A nucleotide sequence that hybridizes with the sequence shown in SEQ ID NO:1 under stringent conditions, wherein the stringent conditions are hybridization at 65°C in 0.1×SSPE containing 0.1% SDS or 0.1×SSC containing 0.1% SDS, followed by washing the membrane with the same solution; or, iv) Nucleotide sequences that are more than 90% homologous to and have the same function as nucleotide sequences of i), ii) or iii).

[0008] Secondly, the present invention provides a product containing the tea tree transcription factor. CsMYB5 Gene-derived biological materials, including but not limited to expression cassettes, transposons, plasmid vectors, viral vectors, or engineered bacteria.

[0009] Thirdly, the present invention provides the tea plant transcription factor. CsMYB5 The application of the gene or its encoded MYB5 transcription factor, or biological material containing the gene, in regulating chlorophyll accumulation and / or leaf color changes in low-temperature sensitive albino tea varieties.

[0010] Furthermore, the regulation activates the tea plant through the MYB5 transcription factor. CsLHCB1.1 This is achieved through gene expression.

[0011] The tea tree CsLHCB1.1 The gene's reference sequence number in NCBI is XM_028241743.1.

[0012] Furthermore, the MYB5 transcription factor and the tea plant CsLHCB1.1 Gene promoter sequences bind and activate expression.

[0013] In this invention, the tea tree is derived from a low-temperature sensitive albino tea variety. CsLHCB1.1 The promoter sequence of the gene is as follows: I) The nucleotide sequence shown in SEQ ID NO:4; or, II) A nucleotide sequence with one or more nucleotides substituted, deleted, and / or added to the nucleotide sequence shown in SEQ ID NO:4, and having the same function; or, III) A nucleotide sequence that hybridizes with the sequence shown in SEQ ID NO:4 under stringent conditions, wherein the stringent conditions are hybridization at 65°C in 0.1×SSPE containing 0.1% SDS or 0.1×SSC containing 0.1% SDS, followed by washing the membrane with the same solution; or, IV) Nucleotide sequences that are more than 90% homologous to and have the same function as nucleotide sequences of I), II) or III).

[0014] In one specific embodiment of the present invention, chlorophyll accumulation in low-temperature sensitive albino tea varieties is promoted under low-temperature conditions by increasing the expression level or activity of the endogenous or exogenous MYB5 transcription factor in the tea plant.

[0015] Furthermore, the expression level of the MYB5 transcription factor can be increased by any one of 1) - 5) or any combination thereof: 1) By importing a plasmid containing the gene; 2) By increasing the copy number of the aforementioned genes on plant chromosomes; 3) By altering the promoter sequence of the aforementioned genes on plant chromosomes; 4) By operatively linking a strong promoter to the gene; 5) By importing enhancers.

[0016] In another specific embodiment of the present invention, chlorophyll accumulation in low-temperature sensitive albino tea varieties is inhibited by reducing the expression level or activity of the MYB5 transcription factor.

[0017] Furthermore, the application includes the step of reducing the expression level or activity of the MYB5 transcription factor using antisense oligonucleotides, RNA interference, or gene editing techniques.

[0018] In embodiments of the present invention, for albino tea trees, low temperature or low temperature conditions generally refer to ambient temperatures below 25°C, more commonly below 22°C, such as ambient temperatures of 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, or 22°C, 23°C, 24°C, or 25°C.

[0019] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects: (I) This invention is the first to simultaneously isolate and obtain tea leaves of different colors. CsMYB5 The gene was identified, and its transient silencing and overexpression were used to verify that it could alter chlorophyll accumulation levels in tea plants under low-temperature treatment. This provides a novel transcription factor resource with a clear positive regulatory function for the breeding of low-temperature-sensitive albino varieties and the investigation of albino mechanisms, filling a gap in existing technologies regarding... CsMYB5 The gap in the role of genes in chlorophyll accumulation.

[0020] (II) Tea Tree CsMYB5 The gene encodes a MYB family transcription factor that can interact with different leaf color varieties. CsLHCB1.1 Promoter sequences bind to and activate expression; during temperature reduction, in green varieties... CsMYB5 The expression was adjusted upwards, while the expression of albino varieties was adjusted downwards, leading to downstream... CsLHCB1.1 The expression level was significantly downregulated, resulting in a reduction in bound chlorophyll and a change in leaf color.

[0021] (III) The key regulatory transcription factors provided by this invention help to conduct in-depth research on leaf whitening in response to low temperature and leaf greening in response to warming at the transcriptional level. This provides technical support for further understanding the leaf color variation mechanism of albino tea trees during the spring shoot development stage, and at the same time helps to improve field management measures for albino tea tree varieties, thereby better stabilizing quality. Attached Figure Description

[0022] Figure 1This invention presents the changes in pigment content of various samples after short-term temperature variation treatment in a preferred embodiment. Different lowercase letters indicate significant differences. P <0.05).

[0023] Figure 2 The following are the results of transcriptome data analysis and weighted gene co-expression network analysis (WGCNA) in a preferred embodiment of the present invention. Specifically, (A) principal component analysis (PCA); (B) association analysis between pigment content and differentially expressed genes (DEGs) in 'Longjing 43' (LJ43); and (C) association analysis between pigment content and DEGs in 'Ruixue 2' (RX2).

[0024] Figure 3 This is a preferred embodiment of the present invention for transcription factor analysis. (A) Phylogenetic tree; (B) Tertiary structure; (C) Amino acid secondary structure distribution.

[0025] Figure 4 In a preferred embodiment of the present invention CsLHCB1.1 Promoter sequence information. Among them, 4-(A) Comparison of promoter sequences in LJ43 and RX2; 4-(B) Analysis of cis-acting elements.

[0026] Figure 5 The interaction verification is for a preferred embodiment of the present invention. Among them, (A) yeast one-hybrid results; (B) dual-luciferase reporter assay results; (C) probe localization; (D) SDS-PAGE colorimetric results of pCold-TF-MYB5 recombinant protein; (E) chemiluminescent gel migration assay (EMSA) results.

[0027] Figure 6 In a preferred embodiment of the present invention CsMYB5 Gene function validation results. A and B represent transient overexpression and silencing in LJ43, respectively; C and D represent transient overexpression and silencing in RX2, respectively; E and F represent transient overexpression and silencing in 'Ruixue 1' (RX1), respectively; G and H represent transient overexpression and silencing in 'Baiye 1' (BY1), respectively; and I and J represent transient overexpression and silencing in 'Jingbai 2' (JB2), respectively. * indicates... P <0.05, ** indicates P <0.01, *** indicates P<0.001.

[0028] Figure 7 This invention presents the subcellular localization results of CsMYB5 and its expression levels under different temperature treatments in two varieties, as shown in the preferred embodiment. (A) Subcellular localization results, with GFP as the empty vector plasmid control; (B) Cooling treatment process; (C) Heating treatment process; (D) Treatment at 13℃ and 28℃ for 20 days. Different lowercase letters indicate significant differences. P <0.05). Detailed Implementation

[0029] This invention provides the tea plant MYB5 transcription factor or the gene encoding said transcription factor. CsMYB5 Application in regulating leaf color changes in low-temperature sensitive albino tea varieties.

[0030] This invention first uses combined physiological and biochemical data and transcriptome data to study the phenotypic performance of leaves in green and low-temperature-sensitive albino varieties during temperature changes, and identifies key regulatory transcription factors to construct a transcription factor regulatory network. Furthermore, it performs functional validation on the key regulatory factor MYB5, and analyzes the expression and phenotypic characteristics of materials that are transiently silenced / overexpressed.

[0031] The present invention adopts the following technical solution: In a first aspect, the present invention provides a tea plant MYB5 transcription factor having the nucleotide sequence shown in SEQ ID NO:1.

[0032] Secondly, the present invention provides the above-mentioned tea tree MYB5 A gene-encoded protein having the amino acid sequence shown in SEQ ID NO:2.

[0033] Thirdly, this invention provides the function of transcription factor MYB5. CsLHCB1.1 The promoter of the gene in different leaf color varieties has the nucleotide sequence SEQ ID NO:3 in green varieties ('Longjing 43', etc.) and the nucleotide sequence SEQ ID NO:4 in albino varieties ('Ruixue 2', etc.).

[0034] Fourthly, this invention provides the expression patterns of the MYB5 transcription factor in tea plants under different temperature treatments in different leaf color varieties.

[0035] Fifthly, this invention provides a tea plant MYB5 transcription factor that activates... CsLHCB1.1 Applications of increased gene expression levels promoting chlorophyll accumulation. CsMYB5 Different leaf color varieties showed differential expression after being subjected to cooling treatment. In albino varieties, the downregulation of expression during cooling and the upregulation during warming were the main reasons for this. CsLHCB1.1Regulatory factors affecting expression level and leaf color changes; the consistently lower expression level in albino varieties compared to green varieties may be a contributing factor even after leaf color reverts to green. This study used 'Longjing 43' as a green control variety and 'Ruixue 2' as a low-temperature-sensitive albino variety.

[0036] Sixthly, the present invention provides methods for promoting and inhibiting chlorophyll accumulation in albino tea plants under low temperatures, respectively by enhancing or weakening chlorophyll accumulation in the tea plants. MYB5 The expression level was achieved. The temperature treatment was 13℃, and the varieties were the green variety 'Longjing 43', the albino varieties 'Ruixue No. 1', 'Ruixue No. 2', 'Baiye No. 1', and 'Jingbai No. 2'.

[0037] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0038] Example 1: Application of transcription factor MYB5 in regulating leaf color changes in low-temperature sensitive albino tea varieties I. Sample processing and pigment content detection Two-year-old cuttings of 'Ruixue 2' (RX2) were used as the material, and two-year-old cuttings of 'Longjing 43' (LJ43) were used as the control, with 180 plants of each variety. All materials were placed in a climate chamber at 20℃ for two months to acclimatize. Each variety was then divided into two equal groups and cultured at 13℃ (11℃ at night) and 28℃ (26℃ at night), respectively. After three days of culture, the first sampling was performed, collecting one bud and two leaves. Samples of RX2 at 13℃ and 28℃ were named RXlo and RXho, respectively (corresponding treatment states were defined as lo and ho). Similarly, samples of LJ43 at 13℃ and 28℃ were named LJlo and LJho, respectively. Subsequently, the temperature was changed from 13℃ to 28℃. A second sampling was conducted 6 hours later. Sample RX2 was named RXlm (time point lm, the same below), and sample LJ43 was named LJlm. A third sampling was conducted 3 days later. Sample RX2 was named RXle (time point le, the same below), and sample LJ43 was named LJle. Similarly, the temperature was lowered from 28℃ to 13℃. A second sampling was conducted 6 hours later. Sample RX2 was named RXhm (time point hm, the same below), and sample LJ43 was named LJhm. A third sampling was conducted 3 days later. Sample RX2 was named RXhe (time point he, the same below), and sample LJ43 was named LJhe. In addition, LJ43 and RX2 cuttings were cultured at 13℃ (11℃ at night) and 28℃ (26℃ at night) for 20 days, respectively, and then samples were taken. The RX2 samples taken at 13℃ and 28℃ were named RXl and RXh, respectively, and the LJ43 samples were named LJl and LJh, respectively. All samples were fixed with liquid nitrogen immediately after collection and stored at -80℃.

[0039] The material subjected to temperature-controlled processing was ground into powder. 200 mg of the powder was weighed and placed in a 15 mL centrifuge tube. 10 mL of pre-cooled acetone was quickly added, and the mixture was ultrasonically extracted for 30 min in a light-protected ice-water bath using a Jiemeng JP-060S ultrasonic cleaner (China). Subsequently, PVPP was added at a ratio of 100 mg / 2 mL acetone, vortexed, and centrifuged at 12000 rpm for 15 min at 4°C. The supernatant was collected for analysis using a Shimadzu LC-20AD high-performance liquid chromatograph (Japan) with a TC-C column. 18A 4.6 mm × 250 mm analytical column (Agilent Technologies) was used. Specific conditions included: column temperature 35℃; injection volume 10 μL; detection wavelength 440 nm; mobile phase A: acetonitrile / acetic acid / water = 3 / 0.5 / 96.5 (v / v / v); mobile phase B: acetonitrile / methanol / chloroform = 75 / 20 / 5 (v / v / v); flow rate 1 mL / min. Elution conditions: phase B linearly increased from 90% to 100% within 15 min and held for 20 min, then linearly decreased to 90% within 5 min and held for 5 min. The qualitative and quantitative analysis of pigments in the samples was performed based on the retention time and peak area of ​​pigment standards. Chlorophyll a, chlorophyll b, neoxanthin, amethyst, xanthophyll, and β-carotene were all purchased from Sigma-Aldrich (USA). All samples underwent three biological replicate assays. Results showed (…). Figure 1 During the temperature reduction process, in LJ43, except for a significant decrease in xanthophyll content, the other five pigments showed no significant changes. Conversely, in RX2, except for no significant change in xanthophyll, the contents of the other five pigments showed a significant decrease after 6 hours of low-temperature treatment, and the decrease in chlorophyll a and chlorophyll b contents was even more significant after 3 days. During the temperature increase process, in LJ43, except for an increase in xanthophyll, the contents of the other five pigments decreased. In RX2, the contents of chlorophyll a, β-carotene, and xanthophyll increased, while chlorophyll b and neoxanthophyll showed no significant changes. This indicates that the albino tea variety RX2 has a strong temperature sensitivity; the chlorophyll content decreases significantly when the temperature decreases and increases significantly when the temperature increases, thus affecting the leaf color phenotype.

[0040] II. Transcriptome Data Analysis and WGCNA Results RNA-seq analysis was performed on samples treated with short-term temperature changes, and sequencing was completed by Beijing Biomarker Biotechnology Co., Ltd. PCA analysis showed good intra-sample reproducibility of transcriptome data, with significant differences between different varieties and temperature treatments, indicating that temperature changes led to substantial changes in gene expression in both varieties. Figure 2 (Part A)). To clarify the temperature-sensitive metabolic pathways in two leaf color varieties, we compared the significant differences in their gene expression profiles before and after temperature changes, using Fold Change ≥ 2 and FDR < 0.01 as the screening criteria for differentially expressed genes. Pigment content and DEGs were analyzed by WGCNA (Wide Gamma-Gamma Analysis). Figure 2 (Parts (B) and (C) in the text). A module member-ship threshold of 0.7 was set to screen for the core regulatory genes of each co-expressed module from the two varieties. In LJ43, MYB5 In the turquoise module, it is positively correlated with pigment content; in RX2, MYB5In the pink module, it is also positively correlated with pigment content.

[0041] III. MYB5 Sequence Analysis Based on transcriptome sequencing CsMYB5 Primers MYB5-1F / MYB-1R (Table 1) were designed to clone complete open reading frames (ORFs) from candidate gene sequences. All primers were synthesized by Yocon Biotechnology Co., Ltd.; EasyPure was used. ® RNA was extracted from the leaves of LJ43 and RX2 plants using a universal plant total RNA extraction kit (Beijing TransGen Biotech Co., Ltd.). cDNA synthesis was performed using the HiScript IV 1st Strand cDNA Synthesis Kit (+gDNAwiper) (purchased from Nanjing Novizan Biotechnology Co., Ltd.), which served as templates for subsequent gene cloning. KOD One was used... TM PCR Master Mix reagent (Toyobo (Shanghai) Biotechnology Co., Ltd.) was used to amplify the sequence of candidate gene ORFs via RT-PCR. Specific procedures should be followed according to the reagent instructions. QuickCut was used to amplify the sequence of candidate gene ORFs. TM EcoRⅠ and QuickCut TM XhoⅠ (Baori Biotechnology (Beijing) Co., Ltd.) was used to perform double restriction enzyme digestion of pB42AD at 37℃ for 3 h. The amplified products were electrophoresed on a 1.0% agarose gel, and the target band was excised and recovered (kit purchased from Beijing Qingke Biotechnology Co., Ltd.). The amplified products were inserted into the pB42AD vector (Wuhan Miaoling Biotechnology Co., Ltd.) under the action of homologous recombination ligase. The one-step cloning kit of In-Fusion HD Cloning Kit (Nanjing Novizan Biotechnology Co., Ltd.) was used to construct the recombinant vector pB42AD-MYB5 containing MYB5, which was then transformed into DH5α competent Escherichia coli (Beijing Qingke Biotechnology Co., Ltd.). The cells were plated on LB solid medium containing 100 μg / mL ampicillin sodium for antibiotic selection. Positive colonies were picked and propagated for 16 h. Colony PCR was performed using 2×Hieff® Ultra-Rapid II HotStart PCR Master Mix reagent (Yisheng Biotechnology (Shanghai) Co., Ltd.). Positive clones were sequenced by Sanger sequencing by Youkang Biotechnology Co., Ltd. Validated by sequencing MYB5The ORF of gene (SEQ ID NO:1) is 879 bp in length and has the same nucleotide sequence in LJ43 and RX2. The amino acid sequence encoded by the ORF was deduced using EditSeq in the DNAStar software package. SEQ ID NO:2 contains 292 amino acid residues (SEQ ID NO:2) and has a relative molecular weight of 33 kDa. The sequence was confirmed to belong to the MYB transcription factor family using the NCBI conserved domain database (CDD). MYB transcription factor member sequences were extracted from the Arabidopsis thaliana database, and a phylogenetic tree was constructed using MEGA software. Figure 3 (A) portion of the sequence was used to ultimately name the sequence MYB5. The theoretical isoelectric point (pI), molecular weight (MW), total average hydrophilicity (GRAVY), and aliphatic amino acid index (AI) of the CsMYB protein were predicted using ProtParam. The secondary structure of the protein was predicted using NPSA. The tertiary structure of the protein was predicted using AlphaFold3, and the results were visualized using PyMOL software. CsMYB5 The encoded protein has a theoretical isoelectric point (pI) of 9.34, an instability coefficient (II) of 46.54, a total average hydrophilicity / hydrophobicity (GRAVY) of -0.757, and an aliphatic amino acid index of 80.41; the predicted template modeling score (pTM) for the transcription factor's three-dimensional structure is relatively low, at 0.46. Figure 3 (Part B) of the MYB5 structure. The MYB5 secondary structure contains only 32.88% α-helices (h), with the remaining structure consisting of random coils (c). Figure 3 (Part (C) in the text).

[0042] Table 1 PCR primer sequences

[0043] IV. Promoter sequence cloning and cis-acting element analysis Genomic DNA was extracted from leaves of LJ43 and RX2 plants using a polysaccharide-polyphenol plant genomic DNA extraction kit (Tiangen Biotech (Beijing) Co., Ltd.). Primers SP1, SP2, and SP3 were designed (Table 1), and the genome walking kit (Baori Biotechnology (Beijing) Co., Ltd.) was used for extraction. CsLHCB1.1The promoter sequence was cloned, the PCR fragment was ligated into the pMD18-T vector (Baori Biotechnology (Beijing) Co., Ltd.), and transformed into DH5α competent Escherichia coli. The cells were plated on LB solid medium containing 100 μg / mL ampicillin sodium for resistance screening. Positive colonies were picked and propagated for 16 h, followed by bacterial PCR. Positive clones were then sequenced by Sanger sequencing at Youkang Biotechnology Co., Ltd. CsLHCB1.1 The promoter sequence in the green variety LJ43 is shown in SEQ ID NO:3, and the promoter sequence in the albino variety RX2 is shown in SEQ ID NO:4. Sequence alignment was performed, and PlantCARE analysis was used to analyze the cis-acting elements of the promoter sequences. The sequence alignment results showed that the first 1000 bp of the promoter sequence differed significantly between the two varieties, while the latter 1000 bp sequence was basically the same. Figure 4 (Part A)). Cis-acting element analysis showed that the last 1000 bp of the sequence contained more photoresponsive elements, while the cis-acting elements in the first 1000 bp of the sequence differed significantly between the two varieties. Figure 4 (Part B)). Compared to RX2, LJ43 contains more MYB recognition and binding elements, as well as more hormone response and stress response elements, which suggests that LJ43 has a more effective defense response mechanism in response to low temperatures. CsLHCB1.1 The expression.

[0044] V. Mutual Verification (I) Yeast One-Hybrid Experiment Using QuickCut TM EcoRⅠ and QuickCut TM XhoⅠ was used to perform double restriction enzyme digestion on the pLacZi2μ vector (plasmid purchased from Wuhan Miaoling Biotechnology Co., Ltd.) at 37℃ for 3 h. The target band was recovered after gel electrophoresis. Design proLHCB1.1 -LJ43-1F / 1R (Table 1) and proLHCB1.1 Using the RX2-1F / 1R primers (Table 1) and genomic DNA as a template, promoter sequences were cloned from LJ43 and RX2, respectively. pLacZi2μ-LJ43- was then constructed using homologous recombination ligase. proLHCB1.1 and pLacZi2μ-RX2- proLHCB1.1 The recombinant plasmid was transformed, cultured PCR was performed, and sequencing was conducted to obtain the correct positive bacteria. pB42AD-MYB5 and pLacZi2μ-LJ43- were extracted using the AFT Spin EndoFree Plasmid Midi Kit (Aibote Biotechnology Co., Ltd.). proLHCB1.1 and pLacZi2μ-RX2- proLHCB1.1The recombinant plasmids were prepared according to the instruction manual. The pB42AD and pLacZi2μ recombinant plasmids were co-transformed into EGY48 yeast competent cells, plated on SD / -Ura / -Trp solid medium, and incubated upside down at 29°C for 48-96 h. Single colonies from the SD / -Ura / -Trp solid medium were picked and placed in 0.9% NaCl solution. The OD of the bacterial culture was measured. 600 Adjust the pH to approximately 0.2, and drop 10 μL of bacterial culture onto SD / Gal / Raf / -Ura / -Trp / +X-gal selection medium containing 5-bromo-4-chloro-3-indolyl-D-galactopyranoside (X-gal). Incubate at 29℃ for 4-6 days, observing yeast growth. Simultaneously, perform self-activation detection. Results showed that MYB5 could bind to the promoter sequences of both varieties. Figure 5 (Part A) in the text.

[0045] (II) Dual-luciferase assay Using QuickCut TM EcoRⅠ and QuickCut TM XhoⅠ was used to perform double restriction enzyme digestion of pGreenⅡ 62-SK, and QuickCut was used to further digest the enzymes. TM KpnⅠ and QuickCut TM XhoⅠ was used to perform double restriction enzyme digestion on pGreenⅡ 0800-LUC (plasmid purchased from Wuhan Miaoling Biotechnology Co., Ltd.). Design proLHCB1.1 -LJ43-2F / 2R and proLHCB1.1 Using the RX2-2F / 2R primers (Table 2) and the pLacZi2μ recombinant plasmids of various varieties as templates, pGreenⅡ 0800-LUC-LJ43- proLHCB1.1 and pGreenⅡ 0800-LUC-RX2- proLHCB1.1 Recombinant plasmids were constructed. Primers MYB5-2F / 2R were designed, and the pGreenⅡ 62-SK-MYB5 recombinant plasmid was constructed using the pB42AD recombinant plasmid as a template. These recombinant plasmids and the empty vector plasmid were transformed into GV3101 (pSoup-p19) Agrobacterium competent cells and plated on LB agar plates containing 50 μg / mL kanamycin and 25 μg / mL rifampin. Single colonies grew after 2-3 days of incubation at 28°C. Single Agrobacterium colonies with a diameter of approximately 1 mm-2 mm were picked from the plates and transferred to 15 mL of LB liquid medium (containing 50 μg / mL kanamycin and 25 μg / mL rifampin). The culture was maintained at 28°C and 200 rpm until the logarithmic growth phase (OD50) of Agrobacterium. 600=0.8-1.0). Collect bacterial cells by centrifugation at 5000 rpm for 10 min at room temperature. Resuspend the Agrobacterium cells in staining buffer (containing 10 mM MgCl2, 10 mM MES, 200 μM acetylsylcholine, pH=5.7) to OD. 600 =1.0. Two bacterial cultures were mixed at a ratio of transcription factor:promoter 1:9 (v / v) and allowed to stand at room temperature for 2-3 hours. Tobacco plants in good growth condition were selected, and the bacterial culture was drawn into the underside of tobacco leaves using a 1 mL sterile syringe (without the needle) and injected into the leaves, marking the injection site with a marker. The injected tobacco plants were cultured in the dark for 1 day, then transferred to light for 1-2 days. An appropriate amount of fluorescein potassium solution (Yisheng Biotechnology (Shanghai) Co., Ltd.) was injected into the Agrobacterium-injected area of ​​the tobacco leaves, and the leaves were placed in the dark for 10 minutes. The luminescence of the tobacco leaves was observed and photographed using an SH 523 chemiluminescence imaging system (Hangzhou Shenhua Technology Co., Ltd.). The results showed that the transcription factor bound to the promoter sequences in both varieties and activated downstream expression (…). Figure 5 (Part B) in the text.

[0046] (III) EMSA Experiment Using QuickCut TM KpnⅠ and QuickCut TM XbaⅠ was used to perform double restriction enzyme digestion on the pCold-TF vector (purchased from Wuhan Miaoling Biotechnology Co., Ltd.) to design... proLHCB1.1 The MYB5 fragment was amplified using primers -LJ43-3F / 3R (Table 1), and the recombinant plasmid pCold-TF-MYB5 was constructed. This plasmid was transformed into competent Rosetta (DE3) cells (Beijing Bomeide Gene Technology Co., Ltd.) and plated on LB solid medium containing 100 μg / mL ampicillin sodium and 34 μg / mL chloramphenicol for resistance selection. Positive clones were picked and seeded into 500 mL LB liquid medium and cultured at 37°C with shaking until OD... 600After the pH value reached 0.6-0.8, IPTG was added to the culture medium to a final concentration of 1 mM and recombinant protein expression was induced at 16℃. After 20 h, the protein was recovered, centrifuged at 4000 rpm for 10 min, and resuspended in 50 mL of 50 mM phosphate buffer (10 mM imidazole, pH 8.0) containing imidazole. The engineered bacteria were sonicated for 30 min, centrifuged at 8000 rpm for 20 min to remove residue, and the supernatant was passed through a nickel column. The supernatant was resuspended in 10 column volumes of 50 mM phosphate buffer (20 mM imidazole, pH 8.0) to wash away impurities. Recombinant MYB5 was then eluted with 5 column volumes of 50 mM phosphate buffer (250 mM imidazole, pH 8.0) containing imidazole. This process used His-tagged protein agarose purification resin (Yisheng Biotechnology (Shanghai) Co., Ltd.). Specific procedures were performed according to the relevant kit instructions. The recombinant MYB5 protein is estimated to have a molecular weight of approximately 85.8 kDa. SDS-PAGE analysis showed that the target gene fused with the TF factor (NCBI ID: AB213654.1) is... MYB5 To obtain high expression and high solubility ( Figure 5 (Part (D)). Three probes, P1, P2, and P3, were designed (Table 1). The probe sequences are located on the promoter as follows: Figure 5 As shown in section (C). The 5' end was modified with biotin labeling, and the corresponding cold probe was synthesized. Using a chemiluminescent EMSA kit (Beyotime Biotechnology Co., Ltd.), the reactants were added in the following order: probe, protein, binding buffer, and gently mixed. Specific procedures were performed according to the relevant kit instructions. Results showed that MYB5 could interact with two sequences (P1 and P2) on the LJ43 promoter and one sequence (P3) on the RX2 promoter. Figure 5 (part E in the text).

[0047] VI. Functional Verification (one) CsMYB5 Gene overexpression experiment Using QuickCut TM SmaⅠ and QuickCut TMXbaⅠ was used to perform double restriction enzyme digestion on the pCAMBIA2300 vector (Wuhan Miaoling Biotechnology Co., Ltd.). MYB5-4F / 4R primers (Table 1) were designed to clone the MYB5 fragment, constructing the pCAMBIA2300-MYB5 recombinant plasmid. This plasmid was then transformed into Agrobacterium AGL1 (pSoup) according to the product instructions from Beijing Coolerbot Technology Co., Ltd. The bacterial culture containing the target gene was streaked onto LB solid medium containing kanamycin (50 μg / mL) and rifampin (25 μg / mL) and cultured at 28℃ for 2-3 days. Single colonies of Agrobacterium were picked and transferred to 15 mL of LB liquid medium containing kanamycin (50 μg / mL) and rifampin (25 μg / mL) and cultured at 28℃ and 200 rpm until OD (digestion occurred). 600 =0.8-1.0. Collect bacteria by centrifugation at 5000 rpm for 10 min. Subsequently, suspend the bacteria in the infection solution (15 mM MES, 10 mM MgCl2, 0.5% D-glucose, 2 mM Na3PO4, 200 μM acetylsylcholine, pH 5.7) to OD. 600 =0.8, stand for 3 h. New shoots of field-grown varieties including 'Longjing 43' (LJ43), 'Ruixue 1' (RX1), 'Ruixue 2' (RX2), 'Baiye 1' (BY1), and 'Jingbai 2' (JB2) were harvested. Agrobacterium was injected into the third leaf below the bud, and after culturing at 10℃ in the dark for 4 days, leaves were collected for real-time quantitative PCR (qRT-PCR) analysis and chlorophyll content determination. RNA extraction was performed using the EasyPure® Universal Plant Total RNA Extraction Kit (Beijing TransGen Biotech Co., Ltd.). cDNA synthesis was performed using HiScript II Q RT SuperMix for qPCR (+gDNA wiper), purchased from Nanjing Novizan Biotechnology Co., Ltd. The tea plant actin gene was used as an internal reference gene, and qRT-PCR reactions were performed using a real-time quantitative PCR kit from Novizan Biotechnology Co., Ltd. MYB5 , LHCB1.1 The qRT-PCR primers for expression level determination are shown in Table 2. (Following step 2...) -ΔΔCT The formula calculates the relative expression levels of each gene. Overall, regardless of whether it's a green or albino variety, overexpression is observed in the leaves. CsMYB5 All can significantly improve CsLHCB1.1 The expression level of [something] further increases the pigment content. Figure 6 In terms of overexpression effect, CsMYB5 The highest overexpression fold (23-fold) was observed in LJ43, downstream CsLHCB1.1The expression level increased 44-fold, indicating that CsMYB5 plays a strong activating role in LJ43, with chlorophyll a and chlorophyll b contents increasing by 1.6 and 1.7 times, respectively. In RX2, CsMYB5 Overexpression fold increase of 25, downstream CsLHCB1.1 Expression levels increased sixfold, with chlorophyll a and chlorophyll b contents increasing by 1.7 and 1.5 times, respectively. In the other three varieties... CsMYB5 Overexpression fold increases were between 2.6 and 6, downstream CsLHCB1.1 The expression level increased by 2-6 times, while the chlorophyll a and chlorophyll b contents increased by 2.0-2.4 and 2.1-2.5 times, respectively.

[0048] Table 2 qPCR primers

[0049] (II) Transient Silencing Experiment of CsMYB5 Gene Designing and screening targets using Solido software CsMYB5 Ten pairs of antisense oligonucleotides (AsODNs) and their corresponding sense oligonucleotides (sODNs) were identified (Table 3). A 50 μM AsODN mixture was injected into the third leaf below the tea bud to silence the nucleotides. CsMYB5 Genes were detected, with sODNs solution used as a control. Leaves were collected after 24 h of incubation at 10℃ in the dark for real-time qRT-PCR analysis and chlorophyll content determination. [Silencing] CsMYB5 Afterwards, among the various varieties CsMYB5 The expression levels were downregulated by folds between 0.02 and 0.36. CsLHCB1.1 The expression levels were downregulated by folds ranging from 0.03 to 0.44, while the folds of reduction in chlorophyll a and chlorophyll b content ranged from 0.52 to 0.75 and 0.54 to 0.68, respectively. (Note:) MYB5 The inhibition of expression led to CsLHCB1.1 Downregulation of expression further reduced pigment content and altered leaf color phenotype. Figure 6 ).

[0050] Table 3 Gene Silencing Primer Sequences

[0051] Note: * indicates thiophosphate modification.

[0052] VII. Analysis of MYB5 Subcellular Localization and Expression Pattern Using QuickCut TMKpnⅠ was used to perform restriction enzyme digestion on the pCAMBIA1300 vector (Wuhan Miaoling Biotechnology Co., Ltd.). MYB5-5F / 5R primers (Table 1) were designed to clone the MYB5 fragment, constructing the pCAMBIA1300-MYB5 recombinant plasmid. This plasmid was then transformed into Agrobacterium GV3101 (with the pCAMBIA1300 empty vector plasmid as a control). The procedure was performed according to the product instructions from Beijing Coolerbot Technology Co., Ltd. The bacterial culture containing the target gene was streaked onto LB solid medium containing kanamycin (50 μg / mL) and rifampin (25 μg / mL) and cultured at 28℃ for 2-3 days. Single colonies of Agrobacterium were picked and transferred to 15 mL of LB liquid medium containing kanamycin (50 μg / mL) and rifampin (25 μg / mL) and cultured at 28℃ and 200 rpm until OD. 600 =0.8-1.0. Collect bacteria by centrifugation at 5000 rpm for 10 min. Resuspend Agrobacterium cells in staining buffer (containing 10 mM MgCl2, 10 mM MES, 200 μM acetylsylcholine, pH=5.7) to OD. 600 =1.0, stand at room temperature for 2-3 hours. Select tobacco plants in good growth condition, and use a 1 mL sterile syringe (without the needle) to draw up the bacterial solution and inject it into the underside of the tobacco leaves, marking the injection site with a marker. Incubate the injected tobacco plants in the dark for 1 day, then transfer them to light for further incubation. After 55-60 hours, observe the fluorescence signal on the tobacco leaf epidermis using a laser confocal scanning microscope (Zeiss LSM 880, ZEISS GmbH, Germany). Figure 7 Part (A) shows that CsMYB5 is located on the cell nucleus, confirming its transcriptional regulatory function.

[0053] RNA was extracted from samples treated with short-term variable temperature and long-term temperature using the EasyPure® Universal Plant Total RNA Extraction Kit (Beijing TransGen Biotech Co., Ltd.). cDNA synthesis was performed using HiScript II Q RT SuperMix for qPCR (+gDNA wiper), purchased from Nanjing Novizan Biotechnology Co., Ltd. Tea plant samples were used as an example. actin The gene was used as an internal reference gene. qPCR was performed using a real-time fluorescence kit from Novizan Biotechnology Co., Ltd. The qPCR primers for determining MYB5 expression levels are shown in Table 2. (Following step 2...) -ΔΔCT The formula calculates the relative expression levels of each gene. In LJ43, cooling treatment caused a rapid increase in gene expression levels at 6 h, although it decreased after 3 days, but remained unchanged from the initial state. After heating, the expression levels remained stable without significant change. Figure 7(Parts (B) and (C) in the text). Conversely, in RX2, cooling treatment caused a continuous decrease in CsMYB5 expression levels, which remained lower than those in LJ43. Heating significantly increased expression levels, and after 3 days of heating, the expression levels were no longer significantly different from those in LJ43. We also examined the expression levels of this transcription factor in both varieties under long-term treatment at different temperatures. Figure 7 (Part (D) of the study). It was found that the expression level of CsMYB5 in LJ43 showed no significant difference under both low and high temperatures, and was significantly higher than that of RX2 under all treatment temperatures. Conversely, the expression level of RX2 was significantly lower under low-temperature treatment than under high-temperature treatment. In conclusion, the temperature response of MYB5 in different leaf color varieties is an important reason for leaf color changes.

[0054] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. Tea plant transcription factors CsMYB5 Genes or containing the above CsMYB5 Application of genetic biomaterials in regulating chlorophyll accumulation and / or leaf color changes in low-temperature sensitive albino tea varieties; The CsMYB5 The gene is the gene that encodes the following protein (a): (a) A protein consisting of the amino acid sequence shown in SEQ ID NO:2; The biological material is an expression cassette, transposon, plasmid vector, viral vector, or engineered bacteria.

2. The application according to claim 1, characterized in that, The regulation is achieved by activating the tea plant through the CsMYB5 transcription factor. CsLHCB1.1 This is achieved through gene expression; The tea tree CsLHCB1.1 The gene's reference sequence number in NCBI is XM_028241743.

1.

3. The application according to claim 2, characterized in that, The CsMYB5 transcription factor and the tea tree CsLHCB1.1 The promoter sequence of a gene binds and activates its expression; Among them, those derived from low-temperature sensitive albino tea tree varieties CsLHCB1.1 The promoter sequence of the gene is the nucleotide sequence shown in SEQ ID NO:

4.

4. The application according to claim 1, characterized in that, By increasing the expression level of the endogenous or exogenous CsMYB5 transcription factor in the tea plant, chlorophyll accumulation in low-temperature sensitive albino tea plant varieties is promoted under low-temperature conditions. The low-temperature conditions are 8℃~25℃.

5. The application according to claim 4, characterized in that, The expression level of the CsMYB5 transcription factor can be increased by using any one of 1) - 5) or any combination thereof: 1) By importing a plasmid containing the gene; 2) By increasing the copy number of the aforementioned genes on plant chromosomes; 3) By altering the promoter sequence of the aforementioned genes on plant chromosomes; 4) By operatively linking a strong promoter to the gene; 5) By importing enhancers.

6. The application according to claim 1, characterized in that, By reducing the expression level of the CsMYB5 transcription factor, chlorophyll accumulation in low-temperature sensitive albino tea varieties under low-temperature conditions was inhibited. The low-temperature conditions are 8℃~25℃.

7. The application according to claim 6, characterized in that, The application includes the step of reducing the expression level of the CsMYB5 transcription factor using antisense oligonucleotides, RNA interference, or gene editing techniques.

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