Inc.3024.2-GbMYB2 for regulating and controlling synthesis of gingko flavonoids and application of lnc.3024.2-GbMYB2

Abstract

The invention discloses lnc.3024.2-GbMYB2 for regulating and controlling synthesis of gingko flavonoids and application of the lnc.3024.2-GbMYB2, and belongs to the technical field of genetic engineering, aiming at analyzing the biological functions of lncRNA (lnc.3024.2) and MYB target gene (Gb06719) participating in gingko flavonoids anabolism, by utilizing the technical means of bioinformatics analysis, real-time fluorescence quantification, subcellular localization, prokaryotic expression, genetic transformation and the like, the GbMYB2 can be used for regulating and controlling synthesis of the gingko flavonoids. The regulation mechanism of the transcription factor lnc.3024.2 and the target gene GbMYB2 of the lnc.3024.2 participating in the synthesis of the flavonoids in the ginkgo leaves is discussed. And a gene regulation network model of gingko flavonoid anabolism is further enriched. On the basis, the molecular mechanism of regulating and controlling the synthesis of the gingko flavonoids by the lncRNA targeting MYB is further analyzed on the early-stage basis, the content of the gingko flavonoids is increased by utilizing a genetic engineering technical means, and a new way is provided for researching the synthesis way of the gingko flavonoids.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and specifically relates to lnc.3024.2-GbMYB2 for regulating the synthesis of ginkgo flavonoids and an application thereof. Background Art

[0002] Ginkgo biloba L. is an ancient and romantic deciduous tree of the genus Ginkgo in the family Ginkgoaceae. Its unique leaf shape is the most recognizable symbol of Ginkgo. During plant growth and development, flavonoid compounds help plants resist adverse stress. At the same time, standardized ginkgo leaf extract (EGB) on the market is often used as an additive for medicines and health products. It has antioxidant, antibacterial, and anti-inflammatory functions, and plays an important role in the treatment of cardiovascular and cerebrovascular diseases and the improvement of memory loss. Therefore, increasing the content of ginkgo flavonoids has become a goal of continuous exploration in breeding research. It is well known that the synthesis of plant flavonoids is not only regulated by structural genes, but also by regulatory genes such as lncRNA and transcription factors. MYB transcription factors are the most critical transcription factor family in the synthesis of plant flavonoids. A large number of studies have shown that MYB transcription factors are involved in the biosynthesis of plant flavonoids and regulate the accumulation of plant flavonoids by activating or inhibiting the expression level of downstream target genes. At present, the research on ginkgo flavonoids mainly focuses on functional genes, transcription factors, etc., while there are few reports on the identification and mechanism of action of lncRNA related to ginkgo flavonoid metabolism. Although many potential lncRNAs have been predicted in ginkgo by high-throughput sequencing, no in-depth functional studies have been conducted, especially the molecular mechanism of lncRNA binding to transcription factors and structural genes to regulate the synthesis of ginkgo flavonoids is still unclear. In the early stage, the research group of the inventor of this application conducted transcriptome sequencing of the full length of 8 tissues including ginkgo roots, stems, young leaves, old leaves, male flowers, female flowers, young fruits and mature fruits, and predicted a lncRNA (lnc.3024.2) and a MYB (Gb_06719) target gene involved in flavonoid synthesis (Ye J, Cheng S, Zhou X, et al. A global survey of full-length transcriptome of Ginkgobiloba reveals transcript variants involved in flavonoid biosynthesis [J]. Industrial Crops and Products, 2019, 139: 111547.), but the specific molecular mechanism of action is still unclear. Therefore, this application deeply analyzes the molecular mechanism of lncRNA-MYB regulating the synthesis of ginkgo flavonoids on the basis of the early stage, and uses genetic engineering technology to increase the content of ginkgo flavonoids, providing a new way to study the metabolism of ginkgo flavonoids. In addition to laying a foundation for studying flavonoid synthesis and the molecular mechanism of flavonoid regulation, this application can also provide a reference for the research of other plant flavonoid compounds. Summary of the invention

[0003] The purpose of the present invention is to provide a lnc.3024.2-GbMYB2 for regulating the synthesis of ginkgo flavonoids and an application thereof in view of the existing problems.

[0004] The present invention is achieved through the following technical solutions:

[0005] A lnc.3024.2-GbMYB2 for regulating the synthesis of ginkgo flavonoids and its application, wherein the GbMYB2 target gene has a length of 750 bp, and its nucleotide sequence is shown in SEQ ID NO.1; the lnc.3024.2 target gene has a length of 2173 bp, and its nucleotide sequence is shown in SEQ ID NO.2.

[0006] An application of lnc.3024.2-GbMYB2 for regulating the synthesis of ginkgo flavonoids, wherein the lnc.3024.2 and GbMYB2 are involved in regulating the biosynthesis of flavonoids in ginkgo leaves.

[0007] Furthermore, there is a targeting relationship between GbMYB2 and lnc.3024.2, and lnc.3024.2 targets GbMYB2 to participate in the biosynthesis of ginkgo flavonoids.

[0008] Furthermore, GbMYB2 and lnc.3024.2 showed a negative correlation with the flavonoid content in ginkgo leaves treated with MeJA and ABA.

[0009] Furthermore, GbMYB2 and lnc.3024.2 inhibit the accumulation of flavonoids in Ginkgo biloba leaves.

[0010] Furthermore, GbMYB2 and lnc3024.2 participate in the accumulation of flavonoids by targeting downstream structural genes in the ginkgo flavonoid biosynthesis pathway.

[0011] Furthermore, the downstream structural genes include UFGT, UF3GT, UFGT73, F3'H, LDOX and DNR.

[0012] Compared with the prior art, the present invention has the following advantages:

[0013] 1. The present invention aims to analyze the biological function of lncRNA (lnc.3024.2) and its MYB target gene (Gb_06719) in the synthesis and metabolism of ginkgo flavonoids. By using bioinformatics analysis, real-time fluorescence quantification, subcellular localization, prokaryotic expression, genetic transformation and other technical means, the regulatory mechanism of lnc.3024.2 and its target gene GbMYB2 transcription factor in the synthesis of flavonoids in ginkgo leaves was explored.

[0014] 2. The present invention cloned GbMYB2 and lnc.3024.2 from the "Family Buddha's Hand" Ginkgo variety. Using bioinformatics analysis, it was predicted that lnc.3024.2 does not have coding ability and is a true long non-coding RNA located in the cytoplasm, while Gb_06719 located in the nucleus contains complete R2 and R3 domains, belonging to the R2R3-MYB transcription factor family member R2R3-GbMYB2, which has the function of regulating plant secondary metabolism. At the same time, the GbMYB2 promoter contains a cis-acting element that responds to external environmental stress, and plays an important role in resisting adversity. Using protein function verification technology, the subcellular localization experiment verified that the 35S:GbMYB2-GFP fusion fluorescence was only expressed in the nucleus of the onion epidermis, proving that GbMYB2 is located in the nucleus and has nuclear function. The GbMYB2-pET32a recombinant vector was transformed into BL21 (DE3) Escherichia coli through a prokaryotic expression experiment, and induced for 3 hours under 0.5 mM IPTG conditions for SDS-PAGE detection. Under this condition, the target protein consistent with the theoretical molecular weight was obtained.

[0015] 3. The present invention uses real-time fluorescence quantitative technology to analyze the expression patterns of different tissues and different developmental periods of Ginkgo, and finds that GbMYB2 and lnc.3024.2 are expressed in all tissues, with the highest expression in leaves and the lowest in roots, and the expression trends in various tissues remain highly consistent. When 1mM MeJA and 100μM ABA are used to treat ginkgo leaves, the results show that the expression trends of GbMYB2 and lnc.3024.2 in different time periods are also highly consistent. It is speculated that GbMYB2 and lnc.3024.2 may have a targeting relationship. The HPLC technology was used to measure and analyze the flavonoid content of ginkgo leaves treated with exogenous hormones in different time periods, and it was found that the flavonoid content under 100μM ABA treatment showed a gradually increasing trend. Combined with qRT-PCR to determine the gene expression amount under exogenous hormone treatment for correlation analysis, the results showed that GbMYB2 and lnc.3024.2 were negatively correlated with the flavonoid content in ginkgo leaves treated with MeJA and ABA.

[0016] 4. The present invention uses Arabidopsis genetic transformation technology to perform overexpression transgenic analysis on Landsberg ecotype Arabidopsis, and combines the HPLC method to determine the flavonoid content in the leaves of transgenic Arabidopsis, and finds that the flavonoid content in the leaves of GbMYB2 and lnc.3024.2 transgenic Arabidopsis is significantly reduced compared with the wild type. It shows that GbMYB2 and lnc.3024.2 are negative regulators of flavonoid synthesis in Arabidopsis leaves. Finally, the qRT-PCR technology is used to quantitatively analyze the structural genes of Arabidopsis, and it is found that the expression levels of PAL1, PAL2, C4H, 4CL1, 4CL2, 4CL5, CHS2, CHI and FLS5 structural genes in GbMYB2 and lnc.3024.2 transgenic plants are significantly increased, while the downstream structural genes (UFGT, UF3GT, UFGT73, F3'H, LDOX and DNR) that regulate anthocyanin synthesis in the flavonoid metabolic pathway are significantly downregulated. It was speculated that GbMYB2 and lnc.3024.2 might be involved in the accumulation of flavonoids by targeting the downstream structural genes (UFGT, UF3GT, UFGT73, F3'H, LDOX and DNR) in the ginkgo flavonoid biosynthesis pathway. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Secondary structure prediction for lnc.3024.2 and GbMYB2;

[0018] Figure 2 This is the alignment result of GbMYB2 protein sequence;

[0019] Figure 3 This is the map of cis-acting elements of the GbMYB2 promoter;

[0020] Figure 4 is the PCR electrophoresis diagram;

[0021] Figure 5 The gene expression patterns of lnc.3014.2 and GbMYB2 in different tissues and at different stages of Ginkgo biloba are shown;

[0022] Figure 6 This is the expression pattern of lnc.3014.2 and GbMYB2 genes after Ginkgo biloba hormone treatment;

[0023] Figure 7 Subcellular localization of GbMYB2:GFP fusion protein in onion epidermal cells;

[0024] Figure 8 SDS-PAGE detection of prokaryotic recombinant GbMYB2-pET32a protein;

[0025] Fig. 9Screening for lnc.3024.2 and GbMYB2 transgenic Arabidopsis;

[0026] Fig.10 This is the electrophoresis diagram for the verification of crude DNA from transgenic Arabidopsis;

[0027] Fig.11 GUS staining verification of transgenic Arabidopsis;

[0028] Fig.12 This is the expression pattern of flavonoid content after Ginkgo biloba hormone treatment;

[0029] Fig.13 The expression pattern of flavonoid content in transgenic Arabidopsis thaliana;

[0030] Fig.14 This is a diagram of the expression pattern of structural genes in transgenic Arabidopsis. DETAILED DESCRIPTION

[0031] In order to further explain the present invention, it is described below in conjunction with the following specific embodiments.

[0032] The experimental materials used in this application were collected from the Ginkgo Resource Garden of the West Campus of Yangtze University in Jingzhou City, Hubei Province. They are 36-year-old "Family Buddha's Hand" grafted varieties. From the beginning of March when the ginkgo leaves "emerge", materials were collected every other week until the ginkgo fruits "shrunken" and fell in October. The different tissue materials of ginkgo roots, stems, young leaves, old leaves, young fruits, mature fruits, male flowers, and female flowers were put into marked self-sealing bags, immediately frozen in liquid nitrogen, and stored at -80°C.

[0033] The hormone-treated ginkgo material is one-year-old ginkgo seedlings, which are cultivated from seeds collected in October 2021. Select healthy seeds that are plump, unbroken, and free of mold contamination, soak them in 3% carbendazim for 1 hour, spread them flat on a tray wrapped in relatively moist gauze, and place them in a 25°C incubator for germination. After the ginkgo buds are 2 cm white, they are transplanted into a seedling pot filled with substrate soil: clay: vermiculite in a ratio of 2:2:1, and placed at 25°C for cultivation.

[0034] Arabidopsis thaliana was selected from wild-type seeds of Landsbergerecta (Landsberg) ecological background stored at 4°C in the laboratory. The seeds were sown in slightly moist imported substrate soil covered with plastic wrap and placed in an artificial incubator at 23°C light / 18°C dark, 16h / 8h. After germination, they were transplanted and planted in seedling pots in an incubator. After transplanting, the soil needed to be fully moistened and covered with plastic wrap for one week for seedling determination.

[0035] The synthesis of the primers and the determination of bacterial liquid sequences used in this application were all sent to Wuhan Sangon Biotechnology Co., Ltd. The primer sequences are shown in Table 1 below:

[0036] Table 1 Primer sequences

[0037]

[0038] Example 1

[0039] Gene cloning and bioinformatics analysis of lnc3024.2 and GbMYB2

[0040] 1.1 Bioinformatics analysis

[0041] (1) Target gene sequence analysis:

[0042] The CPC2 website (https: / / cpc2.gao-lab.org / ) was used to predict gene coding capacity online, and the RNAflod online website (http: / / rna.tbi.univie.ac.at / cgi-bin / RNAWebSuite / RNAfold.cgi) was used to predict the secondary structure of proteins. ExPASy (https: / / web.expasy.org / protparam / ) was used to predict the physicochemical properties of proteins of gene family members, such as length, molecular weight, isoelectric point, instability index, and aliphatic index. Cell-PLoc 2.0 (http: / / www.csbio.sjtu.edu.cn / bioinf / Cell-PLoc-2 / ) was used to predict the subcellular localization information of the target gene.

[0043] The coding ability prediction of lnc.3024.2 and Gb_06719 found that lnc.3024.2 had no coding ability and was a true lncRNA (Table 2). ORF prediction of lnc.3024.2 and Gb_06719 revealed that lnc.3024.2 and Gb_06719 had 19 and 5 ORFs, respectively, of which the largest ORF lengths were 125aa and 249aa, respectively. By blasting the ORFs of lnc.3024.2 and Gb_06719, no homologous protein was found in lnc.3024.2, while Gb_06719 had a 99.6% match with the R2R3-MYB2 transcription factor (GenBank: KY703713.1) (Liu X, Yu W, Zhang X, Wang G, Cao F, et al. Identification and expression analysis under abiotic stress of the R2R3-MYB genes in Ginkgo biloba L[J]. Physiology and Molecular Biology of Plants, 2017, 23: 503-516.), and named it GbMYB2, indicating that GbMYB2 belongs to the R2R3-MYB transcription factor family and plays an important regulatory role in plant secondary metabolism. However, lnc.3024.2 has no homologous sequence, and it is speculated that lnc.3024.2 may be a new non-coding RNA with poor conservation and unknown function.

[0044] Table 2 Coding ability prediction

[0045]

[0046]

[0047] The subcellular localization prediction website was used to predict the subcellular localization of lnc.3024.2 and GbMYB2. The results showed that lnc.3024.2 was localized in the cytoplasm (nucleoplasm), and GbMYB2 was localized in the nucleus. The secondary structures of lnc.3024.2 and GbMYB2 were predicted using the RNAfold online website. It was found that the secondary structures of GbMYB2 and lnc.3024.2 were stable and included the stem region, while the secondary structure of lnc.3024.2 was more complex, including not only the stem region, but also the inner ring, convex ring, multi-branched ring and hairpin ring structure. According to the prediction, the minimum free energy of lnc.3024.2 and GbMYB2 were -450.30 kcal / mol and -226.70 kcal / mol, respectively. Figure 1). In addition, the ExPASy website predicted that the GbMYB2 protein is composed of 249 amino acids, with a molecular weight of 28.34 kD, and the theoretical isoelectric point, instability coefficient, fat coefficient and total average hydrophobicity coefficient are 9.63, 30.94, 69.88 and -0.701, respectively.

[0048] (2) Multiple sequence alignment analysis

[0049] The protein sequences of the R2R3-MYB transcription factor family of angiosperms, including Arabidopsis thaliana, European sweet cherry (Prunus avium), European hazel (Corylus avellana), peach (Prunus persica) and gymnosperms, including European spruce (Picea abies), Cryptomeria japonica, and white spruce (Picea glauca), were downloaded from the NCBI official website. The NCBI accession numbers of each protein sequence are as follows: AtMYB106 (AF249309), AtMYB107 (AF249310), AtMYB92 (AF062916), AtMYB20 (AF-062869), AtMYB43 (AF175990), AtMYB42 (AF175999), AtMYB39 (AF175989), AtMYB49 (AF175991), and CjMYB74 (XP_057846786.2), PgMYB11 (ABQ51227.1), CaMYB41 (XP_059447491.1), PaMYB102 (XP_021815397.1), and PpMYB6 (XP_007211464.1). Homology modeling was performed using Clustal X and TBtools software and then uploaded to MEGA6 software for multiple sequence alignment analysis.

[0050] The results show that Figure 2 ), the GbMYB2 transcription factor contains R2 and R3 domains and belongs to the R2R3-MYB family gene.

[0051] Figure 2 Middle: The completely identical parts of the protein sequence alignment are represented by a black background and a white foreground; the relatively conserved protein sequences are represented by a gray background and a white foreground; the weakly similar or dissimilar parts are represented by a white background and a black foreground.

[0052] (3) Prediction of promoter cis-acting elements

[0053] The Ginkgo genome sequence was extracted from the Ginkgo database (https: / / ginkgo.zju.edu.cn / ), and the promoter sequence 2000 bp upstream of the transcription start site of the GbMYB2 (Gb_06719) gene was extracted using TBtools. The cis-acting elements in the promoter region were predicted online using plantCARE (http: / / bioinformatics.psb.ugent.be / webtools / plantcare / html / ). The prediction results were imported into Excel for classification and screening, and TBtools was used for visualization analysis.

[0054] Table 3GbMYB2 promoter prediction information

[0055]

[0056]

[0057] The results are shown in Table 3. Figure 3 As shown in the figure, it can be seen that the 2000bp upstream of the GbMYB2 promoter contains light response elements of Box-4, G-Box, GATA-motif, GT1-motif, I-box, Sp1 and TCCC-motif binding sites, drought response elements of MBS (CAACTG) binding sites, low temperature response elements of LTR (CCGAAA) binding sites, and hormone response elements, including abscisic acid (ABRE binding site), auxin (TGA-element binding site), salicylic acid (TCA-element binding site) and methyl jasmonate (TGACG-motif, CGTCA-motif binding sites). The promoter of this transcription factor also contains the AS-1 motif that activates gene expression in plant green tissues and root tips, the CAAT-box motif that controls the frequency of transcription initiation, and MYB and MYC binding motif elements. It is speculated that the GbMYB2 transcription factor has a responsive effect on abiotic stress, indicating that the GbMYB2 transcription factor is not only involved in drought and low temperature stress responses, but also in the abscisic acid, auxin, salicylic acid and methyl jasmonate hormone signaling pathways.

[0058] 1.2 Ginkgo RNA extraction and reverse transcription

[0059] (1) Extraction of total RNA from Ginkgo biloba

[0060] The mortar, pestle, gun tip, centrifuge tube and other tools required for the experiment were treated with 0.1% DEPC solution, wrapped in newspaper for sterilization and used for later use. The RNA extraction method of ginkgo tissue material was operated according to the instructions of TaKaRa miniBEST Plant RNAExtraction Kit. The concentration and integrity of the RNA product were detected by ultra-micro spectrophotometer and 1% gel electrophoresis, respectively, and the product was stored at -80℃.

[0061] (2) Reverse transcription of RNA

[0062] The qualified RNA products will be referenced III 1st Strand cDNA Synthesis Kit (+gDNAwiper) Kit Instructions Perform PCR reverse transcription or qPCR reverse transcription as required, and store the product at -40°C.

[0063] 1.3 Gene cloning and vector construction

[0064] (1) Cloning of target gene

[0065] The experimental materials of different tissues of Ginkgo biloba were used to refer to the template cDNA obtained above, and the target band products were amplified by PCR using 2×RapidTaq Master Mix by in vitro transcription. According to the reaction system in Table 4, the samples were added and mixed, and then centrifuged and placed on a PCR instrument for in vitro amplification reaction. The reaction procedure is shown in Table 5. Then, the length of the target gene band was detected by agarose gel electrophoresis. The correct amplification product was further purified and recovered, and the target gene product was finally obtained and stored at -40°C for later use.

[0066] The above PCR products refer to pMD TM 19-T Vetor Cloning Kit Instructions, construct the target gene into pMD TM On the 19-T vector, the recombinant product was transformed into DH5α Escherichia coli competent cells, placed in a 37°C shaking incubator for incubation for 2 hours, coated on an LB medium plate containing 50μg / mlAmp resistance, and inverted in a 37°C constant temperature incubator for overnight incubation. In the clean bench, full single colonies were randomly selected and mixed in a 1.5ml EP tube containing LB liquid culture medium (containing 50μg / mlAmp). After shaking and incubating at 37°C for 3 hours, PCR positive detection was performed using M13-47 and M13-48 primers. The correct bacterial solution was generally stored in a refrigerator with 50% propylene glycol at a ratio of 1:1, and some were sent to the company for sequencing verification.

[0067] Table 4 PCR reaction system

[0068]

[0069] Table 5 PCR reaction program

[0070]

[0071]

[0072] Note: Tm is set according to the average temperature of different primers, and 15sec / kb is set according to the extension time of the primer. Steps 2 to 4 (95℃, 15s; Tm, 15s; 72℃, 15sec / kb) are set for 30-35 cycles, and steps 1 and 5 are set for 1 cycle.

[0073] (2) Construction of expression vector of target gene

[0074] The correct bacterial solution was activated at 37°C overnight. Extract plasmid DNA from E. coli according to the instructions of PlasmidminiPrep Kit, detect the concentration of plasmid DNA using an ultra-micro spectrophotometer, and store it at -40℃ for future use.

[0075] The homology arm recombination method was used to construct the overexpression recombinant vector and the subcellular localization recombinant vector with reference to the instructions of the Nimble Cloning kit. The reaction system is shown in Table 6. The reaction solution was gently pipetted and mixed, then centrifuged instantly and placed on a PCR instrument at 50°C for 45 minutes. The obtained recombinant product was immediately transformed into DH5α, spread on a 50μg / ml Kan resistance plate and cultured at 37°C for 12 hours. Subsequently, the bacterial solution was verified by PCR and the samples were sent to the company for sequencing verification.

[0076] Table 6 NC recombination reaction system

[0077]

[0078] Extract and verify the correct bacterial solution plasmid, transfer it into GV3101 Agrobacterium competent cells by chemical transformation, spread it on YEP medium plates containing 50μg / ml Kan and 50μg / ml Rif, and invert and culture it in a 28℃ constant temperature incubator for 48h for bacterial solution PCR verification. The correct bacterial solution is added with 50% glycerol at a ratio of 1:1 and stored in a -40℃ refrigerator for later use.

[0079] The target gene was cloned and amplified by in vitro PCR with specific primers, and Ginkgo biloba leaves were used as template cDNA. Figure 4The results showed that the length of the band of the cloned product was consistent with that of the target gene, and the lengths of the target genes GbMYB2 and lnc3024.2 were 750 bp and 2173 bp, respectively. The target gene was cloned, purified and recovered, and then recombinant vectors were constructed together with the overexpression vectors, which were transformed into DH5α Escherichia coli and used as bacterial liquid templates for PCR detection ( Figure 4 ). The positive detection of overexpression recombinant vectors used M13-47 and M13-48 as primers, and the length of the positive GbMYB2 recombinant vector solution was 2040bp, while the length of the lnc3024.2 recombinant vector solution was 3451bp. The positive verification of subcellular localization recombinant vectors used the target gene specific upstream primer and PEGFP-N3 as detection primers, and the length of the positive GbMYB2 recombinant vector solution was 877bp. The positive verification of prokaryotic expression recombinant vectors was detected by T7 and T7-ter universal primers, and the length of the positive GbMYB2 recombinant vector solution was 1444bp. The PCR cloning results of these recombinant expression vectors containing the target gene were correct and consistent with the expected band length.

[0080] Figure 4 Middle: (A) PCR electrophoresis of target gene; (B) PCR electrophoresis of overexpression recombinant vector bacterial solution; (C) PCR electrophoresis of subcellular localization recombinant vector bacterial solution; (D) PCR electrophoresis of prokaryotic expression recombinant vector bacterial solution.

[0081] Example 2

[0082] Analysis of expression levels of GbMYB2 and lnc3024.2 genes in different tissues and at different stages

[0083] The samples required for the experiment were selected to determine the expression changes of the target gene in six tissues of Ginkgo (roots, stems, leaves, female flowers, male flowers and fruits). From March 18 when the leaves of the ginkgo trees began to germinate to September 3 when the fruits shrank and began to fall, female flowers and male flowers of 2 time periods, ginkgo stems of 5 time periods, ginkgo fruits of 8 time periods, ginkgo leaves of 9 time periods and ginkgo root tissue of 1 time period were collected, and the collected materials were immediately frozen in liquid nitrogen and stored at -80°C. Total RNA from different tissues was extracted and reverse transcribed into template cDNA, and the operation was performed according to the instructions of ChamQ Universal SYBR qPCRMaster Mix (Table 7). GbGADPH (L26924.1) was used as the internal reference gene of Ginkgo, and 3 biological replicates and 2 -ΔΔCt The Duncan multiple test method was used for data analysis.

[0084] Table 7 qRT-PCR reaction system

[0085]

[0086] In this application, experimental samples were collected from "leaf bud expansion" to "fruit falling", RNA from roots, stems, leaves, male flowers, female flowers and fruits at different stages of Ginkgo biloba were extracted, and the gene expression patterns of different tissues and different stages of Ginkgo biloba were analyzed by qRT-PCR ( Figure 5 ). The results showed that GbMYB2 and lnc3024.2 genes were expressed in all tissues of Ginkgo biloba, especially in leaves, where the highest expression was found, followed by fruit, and the lowest expression was found in roots. Correlation analysis showed that the expression level of GbMYB2 was positively correlated with the expression level of lnc3024.2 gene in ginkgo leaves at different stages (r=0.8221 calculated by Pearson method, P<0.001); the expression level of GbMYB2 was positively correlated with the expression level of lnc3024.2 gene in ginkgo fruits at different stages (r=0.349 calculated by Pearson method, P<0.05); the expression level of GbMYB2 was positively correlated with the expression level of lnc3024.2 gene in ginkgo flower development at different stages (r=0.603 calculated by Pearson method, P<0.01); the expression level of GbMYB2 was positively correlated with the expression level of lnc3024.2 gene in ginkgo stems and roots at different stages (r=0.692 calculated by Pearson method, P<0.001). Based on this, it was found that the expression of lnc.3024.2 and GbMYB2 in different parts of ginkgo at different times was relatively consistent, both showing a trend of "you increase, I also increase; you decrease, I also decrease". Among them, the gene in different periods of ginkgo leaves and fruits showed a trend of rising to falling, and then rising to falling; in flowers and stems, it showed a trend of first rising and then falling. These results indicate that the expression activity of lnc.3024.2 may be related to GbMYB2.

[0087] Figure 5 Middle: Figure A shows different stages of Ginkgo leaves; Figure B shows different stages of Ginkgo fruit; Figure C shows different stages of Ginkgo male and female flowers; Figure D shows different stages of Ginkgo stems and the expression levels of lnc.3014.2 and GbMYB2 genes in Ginkgo roots. All data are expressed as mean ± standard deviation (n = 3), and variance analysis was performed using Duncan's multiple comparison test. Different letters represent significant differences at P < 0.05.

[0088] Example 3

[0089] Analysis of lnc3024.2 and GbMYB2 gene expression under exogenous hormone treatment

[0090] After the cultivated one-year-old seedlings have grown to 10-12 leaves, the ginkgo seedlings with the same growth trend are selected for hormone treatment. 1mM methyl jasmonate (MeJA) and 100μM abscisic acid (ABA) are used for leaf spraying, and clean water is used as a control. Three replicates are set for each treatment, and each replicate has 10 seedlings. Leaves of the treated plants are collected at 0, 3, 6, 12, 24, and 48h, and quickly frozen in liquid nitrogen at -80℃. GbGADPH (L26924.1) is used as the internal reference gene, and each experiment is set up with 3 biological replicates, 2 -ΔΔCt The Duncan multiple test method was used for data analysis.

[0091] In this study, we predicted the promoter of MYB transcription factors online and found that there were hormone response elements such as ABA, IAA, SA and MeJA in the promoter sequence of GbMYB2 gene ( Figure 3). Studies have shown that ABA and MeJA hormone treatments are beneficial to the accumulation of ginkgo flavonoids. The ABA-induced MdbZIP44 transcription factor in apple promotes anthocyanin accumulation by enhancing the binding of MdMYB1 to downstream DFR genes (An JP, Yao JF, Xu RR, You CX, Wang XF, et al. Apple bZIP transcription factor MdbZIP44 regulates abscisic acid-promoted anthocyanin accumulation [J]. Plant, Cell & Environment, 2018, 41 (11): 2678-2692.); safflower was treated with 100 μM MeJA hormone for 6 hours, and two significantly upregulated pCtCHI and pCtHCT promoter genes were discovered (Chen J, Wang J, Wang R, **an B, Ren C, et al. Integrated metabolomics and transcriptome analysis on flavonoid biosynthesis in safflower (Carthamus tinctorius L.) under MeJA treatment [J]. BMC Plant Biology, 2020, 20: 1-12.); MeJA treatment leads to the up-regulation of structural genes (PcCHS, PcCHI, PcF3H, PcDFR, PcANS and PcLAR1) in the flavonoid biosynthesis pathway, thereby promoting the accumulation of flavonoid compounds in pear callus (Premathilake AT, Ni J, Shen J, Bai S, Teng Y. Transcriptome analysis provides new insights into the transcriptional regulation of methyljasmonate-induced flavonoid biosynthesis in pear calli [J]. BMC Plant Biology, 2020, 20: 1-14.). In order to explore whether there is a potential interaction between GbMYB2 and lnc3024.2 genes, 1 mM MeJA and 100 μM ABA were used to treat Ginkgo leaves with exogenous hormones at different times, and the gene expression patterns of lnc.3024.2 and GbMYB2 in leaves treated with hormones at different times were analyzed.Correlation analysis showed that the expression level of GbMYB2 was significantly positively correlated with the expression level of lnc3024.2 gene in different time periods of 100μM ABA hormone treatment of ginkgo leaves (r=0.91 calculated by Pearson method, P<0.0001); while the expression level of GbMYB2 was also positively correlated with the expression level of lnc3024.2 gene in different time periods of 1mM MeJA hormone treatment of ginkgo leaves (r=0.693 calculated by Pearson method, P<0.003). The experimental results showed that lnc.3024.2 and GbMYB2 showed a trend of "decline, rise, and then decline" under 1mM MeJA hormone treatment. However, under 100μM ABA hormone treatment, it showed a trend of "first rise, then decline", and the expression levels of the two genes reached a peak at 3h treatment. In addition, it was found that lnc.3024.2 and GbMYB2 still showed a significantly positive correlation under two different hormone treatments. Therefore, it was further inferred that lnc.3024.2 and GbMYB2 had an interactive relationship (. Figure 6 ).

[0092] Figure 6 Middle: Figure A shows different time periods of 100 μM ABA hormone treatment: it shows a trend of first rising, then falling. Figure B shows different time periods of 1 mM MeJA hormone treatment: it shows a trend of first falling, then rising, and then falling. All data are expressed as mean ± standard deviation (n = 3), and variance analysis was performed using Duncan's multiple comparison test. Different letters represent significant differences with P < 0.05.

[0093] Example 4

[0094] Subcellular localization analysis of GbMYB2

[0095] (1) Select fresh and intact onion tubers as experimental materials. Soak the fresh inner epidermis of onions in 75% ethanol for 10 min, wash with sterile water 5 times, cut into cubes of about 1 cm in width on sterile filter paper, and lay the torn inner epidermis with the mesophyll surface facing down on the preculture medium. Incubate in the dark at 25°C for 24 h and set aside.

[0096] (2) GbMYB2 was constructed into the subcellular localization expression vector pNC-Cam1304-SubC using the Nimble Cloning kit, and the constructed 35S:GbMYB2-GFP vector was transformed into the GV3101 Agrobacterium strain, with the empty vector as the control. The subcellular vector bacterial solution containing the recombinant product was prepared in a clean bench, inoculated into 30 ml YEP liquid culture medium of the corresponding resistance, and cultured at 28°C with shaking until the OD600 value reached 0.6-0.8. Centrifuge at 5000 rpm for 5 minutes and collect the bacteria. Resuspend the bacteria with MS solution until the OD600 value is 0.5-0.6. Before infection, add 100 μM acetosyringone (AS) to the infection solution, and immerse the pre-cultured onion inner epidermis in the infection solution (shake and infect for 20 minutes at 28°C). The co-culture medium was used and cultured in the dark at 25°C for 48 h. The onion inner epidermis was placed on a glass slide and the expression of 35S:GbMYB2-GFP and 35S:GFP was detected under 480 nm fluorescence using a confocal microscope (LSM880, Zeiss).

[0097] The prediction of the subcellular localization online website shows that GbMYB2 is located in the nucleus. Therefore, the present application constructs the GbMYB2 fusion expression vector 35S: GbMYB2-GFP, transforms GV3101 Agrobacterium cells to infect onion epidermal cells, and uses the pNC-Cam1304-subC empty vector as the control group. The fluorescence signal of the Gb06719-GFP fusion protein is observed under GFP excitation light at wavelengths of 405nm and 520nm to verify the distribution position of GbMYB2 in the organelles ( Figure 7 ). The experimental results showed that the GFP fluorescence signal could be detected in the nucleus, cell membrane and cell wall of the empty vector 35S: GFP control group, but the 35S: GbMYB2-GFP fusion gene only observed a strong green GFP fluorescence signal in the nucleus of onion epidermal cells, which was consistent with the predicted subcellular localization results. Therefore, it was determined that GbMYB2 was mainly localized in the nucleus, indicating that the gene has nuclear function.

[0098] Figure 7 Middle: 35S:GFP represents the empty vector of pNC-Cam1304-subC, which is used as the control group; 35S:GbMYB2-GFP represents the fusion protein constructed by the pNC-Cam1304-subC vector and GbMYB2, which is used as the experimental group. DAPI: nuclear marker, the scale bar is 100 μm.

[0099] Example 5

[0100] Prokaryotic expression analysis of GbMYB2

[0101] (1) Construction of a recombinant vector containing the target gene

[0102] To construct the pET32a-GbMYB2 recombinant vector, first select the EcoRⅠ and XhoI restriction sites of the prokaryotic expression vector pET-32a, and design the GbMYB2 primers with EcoRⅠ and XhoI restriction site adapters. Use Ginkgo cDNA as a template to amplify the target gene, and purify and recover the target band for later use.

[0103] First, perform restriction digestion reaction on the prokaryotic expression empty vector plasmid (Table 8), gently blow and mix the reaction solution in the previous step, and place it in the PCR instrument. Set the reaction program: 37℃ restriction digestion for 45min, 60℃ heat inactivation for 15min, and 80℃ inactivation for 15min. The obtained restriction digestion product is connected to the target gene with seamless cloning enzyme. The method refers to the Ready-to-Use Seamless Cloning Kit Kit Instructions for Operation (Table 9). The constructed pET32a-GbMYB2 recombinant vector, the reaction solution is reacted at 50℃ for 30min and immediately placed on ice for 2min for subsequent transformation to DH5α Escherichia coli. Take 30μl of bacterial solution and spread it on LB medium with 50μg / ml kan, invert and culture at 37℃ overnight. Use T7 and T7-ter as primers for colony PCR verification. The verified colonies were sent to Shanghai Shenggong for testing, and some were taken and stored in a -40℃ refrigerator with glycerol.

[0104] Table 8 Linearization vector reaction solution

[0105]

[0106] Table 9 Recombinant vector reaction solution

[0107]

[0108] (2) Transformation of the recombination reaction product into BL21(DE3) Escherichia coli

[0109] Extract plasmids from the correctly sequenced recombinant bacterial solution, and add 5μl of recombinant plasmids and empty vector plasmids to 50μl of BL21 (DE3) competent cells respectively. After placing on ice for 30 minutes, heat shock in a 42℃ water bath for 90s and incubate in ice for 2 minutes. Subsequently, add 700μl LB liquid culture medium and shake and culture at 37℃, 150rpm for 2h. Take 30μl of the transformed bacterial solution and spread it on an LB plate containing 50μg / ml kan, and invert and culture at 37℃ for 12h. Select single colonies and perform colony PCR verification using T7 primers. Take part of the verified colonies and add glycerol to store them in a -40℃ refrigerator, and the remaining part will be used for subsequent experimental operations.

[0110] (3) Inducing GbMYB2 protein expression

[0111] The overnight activated culture was diluted 100 times into LB with 50μg / ml kan, and cultured at 37℃, 200rpm until OD600 was about 0.6. 1ml of the culture was taken as the sample before induction, and the remaining culture was divided into 15ml for induction treatment under different conditions. IPTG was added to the final concentrations of 0.1, 0.5, and 1.0mM, respectively, and cultured at 16℃, 28℃, and 37℃ for 3, 6, 12, and 24h, respectively, and the empty vector pET32a was used as a control.

[0112] (4) Extraction of GbMYB2 protein

[0113] The induced bacterial solution was centrifuged at 12000rpm for 10min, and the supernatant was discarded. 300μl of buffer was added for re-suspending, ultrasonically disrupted for 30min (200W, working 2s, rest 2s), heated at 98℃ for 10min, centrifuged at 12000rpm for 20min, and finally 10μl of the supernatant was taken out for 12% SDS-PAGE electrophoresis.

[0114] The EcoRⅠ and XhoI double restriction sites were selected to obtain the linearized cloning vector, and the recombinant plasmid was constructed by seamless cloning and transferred into BL21 (DE3) Escherichia coli. GbMYB2-pET32a was used as the experimental group and the PET-32a empty vector was used as the control group. After induction with 0.5mM IPTG for 3h, SDS-PAGE electrophoresis was performed to detect protein expression ( Figure 8 ). The experimental results showed that both the experimental group and the control group induced a specific target protein at 28°C. The molecular weight of the protein was about 20.35kDa and 47.5kDa, respectively, which was consistent with expectations. Prokaryotic expression verified that GbMYB2 could translate into a complete protein in BL21 Escherichia coli, confirming the integrity of the cloned GbMYB2 gene sequence.

[0115] The PET-32a prokaryotic expression vector carries a His tag. The magnetic bead protein purification method with a His tag was used to recover the target protein. Finally, after elution with 100 mM imidazole, the target protein was concentrated and recovered using a protein concentrator at 5000 rpm for 30-60 min at 4 °C. The protein purification was detected by SDS-PAGE electrophoresis ( Figure 8 ). The experimental results showed that pET32a-GbMYB2 protein purified a specific target protein without any mixed bands. The molecular weight of the target protein was about 47.5 kDa, which was consistent with the expected band length.

[0116] Figure 8Middle: Figure A is the electrophoresis of protein induction. 1: pET-32a before induction; 2: pET32a-GbMYB2 before induction; 3: pET-32a induced by 0.5mM IPTG for 3h; 4: pET32a-GbMYB2 induced by 0.5mM IPTG for 3h; 5: pET-32a induced by 1.0mM IPTG for 3h; 6: pET32a-GbMYB2 induced by 1.0mM IPTG for 3h. Figure B is the electrophoresis of protein purification. 1: GbMYB2-pET32a protein induced by 0.5mM IPTG for 3h at 28℃; 2: GbMYB2-pET32a protein eluted by 0mM imidazole; 3: GbMYB2-pET32a protein eluted by 12mM imidazole; 4: GbMYB2-pET32a protein eluted by 30mM imidazole; 5: GbMYB2-pET32a protein eluted by 50mM imidazole; 6: GbMYB2-pET32a protein eluted by 120mM imidazole. M: Standard protein marker.

[0117] Example 6

[0118] Identification and analysis of GbMYB2 and lnc3024.2 positive transgenic Arabidopsis

[0119] (1) Infection with wild-type Arabidopsis

[0120] Landsberg wild-type Arabidopsis thaliana was selected as the experimental material. GbMYB2 and lnc.3024.2 were constructed into the overexpression vector pNC-Cam1304-MCS35S using the vector construction method described in Example 1. The constructed vectors 35S: GbMYB2 and 35S: lnc.3024.2 were transformed into the GV3101 Agrobacterium strain, and the wild-type plants were infected by the inflorescence infection method. Preparation of Agrobacterium engineering bacteria: Take 300μl of activated Agrobacterium bacterial solution (containing the overexpression vector bacterial solution of the recombinant product) on the clean bench, and incubate it in 30ml YEP liquid medium containing 30μg / ml Hygromycin B and 50μg / ml Rif at 200rpm, 28℃, shaking to make its OD600 value 0.8-1.0; then centrifuge at 4000rpm, room temperature for 10min to collect the bacteria, resuspend the bacteria in the infection solution until the OD600 value is 0.8-0.9, mix well, wrap it tightly with newspaper, and let it stand in the dark for 3h to prepare for infection of Arabidopsis thaliana flowers. Before infection, cut off the blooming flowers on the Arabidopsis plants, gently and completely immerse the trimmed flowers in the infection solution for 30s, wrap the Arabidopsis plants with black plastic bags, place them on their sides in a dark environment for 24h, spray them with clean water, and place them in a light incubator for normal culture. Repeat the infection once a week later.

[0121] (2) Screening and verification of transgenic Arabidopsis

[0122] Take an appropriate amount of seeds from the clean bench and sterilize them in a 1.5ml EP tube. Use 75% ethanol and 3% sodium hypochlorite for 30s and 5min respectively, blow and aspirate with sterile water 5 times, quickly add anhydrous ethanol after the liquid is sucked out, blow the seeds on a plate with filter paper, and sow them on MS medium containing 30μg / ml Hygromycin B after the seeds are air-dried, and place them in a light culture room for normal culture. After the seeds germinate, T0-generation transgenic plants are obtained, and single-plant green leaf seedlings are transplanted and cultured in a constant temperature incubator. Then, cut the young leaves of mature plants for further PCR detection of DNA.

[0123] The correct positive plants were marked for individual harvesting, and the obtained T1 generation seeds were dried in the sun and stored in a refrigerator at 4°C for future use. The T3 generation seeds obtained by this method were also verified by PCR after maturity, and the positive plants were further verified by GUS staining kit for subsequent experiments.

[0124] Agrobacterium-mediated floral infection was used to transfect Landsberg wild-type Arabidopsis plants, and T0 seeds were collected after the plants matured. T0 seeds were sown on MS medium containing 30 μg / ml Hygromycin B after resistance screening, and green leaf seedlings were transplanted and placed in an incubator for normal culture after 2 weeks. T1, T2 and T3 positive transgenic Arabidopsis plants were screened, with Landsberg wild-type plants as negative control groups and pNC-Cam1304-MCS35S empty vector transgenic plants as positive control groups ( Fig. 9 Arabidopsis leaves or plants were randomly cut and crude plant DNA and GUS staining were used to verify the T1, T2 and T3 generations of Arabidopsis transgenic plants ( Fig.10 , Fig.11 ). DNA from wild-type and transgenic Arabidopsis thaliana was extracted and PCR was performed using primers M13-47 and M13-48. Fig.10 The results showed that no bands were cloned in the wild-type plants, while the empty vector, GbMYB2 and lnc3024.2 transgenic plants all amplified the correct target bands. Fig.11 The results showed that the wild-type plants failed to stain, while the leaves, stems and catkins of the empty vector, GbMYB2 and lnc3024.2 transgenic plants all showed blue, indicating that Agrobacterium-mediated GbMYB2 and lnc3024.2 were successfully expressed in transgenic plants. Therefore, the empty vector, GbMYB2 and lnc3024.2 transgenic Arabidopsis were all positive plants.

[0125] Fig.11Middle: (A) Landsberg wild-type Arabidopsis; (B) pNC-Cam1304-MCS35S empty vector transgenic Arabidopsis plants; (C) lnc.3024.2 transgenic Arabidopsis plants; (D) GbMYB2 transgenic Arabidopsis plants.

[0126] Example 7

[0127] Analysis of flavonoid content in GbMYB2 and lnc3024.2

[0128] (1) Extraction of flavonoids from transgenic Arabidopsis

[0129] Preparation of Arabidopsis flavonoid crude extract: Grind the Arabidopsis leaves in a -80℃ refrigerator with liquid nitrogen until they are powdered and then quickly dispensed into 50ml centrifuge tubes. After precooling the centrifuge tubes in a low temperature environment, place them in a freeze dryer and continue freeze drying for 24 hours until constant weight. Use a 60-mesh sample sieve to screen the sample powder, and each sample is repeated 3 times. Weigh 0.1g of powder, add 2ml75% methanol, and use an ultrasonic cell disruptor to break for 30 minutes, with a power of 360W (ultrasonic work for 5s, intermittent for 8s), and then centrifuge at 10000xg for 10 minutes at room temperature. Take 800μl of supernatant, add 200μl of 36% hydrochloric acid, and place in a water bath at 85℃ for 1h. After naturally cooling to room temperature, seal and store at -40℃ for later use.

[0130] (2) Ginkgo leaf flavonoids extraction

[0131] Preparation of crude extract of ginkgo flavonoids: Grind the hormone-treated ginkgo leaves in a -80℃ refrigerator with liquid nitrogen and quickly dispense them into 50ml centrifuge tubes. Pre-cool at low temperature and freeze-dry in a freeze dryer for 24h to constant weight. Pass the sample powder through a 60-mesh sieve, and repeat 3 times for each sample. Weigh 0.5g of powder for Soxhlet extraction, add 65ml of petroleum ether to the sample and extract it at 65℃ for 3h, dry the sample; then add 65ml of methanol at 90℃ and extract it for 4h to evaporate the sample. Finally, add methanol and 25% hydrochloric acid in a 4:1 mixture to elute the sample for 0.5h, cool it at room temperature and set aside.

[0132] (3) Determination of flavonoid content (HPLC method)

[0133] High performance liquid chromatography (HPLC) was used to detect the content of flavonoids: 1 ml of the extract was taken for the determination of the content of flavonoids, filtered through a 0.22 μl organic phase microporous filter membrane into a brown injection bottle and marked. A Venusil XBP C18 (4.6×250 mm, 4 μm) chromatographic column was used to separate the flavonoid compounds, and high performance liquid chromatography (HPLC) was used for detection under the conditions of a detection wavelength of 370 nm and a column temperature of 30°C. The reaction procedure was as follows: the elution time was 0-27 min, the ratio of methanol (mobile phase A) and 0.3% phosphoric acid aqueous solution (mobile phase B) was 55:45, the flow rate was 1.0 ml / min, the injection volume was 10 μl, and the reaction time was 27 min. The standard solution (quercetin, kaempferol and isorhamnetin were used as standard substances) was diluted with methanol to form a gradient, and the substance content was calculated after the standard curve was made, and the total flavonoid content (μg·g -1 The calculation formula of DW was (quercetin content + kaempferol content + isorhamnetin content) × 2.51.

[0134] Promoter prediction revealed that the GbMYB2 and lnc3024.2 genes contained a large number of ABA and MeJA response elements. Combined with quantitative analysis of different tissues and at different times of Ginkgo biloba, it was found that the expression levels of GbMYB2 and lnc3024.2 genes were highest in Ginkgo biloba leaves. Therefore, the Ginkgo biloba leaves were treated with ABA and MeJA hormones, and the total flavonoid contents of kaempferol, quercetin and isorhamnetin in the Ginkgo biloba leaves were determined respectively ( Fig.12 ). The results of HPLC determination showed that the flavonoid content measured under 1mM MeJA treatment showed an upward and downward cyclic change trend, which was opposite to the expression levels of GbMYB2 and lnc3024.2 genes. In addition, the flavonoid content measured under 100μM ABA hormone treatment showed a gradual upward trend, reaching a peak at 48h, while the expression levels of GbMYB2 and lnc3024.2 genes also showed an opposite trend. The experimental results show that 1mM MeJA and 100μM ABA hormone treatment may be conducive to the accumulation of flavonoid content in leaves, while GbMYB2 and lnc3024.2 genes may negatively regulate the expression of flavonoid content in Ginkgo leaves.

[0135] Fig.12Middle: Figure A shows the flavonoid content and GbMYB2 gene expression level at different times of 100μM ABA hormone treatment; Figure B shows the flavonoid content and lnc3024.2 gene expression level at different times of 100μM ABA hormone treatment; Figure C shows the flavonoid content and GbMYB2 gene expression level at different times of 1mM MeJA hormone treatment; Figure D shows the flavonoid content and lnc3024.2 gene expression level at different times of 1mM MeJA hormone treatment. All data are expressed as mean ± standard deviation (n = 3), and variance analysis was performed using Duncan's multiple comparison test. Different letters represent significant differences with P < 0.05.

[0136] In order to study the changes in flavonoid content of GbMYB2 and lnc3024.2 genes in Arabidopsis, the leaves of T3 transgenic Arabidopsis positive seedlings were used as experimental materials. The contents of kaempferol, quercetin and isorhamnetin in the leaves of transgenic Arabidopsis were determined by HPLC. The total flavonoid content ( Fig.13 ). HPLC test results showed that GbMYB2 and lnc3024.2 genes would cause changes in the content of flavonoids in transgenic plants. The total flavonoid content of wild-type Arabidopsis was measured to be 0.53μg / g; the total flavonoid content of the three strains of GbMYB2 transgenic positive seedlings was measured to be 0.46μg / g, 0.19μg / g and 0.20μg / g, respectively; and the total flavonoid content of the three strains of lnc3024.2 transgenic Arabidopsis reached 0.09μg / g, 0.09μg / g, and 0.10μg / g, respectively. The results showed that the flavonoid content in GbMYB2 and lnc3024.2 transgenic Arabidopsis was 47.17% and 83.02% lower than that in the wild type, respectively, indicating that GbMYB2 and lnc3024.2 genes negatively regulated the accumulation of flavonoid content in Arabidopsis leaves.

[0137] Fig.13 Middle: CK: Landsberg wild-type Arabidopsis (control group); M: GbMYB2 Arabidopsis transgenic plants (treatment group); L: lnc.3024.2 Arabidopsis transgenic plants (treatment group). Data are the mean ± SD of three biological replicates, and different letters represent significant differences at P < 0.05.

[0138] Example 8

[0139] Analysis of the expression of key flavonoid genes in transgenic Arabidopsis

[0140] The mature leaves of wild-type Landsberg Arabidopsis and transgenic Arabidopsis were used as experimental materials. Total RNA was extracted and reverse transcribed into template cDNA. The operation was performed according to the instructions of ChamQ Universal SYBR qPCR Master Mix (the amplification program was divided into 3 steps of reaction, which were 95℃, 30s, and 1 cycle of constant temperature. 95℃, 10s; 60℃, 30s were set for 40 cycles. 95℃, 15s; 60℃, 60s; 95℃, 15s were set for 1 cycle of melting). Quantitative primers were designed for 36 structural genes in the flavonoid metabolic pathway of Arabidopsis, and AtPP2A (AT1G13320) was used as the internal reference gene of transgenic Arabidopsis (Appendix Table 10). Three biological replicates and 2 -ΔΔCt The Duncan multiple test method was used for data analysis.

[0141] Table 10 Transgenic Arabidopsis quantitative primer sequences

[0142]

[0143]

[0144]

[0145]

[0146] This experiment collected 36 structural genes that were verified by researchers to be involved in regulating flavonoid biosynthesis in Arabidopsis from the literature, and used qRT-PCR technology to perform gene quantitative analysis on wild-type and transgenic Arabidopsis to further identify potential target genes in GbMYB2 and lnc3024.2 transgenic Arabidopsis plants ( Fig.14). The results showed that compared with wild-type Arabidopsis plants, GbMYB2 and lnc3024.2 transgenic Arabidopsis plants expressed the first key enzyme PAL in the phenylalanine metabolic pathway (PAL1 and PAL2 genes were upregulated in transgenic plants, while PAL3 and PAL4 were downregulated). The 4CL genes in the synthesis of coumaroyl-CoA and malonyl-CoA in the flavonoid metabolic pathway (except for the 4CL8 gene, the expression levels of 4CL1, 4CL2 and 4CL5 genes were significantly increased). The first CHS key enzyme in the flavonoid synthesis pathway (the expression levels of the CHS gene and CHS1 gene in the transgenic plants were reduced, and the CHS2 gene was slightly upregulated). In addition, the key enzyme FLS that catalyzes flavonol synthesis (the expression of FLS1 gene was reduced in transgenic plants, while the expression level of FLS5 gene was increased), and the AOMT transporter that transports to the vacuole for storage (AOMT1 and OMT1 were upregulated in transgenic plants, while AOMT7 was the opposite), all of these genes were upregulated or downregulated to varying degrees in transgenic plants. More obviously, the expression of UFGT, UF3GT, UFGT73, F3H, F3'H, LDOX and DNR structural genes were all downregulated in transgenic plants, while the C4H structural gene was the opposite. Based on this, it is speculated that GbMYB2 and lnc3024.2 may target the expression of UFGT, UF3GT, UFGT73, F3H, F3'H, LDOX and DNR structural genes to regulate the accumulation of flavonoid content in Arabidopsis.

[0147] Fig.14 Middle: WT: Landsberg wild-type Arabidopsis thaliana (control group). Figure A shows the expression of 4CL gene in transgenic plants; Figure B shows the expression of FLS gene in transgenic plants; Figure C shows the expression of CHS gene in transgenic plants; Figure D shows the expression of UFGT and LDOX gene in transgenic plants; Figure E shows the expression of AOMT and DNR gene in transgenic plants; Figure F shows the expression of PAL gene in transgenic plants; Figure G shows the expression of FAP gene in transgenic plants; Figure H shows the expression of ACC and C4H gene in transgenic plants; Figure I shows the gene expression of CHI, F3H and F3'H gene in transgenic plants. All data are the mean ± SD of three biological replicates, and different letters represent significant differences with P < 0.05.

Claims

1. A lnc.3024.2-GbMYB2 for regulating the synthesis of ginkgo flavonoids and its application, characterized in that: The length of the GbMYB2 target gene is 750 bp, and its nucleotide sequence is shown in SEQ ID NO.1; the length of the lnc.3024.2 target gene is 2173 bp, and its nucleotide sequence is shown in SEQ ID NO.

2.

2. An application of lnc.3024.2-GbMYB2 for regulating the synthesis of ginkgo flavonoids, characterized in that: The lnc.3024.2 and GbMYB2 are involved in regulating the biosynthesis of flavonoids in Ginkgo leaves.

3. The use of lnc.3024.2-GbMYB2 for regulating the synthesis of ginkgo flavonoids according to claim 2, characterized in that: There is a targeting relationship between GbMYB2 and lnc.3024.2, and lnc.3024.2 targets GbMYB2 to participate in the biosynthesis of ginkgo flavonoids.

4. The use of lnc.3024.2-GbMYB2 for regulating the synthesis of ginkgo flavonoids according to claim 3, characterized in that: The GbMYB2 and lnc.3024.2 showed a negative correlation with the flavonoid content in the ginkgo leaves treated with MeJA and ABA.

5. The use of lnc.3024.2-GbMYB2 for regulating the synthesis of ginkgo flavonoids according to claim 4, characterized in that: The GbMYB2 and lnc.3024.2 inhibit the accumulation of flavonoids in Ginkgo leaves.

6. The use of lnc.3024.2-GbMYB2 for regulating the synthesis of ginkgo flavonoids according to claim 5, characterized in that: GbMYB2 and lnc3024.2 are involved in the accumulation of flavonoids by targeting downstream structural genes in the ginkgo flavonoid biosynthesis pathway.

7. The use of lnc.3024.2-GbMYB2 for regulating the synthesis of ginkgo flavonoids according to claim 6, characterized in that: The downstream structural genes include UFGT, UF3GT, UFGT73, F3'H, LDOX and DNR.

Images