Lycium ruthenicum MYB transcription factor LrMYB77 and coding gene and application thereof

Abstract

The invention relates to the technical field of gene engineering, and provides a lycium ruthenicum MYB transcription factor LrMYB77, the amino acid sequence of which is as shown in SEQ ID NO.3. The invention also provides a gene LrMYB77 for coding a transcription factor, and a recombinant expression vector and host bacteria containing the gene. Meanwhile, the invention further provides application of the transcription factor or the gene in promoting synthesis of lycium ruthenicum anthocyanin and a specific method for increasing the synthesis amount of the lycium ruthenicum anthocyanin. According to the invention, the LrMYB77 gene with a regulation effect on anthocyanin synthesis is screened from lycium ruthenicum for the first time, and experiments verify that the expression or overexpression of the gene in a plant is regulated, so that the synthesis and accumulation of anthocyanin in the plant can be remarkably promoted, and the LrMYB77 gene has important significance on germplasm resource creation and molecular breeding of lycium ruthenicum rich in anthocyanin.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to a black goji berry MYB transcription factor LrMYB77 and its encoding gene and applications. Background Technology

[0002] Anthocyanins are a class of flavonoid compounds widely found in plants, possessing various effects such as antioxidant, anti-aging, anti-inflammatory, and cardiovascular and neuroprotective activities. Black goji berries (…) Lycium ruthenicum As a distinctive plant of the Lycium genus in the Solanaceae family, Murr. is rich in anthocyanins in its fruits, flowers, leaves and other tissues. 37 related anthocyanin components have been isolated and identified, earning it the reputation of "King of Anthocyanins". It is an excellent model plant for studying anthocyanin synthesis and metabolism, and also an important breeding material for creating germplasm resources rich in anthocyanins.

[0003] Anthocyanin synthesis in plants relies on complex secondary metabolic pathways, and its efficiency is primarily regulated by a multi-level regulatory network, with transcription factors playing a crucial role. Transcription factors bind to specific DNA elements upstream of target genes, activating the expression of key structural genes in the plant's secondary metabolic pathway and effectively controlling the synthesis and accumulation of secondary metabolites. Currently, several transcription factors involved in anthocyanin metabolism regulation, such as MYB, bHLH, and WRKY, have been identified in species like Arabidopsis thaliana, apple, rhododendron, and potato. Among these, MYB transcription factor has become a research hotspot due to its core regulatory role in the anthocyanin synthesis pathway.

[0004] However, research on the molecular regulatory mechanisms of anthocyanin synthesis in black goji berries is still in its early stages. Although it is known to be rich in anthocyanins, the screening and functional studies of key regulatory factors (especially MYB transcription factors) in the anthocyanin synthesis pathway have not been in-depth, and the core question of which MYB transcription factors are involved in the regulation of anthocyanin synthesis remains unclear.

[0005] Therefore, screening and identifying MYB transcription factors involved in the regulation of anthocyanin synthesis in black goji berries and clarifying their functions has become an urgent problem to be solved in current molecular breeding and secondary metabolism research of black goji berries. Summary of the Invention

[0006] The technical problem to be solved by this invention is to provide a black goji berry MYB transcription factor LrMYB77 and its encoding gene and application. By regulating the expression (or overexpression) of this transcription factor in the plant, the synthesis and accumulation of anthocyanins in the plant can be significantly promoted, which is of great significance for the creation of black goji berry germplasm resources rich in anthocyanins and molecular breeding.

[0007] The present invention solves the above-mentioned technical problems by adopting the following technical solutions: A black goji berry MYB transcription factor LrMYB77, the amino acid sequence of which is shown in SEQ ID NO.3.

[0008] As one of the preferred embodiments of the present invention, the transcription factor LrMYB77 can promote the synthesis of anthocyanins in black goji berries.

[0009] A gene encoding the MYB transcription factor LrMYB77 of the aforementioned black goji berry. LrMYB77 The coding region nucleotide sequence of the gene is shown in SEQ ID NO.2.

[0010] As one of the preferred embodiments of the present invention, the full-length nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0011] As one of the preferred embodiments of the present invention, the expression level of the gene is related to the anthocyanin synthase gene of black goji berries. LrANS There is a positive correlation between the expression levels of these substances.

[0012] A recombinant expression vector containing the above-mentioned gene LrMYB77 .

[0013] A host bacterium containing the above-mentioned recombinant expression vector.

[0014] One of the above transcription factors LrMYB77 or genes LrMYB77 Application in promoting anthocyanin synthesis in plants.

[0015] As one of the preferred embodiments of the present invention, the plant is black goji berry or tobacco.

[0016] A method to increase anthocyanin synthesis in black goji berries by overexpressing the aforementioned gene. LrMYB77 This allows the transcription factor LrMYB77 protein to be expressed efficiently in black goji berries, promoting the synthesis and accumulation of anthocyanins.

[0017] As one of the preferred embodiments of the present invention, the overexpression is achieved by expressing the gene... LrMYB77 The expression is constructed into a plant expression vector and then transformed into target cells or plants.

[0018] The advantages of this invention compared to the prior art are: (1) This invention is the first to screen out a substance from black wolfberry that has a regulatory effect on anthocyanin synthesis. LrMYB77 The gene was identified, clarifying the amino acid sequence of the LrMYB77 transcription factor it encodes and the nucleotide sequence of the encoding gene; big data analysis and qRT-PCR technology confirmed that... LrMYB77The gene was significantly upregulated in three different fruit development stages of black goji berries. This discovery not only fills the gap in key gene resources for the molecular regulatory mechanism of anthocyanin synthesis in black goji berries, but also provides new gene targets for elucidating the regulatory mechanism of anthocyanin synthesis in this field, and provides important scientific basis for improving the anthocyanin synthesis characteristics of plants through genetic engineering.

[0019] (2) This invention thoroughly reveals LrMYB77 The gene regulatory mechanism has been clarified, and its expression product (LrMYB77 transcription factor) has been identified as being able to effectively regulate key genes in anthocyanin synthesis. LrANS The transcription process, specifically, involves the transcription factor's ability to interact with... LrANS Gene promoter-specific binding, thereby regulating LrANS Gene expression, and LrMYB77 Gene expression level and LrANS Gene expression levels showed a positive correlation; furthermore, through mature technologies such as transient expression and heterologous overexpression, the specific expression levels were further clarified. LrMYB77 The function of genes in anthocyanin synthesis in black goji berry fruit.

[0020] (3) The present invention provides a method for overexpression LrMYB77 The method for creating high-anthocyanin black goji berry germplasm through gene creation can be directly used for molecular breeding of black goji berries, while also providing technical support for anthocyanin-related breeding and molecular marker development in plants. This gene and related technology can also directionally improve the anthocyanin synthesis characteristics of black goji berries, helping to cultivate superior new varieties and enhance their economic and nutritional value, which is of great practical significance for promoting the industrialization of black goji berries. Attached Figure Description

[0021] Figure 1 The gene in Example 1 LrANS and LrMYBs The co-expression network diagram; Figure 2 In Example 1 LrANS Analysis diagram of the cis-acting elements of the promoter; Figure 3 In Example 2, black goji berries at different developmental stages were found in... LrMYB77 Relative gene expression results (in the figure, * indicates P<0.05, *** P<0.001, **** P<0.0001); Figure 4 The LrMYB77 protein in Example 4 and LrANS The results of yeast one-hybrid validation of promoter interactions (Figure A shows the growth of bait strains on different concentrations of AbA; Figure B shows the growth of co-transformed strains on gradient dilutions of 100 ng / mL AbA; pGADT7 is the negative control). LrMYB77(for the experimental group) Figure 5 The activation of LrMYB77 protein in Example 5 LrANS Transient transformation verification results of the promoter (Figure A shows the fluorescence signal distribution of tobacco leaves after transient transformation, 1 represents Pro) LrANS Group 2 is LrMYB77+Pro LrANS Group B; Figure B shows the statistics of the relative LUC / REN ratio, *** indicates P<0.001). Detailed Implementation

[0022] The embodiments of the present invention are described in detail below. These embodiments are implemented based on the technical solution of the present invention, and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments. Furthermore, unless otherwise specified, the reagents and materials used in the following embodiments are all commercially available reagents and materials conventional in the art. Unless otherwise specified, the experimental methods used in the following embodiments are all conventional methods in the art and will not be described further.

[0023] Example 1: Screening for transcription factor LrMYB77: Genome data of black goji berries was downloaded from the NCBI Genomics Database. A Hidden Markov Model (http: / / hmmer.org) MYB(PF:00279) was used to query sequences, and BLAST analysis was performed on the black goji berry genome. Transcriptome sequencing was performed on black goji berry fruits at 7, 21, and 35 days post-flowering. Combined with the transcriptome data, candidate gene sequences were compared with the anthocyanin synthase gene of black goji berries using R language. LrANS Perform correlation analysis: Co-expression network analysis: A network was constructed through gene co-expression analysis (see Figure 1), with the core node being the anthocyanin structural gene. LrANS (LOC132634997) is co-expressed with multiple MYB genes, among which LrMYB77 (LOC132603677) and LrANS Highest correlation ， This suggests that it plays a regulatory role in metabolic pathways.

[0024] LrANS Promoter cis-acting element prediction: LrANS Cis-acting element analysis of promoter sequences (see) Figure 2 The results showed that the promoter region contained MYB binding elements (MYB, MYB-like sequence), which will be helpful for further verification. LrMYB77 and LrANS Promoter interactions provide a sequence basis.

[0025] Based on the above results, the following were selected: LrANS Highly correlated LrMYB77 The genes were used in subsequent experiments. The selected genes... LrMYB77 The gene, whose full-length nucleotide sequence is shown in SEQ ID NO.1, whose coding region nucleotide sequence is shown in SEQ ID NO.2, and whose amino acid sequence encoding the protein is shown in SEQ ID NO.3.

[0026] Example 2: Black goji berry fruits at different developmental stages LrMYB77 Comparison of gene expression levels: Black goji berry fruits were selected as plant samples at 7, 21, and 35 days after flowering. Quantitative real-time PCR was used to determine the levels of phytochemicals in the corresponding plants at different time points. LrMYB77 Gene expression levels are determined through the following steps: Primers (upstream primers) were designed using Primer Premier 5.0 software. YMYB77 -F, downstream primer YMYB77 -R, sequences as shown in SEQ ID NO.4 and SEQ ID NO.5 respectively, were synthesized by Shanghai Sangon Biotech Co., Ltd.; at the same time, RNA was extracted from the plant fruit using a plant RNA extraction kit (Nanjing Novizan Biotechnology Co., Ltd., catalog number RC401-01), and cDNA was synthesized by reverse transcription using the EasyScript® One-Step gDNA Removal and cDNA Synthesis SuperMix kit.

[0027] Add reagents according to the system in Table 1, set up three biological replicates, and carry out the reaction according to the reaction conditions in Table 1. Calculate the relative expression level using the 2-ΔΔCt method.

[0028] Table 1. Reaction system and reaction conditions for real-time PCR

[0029] The results of the expression level measurement are shown in Figure 3 The results showed that as the fruit ripened, LrMYB77 Gene expression levels increased significantly.

[0030] Example 3, Gene LrMYB77 Clones: RNA was extracted from the fruit of *Lycium barbarum* 35 days after flowering using a plant RNA extraction kit. The RNA was reverse transcribed into cDNA using a reverse transcription kit (EasyScript® One-Step gDNA Removal and cDNA Synthesis SuperMix) according to the instructions. The cDNA was then obtained from the *Lycium barbarum* genome (V4.0).LrMYB77 The complete sequence of the gene's CDS (coding region) was used to design full-length primers (upstream primers) using Primer Premier 5.0 software. LrMYB77 -F1, downstream primer LrMYB77 -R2, the sequences are shown in SEQ ID NO.6 and SEQ ID NO.7 respectively.

[0031] Using cDNA as a template, PCR amplification was performed. The reaction system and reaction conditions are shown in Table 2.

[0032] Table 2. PCR amplification reaction system and reaction conditions

[0033] Example 4, Gene LrMYB77 and LrANS Promoter interaction yeast one-hybrid assay: (1) Select SmaⅠ and XhoⅠ as the restriction enzyme sites for the pAbAi vector, and design a vector with overlapping sequences of these restriction enzyme sites. LrANS Promoter homologous recombination primers ( LrANS Pro-pAbAi-F, LrANS Pro-pAbAi-R (sequences shown in SEQ ID NO. 8 and SEQ ID NO. 9, respectively). Using black goji berry genomic DNA as a template, PCR amplification was performed to obtain pro-pAbAi-R with matching overlapping sequences. LrANS Promoter fragment (PCR amplification system and reaction conditions are the same as in Table 2). After PCR product detection by gel electrophoresis and purification using the EZ-10 column DNA gel extraction kit, homologous recombination was performed using the pEASY®-Basic Seamless Cloning and Assembly Kit. LrANS The promoter fragment was ligated into the pAbAi vector digested with SmaⅠ and XhoⅠ to construct the bait vector pAbAi- LrANS Pro (The double enzyme digestion reaction system and reaction conditions are shown in Table 3, and the connection reaction system and reaction conditions are shown in Table 4).

[0034] (2) EcoRI and BamHI were selected as the restriction enzyme sites for the pGADT7 AD vector, and a vector with overlapping sequences of these restriction enzyme sites was designed. LrMYB77 Gene homologous recombination primers ( LrMYB77 -pGADT7 AD-F1、 LrMYB77 -pGADT7 AD-R1 (sequences shown in SEQ ID NO.10 and SEQ ID NO.11, respectively). Obtained by cloning in Example 3. of LrMYB77 Using the gene coding region sequence as a template, PCR amplification was performed to obtain sequences with matching overlapping sequences. LrMYB77Gene fragments (PCR amplification system and reaction conditions are the same as in Table 2). After gel electrophoresis detection and purification, the PCR products were used to perform homologous recombination using the pEASY®-Basic Seamless Cloning and Assembly Kit. LrMYB77 The gene fragment was ligated into the pGADT7 AD vector, which had been digested with BamHI and EcoRI, to construct the prey vector pGADT7 AD- LrMYB77 (The double enzyme digestion reaction system and reaction conditions are shown in Table 3, and the ligation reaction system and reaction conditions are shown in Table 4).

[0035] Table 3. Double enzyme digestion reaction system and reaction conditions

[0036] Table 4. Connection Reaction System and Reaction Conditions

[0037] (3) Streak the Y1HGold strain of Saccharomyces cerevisiae stored at -80℃ on YPDA medium and incubate at 28℃ for 2-3 days until single colonies appear; pick a single yeast colony and put it into 5mL of YPDA liquid medium, incubate at 28℃ overnight at 180r / min until OD 600 >1.5; Inoculate 1 mL of culture medium into 50 mL of YPDA liquid medium and incubate at 28℃ and 180 r / min until OD... 600 =0.4~0.6, centrifuge at 6000 r / min for 5 min at room temperature to collect bacterial cells; resuspend the bacterial cells in 10 mL ddH2O, centrifuge at 6000 r / min for 5 min, remove the supernatant, and resuspend the bacterial cells in 1.5 mL 1×LiAc to obtain competent cells.

[0038] (4) The above-mentioned bait carrier pAbAi- LrANS The Pro plasmid was linearized using the restriction enzyme BstBI and then transformed into the yeast competent cells prepared in step (3). The cells were then plated onto SD / -Ura solid medium and cultured at 28°C until single colonies appeared. Colony PCR was then performed to screen for positive clones, i.e., positive yeast strains (named Y1HGold-pAbAi-) whose bait vector had successfully integrated into the Y1HGold genome. ).

[0039] (5) Resuspend the positive bait yeast strain monoclonal strain prepared in step (4) in 0.9% NaCl to OD. 600=0.002, take 100µL of the resuspended bacterial solution and spread it on SD / -Ura deficient medium with different concentrations (0ng / mL, 50ng / mL, 100ng / mL, 200ng / mL, 300ng / mL, 400ng / mL, 500ng / mL, 600ng / mL) of aureobasidin A (AbA), and incubate at 28℃ for 2-3 days. Count the number of colonies on each plate and take pictures to determine the minimum inhibitory concentration of AbA.

[0040] (6) The prey carrier pGADT7 AD- LrANS Pro The cells were directly transformed into the aforementioned positive yeast strains, plated onto SD / -Leu solid medium, and incubated upside down at 28°C for 3–5 days until single colonies appeared. Co-transformed positive clones were screened by colony PCR. Simultaneously, the empty pGADT7 AD vector was transformed into the aforementioned positive yeast strain Y1HGold-pAbAi- LrMYB77 In Pro, as a negative control, it was also plated onto SD / -Leu solid medium for screening.

[0041] (7) After serially diluting the selected co-transformed positive clones and negative control clones, they were plated on SD / -Leu solid medium containing 100 ng / mL AbA.

[0042] Result Interpretation: If co-transformed positive clones can grow normally on culture medium containing AbA, while negative controls cannot grow, it indicates that LrMYB77 protein can interact with... LrANS The promoter binds specifically to the two, and they interact.

[0043] See results LrANS The results showed that the LrMYB77 protein can effectively bind to... Figure 4 The initiation subregion, regulation LrANS Gene expression.

[0044] Example 5: Construction of recombinant vectors for transient conversion and LrANS Instantaneous transformation verification: (1) The multiple cloning sites of the plant overexpression vector pGreenII 62-SK and the luciferase reporter vector pGreenII 0800-LUC were analyzed using SnapGene software to determine the BamHI and EcoRI restriction sites for the pGreenII 62-SK vector (for insertion). LrMYB77 (Gene), KpnⅠ and BamHI were selected as restriction sites for the pGreenII 0800-LUC vector (for insertion). LrMYB77 (Promoter sequence).

[0045] (2) Design homologous recombination primers containing overlapping sequences of 15-25 bp plasmid restriction enzyme sites, specifically as follows: LrANS -62SK-F1 LrMYB77 -62SK-R2 LrMYB77 Pro-0800-F1, LrANS Pro-0800-R2, the sequences are shown in SEQ ID NO. 12~15 respectively.

[0046] (3) Using a 25 μL PCR reaction system, the clones obtained in Example 3 were respectively... LrANS The gene coding region sequence serves as a template (for amplification). LrMYB77 The target fragment), black goji berry genomic DNA as a template (amplification) LrMYB77 The promoter fragment was used to perform PCR amplification with corresponding primers, resulting in an amplified product containing overlapping sequences that match the restriction enzyme sites on the vector. After confirming the presence of a single target band by gel electrophoresis, the PCR product was purified using an EZ-10 column-based DNA gel extraction kit.

[0047] (4) The vector was constructed using the pEASY®-Basic Seamless Cloning and Assembly Kit (TransGenBeijing, China) via homologous recombination. The pGreenII 62-SK vector was first treated with the enzyme digestion system shown in Table 1, and then the purified vector was digested using the homologous recombination enzyme according to the reaction system shown in Table 2. LrANS The target fragment was recombined with the enzyme-digested pGreenII 62-SK vector to construct the recombinant overexpression vector pGreenII 62-SK- LrMYB77 .

[0048] Simultaneously, vectors were constructed using the pEASY®-Basic Seamless Cloning and Assembly Kit (TransGenBeijing, China) via homologous recombination. First, pGreenII 62-SK and pGreenII 0800-LUC vectors were treated with the enzyme digestion system shown in Table 1. Then, using homologous recombination enzymes, the purified vectors were processed according to the reaction system shown in Table 2. LrMYB77 The promoter fragment was recombinated with the enzyme-digested pGreenII 0800-LUC vector to construct the recombinant reporter vector pGreenII 0800- LrANS Pr o -LUC.

[0049] (5) Extract the recombinant overexpression vector pGreenII 62-SK- LrANS and the reorganization report carrier pGreenII0800- The Pro-LUC plasmid was transformed into Agrobacterium GV3101 (P19) strain; after screening for positive single colonies and culturing them to a suitable concentration, the two bacterial solutions were mixed in equal volumes. The mixed bacterial solution was injected into the right side of a Tobacco Benzovia leaf using a needleless syringe, while a control group with an empty vector was set up (left side of the leaf: pGreenII 62-SK empty vector and pGreenII 0800- LrMYB77 After culturing a mixed bacterial culture containing the Pro-LUC reporter vector for 72 hours, the activities of firefly luciferase and kidney luciferase were detected using a dual-luciferase reporter assay system (E710, Promega) and a Modulus luminescence analyzer (GloMax96, Promega). The LUC / REN ratio was calculated to verify the results. LrANS right LrANS The activation effect of promoters.

[0050] See results LrMYB�7 The results showed that LrMYB77 protein and LrANS Promoter-driven LUC in tobacco ( N. Figure 5 LrMYB77 protein is co-expressed in the leaves of [various species], and its expression can be significantly upregulated. LrANS Gene expression.

[0051] In summary, this invention has successfully screened and cloned the transcription factor gene of black goji berry. tabacum Gene co-expression analysis and promoter cis-elements prediction identified its connection with key genes in anthocyanin synthesis. LrANS The association; yeast one-hybrid assay confirmed that the LrMYB77 protein can specifically bind to... LrMYB77 Promoter; further confirmation through transient conversion experiments showed that LrMYB77 protein can significantly activate LrANS The transcriptional activity of the promoter regulates anthocyanin synthesis. This indicates that... LrANS LrANS LrMYB77 It can serve as a key transcription factor gene for regulating anthocyanin synthesis in black goji berries, providing important genetic resources and theoretical basis for improving the anthocyanin content of black goji berries through genetic engineering.

[0052] 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, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A black goji berry MYB transcription factor LrMYB77, characterized in that, The amino acid sequence of the transcription factor LrMYB77 is shown in SEQ ID NO.

3.

2. The transcription factor LrMYB77 according to claim 1, characterized in that, The transcription factor LrMYB77 can promote the synthesis of anthocyanins in black goji berries.

3. A gene encoding the LrMYB77 transcription factor of black goji berry as described in claim 1 or 2. LrMYB77 Its characteristics are, The coding region nucleotide sequence of the gene is shown in SEQ ID NO.

2.

4. The gene according to claim 3 LrMYB77 Its characteristics are, The full-length nucleotide sequence of the gene is shown in SEQ ID NO.

1.

5. The gene according to claim 3 LrMYB77 Its characteristics are, The expression level of the gene is related to the anthocyanin synthase gene in black goji berries. LrANS There is a positive correlation between the expression levels of these substances.

6. A recombinant expression vector, characterized in that, Contains the gene described in any one of claims 3 to 5 LrMYB77 .

7. A host bacterium, characterized in that, The recombinant expression vector as described in claim 6.

8. A transcription factor LrMYB77 as described in any one of claims 1 to 2, or a gene as described in any one of claims 3 to 5. LrMYB77 Its application in promoting anthocyanin synthesis in plants.

9. The application according to claim 8, characterized in that, The plant in question is either black goji berry or tobacco.

10. A method for increasing the anthocyanin synthesis in black wolfberry, characterized in that, By overexpressing the gene according to any one of claims 3 to 5 LrMYB77 This allows the transcription factor LrMYB77 protein to be expressed efficiently in black goji berries, promoting the synthesis and accumulation of anthocyanins.

Images