Application of Citrus ERF transcription factor CitERF92 in regulating the content of polymethoxyflavones in citrus

Abstract

This invention belongs to the field of genetic engineering technology, specifically involving the application of the citrus ERF transcription factor CitERF92 in regulating the content of polymethoxyflavones in citrus. This invention, through transient expression of CitERF92 in the peel of sugar oranges, found that compared with the control group, the contents of sweet orange flavonoids, norihesperidin, 5,6,7,4'-tetramethoxyflavonoids, heptamethoxyflavonoids, and hesperidin were all upregulated to varying degrees in the overexpression group, indicating that transient overexpression of CitERF92 can promote the accumulation of citrus polymethoxyflavones. Therefore, the transcription factor CitERF92 described in this invention participates in and regulates the synthesis of polymethoxyflavones, which is of great significance for improving the regulatory network of citrus flavonoid biosynthesis and metabolism and for realizing the industrialized synthesis of polymethoxyflavones.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically involving the application of citrus ERF transcription factor CitERF92 in regulating the content of polymethoxyflavones in citrus. Background Technology

[0002] Citrus fruits are among the most widely cultivated fruits in the world. They are not only nutritious and flavorful but also rich in various bioactive substances, among which flavonoids have attracted much attention due to their wide range of physiological functions. Polymethoxyflavones are a special class of flavonoid compounds in citrus fruits, specifically found in the peel and leaf tissues. The accumulation of polymethoxyflavones in Lamiaceae plants is correlated with their habitat, helping plants adapt to their ecological environment. Alfalfa-based compounds (5,7,4'-trihydroxy-3',5'-dimethoxyflavonoids) accumulated in rice can effectively reduce the spread of soil-borne fungal-mediated root rot. Polymethoxyflavones in citrus fruits can inhibit the growth of anthracnose and Penicillium fingernail, common pathogens in post-harvest fruit, playing an important role in pathogen defense. Simultaneously, polymethoxyflavones have been widely reported to help maintain human health, playing 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 identification of 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 industrialized synthesis of polymethoxyflavones. Summary of the Invention

[0003] The purpose of this invention is to provide the application of the citrus ERF transcription factor CitERF92 in regulating the content of polymethoxyflavones in citrus. This invention finds 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] This invention provides the application of citrus ERF transcription factor CitERF92 in regulating the content of polymethoxyflavones in citrus fruits, and the amino acid sequence of the citrus ERF transcription factor CitERF92 is shown in SEQ ID NO.1.

[0005] As a preferred embodiment, the nucleotide sequence of the citrus ERF transcription factor CitERF92 is shown in 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 polymethoxyflavonoids include one or more of sweet orange flavonoids, norihesperidin, 5,6,7,4'-tetramethoxyflavonoids, heptamethoxyflavonoids, and hesperidin.

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

[0009] 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.

[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 shown in SEQ ID NO.2.

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

[0013] This invention also provides the application of the primer set or the biological material described herein in regulating the content of polymethoxyflavonoids in citrus and / or creating new germplasm.

[0014] 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 genome of the target citrus;

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

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

[0017] Beneficial Effects: This 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 peel of sugar oranges, this invention found that compared with the control group, the contents of sweet orange flavonoids, norihesperidin, 5,6,7,4'-tetramethoxyflavonoids, heptamethoxyflavonoids, and hesperidin were all upregulated to varying degrees in the overexpression group, indicating that transient overexpression of CitERF92 can promote the accumulation of polymethoxyflavones in citrus. Therefore, the transcription factor CitERF92 described in this 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 industrialized synthesis of polymethoxyflavones. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

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

[0020] Figure 2 This refers to the system evolution analysis in Example 1;

[0021] Figure 3 The expression levels of CitERF92 in the oil cell layer and white peel of the sugar orange at different developmental stages, as shown in Example 2.

[0022] Figure 4 In Example 3, transient overexpression of CitERF92 promoted the accumulation of PMFs (polymethoxyflavones) in the peel of rock sugar oranges. A represents the expression level of CitERF92, and B represents the content of polymethoxyflavones.

[0023] Figure 5 This is an example of the 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 The image shows the 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 CitERF92-GST recombinant protein;

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

[0026] This invention provides the application of citrus ERF transcription factor CitERF92 in regulating the content of polymethoxyflavones 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 one specific embodiment, the nucleotide sequence of the citrus ERF transcription factor CitERF92 is shown in SEQ ID NO.2: 5'-ATGATTTCTCATCTTCACCATCATCAATATCATGGA-3'.

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

[0029] The polymethoxyflavonoids described in this invention include one or more of sinensetin, nobiletin, 5,6,7,4'-tetramethoxyflavonoids (5,6,7,4'-TMF), heptamethoxyflavonoids (HMF), and tangeretin. As a specific embodiment, compared with the control group, overexpression of the citrus ERF transcription factor CitERF92 resulted in varying degrees of upregulation of the contents of sinensetin, nobiletin, 5,6,7,4'-tetramethoxyflavonoids (5,6,7,4'-TMF), heptamethoxyflavonoids (HMF), and tangeretin, with upregulation rates of 24.0%, 28.0%, 24.7%, 30.1%, and 27.2%, respectively, confirming that CitERF92 can promote the accumulation of citrus polymethoxyflavonoids.

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

[0031] 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.

[0032] The sequences of the primer set described in this invention are as follows:

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

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

[0035] This invention also provides 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; the nucleotide sequence of the citrus ERF transcription factor CitERF92 is shown in SEQ ID NO.2. The basic framework of the recombinant expression vector containing the citrus ERF transcription factor CitERF92 of this 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 one embodiment, the recombinant microorganism containing the citrus ERF transcription factor CitERF92 includes Agrobacterium EHA105 containing the citrus ERF transcription factor CitERF92.

[0036] This invention also provides the application of the primer set or the biological material described herein in regulating the content of polymethoxyflavonoids in citrus and / or creating new germplasm.

[0037] This invention also provides a method for increasing the content of polymethoxyflavonoids in citrus fruits, comprising the following steps: overexpressing the citrus ERF transcription factor CitERF92 in the genome of a target citrus fruit; the nucleotide sequence of the citrus ERF transcription factor CitERF92 is shown in SEQ ID NO.2. As a specific embodiment, the citrus fruit can be a sugar orange. In a specific embodiment, after transforming the CitERF92-pBI121 recombinant expression vector into Agrobacterium EHA105 strain, the bacterial solution containing the CitERF92-pBI121 recombinant expression vector is injected into the citrus peel, resulting in citrus peel with increased polymethoxyflavonoid content.

[0038] This invention, through transient expression of CitERF92 in the peel of sugar oranges, found that compared with the control group, the contents of sweet orange flavonoids, norihesperidin, 5,6,7,4'-tetramethoxyflavonoids, heptamethoxyflavonoids, and hesperidin were all upregulated to varying degrees in the overexpression group, indicating that transient overexpression of CitERF92 can promote the accumulation of polymethoxyflavonoids in citrus. It is evident that the transcription factor CitERF92 described in this invention participates in and regulates the synthesis of polymethoxyflavonoids, which is of great significance for improving the regulatory network of citrus flavonoid biosynthesis and metabolism and for realizing the industrialized synthesis of polymethoxyflavonoids.

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

[0040] Unless otherwise specified, the present invention does not have special requirements for the raw materials used in the preparation, 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] Total RNA was extracted from the peel tissue of sweet oranges using the CTAB method, and its quality was assessed by agarose gel electrophoresis and NanoDrop 2000 (Thermo Fisher Scientific, USA). Genomic DNA was removed and first-strand cDNA was synthesized using the HiScript III 1st Strand cDNA Synthesis Kit (Novizan, Nanjing) with gDNAwiper, following the manufacturer's instructions. CDS sequence amplification primers for CitERF92 were designed based on the sweet orange 1.0 reference genome data (PRJNA86123), and amplification was performed using the peel cDNA as a template. The CDS amplification primers for CitERF92 were:

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

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

[0046] Amplification was performed using the 2×Phanta Max MasterMix (Dye Plus) kit (NovaZeneca, Nanjing). The reaction mixture consisted of 15 μL of 2×Phanta Mix, 1.2 μL of 10 μM upstream primer, 1.2 μL of 10 μM downstream primer, 2 μL of cDNA template, and RNase-free H2O to a final volume of 30 μL.

[0047] After mixing, the product was reacted in a PCR instrument according to the following program: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 sec, 58℃ annealing for 15 sec, 72℃ extension for 1 min, 35 cycles; 72℃ final extension for 5 min; stored at 4℃. The amplified product was analyzed by agarose gel electrophoresis and purified using a DNA recovery kit (EasyDNA, 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. Sequence alignment was performed, and a phylogenetic tree was constructed using ERF genes homologous to those in Arabidopsis thaliana and ERF members in citrus reported to be involved in the regulation of polymethoxyflavonoid synthesis. Multiple sequence alignment analysis of the amino acids in the ERFs was performed using Clustal X software (Clustal X Software, Ireland). The aligned .aln files were imported into MEGAX software (MEGA Software, USA), and the Neighbor-Joining method was used to construct the phylogenetic tree. Information on the ERF members used to construct the phylogenetic tree is summarized in Table 1.

[0050] Table 1 Summary of ERFs Member Information for Constructing the Evolutionary Tree

[0051]

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

[0053] 3. Experimental Results

[0054] The full-length coding sequence of CitERF92, 624 bp, was obtained by cloning, as shown in SEQ ID NO.2; it encodes 208 amino acids, as shown in SEQ ID NO.1. Analysis using NCBI Conserved Domain in Search revealed that CitERF92 possesses an AP2 domain, belonging to the ERF subfamily within the AP2 / ERF transcription factor family. Figure 1 Further phylogenetic analysis showed that CitERF92 belongs to the ERF B-3 subgroup and is most closely related to Arabidopsis thaliana AtERF1A (At4g17500). Figure 2 ).

[0055] Example 2: Expression analysis of CitERF92 in the peel of sugar oranges at different developmental stages

[0056] 1. Sample collection and transcriptome sequencing

[0057] Eight developing sugar 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). The peel was separated into the oil cell layer and the white peel layer (the S0 stage fruits were too small to be separated and were measured together). All samples were cut into small pieces, flash-frozen in liquid nitrogen, and then ground into powder for later use. 0.3g of fresh sample powder was weighed, and total RNA was extracted from the sample using the CTAB method. RNA-seq was performed by BGI Genomics on the BGISEQ-500RS sequencing platform. After the raw data was downloaded, filtered, and cleaned, it was aligned to the Citrus sweet orange v1.0 reference genome (https: / / www.citrusgenomedb.org / citrus_downloads / Citrus_sinensis / C.sinensis_Hzau_v1.0_genome / ), and the gene expression levels were measured using the FPKM value (fragments per kilobase of exon per million fragments mapped).

[0058] 2. Experimental Results

[0059] like Figure 3As shown in Table 2, the expression level of CitERF92 in the peel exhibits a trend of first increasing and then decreasing, with the highest expression level at stage S3 (80 days after flowering), consistent with the accumulation pattern of flavonoids in citrus peel. Furthermore, the expression levels of CitERF92 in the oil cell layer and albedo layer are similar at stages S1 and S2, while the expression level in the oil cell layer is higher than that in the albedo layer at stages S3 and beyond, which is positively correlated with the abundant accumulation of polymethoxyflavonoids in the oil cell layer. Therefore, it is speculated that the transcription factor CitERF92 responds to the fruit development process and participates in the regulation of citrus polymethoxyflavonoid synthesis.

[0060] Table 2. Expression levels of CitERF92 in the oil cell layer and white peel of sugar oranges at different developmental stages.

[0061]

[0062] Example 3: Transient overexpression of CitERF92

[0063] 1. Vector construction and Agrobacterium-mediated transformation

[0064] After removing the terminator from the CDS sequence of CitERF92, homologous recombination was used to construct 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 particulates in the biosynthesis of polymethoxylated flavones in citrus. Plant Physiology 192:2049–2066.). The cloning primers are as follows:

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

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

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

[0068] After thorough mixing, ligation was performed at 37°C for 30 min. Then, 5 μL of the ligation product was added to 20 μL of DH5α competent cells, and transformation was performed following the procedure of incubation on ice for 25 min, heat shock at 42°C for 90 sec, and incubation 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 selection was performed using kanamycin (Kan; 50 μg / mL) and rifampicin (Rif; 20 μg / mL). Positive clones were picked for electrophoresis detection and then stored in 25% glycerol at 80°C.

[0069] 2. Transient overexpression in citrus peel

[0070] 10 μL of Agrobacterium tumefaciens culture containing the CitERF92-pBI121 recombinant plasmid was streaked onto LB solid medium for activation. After incubation at 28°C for 2 days, positive clones were picked and added to 200 mL of LB liquid medium, and cultured at 28°C with shaking until OD reached. 600 =0.8. Collect bacterial cells by centrifugation at 6000 rpm for 10 min, and resuspend the cells in an equal volume of osmotic buffer (10 mM MES, 10 mM MgCl2, 150 μM acetylsylcholine, pH = 5.6). Mature sugar oranges of uniform maturity and size, free from mechanical damage and pests / diseases, were selected as materials. The peel tissue on opposite sides of the equatorial plane of the same fruit was injected. One side was injected with Agrobacterium tumefaciens containing CitERF92-pBI121, and the other side was injected with pBI121 empty vector as a control. The injection volume on each side was approximately 5 mL; each fruit constituted a biological replicate, with a total of 5 replicates. Injected fruits were placed in a 25℃ climate chamber and kept in the dark for 24 h, then cultured under a 16 h light / 8 h dark light cycle. After 5 days, the injected peel tissue was harvested, cut into small pieces, flash-frozen in liquid nitrogen, and stored at -80℃ for later use.

[0071] 3. Expression analysis of CitERF92

[0072] After grinding the overexpression and control samples with liquid nitrogen, total RNA was extracted using 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 product melting curves and sequencing verification were performed. The RT-qPCR reaction system was 20 μL, containing 10 μL of 2×ChamQ Universal SYBR qPCRMaster Mix (Novizan, Nanjing), 2 μL of cDNA template, 0.4 μL each of forward and reverse primers (10 μmol / L), and 7.2 μL of RNase-free water. The reaction was performed using a CFX96 real-time quantitative PCR instrument (Bio-rad, USA). The reaction program was as follows: (1) 95℃, 30s; (2) 95℃, 10s; (3) 60℃, 30s; (2) to (3) 40 cycles; (4) 95℃, 10s; (5) from 65℃ to 95℃, increasing by 0.5℃ every 5s and collecting fluorescence values ​​once. Citrus β-actin was used as an internal reference gene to normalize the expression level.

[0073] 4. Extraction and Detection of Citrus Polymethoxyflavonoids

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

[0075] After centrifuging at 12,000 rpm for 20 min, 100 μL of the extract was used for analysis. Polymethoxyflavones in the sample were detected using a Waters HPLC-DAD system (2695 four-stage pump, 2996 diode array detector) (Waters Crop., USA) and a Sunfire C18 ODS column (4.6 × 250 mm, 5 μm; Waters). The mobile phase consisted of chromatographic acetonitrile (phase A) and purified water containing 0.1% formic acid (phase B), with linear gradient elution. The elution concentration 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. A standard curve was established using polymethoxyflavonoid standards for qualitative and quantitative analysis of flavonoids in the samples. The standard curves are shown in Table 3.

[0076] Table 3 Standard curves for qualitative and quantitative analysis of flavonoids in the samples.

[0077]

[0078] 5. Experimental Results

[0079] Compared with the control pericarp injected with no vector, the CitERF92 expression level in the overexpression group was significantly increased, confirming the effectiveness of this transient overexpression system. Figure 4 (A). Further analysis was conducted on the content of five polymethoxyflavonoid monomers, such as... Figure 4 As shown in Table 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 were all upregulated to varying degrees, with upregulation rates of 24.0%, 28.0%, 24.7%, 30.1%, and 27.2%, respectively. Transient overexpression experiments confirmed that CitERF92 promotes the accumulation of polymethoxyflavones in citrus fruits.

[0080] Table 4. Accumulation of polymethoxyflavonoid monomers in the peel of rock sugar oranges after transient overexpression of CitERF92 (mg / g)

[0081]

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

[0083] 1. Recombinant plasmid construction and Agrobacterium-mediated transformation

[0084] Genomic DNA was extracted from leaves of *Citrus aurantiacus* using the Ezup column-based plant genomic DNA extraction kit (Sangon Biotech, Shanghai). Using this DNA as a template, the promoter region of CitFNSII-1 was cloned and constructed into 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 BiotechnolJ 19:671–688.). The resulting CitFNSII-1 promoter-2000 bp sequence was obtained, and the constructed recombinant plasmid was named CitFNSII-1pro-LUC. The CDS sequence of CitERF92 was cloned into the pGreen II 002962-SK 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 Biote chnol J 19:671–688.). 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 ID NO. 12).

[0091]

[0092] The constructed recombinant plasmid was transformed into Agrobacterium GV3101::pSoup competent cells (Weidi, Shanghai; CAT#:AC1002), and 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 fluorescence detection

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

[0095] Using a 4mm diameter punch, samples were taken from the injection area of ​​tobacco leaves and homogenized with 100μL of 1×PBS buffer (pH=7.4). 50μL of the homogenate was transferred to 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 reagents (Promega, USA). The REN value was used as a control, and the LUC / REN ratio reflected the regulatory effect of transcription factors on the promoter. The LUC / REN ratio for the negative control was set to 1.

[0096] 3. Experimental Results

[0097] The results of the tobacco dual-luciferase assay are as follows: Figure 5 As shown, CitERF92 can act as a transcription activator, activating the promoter activity of CitFNSII-1 by 1.96-fold.

[0098] Example 5: Purification and EMSA Analysis of CitERF92 Protein

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

[0100] The CDS sequence of CitERF92 after the terminator was removed was amplified and constructed into the pGEX-4T-1 vector with a GST tag using homologous recombination (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 BiotechnolJ 19:2082–2096.). The amplification primers with the pGEX-4T-1 vector adapter were:

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

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

[0103] The amplification system was the same as in Example 1, and the ligation into the pGEX-4T-1 vector and the transformation system for DH5α were the same as in Example 3. The correctly sequenced CitERF92-pGEX recombinant plasmid was transformed into the BL21(DE3)pLysS expression strain by heat shock, and positive clones were picked and preserved with glycerol.

[0104] 2. Prokaryotic expression and recombinant protein purification

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

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

[0107] 3. EMSA test

[0108] EMSA assays were performed using the LightShift Chemiluminescent EMSA Kit (Thermofisher Scientific, USA). Probes labeled with 3'-Biotin were synthesized by Hangzhou Jinbaiao Biotechnology Co., Ltd. Primers without 3'-Biotin labeling of the same sequence were used as competing probes. The binding probe sequences for the CitFNSII-1 promoter region are as follows:

[0109] Upstream probe:

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

[0111] Downstream probe:

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

[0113] The recombinant protein-probe binding reaction system was as follows: 1 μL 10× Binding buffer, 0.5 μL MgCl2, 0.5 μL poly(dI-dC), 0.5 μL 1% NP-40, 0.5 μL 50% Glycerol, 4 μL recombinant protein, 1 μL double-stranded probe (10 nM), and water to bring the volume to 10 μL. The reaction was incubated at room temperature for 30 min. For the competition experiment, 1 μL of the competition probe was added first and reacted for 10 min. After the reaction, 1 μL of loading buffer was added, and electrophoresis was performed using a 6% Acr-Bis non-denaturing gel with 0.5× TBE buffer at 160 V for 35 min. The binding strip was then transferred to a positively charged nylon membrane (Millipore, USA) using a transfer apparatus (0.3A, 30 min). After UV crosslinking for 30 min, a luminescence reaction was performed using a kit, and images were taken using a ChemiDoc™ XRS+ (Bio-rad, USA) instrument.

[0114] 4. Experimental Results

[0115] Purified CitERF92-GST as Figure 6 As shown, a recombinant protein of approximately 48 kDa was obtained (GST protein predicted to be 26.5 kDa), consistent with the CitERF92 prediction of 22.9 kDa. Figure 6 )

[0116] The CitERF92-GST recombinant protein can bind to the biotin-labeled CitFNSII-1 promoter probe, producing a migration arrest band, and the band gradually weakens with increasing competitive probe concentration. These results indicate that the CitERF92 protein can bind to the promoter region of CitFNSII-1 (…). Figure 7 ).

[0117] Therefore, this invention demonstrates that transient expression of CitERF92 in the peel of sugar oranges resulted in varying degrees of upregulation of the contents of sweet orange flavonoids, norihesperidin, 5,6,7,4'-tetramethoxyflavonoids, heptamethoxyflavonoids, and hesperidin in the overexpression group compared to the control group. This indicates that transient overexpression of CitERF92 can promote the accumulation of polymethoxyflavonoids in citrus. It is evident that the transcription factor CitERF92 described in this invention participates in and regulates the synthesis of polymethoxyflavonoids, which is of great significance for improving the regulatory network of citrus flavonoid biosynthesis and for realizing the industrialized synthesis of polymethoxyflavonoids.

[0118] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Overexpression of citrus ERF transcription factors CitERF92 Its application in increasing the content of polymethoxyflavonoids in citrus fruits is characterized by... Citrus ERF transcription factors CitERF92 The amino acid sequence is shown in SEQ ID NO.1; The citrus fruit is rock sugar orange; The polymethoxyflavonoids are one or more of sweet orange flavonoids, noriheptacortin, 5,6,7,4'-tetramethoxyflavonoids, heptamethoxyflavonoids, and hesperidin.

2. The application according to claim 1, characterized in that, Citrus ERF transcription factors CitERF92 The nucleotide sequence is shown in SEQ ID NO.

2.

3. Overexpression of citrus ERF transcription factors CitERF92 The application of biomaterials in increasing the content of polymethoxyflavonoids in citrus fruits is characterized by, The biological material includes overexpression of the citrus ERF transcription factor. CitERF92 Recombinant expression vectors and / or overexpression of the citrus ERF transcription factors CitERF92 Recombinant microorganisms; Citrus ERF transcription factors CitERF92 The nucleotide sequence is shown in SEQ ID NO.2; The citrus fruit is rock sugar orange; The polymethoxyflavonoids are one or more of sweet orange flavonoids, noriheptacortin, 5,6,7,4'-tetramethoxyflavonoids, heptamethoxyflavonoids, and hesperidin.

4. The application according to claim 3, characterized in that, Overexpression of the citrus ERF transcription factor CitERF92 The basic framework of the recombinant expression vector includes the pBI121 vector.

5. A method for increasing the content of polymethoxyflavonoids in citrus fruits, characterized in that, The steps include: overexpressing citrus ERF transcription factors in the target citrus genome. CitERF92 ; Citrus ERF transcription factors CitERF92 The nucleotide sequence is shown in SEQ ID NO.2; The citrus fruit is rock sugar orange; The polymethoxyflavonoids are one or more of sweet orange flavonoids, noriheptacortin, 5,6,7,4'-tetramethoxyflavonoids, heptamethoxyflavonoids, and hesperidin.

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