Application of citrus ERF transcription factor CitERF92 in regulation and control of content of citrus polymethoxylated flavonoids

Abstract

The invention belongs to the technical field of genetic engineering, and particularly relates to application of a citrus ERF transcription factor CitERF92 in regulating and controlling the content of citrus polymethoxylated flavonoids. Through transient expression of CitERF92 in rock candy orange peel, it is found that compared with a control group, the content of citrus flavones, nobiletin, 5, 6, 7, 4 '-tetramethoxyflavone, heptamethoxyflavone and tangeretin in an overexpression group is up-regulated to different degrees, and it is indicated that accumulation of citrus polymethoxyflavone can be promoted through transient overexpression of CitERF92. Therefore, the transcription factor CitERF92 participates in and regulates synthesis of the polymethoxylated flavonoids, and is of great significance in perfecting a regulation network of anabolism of the citrus flavonoids and realizing industrialized synthesis of the polymethoxylated flavonoids.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and particularly relates to the application of citrus ERF transcription factor CitERF92 in regulating the content of polymethoxyflavones in citrus. Background Art

[0002] Citrus is one of the most widely cultivated fruits in the world. It not only has excellent nutritional flavor but also is rich in a variety of bioactive substances, among which flavonoids have attracted much attention due to their wide range of physiological functions. Polymethoxyflavones are a special type of flavonoid compounds in citrus, specifically present in citrus peel and leaf tissues. The accumulation of polymethoxyflavones in Lamiaceae plants is correlated with their habitats, helping plants adapt to the ecological environment. The accumulation of medicarpin (5,7,4'-trihydroxy-3',5'-dimethoxyflavone) in rice can effectively reduce the spread of root rot mediated by soil-borne fungi. Polymethoxyflavones in citrus can inhibit the growth of common anthracnose pathogens and Penicillium digitatum during postharvest of fruits, playing an important role in pathogen defense. At the same time, polymethoxyflavones are widely reported to be able to help maintain human health and play an important role in reducing the risk of chronic diseases and enhancing the immune system. However, the upstream regulatory factors for the biosynthesis of polymethoxyflavones are not clear. Therefore, further identifying the transcription factors involved in the regulation of polymethoxyflavone synthesis is of great significance for improving the regulatory network of citrus flavonoid synthesis metabolism and realizing the industrial synthesis of polymethoxyflavones. Summary of the Invention

[0003] The purpose of the present invention is to provide the application of citrus ERF transcription factor CitERF92 in regulating the content of polymethoxyflavones in citrus. The present invention discovers that transient overexpression of CitERF92 can promote the accumulation of polymethoxyflavones in citrus, which is of great significance for improving the regulatory network of citrus flavonoid synthesis metabolism and realizing the industrial synthesis of polymethoxyflavones.

[0004] The present invention provides the application of citrus ERF transcription factor CitERF92 in regulating the content of polymethoxyflavones in citrus, and the amino acid sequence of the citrus ERF transcription factor CitERF92 is shown as SEQ ID NO.1.

[0005] As a preferred embodiment, the nucleotide sequence of the citrus ERF transcription factor CitERF92 is shown as SEQ ID NO.2.

[0006] As a preferred embodiment, the regulation includes overexpressing the citrus ERF transcription factor CitERF92 in the citrus genome to increase the content of polymethoxyflavones in citrus.

[0007] As a preferred embodiment, the polymethoxyflavones include one or more of sinensetin, nobiletin, 5,6,7,4'-tetramethoxyflavone, heptamethoxyflavone, and tangeretin.

[0008] The present invention also provides a primer set for amplifying the citrus ERF transcription factor CitERF92, which includes a forward primer and a reverse primer;

[0009] The nucleotide sequence of the forward primer is as shown in SEQ ID NO.3; the nucleotide sequence of the reverse primer is as shown in SEQ ID NO.4; the nucleotide sequence of the citrus ERF transcription factor CitERF92 is as shown in SEQ ID NO.2.

[0010] The present invention also provides a biological material containing the citrus ERF transcription factor CitERF92, including a recombinant expression vector containing the citrus ERF transcription factor CitERF92 and / or a recombinant microorganism containing the citrus ERF transcription factor CitERF92;

[0011] The nucleotide sequence of the citrus ERF transcription factor CitERF92 is as shown in SEQ ID NO.2.

[0012] As a preferred embodiment, the basic backbone of the recombinant expression vector containing the citrus ERF transcription factor CitERF92 includes the pBI121 vector.

[0013] The present invention also provides the application of the primer set or the biological material in regulating the content of polymethoxyflavones in citrus and / or creating new germplasms.

[0014] The present invention also provides a method for increasing the content of polymethoxyflavones in citrus, including the following steps: overexpressing the citrus ERF transcription factor CitERF92 in the target citrus genome;

[0015] The nucleotide sequence of the citrus ERF transcription factor CitERF92 is as shown in SEQ ID NO.2.

[0016] As a preferred embodiment, the citrus includes Bingtangcheng orange.

[0017] Beneficial effects: The present invention provides the application of the citrus ERF transcription factor CitERF92 in regulating the content of polymethoxyflavones in citrus. By transiently expressing CitERF92 in the pericarp of Bingtangcheng orange, it is found that compared with the control group, the contents of sinensetin, nobiletin, 5,6,7,4'-tetramethoxyflavone, heptamethoxyflavone and tangeretin in the overexpression group are all up-regulated to varying degrees, indicating that transient overexpression of CitERF92 can promote the accumulation of polymethoxyflavones in citrus. It can be seen that the transcription factor CitERF92 of the present invention participates in and regulates the synthesis of polymethoxyflavones, which is of great significance for improving the regulatory network of citrus flavonoid synthesis metabolism and realizing the industrial synthesis of polymethoxyflavones. Brief Description of the Drawings

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments.

[0019] Figure 1 Amino acid sequence alignment of CitERF92 in Example 1;

[0020] Figure 2 Phylogenetic analysis in Example 1;

[0021] Figure 3 Expression levels of CitERF92 in the oil cell layer and albedo of Bingtangcheng orange at different developmental stages in Example 2;

[0022] Figure 4 Transient overexpression of CitERF92 promotes the accumulation of PMFs (i.e., polymethoxyflavones) in the pericarp of Bingtangcheng orange in Example 3, where A is the expression level of CitERF92 and B is the determination of the content of polymethoxyflavones;

[0023] Figure 5 Regulatory effect of CitERF92 on the CitFNSII-1 promoter in Example 4, where A is a schematic diagram of the effector and reporter, and B is the regulatory effect of the transcription factor on the promoter;

[0024] Figure 6 SDS-PAGE electrophoresis detection of CitERF92 recombinant protein in Example 5, where A is a schematic diagram of the CitERF92-pGEX vector and B is the detection result of the CitERF92-GST recombinant protein;

[0025] Figure 7 Analysis of the binding of CitERF92 protein to the CitFNSII-1 promoter in Example 5. Detailed Embodiments

[0026] The present invention provides the application of the citrus ERF transcription factor CitERF92 in regulating the polymethoxyflavone content in citrus. As a specific embodiment, the amino acid sequence of the citrus ERF transcription factor CitERF92 is shown in SEQ ID NO.1: MISHLHHHQYHGSSSSNKNDGLVISSCLTENW GDLPLRVNDSEDMIIFNYLHDAVNSGWSPLDSTATSSVKAESVSDNYKNNNNINNQKMCRGGRHYRGVRQRPWGKFAAEIRDPAKNGARVWLGTYETAEEAALAYDRAAFDIRGSKALLNFPHRIGSDEPPPVRVTAKRSHSVDSAAAAKRRKDSAAAKEHQLTMLPAGEHLLVN.

[0027] As a specific embodiment, the nucleotide sequence of the citrus ERF transcription factor CitERF92 is shown in SEQ ID NO.2: 5'-ATGATTTCTCATCTTCACCATCATCAATATCATGGA AGCTCAAGCTCCAATAAAAATGATGGTCTCGTGATCAGTTCTTGTTTAACAGAGAACTGGGGAGACTTGCCGTTAAGAGTTAACGACTCCGAGGACATGATCATCTTCAACTACCTCCATGACGCCGTCAACTCAGGCTGGTCTCCGTTAGATTCAACGGCAACGTCTAGCGTAAAAGCCGAGTCAGTAAGTGATAATTACAAAAATAATAATAATATCAATAATCAGAAGATGTGTAGAGGAGGGAGGCACTATAGAGGAGTGAGGCAGAGGCCATGGGGTAAGTTCGCGGCTGAGATAAGGGACCCAGCTAAGAATGGAGCTAGGGTTTGGCTTGGAACTTACGAGACGGCTGAGGAAGCTGCTCTGGCTTATGATCGAGCCGCTTTTGATATCCGTGGCTCGAAGGCTCTGCTTAATTTTCCCCACAGGATTGGCTCCGACGAGCCTCCGCCAGTCAGAGTTACGGCTAAGCGAAGTCATTCGGTAGACTCGGCGGCTGCGGCTAAGAGGAGAAAAGATTCAGCGGCGGCTAAGGAGCATCAATTGACCATGCTGCCAGCTGGCGAGCACTTATTGGTAAATTGA-3'.

[0028] The regulation described in the present invention includes overexpressing the citrus ERF transcription factor CitERF92 in the citrus genome to increase the content of polymethoxyflavones in citrus.

[0029] The polymethoxyflavones described in the present invention include one or more of sinensetin, nobiletin, 5,6,7,4'-tetramethoxyflavone, heptamethoxyflavone, and tangeretin. As a specific embodiment, compared with the control group, after overexpressing the citrus ERF transcription factor CitERF92, the contents of sinensetin, nobiletin, 5,6,7,4'-tetramethoxyflavone (5,6,7,4'-TMF), heptamethoxyflavone (HMF), and tangeretin were all up-regulated to varying degrees, and the up-regulation amplitudes were 24.0%, 28.0%, 24.7%, 30.1%, and 27.2% respectively, which confirmed that CitERF92 can promote the accumulation of citrus polymethoxyflavones.

[0030] The present invention also provides a primer set for amplifying the citrus ERF transcription factor CitERF92, and the primer set includes a forward primer and a reverse primer;

[0031] The nucleotide sequence of the forward primer is as shown in SEQ ID NO.3; the nucleotide sequence of the reverse primer is as shown in SEQ ID NO.4; the nucleotide sequence of the citrus ERF transcription factor CitERF92 is as shown in SEQ ID NO.2.

[0032] The sequence of the primer set described in the present invention is as follows:

[0033] Forward primer (SEQ ID NO.3): 5'-ATGATTTCTCATCTTCACCATC-3',

[0034] Reverse primer (SEQ ID NO.4): 5'-TCAATTTACCAATAAGTGCTCG-3'.

[0035] The present invention also provides a biological material comprising the citrus ERF transcription factor CitERF92, including a recombinant expression vector containing the citrus ERF transcription factor CitERF92 and / or a recombinant microorganism containing the citrus ERF transcription factor CitERF92; the nucleotide sequence of the citrus ERF transcription factor CitERF92 is as shown in SEQ ID NO.2. The basic backbone of the recombinant expression vector containing the citrus ERF transcription factor CitERF92 in the present invention includes the pBI121 vector. As a specific embodiment, the recombinant expression vector containing the citrus ERF transcription factor CitERF92 includes inserting the citrus ERF transcription factor CitERF92 into the pBI121 vector to obtain the CitERF92-pBI121 recombinant expression vector. As an embodiment, the recombinant microorganism containing the citrus ERF transcription factor CitERF92 includes the Agrobacterium tumefaciens EHA105 containing the citrus ERF transcription factor CitERF92.

[0036] The present invention also provides the use of the primer set or the biological material in regulating the content of polymethoxyflavones in citrus and / or creating new germplasms.

[0037] The present invention also provides a method for increasing the content of polymethoxyflavones in citrus, comprising the following steps: overexpressing the citrus ERF transcription factor CitERF92 in the target citrus genome; the nucleotide sequence of the citrus ERF transcription factor CitERF92 is as shown in SEQ ID NO.2. As a specific embodiment, the citrus can be Bingtangcheng orange. In a specific example, after transferring the CitERF92-pBI121 recombinant expression vector into the Agrobacterium tumefaciens EHA105 strain, the bacterial solution containing the CitERF92-pBI121 recombinant expression vector is injected into the citrus peel to obtain a citrus peel with an increased content of polymethoxyflavones.

[0038] By transiently expressing CitERF92 in the peel of Bingtangcheng orange, the present invention found that compared with the control group, the contents of sinensetin, nobiletin, 5,6,7,4'-tetramethoxyflavone, heptamethoxyflavone and tangeretin in the overexpression group were all up-regulated to varying degrees, indicating that transient overexpression of CitERF92 can promote the accumulation of polymethoxyflavones in citrus. It can be seen that the transcription factor CitERF92 described in the present invention participates in and regulates the synthesis of polymethoxyflavones, which is of great significance for improving the regulatory network of citrus flavonoid synthesis metabolism and realizing the industrial synthesis of polymethoxyflavones.

[0039] To further illustrate the present invention, the application of the citrus ERF transcription factor CitERF92 provided by the present invention in regulating the polymethoxyflavone content of citrus will be described in detail below in conjunction with embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0040] Unless otherwise specified, the present invention has no special requirements for the preparation raw materials, and commercially available products well-known to those skilled in the art can be used.

[0041] Example 1 Cloning and bioinformatics analysis of the CitERF92 gene

[0042] 1. Cloning of the CitERF92 gene

[0043] Using the pericarp tissue of Bingtangcheng as the material, total RNA of the sample was extracted by the CTAB method, and the quality of the total RNA was detected by agarose gel electrophoresis and NanoDrop 2000 (Thermo Fisher Scientific, USA). The HiScript III 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing) with gDNAwiper was used to remove genomic DNA and synthesize the first-strand cDNA according to the instructions. Primers for amplifying the CDS sequence of CitERF92 were designed according to the sweet orange 1.0 reference genome data (PRJNA86123), and the pericarp cDNA was used as the template for amplification. The CDS amplification primers for CitERF92 are:

[0044] Forward primer: 5'-ATGATTTCTCATCTTCACCATC-3' (as shown in SEQ ID NO.3);

[0045] Reverse primer: 5'-TCAATTTACCAATAAGTGCTCG-3' (as shown in SEQ ID NO.4).

[0046] The 2×Phanta Max MasterMix (Dye Plus) (Vazyme, Nanjing) kit was used for amplification, and the reaction system was: 2×Phanta Mix 15 μL, 10 μM forward primer 1.2 μL, 10 μM reverse primer 1.2 μL, cDNA template 2 μL, and RNase-free H2O was added to make up to 30 μL.

[0047] After mixing, the reaction was carried out in a PCR instrument according to the following program: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 sec, annealing at 58°C for 15 sec, extension at 72°C for 1 min, for 35 cycles; full extension at 72°C for 5 min; storage at 4°C. The amplified product was analyzed by agarose gel electrophoresis and purified using a DNA recovery kit (Eastep, Hangzhou). After sequencing, the CDS nucleotide sequence of CitERF92 was obtained.

[0048] 2. Bioinformatics analysis

[0049] The CDS sequence of CitERF92 was analyzed using the online software Expasy (https: / / web.expasy.org / translate / ) to obtain its encoded amino acid sequence. After sequence alignment, a phylogenetic tree was constructed with the homologous ERF genes in Arabidopsis thaliana and the ERF members reported to be involved in the regulation of polymethoxyflavone synthesis in citrus. The Clustal X software (Clustal X Software, Ireland) was used to perform multiple sequence alignment analysis of the amino acids of ERFs; the completed.aln suffix file after alignment was imported into the MEGA X software (MEGA Software, USA), and the neighbor-joining method was selected to construct the phylogenetic tree. The information of ERF members used to construct the phylogenetic tree is summarized in Table 1.

[0050] Table 1 Summary of information of ERF members for constructing the phylogenetic tree

[0051]

[0052] To further analyze the protein structure of CitERF92, multiple comparative analyses were performed between CitERF92 and members of ERF IXa from other plants. The alignment between protein sequences was completed by the Expresso tool in the T-Coffee Server (https: / / tcoffee.crg.eu / apps / tcoffee / do:expresso), and the sequence similarity and secondary structure information were visualized through ESPript 3.0 (https: / / espript.ibcp.fr / ).

[0053] 3. Experimental results

[0054] The full-length coding sequence of CitERF92 was obtained by cloning, which is 624 bp in length as shown in SEQ ID NO.2; it encodes 208 amino acids in total as shown in SEQ ID NO.1. Through NCBI Conserved Domain in Search analysis, it was found that CitERF92 has an AP2 domain and belongs to the ERF subfamily of the AP2 / ERF transcription factor family( Figure 1 ); further phylogenetic tree analysis showed that CitERF92 belongs to the ERF B-3 subgroup and has the closest genetic relationship with Arabidopsis thaliana AtERF1A (At4g17500). Figure 2 )

[0055] Example 2 Expression analysis of CitERF92 in the pericarp of Bingtang orange at different developmental stages

[0056] 1. Sample collection and transcriptome sequencing

[0057] Eight developing Bingtang orange fruits were collected, including S0 (10 days after flowering), S1 (30 days after flowering), S2 (60 days after flowering), S3 (80 days after flowering), S4 (100 days after flowering), S5 (120 days after flowering), S6 (150 days after flowering) and S7 (180 days after flowering); and the pericarp was divided into the oil cell layer and the white peel layer (the S0 stage was too small to be separated and was measured together). All samples were cut into small pieces, quickly frozen in liquid nitrogen and ground into powder for later use. 0.3 g of fresh sample powder was weighed, and total RNA in the sample was extracted by the CTAB method. BGI Genomics was commissioned to conduct RNA-seq on the BGISEQ-500RS sequencing platform. After downloading the raw data and filtering and cleaning it, it was aligned to the Citrus sinensis v1.0 reference genome (https: / / www.citrusgenomedb.org / citrus_downloads / Citrus_sinensis / C.sinensis_Hzau_v1.0_genome / ), and the FPKM value (fragments per kilobase of exon per million fragments mapped) was used to measure the gene expression level of the measurement.

[0058] 2. Experimental results

[0059] As Figure 3As shown in Table 2, the expression level of CitERF92 in the pericarp showed a trend of first increasing and then decreasing, reaching the highest level at the S3 stage (80 days after flowering), which was consistent with the accumulation pattern of flavonoids in citrus pericarp. In addition, the expression levels of CitERF92 in the oil gland layer and albedo were similar at the S1 and S2 stages, and the expression level in the oil gland layer was higher than that in the albedo at the S3 stage and after the S3 stage, which was positively correlated with the abundant accumulation of polymethoxylated flavones in the oil gland layer. Therefore, it was speculated that the transcription factor CitERF92 responded to the fruit development process and participated in the regulation of the synthesis of polymethoxylated flavones in citrus.

[0060] Table 2 Expression levels of CitERF92 in the oil gland layer and albedo of Bingtangcheng at different development stages

[0061]

[0062] Example 3 Transient overexpression of CitERF92

[0063] 1. Vector construction and Agrobacterium transformation

[0064] After removing the terminator from the CDS sequence of CitERF92, it was constructed into the pBI121 vector (Liao, Z., Liu, X., Zheng, J., Zhao, C., Wang, D., Xu, Y., Sun, C., (2023) A multifunctional true caffeoyl coenzyme A O-methyltransferase enzyme participates in the biosynthesis of polymethoxylated flavones in citrus. Plant Physiology 192:2049–2066.) using homologous recombination. The cloning primers were as follows:

[0065] Forward primer: 5'-GGTTCCGCGTGGATCCATGATTTCTCATCTTCACCATC-3' (shown as SEQ ID NO.5);

[0066] Reverse primer: 5'-GGACTCTAGAGGATCCATTTACCAATAAGTGCTCGCC-3' (shown as SEQ ID NO.6).

[0067] Ligation was carried out using the ClonExpress II One Step Cloning Kit (Vazyme, Nanjing). The ligation system was as follows: 2 μL of 5×CE II buffer, 1 μL of Exnase II, 1 μL of the CitERF92 amplification product, 1 μL of the linearized pBI121 vector, and RNase-free H2O was added to make up to 10 μL.

[0068] After thorough mixing, ligation was performed at 37 °C for 30 min. Subsequently, 5 μL of the ligation product was added to 20 μL of DH5α competent cells, and transformation and plating were carried out according to the steps of placing on ice for 25 min, heat shock at 42 °C for 90 sec, and placing on ice for 2 min. Positive colonies were picked for sequencing verification. The correctly sequenced CitERF92-pBI121 recombinant plasmid was transformed into Agrobacterium tumefaciens EHA105, and screening was performed using kanamycin (Kan; 50 μg / mL) and rifampicin (Rif; 20 μg / mL). After picking positive clones for electrophoresis detection, they were stored at -80 °C in 25% glycerol.

[0069] 2. Transient overexpression in citrus peel

[0070] 10 μL of the Agrobacterium tumefaciens EHA105 bacterial solution containing the CitERF92-pBI121 recombinant plasmid was streaked and activated on LB solid medium. After culturing at 28 °C for 2 d, positive clones were picked and added to 200 mL of LB liquid medium, and cultured with shaking at 28 °C until OD 600 = 0.8. The bacteria were collected by centrifugation at 6000 rpm for 10 min, and the cell pellet was resuspended in an equal volume of infiltration solution (10 mM MES, 10 mM MgCl2, 150 μM acetosyringone, pH = 5.6). Mature Bingtangcheng oranges with uniform maturity, size, no mechanical damage, and no pests and diseases were selected as materials. The peel tissues on the opposite sides of the equatorial plane of the same fruit were injected. One side was injected with Agrobacterium tumefaciens containing CitERF92-pBI121, and the other side was injected with the empty pBI121 vector as a control. The injection volume for each side was approximately 5 mL; each fruit was a biological replicate, and there were 5 replicates in total. The injected fruits were placed in a climate chamber at 25 °C, kept in the dark for 24 h, and then cultured under a 16 h light / 8 h dark photoperiod. After 5 d, the injected peel tissues were removed, cut into small pieces, quickly frozen in liquid nitrogen, and stored at -80 °C for later use.

[0071] 3. Expression analysis of CitERF92

[0072] After grinding the overexpression and control group samples with liquid nitrogen, total RNA was extracted by the CTAB method and reverse-transcribed into cDNA. Specific primers for CitERF92 were designed using NCBI (forward primer: 5'-TTTCCCCACAGGATTGGCTC-3' (as shown in SEQ ID NO.7); reverse primer: 5'-CGCCGCTGAATCTTTTCTCC-3' (as shown in SEQ ID NO.8)), and the product melting curve and sequencing verification were performed. The reaction system for RT-qPCR was 20 μL, including 10 μL of 2×ChamQ Universal SYBR qPCR Master Mix (Novoprotein, Nanjing), 2 μL of cDNA template, 0.4 μL of each forward and reverse primer (10 μmol / L), and 7.2 μL of RNase-Free water. The reaction was completed using a CFX96 real-time fluorescence quantitative PCR instrument (Bio-rad, USA), and the reaction program was: (1) 95°C, 30 s; (2) 95°C, 10 s; (3) 60°C, 30 s; (2)-(3) 40 cycles; (4) 95°C, 10 s; (5) from 65°C to 95°C, increasing by 0.5°C every 5 s and collecting fluorescence values once. The expression level was normalized using citrus β-actin as an internal reference gene.

[0073] 4. Extraction and Detection of Citrus Polymethoxyflavones

[0074] Accurately weigh 0.1 g of well-ground fresh citrus sample powder, add 1 mL of 80% ethanol, vortex thoroughly, and then perform ultrasonic-assisted extraction for 45 min. Centrifuge at 6000 rpm for 10 min to collect the supernatant. Repeat the extraction twice and combine the supernatants (about 2 mL) for subsequent flavonoid detection.

[0075] After the extract was centrifuged at 12,000 rpm for 20 min, 100 μL was taken for on-machine detection. The polymethoxyflavones in the sample were detected using a Waters HPLC-DAD system (2695 quaternary pump, 2996 diode array detector) (Waters Crop., USA) and a Sunfire C18 ODS chromatographic column (4.6×250 mm, 5 μm; Waters). The mobile phase was chromatographic acetonitrile (phase A) and pure water containing 0.1% formic acid (phase B), and linear gradient elution was performed. The elution concentration change and flow rate were as follows: 0 - 5 min, A (20%); 5 - 10 min, A (20% - 27%); 10 - 15 min, A (27%); 15 - 25 min, A (27% - 40%); 25 - 35 min, A (40% - 60%); 35 - 40 min, A (60% - 80%); 40 - 42 min, A (80% - 100%); 42 - 45 min, A (100% - 20%); 45 - 50 min, A (20%). The sample injection volume was 10 μL, and the detection wavelength was 200 - 400 nm. The standard curve was determined using a polymethoxyflavone standard, and the qualitative and quantitative analysis of flavonoids in the sample was carried out. The standard curve is shown in Table 3.

[0076] Table 3 Standard curve for the qualitative and quantitative analysis of flavonoids in the sample

[0077]

[0078] 5. Experimental results

[0079] Compared with the control pericarp injected with the empty vector, the expression level of CitERF92 in the overexpression group increased significantly, confirming the effectiveness of this transient overexpression system ( Figure 4 in A). Further analysis of the contents of 5 polymethoxyflavone monomers showed that, as Figure 4 shown in B and Table 4, compared with the control group, the contents of sinensetin, nobiletin, 5,6,7,4'-tetramethoxyflavone (5,6,7,4'-TMF), heptamethoxyflavone (HMF), and tangeretin all increased to varying degrees, with the upregulation amplitudes being 24.0%, 28.0%, 24.7%, 30.1%, and 27.2% respectively. The transient overexpression experiment confirmed that CitERF92 promoted the accumulation of citrus polymethoxyflavones.

[0080] Table 4 Accumulation of polymethoxyflavone monomer contents in the pericarp of Bingtangcheng oranges after transient overexpression of CitERF92 (mg / g)

[0081]

[0082] Example 4 Tobacco dual luciferase verification of CitERF92 transcriptional activation of CitFNSII-1 promoter

[0083] 1. Recombinant plasmid construction and Agrobacterium transformation

[0084] The genomic DNA of the rock sugar orange leaves was extracted using the Ezup column plant genomic DNA extraction kit (Shengong, Shanghai), and the promoter region of CitFNSII-1 was cloned using this DNA as a template and constructed on the pGreen II 0800-LUC vector (Zhao, C., Liu, X., Gong, Q., Cao, J., Shen, W., Yin, X., Grierson, D., Zhang, B., Xu, C., Li, X., Chen, K., Sun, C., (2021) Three AP2 / ERF family members modulate flavonoid synthesis by regulating type IV chalcone isomerase in citrus. Plant Biotechnol J 19: 671–688.) to obtain the Cit FNSII-1 promoter-2000bp sequence, and the constructed recombinant plasmid was named CitFNSII-1pro-LUC. The CDS sequence of CitERF92 was cloned into pGreen II 002962-SK (Zhao, C., Liu, X., Gong, Q., Cao, J., Shen, W., Yin, X., Grierson, D., Zhang, B., Xu, C., Li, X., Chen, K., Sun, C., (2021) Three AP2 / ERF family members modulate flav onoid synthesis by regulating type IV chalcone isomerase in citrus. Plant Biote chnol J 19: 671–688.) vector and the constructed recombinant plasmid was named CitERF92-SK. The PCR reaction system was the same as in Example 1, the ligation system was the same as in Example 3, and the cloning primers were as follows:

[0085] CitERF92-SK:

[0086] Upstream primer: 5'-CGGGCTGCAGGAATTCATGATTTCTCATCTTCACCATC-3' (as shown in SEQ ID NO.9),

[0087] Downstream primer: 5'-CGGGCCCCCCCTCGAGTCAATTTACCAATAAGTGCTCG-3' (as shown in SEQ ID NO.10).

[0088] CitFNSII-1pro-LUC:

[0089] Upstream primer: 5'-GCAGCCCGGGGGATCCAACTGGCCTTTAGGCACTTCT-3' (as shown in SEQ ID NO.11),

[0090] Downstream primer: 5'-TTGGCGTCTTCCATGGGGCTAGAAAAGATCTCTAGGCTTC T-3' (as shown in SEQ IDNO.12).

[0091]

[0092] The constructed recombinant plasmid was transformed into Agrobacterium tumefaciens GV3101::pSoup competent cells (Vidy, Shanghai; CAT#: AC1002). Positive clones were screened and preserved in LB medium containing 50 μg / mL Kan and 25 μg / mL Get.

[0093] 2. Tobacco leaf infection and dual-luciferase detection

[0094] The bacterial suspension was prepared according to the method described in Example 3. Subsequently, the CitERF92-SK and CitFNSII-1pro-LUC bacterial solutions were mixed at a ratio of 10:1 (v / v) (the resulting mixture was named CitERF92-SK::CitFNSII-1pro-LUC mixed bacterial solution). The bacterial solution of empty vector SK (i.e., pGreen II 002962-SK vector) and CitFNSII-1pro-LUC (i.e., pGreen II0800-LUC vector) bacterial solution were mixed at a ratio of 10:1 (v / v) as a negative control. Four-week-old Nicotiana benthamiana with good growth status and flat leaf surfaces were selected for half-leaf injection. One half was injected with the negative control, and the other half was injected with the CitERF92-SK::CitFNSII-1pro-LUC mixed bacterial solution. Three leaves of each tobacco plant were injected. After culturing in an artificial climate chamber at 25 °C for 3 d, fluorescence detection was performed.

[0095] A puncher with a diameter of 4 mm was used to sample the injection area of the tobacco leaf, and 100 μL of 1×PBS buffer (pH = 7.4) was added and ground until completely homogenized. 50 μL was aspirated into a 96-well white microplate, and the luminescence of Firefly luciferase (LUC) and Renilla luciferase (REN) was detected using a GloMax96 instrument (Promega, USA) and Dual-Luciferase Reporter Assay System reagent (Promega, USA). Taking the REN value as a control, the LUC / REN value reflects the regulatory effect of the transcription factor on the promoter, and the LUC / REN value of the negative control was set to 1.

[0096] 3. Experimental results

[0097] The results of the tobacco dual-luciferase assay were as Figure 5 shown. CitERF92 could act as a transcriptional activator and activate the promoter activity of CitFNSII-1, with an activation effect of 1.96-fold.

[0098] Example 5 Purification and EMSA analysis of CitERF92 protein

[0099] 1. Construction of recombinant vector and transformation of Escherichia coli

[0100] Amplify the CDS sequence of CitERF92 after removing the terminator, and construct it onto the pGEX-4T-1 vector with GST tag by homologous recombination method (Wei, C., Liu, H., Cao, X., Zhang, M., Li, X., Chen, K., Zhang, B., (2021) Synthesis of flavour-related linalool is regulated by PpbHLH1 and associated with changes in DNA methylation during peach fruit ripening. Plant Biotechnol J 19:2082–2096.). The amplification primers with pGEX-4T-1 vector adapter are as follows:

[0101] Forward primer: 5'-GGTTCCGCGTGGATCCATGATTTCTCATCTTCACCATC-3' (shown in SEQ ID NO.14);

[0102] Reverse primer: 5'-GTCGACCCGGGAATTCATTTACCAATAAGTGCTCGCC-3' (shown in SEQ ID NO.15).

[0103] The amplification system is the same as that in Example 1, and the system for ligation to pGEX-4T-1 vector and transformation of DH5α is the same as that in Example 3. Transfer the correctly sequenced CitERF92-pGEX recombinant plasmid into the BL21(DE3)pLysS expression strain by heat shock method, and pick positive clones for glycerol preservation.

[0104] 2. Prokaryotic expression and purification of recombinant protein

[0105] Take 10 μL of glycerol bacteria containing CitERF92-pGEX recombinant plasmid for streaking, pick the monoclonal into 20 mL LB liquid medium (containing 100 μg / mL Amp), and culture overnight in a shaker at 37°C. After culturing for 16 h, add the bacterial liquid to 500 mL of fresh LB liquid medium at a ratio of 1:50, and culture in a shaker at 37°C and 160 rpm for 1.5 h - 2 h. When the OD of the bacterial liquid 600When OD600 = 0.7, IPTG with a final concentration of 1 mM was added, and the cells were cultured at 18 °C and 160 rpm for 20 h to induce the expression of recombinant protein. The cells were collected by centrifugation at 4 °C (4000 g, 15 min), resuspended in 25 mL of 1×PBS (Phosphate Buffered Saline; pH = 7.4) buffer, and then frozen at -80 °C for 48 h.

[0106] The completely frozen resuspended cell suspension was thawed in a water bath at 30 °C, and Escherichia coli cells were lysed using an ultrasonic cell disruptor (XO-650D, Nanjing) under ice bath conditions (ultrasonic for 3.0 s, interval for 2 s, working for 10 min, power 27%). Subsequently, the supernatant was collected by centrifugation at 4 °C for 30 min (10000 rpm), and the supernatant was filtered through a 0.45 μm filter membrane (Millipore, USA). The transcription factor protein was purified using the GST-tagged protein purification kit from Beyotime (Beyotime, Shanghai) according to the instructions. The CitERF92-GST recombinant protein was obtained and stored at -80 °C for later use.

[0107] 3. EMSA assay

[0108] The EMSA assay was performed using the LightShift Chemiluminescent EMSA Kit (Thermofisher Scientific, USA). The probe labeled with 3'-Biotin was synthesized by Hangzhou Jinbaiao Biotechnology Co., Ltd., and the primer with the same sequence without 3'-Biotin label was used as a competitive probe. The binding probe sequences in the CitFNSII-1 promoter region are as follows:

[0109] Upstream probe:

[0110] 5'-AAATGAAGACAGAAACGGCAGCCACTTGCTGCCGTTTTCATTCACTTACC-3' (shown as SEQ ID NO. 16);

[0111] Downstream probe:

[0112] 5'-GGTAAGTGAATGAAAACGGCAGCAAGTGGCTGCCGTTTCTGTCTTCATTT-3' (shown as SEQ ID NO. 17).

[0113] The reaction system for the binding of the recombinant protein and the probe is as follows: 1 μL of 10× Binding buffer, 0.5 μL of MgCl2, 0.5 μL of poly(dI-dC), 0.5 μL of 1% NP-40, 0.5 μL of 50% Glycerol, 4 μL of the recombinant protein, 1 μL of the double-stranded probe (10 nM), and the volume is made up to 10 μL with water. React at room temperature for 30 min. For the competition experiment, 1 μL of the competing probe needs to be added first and reacted for 10 min. After the reaction, 1 μL of Loading buffer is added, and electrophoresis is carried out using a 6% Acr-Bis non-denaturing gel. The buffer is 0.5× TBE solution, and the electrophoresis conditions are 160 V for 35 min. Subsequently, the binding bands are transferred to a positively charged nylon membrane (Millipore, USA) by a transfer membrane instrument (0.3 A, 30 min). After ultraviolet cross-linking for 30 min, a luminescence reaction is carried out using a kit, and photography is completed with a ChemiDocTM XRS+ (Bio-rad, USA) instrument.

[0114] 4. Experimental results

[0115] The purified CitERF92-GST is as Figure 6 shown, and a recombinant protein with a size of approximately 48 kDa is obtained (the predicted size of the GST protein is 26.5 kDa), which is consistent with the predicted size of 22.9 kDa of CitERF92 ( Figure 6 )

[0116] The CitERF92-GST recombinant protein can bind to the biotin-labeled CitFNSII-1 promoter probe to produce a retarded migration band, and as the concentration of the competing probe increases, the band gradually weakens. The results indicate that the CitERF92 protein can bind to the promoter region of CitFNSII-1 ( Figure 7 )

[0117] Thus, it can be seen that in the present invention, by transiently expressing CitERF92 in the pericarp of Ponkan oranges, it is found that compared with the control group, the contents of naringenin, nobiletin, 5,6,7,4'-tetramethoxyflavone, heptamethoxyflavone, and tangeretin in the overexpression group are all up-regulated to varying degrees, indicating that transient overexpression of CitERF92 can promote the accumulation of polymethoxyflavones in citrus. It can be seen that the transcription factor CitERF92 described in the present invention participates in and regulates the synthesis of polymethoxyflavones, which is of great significance for improving the regulatory network of citrus flavonoid synthesis metabolism and realizing the industrial synthesis of polymethoxyflavones.

[0118] Although the above embodiments have made a detailed description of the present invention, they are only a part of the embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments based on this embodiment without creative efforts, and these embodiments all belong to the protection scope of the present invention.

Claims

1. Application of citrus ERF transcription factor CitERF92 in regulating the content of citrus polymethoxyflavonoids, characterized in that: The amino acid sequence of the citrus ERF transcription factor CitERF92 is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that: The nucleotide sequence of the citrus ERF transcription factor CitERF92 is shown in SEQ ID NO.

2.

3. The use according to claim 1, characterized in that: The regulation includes overexpressing the citrus ERF transcription factor CitERF92 in the citrus genome to increase the content of polymethoxyflavonoids in citrus.

4. The use according to any one of claims 1 to 3, characterized in that: The polymethoxyflavonoids include one or more of sweet orange flavonoids, nobiletin, 5,6,7,4'-tetramethoxyflavonoids, heptamethoxyflavonoids and tangeretin.

5. A primer set for amplifying the citrus ERF transcription factor CitERF92, characterized in that: The primer set includes a forward primer and a reverse primer; The nucleotide sequence of the forward primer is shown in SEQ ID NO.3; the nucleotide sequence of the reverse primer is shown in SEQ ID NO.4; and the nucleotide sequence of the citrus ERF transcription factor CitERF92 is shown in SEQ ID NO.

2.

6. A biological material comprising the citrus ERF transcription factor CitERF92, characterized in that: It includes a recombinant expression vector containing the citrus ERF transcription factor CitERF92 and / or a recombinant microorganism containing the citrus ERF transcription factor CitERF92; The nucleotide sequence of the citrus ERF transcription factor CitERF92 is shown in SEQ ID NO.

2.

7. The biomaterial according to claim 6, characterized in that The basic skeleton of the recombinant expression vector containing the citrus ERF transcription factor CitERF92 includes the pBI121 vector.

8. Use of the primer set according to claim 5 or the biological material according to claim 6 or 7 in regulating the content of polymethoxyflavonoids in citrus and / or creating new germplasm.

9. A method for increasing the content of polymethoxyflavonoids in citrus, characterized in that: The method comprises the following steps: overexpressing a citrus ERF transcription factor CitERF92 in a target citrus genome; The nucleotide sequence of the citrus ERF transcription factor CitERF92 is shown in SEQ ID NO.

2.

10. The method according to claim 9, characterized in that The citrus fruits include sugar oranges.

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