Jujube ZjMYB86 gene for regulating flavonoid synthesis as well as expression protein and application of jujube ZjMYB86 gene

Abstract

The invention belongs to the technical field of agricultural biology, and particularly relates to a jujube ZjMYB86 gene for regulating flavonoid synthesis as well as an expression protein and application of the jujube ZjMYB86 gene. The invention provides a ZjMYB86 gene, a coding protein sequence of the ZjMYB86 gene, an overexpression vector, host bacteria and application of the ZjMYB86 gene in regulation and control of accumulation of flavonoids in plant organs. According to the invention, an over-expression vector of the ZjMYB86 gene is constructed, verification of biological functions of the ZjMYB86 gene is carried out through agrobacterium-infected heterologous transformation of tobacco, and a novel gene ZjMYB86 for negative regulation of flavonoid synthesis is provided and can inhibit accumulation of flavonoid in plant organs.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of agricultural biotechnology, and particularly relates to a jujube gene for regulating flavonoid synthesis ZjMYB86 and an expression protein thereof and application. BACKGROUND

[0002] Flavonoids are a class of secondary metabolites widely existing in plants, have multiple biological activities and functions, and play an important role in the growth and development of plants, stress resistance, defense mechanism and interaction with microorganisms. According to the different hetero-oxygen rings and conformations of the basic molecular structure of flavonoids, flavonoids are generally divided into six categories: flavone, flavonol, isoflavone, flavanone, flavanol and anthocyanidin.

[0003] Flavonoid biosynthesis is initiated from the phenylpropanoid pathway. Under the action of phenylalanine ammonia lyase (PhAAL), phenylalanine is deaminated to form trans-cinnamic acid. Then, under the action of cinnamate-4-hydroxylase (C4L), trans-cinnamic acid is converted into 4-coumaric acid. Under the action of 4-coumarate-CoA ligase (C4CoAL), 4-coumaric acid is converted into 4-coumaroyl-CoA. Under the action of chalcone synthase, 4-coumaroyl-CoA and three molecules of 3-malonyl-CoA undergo condensation reaction to generate 2',4',6',4-tetrahydroxychalcone. Under the action of chalcone-flavanone isomerase, the compound is further isomerized into flavanone, thereby starting the flavonoid pathway to generate flavonols, flavones, dihydroflavonols, isoflavones and anthocyanins and other different categories of flavonoids. It can be seen that the plant flavonoid biosynthesis pathway is regulated by a series of transcription factors, mainly involving MYB, bHLH, WD40, WRKY, NAC, bZIP and other transcription factor families.

[0004] Studies have shown that MYB transcription factors regulate flavonoid biosynthesis in the following three ways: (1) by directly binding to the promoters of structural genes in the flavonoid pathway to activate or inhibit the expression of genes in the pathway; (2) forming an MBW complex with bHLH transcription factors and WD40 proteins to cooperatively regulate the expression of structural genes in the flavonoid pathway; and (3) forming a protein complex with ERF, WRKY and MYC transcription factors to cooperatively regulate the expression of structural genes in the flavonoid pathway.

[0005] Existing technologies have identified over 300 flavonoids from jujube fruits, leaves, and seeds. However, research on the molecular regulatory mechanisms of jujube flavonoid biosynthesis is limited, and most studies focus on anthocyanin regulation. Transcription factors ZjMYB5, ZjTT8, and ZjWDR3 can synergistically transcribe and activate anthocyanin structural genes. ZjANS and ZjUGT79B1 The expression of WD40 transcription factor family member ZjFAS2 positively regulates the accumulation of anthocyanins in jujube fruit. MYB86 transcription factor belongs to the R2R3-MYB subfamily, and previous studies have revealed that it has multiple biological functions: (1) The interaction between GhMYB86 and GhMBE2 positively regulates the accumulation of anthocyanins in jujube fruit. GhTUB7 (1) It promotes the elongation of cotton fibers; (2) ZaMYB86 and ZaMYB5 work together to regulate the formation of bristles in *Zanthoxylum bungeanum*; (3) TaMYB86B improves the salt tolerance of wheat by regulating ion balance. However, the existing technology has not provided clear experimental evidence to confirm that MYB86 participates in the regulation of flavonoid biosynthesis. Summary of the Invention

[0006] The purpose of this invention is to provide a flavonoid transcription factor ZjMYB86.

[0007] Another object of the present invention is to provide the encoding gene of the flavonoid transcription factor ZjMYB86.

[0008] Another object of the present invention is to provide a recombinant expression vector containing the above-mentioned coding gene.

[0009] Another object of the present invention is to provide a recombinant strain containing the above-mentioned encoding gene.

[0010] Another object of the present invention is to provide the application of the above-mentioned flavonoid transcription factor ZjMYB86 and its encoding gene.

[0011] This invention provides ZjMYB86 The nucleotide sequence of the gene is shown in SEQ ID NO.1: The sequence contains 1,467 bases and encodes the flavonoid transcription factor ZjMYB86, which has 488 amino acids.

[0012] The amino acid sequence of the flavonoid transcription factor ZjMYB86 is shown in SEQ ID NO.2: MGRHSCCYKQKLRKGLWSPEEDEKLLRHIAKYGHGCWSSVPKQAGLERCGKSCRLRWINYLRPDLKRGTFSREEENLIIELHAVLGNRWSQIAAQLPGRTDNDIKNLWNSCLKKKLRQRGVD PVTHRPISEDKDREAKSSGEKLVSVAISNELNLLETENSKKAAAAAALFGAAQVVEANNSSLCSNKTTNTTTDNNNMMMMKELFLQEQDTSSTTNCQQPPSDYLGQLNSLHQFKYKYGSTTN TGSSTTATTRLPSSNPCSNWFLQPGKSTLDMNSHDDALSNCNSIPIILPLSLPIPIPAPSSTSFPSSTASCIANCKPTSLTFPPTATGSIDNNNSMASFPISNNSSSSSSNCFLENNNMFGW GLVADCSTVSEKEAHIQVNNLMDSQAGAADDIKWAEYLSNPPTFLQTTTTQHQNSHSLLCITSSSSSTSDIKSSETQHSVMWPPPHNSKHQQLQPSQLQTTSSDICAKDIHRLTAALGGHN The amino acid sequence has a molecular weight of 53.500 kDa and an isoelectric point of 7.39.

[0013] The present invention also provides a recombinant expression vector containing the above-mentioned coding gene.

[0014] The present invention also provides recombinant strains containing the above-mentioned coding genes. Preferably, the host strain is Agrobacterium GV3101.

[0015] This invention also provides ZjMYB86 Application of genes in regulating the accumulation of flavonoids in plants.

[0016] Preferably, the plant is tobacco or jujube, and the plant organs in which flavonoids accumulate include flowers, leaves or fruits.

[0017] In this invention ZjMYB86 Gene regulation of plant steroids is mainly negative, meaning that gene regulation is primarily negative. ZjMYB86 It can inhibit the accumulation of flavonoids in plant floral organs. Specifically, it will contain...ZjMYB86 The gene overexpression vector was used to infect plants with Agrobacterium, and it was found that the content of total flavonoids and total anthocyanins in the petals of the plants was significantly reduced.

[0018] This invention also provides a method for preparing transgenic plants with reduced flavonoid content, comprising the following steps: [The method involves]... ZjMYB86 Transgenic plants with reduced flavonoid content were obtained by introducing the target plant and culturing and screening them.

[0019] The method of this invention can prepare plants with reduced steroid content in their flowers, leaves or fruits, preferably jujube or tobacco.

[0020] The beneficial effects of this invention are: This invention provides ZjMYB86 Genes and their encoded protein sequences, overexpression vectors, host bacteria and ZjMYB86 Application of genes in regulating flavonoid accumulation in plant organs. This invention constructs... ZjMYB86 The biological function of the gene overexpression vector was verified by infecting heterologous transformed tobacco with Agrobacterium. This invention provides... ZjMYB86 A technical solution for gene regulation of flavonoid accumulation in plant organs.

[0021] This invention used jujube flowers at the initial flowering stage, full bloom stage, and late flowering stage as experimental materials. Through physiological, metabolomics, and transcriptomics analyses, it was found that the transcription factor ZjMYB86 was positively correlated with early structural genes and negatively correlated with late structural genes in the flavonoid synthesis pathway. Genetic transformation experiments further demonstrated that the transcription factor... ZjMYB86 It negatively regulates the accumulation of flavonoids. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 Amplification ZjMYB86 Agarose gel electrophoresis image of the full-length cDNA PCR product; M is the marker, lane 1 is... ZjMYB86 Full-length cDNA of the gene.

[0024] Figure 2This image shows an agarose gel electrophoresis image of a positive clone of pClone007 Blunt-ZjMYB86 Escherichia coli identified by PCR. M is the marker, and lanes 1-6 are the PCR products of single colonies 1-6 of pClone007 Blunt-ZjMYB86 Escherichia coli.

[0025] Figure 3 This image shows an agarose gel electrophoresis image of a positive clone of Agrobacterium part-CAM-ZjMYB86 identified by PCR. M is the marker, and lanes 1-8 are the PCR products of single colonies of Agrobacterium part-CAM-ZjMYB86.

[0026] Figure 4 This shows the flow chart for the part-CAM-ZjMYB86 to tobacco conversion. Figure A shows the acquisition of SR1 tobacco aseptic seedlings, Figure B shows callus induction, Figure C shows bud differentiation induction, and Figure D shows rooting culture.

[0027] Figure 5 The image shows an agarose gel electrophoresis image of a part-CAM-ZjMYB86 transgenic tobacco plant that was positive for PCR identification; M is the marker, and lanes 1-5 are the PCR products of part-CAM-ZjMYB86 transgenic tobacco.

[0028] Figure 6 The phenotypes of part-CAM-ZjMYB86 tobacco wild-type control and transgenic positive single plants are shown.

[0029] Figure 7 This displays the total flavonoid content in petals of wild-type control plants and transgenic positive plants of part-CAM-ZjMYB86 tobacco. CK represents the total flavonoid content in petals of the wild-type control plant, and OE-1 to OE-5 represent the total flavonoid content in petals 1-5 of transgenic positive plants of part-CAM-ZjMYB86 tobacco (*** indicates...). P <0.001).

[0030] Figure 8 This displays the total anthocyanin content in petals of wild-type control plants and transgenic positive plants of part-CAM-ZjMYB86 tobacco. CK represents the total anthocyanin content in petals of the wild-type control plant, and OE-1 to OE-5 represent the total anthocyanin content in petals 1-5 of transgenic positive plants of part-CAM-ZjMYB86 tobacco (*** indicates...). P <0.001). Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be described in detail below. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other implementation methods obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0032] Example 1: Cloning and Sequence Analysis of the ZjMYB86 Gene Based on the reference genome of the jujube variety 'Winter Jujube' LOC107409758 The gene sequence was used as a reference sequence to design primers for amplification of the ZjMYB86 gene.

[0033] Upstream primer P1: 5'-ATGGGGAGGCACTCTTGCTGTTACAAG-3'; Downstream primer P2: 5'-ATTATGTCCACCAAGGGCCGC-3'.

[0034] Using jujube flowers in full bloom as the experimental material, total RNA was extracted from the flowers according to the instructions of the RNA extraction kit. cDNA was then reverse transcribed into cDNA according to the instructions of the cDNA first-strand synthesis kit. This cDNA was then used as a template for PCR amplification using high-fidelity DNA polymerase.

[0035] The PCR reaction system (50 μL) is as follows:

[0036] The PCR procedure is as follows:

[0037] 100 μL of PCR product was subjected to 1% agarose gel electrophoresis, and a band of approximately 1,500 bp was detected using a UV gel imaging system. Figure 1 (The value is consistent with the expected size.)

[0038] Recovery using PCR product purification kits ZjMYB86 Gene fragment. The recovered and purified DNA fragment was ligated with the cloning vector plasmid pEASY®-Blunt (Beijing Qingke Biotechnology Co., Ltd., pClone007 Blunt Vector Kit, catalog number: T-1111) according to the instructions.

[0039] The recombinant plasmid pClone007 Blunt-ZjMYB86 was transformed into Escherichia coli competent cells (Baori Biotechnology (Beijing) Co., Ltd., Stellar Competent Cells, catalog number: 636763). Positive clones were screened by kanamycin (50 mg / L) and identified by bacterial culture PCR.

[0040] Upstream primer P1: 5'-AGAAGACGTTCCAACCACGT-3'; Downstream primer P2: 5'-CGGTAAGGATCTGAGCTACACA-3'.

[0041] like Figure 2 As shown, a band of approximately 2,000 bp was obtained, and the fragment size was consistent with expectations.

[0042] The bacterial culture containing the positive recombinant plasmid was sent for sequencing. The cDNA sequence of the ZjMYB86 gene is shown in SEQ ID NO.1: The sequence contains 1,467 bases and encodes 488 amino acids, as shown in SEQ ID NO.2: MGRHSCCYKQKLRKGLWSPEEDEKLLRHIAKYGHGCWSSVPKQAGLERCGKSCRLRWINYLRPDLKRGTFSREEENLIIELHAVLGNRWSQIAAQLPGRTDNDIKNLWNSCLKKKLRQRGVD PVTHRPISEDKDREAKSSGEKLVSVAISNELNLLETENSKKAAAAAALFGAAQVVEANNSSLCSNKTTNTTTDNNNMMMMKELFLQEQDTSSTTNCQQPPSDYLGQLNSLHQFKYKYGSTTN TGSSTTATTRLPSSNPCSNWFLQPGKSTLDMNSHDDALSNCNSIPIILPLSLPIPIPAPSSTSFPSSTASCIANCKPTSLTFPPTATGSIDNNNSMASFPISNNSSSSSSNCFLENNNMFGW GLVADCSTVSEKEAHIQVNNLMDSQAGAADDIKWAEYLSNPPTFLQTTTTQHQNSHSLLCITSSSSSTSDIKSSETQHSVMWPPPHNSKHQQLQPSQLQTTSSDICAKDIHRLTAALGGHN The amino acid sequence has a molecular weight of 53.500 kDa and an isoelectric point of 7.39.

[0043] Example 2 ZjMYB86 Construction of gene overexpression vectors Using the bacterial culture of pClone007 Blunt-ZjMYB86 obtained in Example 1 as a template, the following primers were used: Upstream primer P1: 5'-TCATTTGGAGAGGACACGCATGGGGAGGCACTCTTGCTGTTACAAG-3'; Downstream primer P2: 5'-TCGTCATCCTTGTAGTCGAAATTATGTCCACCAAGGGCCGC-3'.

[0044] PCR amplification was performed using high-fidelity DNA polymerase.

[0045] 100 μL of the PCR product was subjected to 1% agarose gel electrophoresis. A band of approximately 1,500 bp was detected using a UV gel imaging system, consistent with the expected size. The ZjMYB86 cDNA fragment was recovered using a PCR product purification kit.

[0046] The plasmid pART-CAM-FLAG-GFP was digested with XhoI and EcoRI. After digestion, the corresponding bands of the large fragments of the vector were obtained by agarose gel electrophoresis, and the digestion products were recovered using a gel recovery kit.

[0047] Double enzyme digestion reaction system:

[0048] Enzyme digestion at 37℃ for 40 min; inactivation at 65℃ for 20 min.

[0049] The target cDNA fragment and the linearized vector were ligated using T4 ligase and transformed into *E. coli* DH5α competent cells (Baori Biotechnology (Beijing) Co., Ltd.) via heat shock. Single clones were picked for colony PCR detection using upstream primer P1 (5'-AGAAGACGTTCCAACCACGT-3') and downstream primer P2 (5'-CGGTAAGGATCTGAGCTACACA-3'), yielding a band containing the expected fragment size of approximately 2,000 bp.

[0050] Three tubes of bacterial culture with the expected band size were randomly selected for sequencing. Based on the sequencing results, single clones with the correct ZjMYB86 cDNA sequence were selected, and the pART-CAM-ZjMYB86 plasmid was recovered according to the plasmid extraction kit instructions and stored at -20℃.

[0051] The pART-CAM-ZjMYB86 plasmid and pART-CAM empty vector were transformed into Agrobacterium GV3101 (Baori Biotechnology (Beijing) Co., Ltd.) using the freeze-thaw method according to the GV3101 instructions. Single clones were picked for colony PCR detection. The upstream primer P1 (5'-AGAAGACGTTCCAACCACG-3') and the downstream primer P2 (5'-TGTGCTGCAATCTGAGACCAC-3') yielded a band containing the expected fragment size of approximately 450 bp (e.g., ...). Figure 3 As shown), this indicates that a device with [missing information] was successfully obtained. ZjMYB86 Agrobacterium cDNA sequence overexpression vector.

[0052] Example 3 ZjMYB86 Obtaining and identifying transgenic tobacco strains Using tobacco SR1 as the experimental material, tobacco seeds were rinsed with sterile water, disinfected with 75% ethanol for 1 min, disinfected with 2% sodium hypochlorite solution for 10-15 min, rinsed with sterile water 3-5 times, and the seeds were dried with sterile filter paper before being inoculated onto 1 / 2 MS solid medium. After incubation in the dark at 25℃ for 3 days, they were then cultured under light. Figure 4 A).

[0053] Agrobacterium pART-CAM-ZjMYB86 and Agrobacterium pART-CAM empty vector obtained in Example 2 were streaked onto LB solid medium (containing 50 mg / L kanamycin + 50 mg / L gentamicin + 50 mg / L rifampin) and cultured at 28 °C for 2 days. Single colonies were picked and inoculated into 5 mL of LB liquid medium (containing 50 mg / L kanamycin + 50 mg / L gentamicin + 50 mg / L rifampin) and cultured overnight at 28 °C with shaking at 220 rpm. When the bacterial concentration OD600 was approximately 0.6, the cells were collected by centrifugation at 5,000 rpm for 10 min. The collected cells were resuspended in MS liquid medium to OD600. 600 =0.4.

[0054] Take tender tobacco leaves that have been sterilely cultured for 1-2 months and cut them into small pieces. Infect the cut explants with a resuspended Agrobacterium solution for 8-10 minutes. Blot the surface of the explants dry with sterile filter paper and place them on co-culture medium (MS + 6-BA 1 mg / L, pH 5.8) and incubate in the dark at 25°C for 2-3 days. Figure 4 B).

[0055] After co-culture, the explants were transferred to shoot induction differentiation medium (MS + 6-BA 1 mg / L + Timentin 300 mg / L + Kan 100 mg / L, pH 5.8). Shoot formation was induced, and the medium was changed every two weeks. Figure 4 C).

[0056] When the resistant shoots reach 2 cm in length, they are cut off and transferred to rooting medium (1 / 2 MS + Timentin 300 mg / L + IAA 0.5 mg / L + Kan 100 mg / L, pH 5.8) until complete plantlets are formed. Figure 4 D).

[0057] Partial leaf sections were harvested, and genomic DNA was extracted using a DNA extraction kit (TransDirect Plant Tissue PCR Kit, catalog number: AD301, Beijing TransGen Biotech Co., Ltd.). Positive plants were detected using upstream primer P1 (5'-AGAAGACGTTCCAACCACG-3') and downstream primer P2 (5'-TGTGCTGCAATCTGAGACCAC-3'). Figure 5 ).

[0058] Example 4: Detection of total flavonoids and total anthocyanins in the petals of transgenic tobacco positive lines and empty vector control lines. The experimental materials were petals of tobacco transgenic positive lines and empty vector control lines 48 hours after flowering. Figure 6 The total flavonoid content in the petals of transgenic positive tobacco lines and empty vector control plants was determined using a plant flavonoid assay kit (Nanjing Jiancheng Bioengineering Institute, catalog number: A142-1-1), and the total anthocyanin content in the petals of transgenic positive tobacco lines and empty vector control plants was determined using the experimental method described in Li et al., 2023.

[0059] result Figure 7 As shown, the total flavonoid content in the petals of the empty vector control plant (CK) was 3.00 mg / g, while the total flavonoid content in the petals of the tobacco transgenic positive plants (OE-1~OE-5) ranged from 2.00 to 2.88 mg / g.

[0060] The results are as follows Figure 8 As shown, the total anthocyanin content in the petals of the empty vector control (CK) was 0.32 mg / g, while the total anthocyanin content in the petals of the tobacco transgenic positive plants (OE-1~OE-5) ranged from 0.18 to 0.25 mg / g. t Tests showed that the total flavonoid and total anthocyanin contents in the petals of the transgenic positive tobacco strains were significantly lower than those in the petals of the empty vector control strains.

[0061] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. Flavonoid transcription factor ZjMYB86, characterized in that, Its amino acid sequence is shown in SEQ ID No.

2.

2. Flavonoid-related genes ZjMYB86 Its characteristics are, Encodes the flavonoid-related transcription factor ZjMYB86 as described in claim 1.

3. The flavonoid-related gene according to claim 2 ZjMYB86 Its characteristics are, Its nucleotide sequence is shown in SEQ ID NO.

1.

4. Contains the flavonoid-related gene as described in claim 2. ZjMYB86 Recombinant expression vectors.

5. Contains the flavonoid-related gene as described in claim 2. ZjMYB86 Recombinant strains.

6. ZjMYB86 The application of genes in regulating the accumulation of flavonoids in plants is characterized by, ZjMYB86 The nucleotide sequence of the gene is shown in SEQ ID No.

1.

7. The application according to claim 2, characterized in that, The plant in question is either tobacco or jujube.

8. A method for preparing a transgenic plant with reduced flavonoid content, characterized in that, Includes the following steps: transferring the flavonoid-related gene as described in claim 2 or 3 ZjMYB86 Transgenic plants with reduced flavonoid content were obtained by introducing the target plant and culturing and screening them.

9. The method according to claim 8, characterized in that, The plant in question is either tobacco or jujube.

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