LU601941B1 - Application of miR858a-GbMYBs regulating the biosynthesis of flavonoids in Ginkgo biloba L. - Google Patents

Abstract

The present invention relates to the field of genetic engineering technology, in particular to an application of miR858a-GbMYBs regulating the biosynthesis of flavonoids in G. biloba. In the present invention, one miR858 and three MYBs target genes were screened from G. biloba miRNA and transcriptome sequencing data, and the function of candidate genes in the flavonoid metabolic pathway of G. biloba was studied by bioinformatics analysis, gene cloning and genetic transformation techniques. The interaction between Gb-miR858a and target genes of GbMYBs was revealed. It was proved that GbMYB1, GbMYB39 and GbMYB41 were all positive regulators of flavonoid synthesis. The regulatory mechanism of Gb-miR858a targeting GbMYBs involved in flavonoid synthesis was preliminarily elucidated, providing a theoretical basis for improving the flavonoid content of G. biloba using genetic engineering technology.

Description

DESCRIPTION LU601941

APPLICATION OF MIR858A-GBMYBS REGULATING THE BIOSYNTHESIS OF

FLAVONOIDS IN GINKGO BILOBA L.

TECHNICAL FIELD

The present invention relates to the technical field of genetic engineering, and in particular to an application of miR858a-GbMY Bs regulating the biosynthesis of flavonoids in Ginkgo biloba L.

BACKGROUND TECHNOLOGY

Ginkgo biloba L. is the only gymnosperm plant belonging to the family G. biloba and genus G. biloba, which is a rare tree species left in the Mesozoic relict and known as a "living fossil" in the plant kingdom. Flavonoids are the main active components of extract of G. biloba (EGB), which have various biological activities such as improving cardiovascular disease, antibacterial, antioxidant free radicals, anti-tumor and anti- inflammatory activities, and can play an important role in various biological processes such as antioxidant, antimicrobial substances, signaling molecules for plant and bacterial interactions, light and pigments, and plant growth, development and stress resistance.

MicroRNAs (miRNAs) are endogenous non-coding small RNAs of 20-25 nucleotides in length that participate in plant flavonoid biosynthesis by negatively regulating key enzyme genes or transcription factors. MYB transcription factor is one of the important determinants regulating flavonoid pathway, which can play a precise role in regulating specific developmental periods and tissues and activate or inhibit the biosynthesis of corresponding compounds. Although it has been found that miR858 targets MYB and participates in the synthesis of different plant flavonoids, its regulatory mechanism in ginkgo is still unknown.

SUMMARY OF THE INVENTION LU601941

The purpose of the present invention is to provide an application of miR858a-GbMYBs regulating the biosynthesis of flavonoids in G. biloba in response to the existing problems.

The present invention is achieved through the following technical solutions:

An application of MIR858a-GbMYBs regulating the biosynthesis of flavonoids in G. biloba, comprising screening and identification of Gb-miR858a-GbMY Bs regulatory components.

Further, a precursor sequence of Gb-miR858a is shown as SEQ ID NO.1;

SEQ ID NO 1:

ACCCCTTGATTAAACTGATCGTCCGTCGAGCCTTTTCCTGAGCTGTATTGGGATTIT

GGGAGCCTTTTCCTGATGTGAGACCGCCAGTAATTCTTAATTTCGTTGTCTGTTCGG

CCTGGCATCTTCTCTGCAATCAAAGACCATCTGCACATGCCACCAAAACATGATCAA

ATAACAGGTAAAAATTGCCCAAAATTTATTTAGCGCGGATAGGTGGCCTTTCATGTAG

AAATGAGAGGCAAAGTCGTCA the GbMYBs comprise GbMYB1, GbMYB39 and GbMYB41, with corresponding nucleotide sequences shown as SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4 respectively.

SEQ ID NO.2:

ATGGGTCGGTCTCCTTGCTGTGAAAAAGCTCATACCAACAAAGGGGCTTGGACCAA

AGAGGAAGACGACAGGCTTATTGCACACATTCAGGCTCATGGAGAAGGATGCTGGA

GGTCCCTCCCCAAGGCTGCAGGGCTGCTGCGTTGTGGGAAGAGTTGCAGGCTCC

GGTGGATAAACTATCTCCGTCCCGACCTCAAGCGTGGGAATTTTTCAGAAGAAGAA

GACGAACTCATCATCAAGCTCCACGCCCTCTTAGGGAACAAGTGGTCTCTCATCGC

AGGAAGATTGCCCGGTCGCACAGATAACGAAATAAAAAATTATTGGAACACCCATAT

AAAAAGAAAATTGATTAGCAGGGGCGTAGACCCACAGTCGCATCAACCCATCCAGA

CCGGCTATCAGTCGGCCAAGCTAATTGCAGCGGATTCTCCACAGATTTTTGCATTCC

AGCGGAATACGCCGGAGATGGCAGACTTCTTCGAATACAATGACATGATGAGTACAT

CAGTGGAGCAGCAGCAGACGGCAGCTCCAGCTAATTCGGATGGTCAGGGCAGTG

GAATCGAAGACCATCCGGACCTCAACCTCGAATTGTGTATCAGTTTGCCCTCTGGC

CCGGCTAGCATAAGCAGAGCCACCACAGCCGATTCAAAGGAGGTTTCCAGTCCAA

ATAGTGCCGGGTCGAAGGCAGACGCTGTGTGCTGTCACATGGGATTGCAAATCAAT

GGAGGAGGCTGCTGTGAGGATAATAGATGCTCACATGAAGACCATGTGGCTGCGA

CGGCTGGCCATTTTAATTACAGGCAACTGCGGGTATAA

SEQ ID NO.3: LU601941

ATGGGTCGAGCTCCGTGCTGTGCCAAGGTTGGATTGAATAGGGGCCGATGGACTG

CTGAAGAAGATGACATTTTAGCTAGATATATTGAAGCCCATGGCGAAGGTTGCTGGC

GAGCACTTCCCAAAAATGCAGGATTGCTGCGGTGCGGAAAGAGTTGCAGGTTGAG

ATGGATCAACTATCTTCGTTCGGATGTGAAGCGTGGCAACATTTGCGAAGAAGAAG

AGGAGCTTATCATCAAACTCCACAATCTTCTGGGCAATCGGTGGTCATTGATTGCAG

GCCGTATGCCTGGTCGAACGGACAAT GAGATAAAAAACTACTGGAATACTCATCTAA

GCCGGAAGCTCGTAGACAGAGGAATCAATCCCGTGACCCACAAGCCATTAGATGCT

AAAGTCATGCCTAAGCCCTCCGCATGTATCAGCAATGCGGCGAAGCTGAAGAAAAC

AGACGAGTGTGGTATTGTTGATCAAGTGTCTGGAATAACCCGACGAGATGTGGTTIT

GCAGAAGCCCGAGAAAGAAGAGAATCCAAGATCTCGAGTAAGCCAGAAAACTGTG

GAAAAAAACGCAGAGGATTCCACTGTTAAAGGCAAAAGGAAGAGGACTTCGTTAAA

ATTGGCTTCCAGCAGTGAAGGCCGAGGTCAGAAGCTGAAAATGTTGGGCGACCTT

AAATTAACTGAACAACCTTCCTCCACAGTTTCAGTGCAGTCGAGCTGTGAATCATGT

TCCGACTTCGATTCCCCTCCCCTGGAGGCCATGACATCTAATCCTAGTTGTTCTGAT

CAGGCTCAGTTGTTTTGTGAAAAAGCTTCACAGAGTGCCCAGGTAGAGGCAAAGG

AAAATGAAGATTACAAGGTACAAGATGCAGGAATTGCGGTAGCATTACAGGAAGATC

CAAGCAGTTCATTTACTGTTAATTCGGATTCTCTGTGGGATAATTCGTTTTCGTCGCA

GATACTTTCTTCAGACATGGACTTGTTTGGATCATTAGAAACGGAAGCTCTGTTCGA

GTATTGTTTACCTAATGCGGATGCGAATTCAAATGCAGAGAGCATGGAGATGTCTGG

CGATCCAGACGAACTGTGGTCTTTCTTTCAGTCTGCAAATGCAGGAGGAGGAGACT

CAACTTATCCCAACTCCTTGGCATGGATTCTTCCTCCGCGTCCCCCATCCGGCTGA

SEQ ID NO.4:

ATGAGCTTTAACGTGTTCCAAGGACAAGAGGGCTCAACTTTACTCCTTGGAAACCG

ATTCATAGATGATTCTGTGAGTTTGCCATTCGCGATTGGAGAAATAAAAGTGAAGTC

GGATCACATGATGGGACGTTCCCCTTGTTGCTCAAAGCATAGTGTTAACAGGGGTG

CTTGGACTGCGGTTGAGGACAGTTTACTGAGAAAATATATTGAAACCCACGGAGAA

GGTGGTTGGAGATCTCTTCCTAAGAAAGCAGGGCTACAACGATGTGGAAAGAGCT

GCAGGTTGAGATGGCTGAATTATCTCCGCCCCAACATCAAGCATGGTAATATTTCTG

CTGATGAAGAAGAGCTTATAATACGAATGCACGGACTCCTTGGGAACAGGTGGTCG

TTAATTGCAGGAAGAGTGCCAGGCCGAACGGACAACGAAATCAAGAATTACTGGAA

CACTCATTTGGGTAAGAAGGTTGCAGCTCTCAAGGGTGATGATACCAAAACGCATAA

AAATGTTGTGCAGAATAGCCCGACTCTTGCTTGTAATATGAATTCTAGTGATGATATG

CCATCGAGTTCAATTAGTAAACTGAAGAAAACAAATATGGACCGAAGTCGATCATCA

GCATTTGTAATCCCTAATGTACACAATTTGAAATATATAGATACCTCAAATCGCTTCTG U601941

CTCTCTTCAAGGAACCGTTCATGTGATGGATGGGGGAAAAAGCCTTCATTGTAATCC

TAAAGCTATAGGTCCGAGTTTGCAAGGAAATAGTCCCATGGATGACAAAATAGACTC

AAACCCAGTGAAGGCAAACTTGATCTCTACTGAACTGCATTCTACGGTGTCCCTAGT

AAAACTCCATGATGTCGAACCTCACGCCTCAGACGTCCTGGAGAATGATGATAATCT

CTATCTCAGTAACACAGTCTCAGAGGATGATGAGCTATTCCTTGATACTGATTGCTCA

ATGGAGGAAAGTCCAGAAATATTAAACTTGTTAAAGCATCATAATAACGTAAATACAG

AAGATGGCTCTAACATCACTCCTGCATCGACACCCGATGACCATATGTTAGACCAGA

TGAATGGCAAGAATATAATTCAGGAACAGAGAAGCCAAATCCTTGATGTCCTCAACT

TCTTTGAAGTTGGAGAAGCAGAGAAAGAGGTGTGCTGCAATATTCATGAGTGGGGG

CAGGATCGGCATTACTTGAAAGGTTCGTCTTCGCGTTTAATGGATGACAGACTAATT

CAATGGCCGGATGATCATGAGGATATGCAGCTGCAGGGAGGAAATGGTTTTGGAAT

TGCTAGTCCTCATCTCGATTATGCAGGTTTGACAGATGGTGCAACGTGGGAAGCAT

CTGTTTGGTATGAAGAGTAA

Further, the GbMYBs positively regulates the biosynthesis of flavonoids in G. biloba.

Further, the Gb-miR858a is involved in the biosynthesis of flavonoids in G. biloba by targeting the GbMYBs.

Further, content of flavonoids in G. biloba and expression level of GbMYBs in G. biloba are increased by exogenous hormone treatment.

Further, the exogenous hormone is MeJA or ABA.

Compared with the existing technology, the present invention boasts for the following advantages: one miR858 and three MYBs target genes are screened from the miRNA and transcriptome sequencing data of G. biloba, and the function of candidate genes in the flavonoid metabolic pathway of G. biloba is studied by bioinformatics analysis, gene cloning and genetic transformation techniques. The interaction between Gb-miR858a and target genes of GbMYBs was revealed. It was proved that GbMYB1, GbMYB39 and

GbMYB41 were all positive regulators of flavonoid synthesis. The regulatory mechanism of Gb-miR858a targeting GbMYBs involved in flavonoid synthesis was preliminarily elucidated, providing a theoretical basis for improving the flavonoid content of G. biloba using genetic engineering technology.

BRIEF DESCRIPTION OF THE DRAWINGS LU601941

Figure 1 illustrates a map of plant expression vectors;

Figure 1 illustrates a diagram showing the mode of regulating flavonoid synthesis by miRNA and its target genes in G. biloba;

Figure 3 illustrates cloning and sequence analysis of Gb-miR858a;

Figure 4 illustrates construction of miRNA overexpression vector;

Figure 5 illustrates in vivo verification of Gb-miR858a targeting GbMYB1;

Figure 6 illustrates the in vivo verification of Gb-miR858a targeting GbMYB39;

Figure 7 illustrates tissue-specific expression of GbMYBs;

Figure 8 illustrates flavonoid content in different organs of G. biloba;

Figure 9 illustrates effects of exogenous treatment on the expression of GbMYBs;

Figure 10 illustrates the effect of exogenous treatment on the flavonoid content of G. biloba;

Figure 11 illustrates electrophoresis of GbMYBs TA clone;

Figure 12 illustrates analysis of GbMYBs characteristics;

Figure 13 illustrates the electrophoresis of bacterial liquid detection of GbMYBs overexpression vector construction;

Figure 14 illustrates transformation of GbMYBs overexpression vector into

Agrobacterium;

Figure 15 illustrates homozygous screening of transgenic Arabidopsis;

Figure 16 illustrates PCR identification of GbMYBs transgenic Arabidopsis;

Figure 17 illustrates GUS staining of transgenic Arabidopsis;

Figure 18 illustrates the flavonoid content of GbMYBs transgenic Arabidopsis;

Figure 19 illustrates qRT-PCR analysis of the structural genes of the flavonoid synthesis pathway of GbMYB1 transgenic Arabidopsis;

Figure 20 illustrates the qRT-PCR analysis of the structural genes of the flavonoid synthesis pathway of GbMYB39 transgenic Arabidopsis;

Figure 21 illustrates the qRT-PCR analysis of the structural genes of the flavonoid synthesis pathway of GbMYB41 transgenic Arabidopsis.

SPECIFIC EMBODIMENTS LU601941

In order to further explain the present invention, the following is elaborated in combination with the following specific embodiments. 1. Materials 1.1 Experimental materials

G. biloba materials: The experimental subjects were 35 years old "bergamot" grafted G. biloba trees, derived from the G. biloba Science Park of the West Campus of Yangtze

University (112.15 ° E, 30.36 ° N).

Arabidopsis: The Arabidopsis ecotype used in the experiment was Landsberg erecta (Ler) wild type.

Tobacco: Nicotiana benthamiana was used in the experiment. 1.2 Experimental reagents 1.2.1 Strains and vectors

Escherichia coli competent Trelief 5a and Agrobacterium competent GV3101 were purchased from Beijing Qingke Biotechnology Co., Ltd.; p-MD19-T vector was purchased from Dalian Bao Biotechnology Co., Ltd.; pNC-Cam2304-MCS35S overexpression vector was donated by the Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences; pICH86988 and plCSL50008 recombinant expression vectors were donated by the Fruit and Tea Research Institute, Hubei Academy of

Agricultural Sciences.

The vector map is shown in Figure 1. 1.2.2 Preparation of culture media and reagents (1) LB medium: Dissolve 5 g yeast extract, 10 g of tryptone, and 10 g of NaCl in 1 L deionized water, adjust the pH to 7.0, autoclave for 20 min, and add the corresponding antibiotics. For solid medium, add 15 g of agar powder. (2) YEP medium: Dissolve 16.5 g of YEP medium powder in 1 L of deionized water, adjust the pH to 7.0, autoclave for 20 min, and add the corresponding antibiotics. For solid medium, add 15 g of agar powder. (3) MS medium: Dissolve 4.74 g of MS medium powder and 30 g of sucrose in 1 L of deionized water, adjust the pH to between 5.8 and 6.0, autoclave for 20 min, and add the corresponding antibiotics. For solid medium, add 8 g of agar powder.

(4) Arabidopsis thaliana inoculum infection solution: Dissolve 50 g of sucrose in 1 L ofjg01941 deionized water and filter the aqueous phase through a membrane filter to sterilize. (5) 2-Morpholineethanesulfonic acid (MES) resuspension stock solution: Dissolve 3.1325 g of MES powder in 10 mL deionized water, adjust the pH to 5.6 with KOH, and filter the aqueous phase through a membrane filter to sterilize to obtain 10 mM MES-KOH (pH=5.6); Dissolve 2.033 g of MgCl particles in 10 mL deionized water to obtain 10 mM

MgCl solution. (6) DNA crude extraction buffer: Add 25 mL of 1 M Tris-HCI (pH=8) solution, 10 mL of 0.5

M EDTA (pH=8) solution, 10 mL of 5 M NaCl solution, and 50 mL of 10% SDS solution to a volume of 500 mL. (7) 5/3 M potassium acetate solution: Add 23 mL of glacial acetic acid and 120 mL of potassium acetate solution to make up to 200 mL with water. 1.2.3 Experimental reagents

Table 1. Reagents

Reagents Manufacturer

DNA marker and 2xRapid Taq Master Mix Nanjing Vazyme Biotech Co.,

Ltd.

Antibiotics such as Ampicillin and Kanamycin Sangon Biotech (Shanghai)

Co., Ltd.

Bsal restriction enzyme, 10X Buffer G and 10 Thermo Fisher Scientific Inc. mM ATP

Diethyl pyrocarbonate (DEPC), quercetin, Shanghai yuanye Bio- kaempferol, isornamnetin, MeJA and ABA Technology Co., Ltd

Unless otherwise specified, the raw materials used in the present invention are all from conventional products purchased on the market.

Kits LU601941

The purification kit EasyPure® PCR Purification Kit and the plasmid extraction kit

EasyPure® Plasmid MiniPrep Kit were purchased from Beijing Quanshijin Biotechnology

Co., Ltd.; the Nimble Cloning kit was purchased from Hainan Nixing Biotechnology Co.,

Ltd.; the ClonExpress® Il One Step Cloning Kit, the reverse transcription kit HiScript® Ill 1st Strand cDNA Synthesis Kit (+gDNA wiper) and the fluorescence quantitative kit

ChamQTM Universal SYBR® gPCR Master Mix were purchased from Nanjing Vazyme

Biotech Co., Ltd.; T4 DNA Ligase, TaKaRa MiniBEST Universal RNA Extraction Kit and

TaKaRa MiniBEST Plant Genomic DNA Extraction Kit were purchased from TaKaRa

Bioengineering (Dalian) Co., Ltd.; the GUS staining kit was purchased from Beijing

Huayueyang Biotechnology Co., Ltd.

Table 2. PCR primer sequences

Name Sequence (5'-3") Description

F: ACCCCTTGATTAAACTGATCG

Gb-miR858a-T TA cloning primers

R: TGACGACTTTGCCTCTCA

F: ATGGGTCGGTCTCCTTGC

GbMYB1-T TA cloning primers

R: TTATACCCGCAGTTGCCTGT

F: ATGGGTCGAGCTCCGTGCTG

GbMYB39-T TA cloning primers

R: TCAGCCGGATGGGGGACG

F: ATGAGCTTTAACGTGTTCCA

GbMYB41-T TA cloning primers

R: TTACTCTTCATACCAAACAGATGC

M13

R: GAGCGGATAACAATTTCACAC primers

F: agtggtctctgtccagtcctATGGGTCGGTCTCCTTGC

R: Overexpression

GbMYB1-o0e ggtctcagcagaccacaagt TTATACCCGCAGTTGCCT Vector construction

GT

F: LU601941 agtggtetctatccagtectATGGGTCGAGCTCCGTGCT

G Overexpression

GbMYB39-0e

R: vector construction ggtctcagcagaccacaagtTCAGCCGGATGGGGGAC

G

F: agtggtctctgtccagtcctATGAGCTTTAACGTGTTCCA

Overexpression

GbMYB41-o0e R: vector construction ggtctcagcagaccacaagtT TACTCTTCATACCAAACA

GATGC pNC-—Cam2304— F: ATGGGTCGGTCTCCTTGC Overexpression

MYB1 R: TCATTAGGCACCCCAGGC bacteria liquid test pNC-Cam2304— F: ATGGGTCGAGCTCCGTGCTG Overexpression

MYB39 R: TGGCACGACAGGTTTCCCGAC bacteria liquid test pNC-—Cam2304— F: ATGAGCTTTAACGTGTTCCA Overexpression

MYB41 R: CCAGGCTTTACACTTTATGC bacteria liquid test pNC—Cam2304- F: TCGTCAACATGGTGGAGCA Overexpression

MCS35S R: CACCAGTCCCTGTTCTCGT bacteria liquid test

F: tacaattatcgatacaatgACCCCTTGATTAAACTGATC miRNA pICH86988- GTCC overexpression miR858a R: vector construction tcattaaagcaggacaagcTGACGACTTTGCCTCTCAT

TTCT

F: miRNA pICH86988— £399 tacaattatcgatacaatgCTTGCTTGTAGATTGTAGAC overexpression mi

AGGGA vector construction

R: LU601941 tcattaaagcaggacaagcGTCTGTGAAGGCTGTTCC

AGAGA

F:

GTGGTCTCAAATGCCGGTCGCACAGATAACGA

A miRNA

MYB1-GFP overexpression

R vector construction

TGGGTCTCACGAATTCGTTATCTGTGCGACCG

G

Embodiment 1

Identification of Gb-miR858a and its target gene GbMYBs by comprehensive sequencing screening 1.1 Screening of miRNAs associated with flavonoid synthesis and their target genes

In this study, based on miRNA data and transcriptome data, 9 enzyme genes as well as 22 transcription factors were found to be potential target genes of miRNAs (Figure 2). Of these, 5 conserved miRNAs and 11 novel miRNAs target 18 MYB transcription factors involved in flavonoid metabolism. Further analysis revealed that three MYBs transcription factors (Gb18153, Gb19333, and Gb22883), which are highly associated with flavonoid synthesis, were all targets of miR858. Members of the miR858 family have been reported to be involved in plant flavonoid biosynthesis in a variety of plants. Based on this, miRNAs predicted to be members of the miR858 family (MIR568) and their MYBs target genes were investigated in this experiment. 1.2 Gb-miR858a cloning and sequence characterization

The precursor sequence of miR858 gene was cloned using G. biloba cDNA as a template using specific primers, and the electrophoresis results showed that the target band was similar to the expected length (Figure 3A). The gel recovery product was ligated into the

T vector, transferred into E. coli, positive clones were picked, PCR amplification was performed with M13 detection primers, and the band length (402 bp) met the expected positive bacterial solution was sent for detection (Figure 3B), and the sequencing results showed that the length of the G. biloba miR858 precursor sequence was 250 bp (SEQ ID

NO.1).

Sequence alignment of the mature sequence of miR858 from G. biloba leaves with th&1601941 mature sequence of miR858 from A. thaliana, Solanum lycopersicum, Citrus, Malus domestica, aspen and European spruce showed that the mature sequence of miR858 in different species was highly consistent, so it was named Gb-miR858a (Figure 3C).

Through the RNA Folding Form website (http://www.unafold.org/mfold/applications/rna- folding-form.php), the secondary structure of Gb-miR858a was predicted, and the results showed that Gb-miR858a had a more stable secondary structure and could form a classical stem-loop structure (Figure 3D).

Embodiment 2

Validation of Gb-miR858a interaction with target gene GbMYBs by transient transformation in tobacco

The precursor sequences of Gb-miR858a and control miRNA (Gb-miR322) were amplified with vector construction primers, and Gb-miR858a (Figure 4A) and Gb-miR322 (Figure 4B) were cloned into the pICH86988 overexpression vector by one-step cloning.

The Golden Gate method was used to construct the overexpression vector of GOMYB1 target site (Figure 5A) fused with green fluorescent protein, the overexpression vector of

GbMYB1 mutant target site fused with GFP (Figure 5B), the overexpression vector of

GbMYB39 target site fused with GFP (Figure 6A), and the overexpression vector of

GbMYB39 mutant target site fused with GFP (Figure 6B), respectively. Tobacco leaves were injected after the vector was transformed into Agrobacterium tumefaciens GV3101 and left for three days to observe green fluorescence under UV light. The results showed that Gb-miR858a could specifically target and cleave GbMYB1 and GbMYB39, resulting in the disappearance of green fluorescence (Figure 5C, Figure 6C). After mutating the target sites of GbMYB1 and GbMYB39, Gb-miR858a could not be cleaved, indicating that

Gb-miR858a interacted with the target genes GbMYB1 and GbMYB39, and Gb-miR858a could regulate the biosynthesis of flavonoids in G. biloba by targeting GbMYB1 and

GbMYB39.

Embodiment 3

Analysis of GbMYBs gene expression and flavonoid content in different organs of G. biloba at different stages 3.1 Expression analysis of GbMYBs in different organs at different stages

In the present application, G. biloba root (R), stem (S), leaf (L), female flower (OS), male flower (M) and fruit (F) at different stages were used as experimental materials to analyze the expression levels of GOMYBs genes (Figure 7). GbMYB1, GbMYB39 and GbMYB41,501941 genes were expressed in different organs at different stages, and the expression levels of the genes in different organs basically showed a decreasing trend over the growth period, that is, the expression levels of the three genes in young organs were higher than those in organs at a later stage of development. In addition, the expression levels of all three genes in male flowers were significantly lower than those in female flowers. 3.2 Analysis of flavonoid content in different organs at different stages

Quercetin, kaempferol and isorhamnetin are three common flavonol glycosides, and their sum of contents is often used to calculate flavonoid content in G. biloba. In this experiment, the three monomers were successfully separated by fine-tuning the extraction method of flavonoids in G. biloba and exploring the determination method. As shown in Figure 8, flavonoids were detected in all organs of G. biloba, with the highest content in leaves (34.56 mg/g) and the lowest content in roots (0.33 mg/g) at L1 stage, and the content of flavonoids in different organs showed a decreasing trend over time.

The expression levels of GbMYB1, GbMYB39 and GbMYB41 genes in roots, stems, leaves and female flowers were similar to the trend of flavonoid content, and the gene expression levels in male flowers were inconsistent with flavonoid content, and it was speculated that the accumulation of flavonoid content in male flowers was dominated by other key genes. In summary, combined with gene expression and flavonoid content changes, it is speculated that GbMYB1, GbMYB39 and GbMYB41 genes play an important role in flavonoid biosynthesis in different organs of G. biloba.

Embodiment 4

Effect of exogenous treatment on expression level of GbMYBs and content of flavonoids in G. biloba

In this study, leaves of annual G. biloba seedlings were treated with 1 mM MeJA and 100

UM ABA, and GbMYBs expression levels are shown in Figure 9. Changes in flavonoid content are shown in Figure 10. It can be seen that the expression of GbMYBs gene was significantly increased after MeJA and ABA treatment, and the flavonoid content in G. biloba leaves gradually increased over time, and it is speculated that the three GbMYBs genes may be involved in the regulation of flavonoid metabolic pathways.

Embodiment 5

Cloning and bioinformatics analysis of G. biloba GbMYBs genes

GbMYB1, GbMYB39 and GbMYB41 genes were cloned from G. biloba cDNA 3$)601941 template, and the electrophoresis results showed that the target bands were similar to the expected length (Figure 11A, C, E). The gel recovery product was ligated into the T- vector, transferred into E. coli, positive clones were picked, and PCR amplification was performed with M13 detection primers, and the band lengths were as expected (Figure 10B, D, F). The positive bacterial fluid was sent for testing, and the results showed that the CDS sequences of GbMYB1, GbMYB39, and GbMYB41 genes were 819 bp (SEQ ID

NO.2), 1182 bp (SEQ ID NO.3), and 1383 bp (SEQ ID NO.4) in length, respectively.

Analysis of gene structure revealed that the GbMYB1, GbMYB39, and GbMYB41 genes all comprised three exons and two introns (Figure 12A). Further analysis revealed (Table 3) that the data indicated that all three GbMYBs proteins were unstable proteins and all were hydrophobic proteins. In addition, subcellular localization results showed that all three GbMYBs proteins were located in the nucleus.

In Figure 11: (A) GbMYB1 PCR amplification electropherogram; (B) GbMYB1 TA clone bacterial solution assay electropherogram; (C) GbMYB39 PCR amplification electropherogram; (D) GbMYB39 TA clone bacterial solution assay electropherogram; (E)

GbMYB41 PCR amplification electropherogram; (F) GbMYB41 TA clone bacterial solution assay electropherogram.

By NCBI comparison, three GbMYBs proteins were found to have the same R2 and R3 repeats as the R2R3 — MYB protein sequences of other species, indicating that these three genes belong to the R2R3 — MYB family members (Figure 12B). To further clarify the functions of the three GbMYBs, a phylogenetic tree was constructed by combining

MYB transcription factors associated with flavonoid synthesis with GbMYBs proteins in species such as Arabidopsis, Malus domestica and Prunus avium. Results As shown in

Figure 12C, GbMYB1 had the highest homology with PgMYB16 and was closely related to Arabidopsis S4 subfamily members AtMYB3, AtMYB4, AtMYB7 and AtMYB32;

GbMYB39 was in the same branch as Arabidopsis S7 subfamily members AtMYB11,

AtMYB12 and AtMYB111 and was closely related to GbMYB1; GbMYB41 was divided into S5 subfamily and had the highest homology with PrMYB2 of gymnosperm Pinus radiata and was closely related to AtMYB123.

Table 3. Basic information of GBMYBs protein

Name Amin | Relative | Total mean | Isoelectri | Instabilit | Fat Subcellu|-Y601941

O molecul | hydrophob | c point y index | coefficie | ar acids | ar ic index nt weight

GbMYB3 | 393 | 43.33 -0.639 5.54 62.17 70.28 Nucleus 9

GbMYB4 | 460 | 51.36 -0.615 5.53 48.67 76.91 Nucleus 1

The promoter regions of GOMYB1, GbMYB39, and GbMYB41 genes were extracted from the G. biloba genome (G. biloba genome data (http://gigadb.org/dataset/100613)) for analysis, and the results are shown in Figure 12D. The promoter sequences of the three genes not only contain basic elements such as the start transcription site and TATA-box, but also contain many cis-acting elements related to gene function. The most abundant elements in the promoter of the GbMYB1 gene are light-responsive elements, including

AE-box, I-box, and chs-CMA1a. In addition, it was found that it also contains a drought- induced element (MBS), a low temperature response element (LTR), an anaerobic induction response element (ARE), a meristem expression-related element (CAT-box), elements related to MeJA response (CGTCA and TGACG motifs), and gibberellin response elements (TATC-box and P-box). The most abundant elements in the promoter of the GbMYB39 gene are also light-responsive elements, including Box 4, G-box, and spl. In addition, one element responding to low temperature, one element involved in meristem expression, and four anaerobic induction response elements were found in its promoter sequence. Compared with GbMYB1, the promoter of GbMYB39 gene comprises elements responding to auxin (TGA) and abscisic acid (ABRE) in addition to elements responding to MeJA; the promoter of GbMYB41 gene comprises light response elements such as Box 4, G-box and GT1 motifs. In addition, multiple elements related to drought induction and anaerobic induction, one element involved in stress (TC-rich repeats), and one element involved in meristem expression were found. The promoter of

GbMYB41 gene also comprises elements responding to MeJA, as well as multiple elements responding to abscisic acid and salicylic acid (TCA).

In Figure 12: (A) GbMYBs gene structure analysis; (B) GbMYBs amino acid sequenggjgo1941 alignment; (C) GbMYBs phylogenetic tree analysis; (D) GbMYBs promoter analysis. At,

Arabidopsis thaliana; Os, Oryza sativa; Md, Malus domestica; Pa, Prunus avium; Vv, Vitis vinifera L.; Pb, Pyrus bretschneideri; Pt, Populus tremula; Pg, Picea aurantiaca; Pr, Pinus radiata; Gb, G. biloba.

Embodiment 6

Construction of Ginkgo GbMYBs gene overexpression vector

The NC cloning kit was used to connect the target genes to the pNC-Cam2304-MCS35S overexpression vector, transform Escherichia coli, and pick single colonies for bacterial liquid PCR verification (Figure 13). The positive clones were sent to the company for sequencing, and the results were consistent with the target gene sequence. The

GbMYB1, GbMYB39 and GbMYB41 genes were successfully recombined into the overexpression vector. The recombinant plasmid was transferred into Agrobacterium by chemical transformation, cultured on YEP solid culture medium plates comprising Kan antibiotics for 2-3 days, and single colonies were randomly picked. The bacterial liquid was verified by PCR using detection primers (Figure 14). The target band was consistent with the expected length, and it can be considered that the recombinant vector was successfully transferred into GV3101 Agrobacterium. The obtained positive bacterial liquid was added with an equal volume of 50% glycerol and stored in an ultra-low temperature refrigerator for later use.

In Figure 13: (A) GbMYB1 NC clone bacterial solution assay electropherogram; (B)

GbMYB39 NC clone bacterial solution assay electropherogram; (C) GbMYB41 NC clone bacterial solution assay electropherogram.

In Figure 14: (A) Agrobacterium tumefaciens transformed with GbMYB1 recombinant vector; (B) Agrobacterium tumefaciens transformed with GbMYB39 recombinant vector; (C) Agrobacterium tumefaciens transformed with GbMYB41 recombinant vector; (D)

Agrobacterium tumefaciens transformed with empty vector.

Embodiment 7

Transformation of target gene into Arabidopsis 7.1 Transgenic Arabidopsis homozygote screening

Recombinant vectors comprising GbMYB1, GbMYB39 and GbMYB41 genes and emptyjgo1941 vectors were prepared to infect wild-type Arabidopsis using Agrobacterium-mediated inflorescence infection, cultured in a light incubator until the seeds matured, and TO generation seeds were collected. The TO generation transgenic seeds were uniformly sown on MS solid medium comprising Kan for resistance screening after disinfection.

After 10-15 d, the plants with normal seed germination, tender green cotyledons color, rapid rooting and robust growth could be preliminarily identified as T1 generation transgenic Arabidopsis plants (Figure 15). Transgenic Arabidopsis seedlings identified as

T1 generation were transplanted into vegetative substrate soil and cultured in a light incubator until seeds matured, and seeds were collected by ramets and screened on resistant plates. Lines with a 3:1 ratio of normally grown seedlings to etiolated seedlings were identified as T2 generation transgenic Arabidopsis plants. The T2 transgenic plants were transplanted and cultured until the seeds matured, and the seeds were collected by ramets and screened for resistance, and the seeds that no longer developed trait separation were homozygous for the T3 generation.

In Figure 15: (A) screening of GbMYB1 transgenic plants at T1, T2, and T3; (B) screening of GbMYB39 transgenic plants at T1, T2, and T3; (C) screening of GbMYB41 transgenic plants at T1, T2, and T3; and (D) screening of empty vector transgenic plants at T1, T2, and T3. 7.2 PCR Positive Identification

After the leaves of transgenic plants grew to a certain size, 1 - 2 leaves were taken to extract DNA, and positive PCR identification was performed using the detection primers constructed by the vector, with wild-type Arabidopsis thaliana under the same growth conditions as the negative control (C) and Agrobacterium tumefaciens bacterial solution comprising the recombinant vector as the positive control (P). Electrophoresis results (Figure 16) showed that the bands amplified from transgenic Arabidopsis were consistent with the positive control PCR results, and there was no target band amplification in wild- type plants. The above results showed that overexpression vectors comprising GbMYB1,

GbMYB39 and GbMYB41 genes were successfully transformed into Arabidopsis.

In Figure 16: (A) PCR identification of GbMYB1 transgenic Arabidopsis; (B) PCR identification of GbMYB39 transgenic Arabidopsis; (C) PCR identification of GbMYB41 transgenic Arabidopsis; (D) PCR identification of empty vector transgenic Arabidopsis. 7.3 GUS staining

The pNC — Cam2304 — MCS35S overexpression vector comprised a GUS reporter geneygo1941 and transgenic plants were further verified by GUS staining of Arabidopsis seedlings. As shown in Figure 17, no GUS gene was expressed in wild-type Arabidopsis leaves, and blue spots were present in roots, stems, and leaves of transgenic plants. The above results indicated that GoMYB1, GbMYB39 and GbMYB41 overexpression vectors were successfully expressed in transgenic plants.

In Figure 17: (A) wild-type Arabidopsis; (B) empty vector transgenic Arabidopsis; (C)

GbMYB1 transgenic Arabidopsis; (D) GbMYB39 transgenic Arabidopsis; (E) GbMYB41 transgenic Arabidopsis.

Embodiment 8

Analysis of flavonoid content in transgenic Arabidopsis

To investigate the effect of GbMYB1, GbMYB39 and GbMYB41 genes on flavonoid biosynthesis in Arabidopsis, flavonoids were extracted from leaves of wild-type and transgenic Arabidopsis thaliana, and the contents of quercetin, kaempferol and isorhamnetin were detected by HPLC. The results showed that there was no significant difference in flavonoid content in empty vector transgenic Arabidopsis compared with wild type, and flavonoid content was significantly increased in GbMYB1, GbMYB39 and

GbMYB41 transgenic Arabidopsis (Figure 18). showed that overexpression of GbMYB1,

GbMYB39 and GbMYB41 genes in Arabidopsis could promote accumulation of flavonoid content.

Embodiment 9

QRT PCR analysis of structural gene of flavonoid synthesis pathway in transgenic

Arabidopsis

To further investigate the effect of GbMYBs gene on flavonoid synthesis, 36 structural genes of flavonoid synthesis pathway in Arabidopsis (Table 4) were downloaded by this experimental search, and AtPP2A (AT1G13320) was used as an internal reference gene to analyze its expression levels in wild-type and transgenic Arabidopsis using gRT — PCR technique.

Table 4. aRT — PCR primer sequences LU601941

Name Sequence (5'—3")

F: ATGGGTCGGTCTCCTTGC

GbGAPDH

R: TTATACCCGCAGTTGCCTGT

F: ATGGGTCGGTCTCCTTGC

GbMYB1-Q

R: TTATACCCGCAGTTGCCTGT

F: ATGGGTCGAGCTCCGTGCTG

GbMYB39-Q

R: TCAGCCGGATGGGGGACG

F: ATGAGCTTTAACGTGTTCCA

GbMYB41-Q

R: TTACTCTTCATACCAAACAGATGC

AtPP2A F: TCATTGCTCGTGCTCTTGGA (AT1G13320) R: CTTCCGCCATAGCCAAAAGC

AtPAL1 F: TCTCGTTCCTCTCTCCTACATC (AT2G37040) R: CGGAGCTGATTCCTGCTAAT

AtPAL2 F: TACCTCTCCGTGGAACCATTA (AT3G53260) R: ACCGGTGGCTTTGGAATTAG

AtPAL3 F: CGACCCACTTAACTGGAATGT (AT5G04230) R: CTGCACCGTTCCTTTCCTATAA

AtPAL4 F: GTGACCTTGTTCCTCTCTCTTAC (AT3G10340) R: GATACTCCGGCGAGCTTAAA

AtC4H F: AGCTACCTCCAGGTCCTATAC (AT2G30490) R: CGCCGAATTTCTTAGCGTAATC

At4CL1 F: GGAGAAACAGAGCAACAACAAC (AT1G51680) R: AGATGTAGTCGTGGAGAGATAGG

At4CL2 F: TCCACGTAACATCTCGGAAAC LU601941 (AT3G21240) R: TCGGGAGGAGGATCATTACA

At4CL3 F: TCCGGAGCCAAACTCATAATC (AT1G65060) R: GGTGTTGGTTCATCGGTAGT

At4CL5 F: AACCAACATGCGGAGGATAG (AT3G21230) R: AGGTAAGCAACGGCAAGAA

At4CL8 F: ACAACATTCATCTCCTCCCAAA (AT5G38120) R: GATCCACGGCCATCCATAAA

AtACC1 F: GTGGAGATGGCTGAAGTAACA (AT1G36160) R: GATGCTGGAGGACCAAGAAATA

AtACC2 F: AGGATCAATGCGGAACATATCA (AT1G36180) R: CCTCAGCCATCTCCACAATAAG

AtCHS F: GTCCCTAAGCTAGGCAAAGAAG (AT5G13930) R: GTAGTGCAGAAGACGACATGAG

AtCHSL2 F: CCGCAAGGGTAGAGAAACAA (AT4G00040) R: GAGATTCTCCTGGGAAACAACA

AtCHSL3 F: TGACCGAGATGGCAGTAGAA (AT4G34850) R: GCTTCGCTTGAGGAGACATAG

AtCHI F: GTAACGCCGTTCCTTCTCTATC (AT3G55120) R: ACGCACCGGTGACTATTTC

AtCHIL F: CGTTTGGCTGAGGAGGATAAG (AT5G05270) R: TGGCTGAGAAATGGTAAGTGATAA

AtFAP1 F: CTCTCACCGTTCGTCTCTTTC (AT3G63170) R: CGAGAAACCGGTCTTAGGTTC

AtFAP2 F: ACGTCTCTGTGTTCCCATTTC LU601941 (AT2G26310) R: GTAGCCTCTCAGCTTCATTAACA

AtFAP3 F: GTGTGTCTCTCTACGCATTTCT (AT1G53520) R: GAGAGACGTAGTTCCCAGTTTG

AtF3H F: AGGAGGATTCATCGTCTCTAGT (AT3G51240) R: CACCGTGAGTAGTCTCTGTTTC

AtF3H F: CCTTACCTTCAGGCGGTTATC (AT5G07990) R: GAGTCGATCCTTTCGGGATATG

AtFLS1 F: CCTGAATACAGGGAGGTGAATG (AT5G08640) R: TCCATCCGAGAGAATCCCTAATA

AtFLS2 F: TCAACACATTTCTCCACCATCT (AT5G63580) R: CCACTCTTCACTCCCTTTCAC

AFLS3 F: CCAGAAGACTCCTTGGACATAG (AT5G63590) R: CCAGATGCGGTGAAAGAGAT

AtFLS5 F: TGGGACGAACATCTCTTTCAC (AT5G63600) R: CCTCCGTCACCTCCCTATATT

AtFLS6 F: TATCTCCACCGTGTCTCCTAAC (AT5G43935) R: CACTCTTCGCTCGCTTTCA

AtDFR F: CGTTCGAGATCCCGGTAATTT (AT5G42800) R: TCGTAGCTTCCTTCCTCAGATA

AtANS F: CTGTGGAAGAGAAGGAGAAGTATG (AT4G22880) R: CTCGCGTTGTTAGCCAATTTAC

AIRT F: AAGCGGCTCCACGTATTT (AT1G30530) R: ACCAGAAGAAGGCATCTGTTAG

AtUFGT F: CTTCCACCGTCTTCTCTTTCTT LU601941 (AT5G17050) R: CCGTCGGCAATATCGTATACTC

AtUF3GT F: CGCAGAGAAAGGTCACAAGA (AT5G54060) R: GGGATAGAGATGGTGTGGAAAG

AtUGT73C6 F: GAGTCTGGTTTGCCCATCAA (AT2G36790) R: GTTATCTGCTCCATCGTGGTAAG

AtOMT1 F: CCTACCGAGATCGCTTCTAAAC (AT5G54160) R: GAGCAGGTTAAGACGGAGTAAG

AtAOMT1 F: AATGAAGGAACTCAGGGAAGTG (AT4G34050) R: CTCCGATCTCCATTGTGTTCTT

AtAOMT7 F: CTCAAGCTGGTATGGCTACTG (AT4G26220) R: AGCAGTGAGGAGAAGAGAGTAT 9.1 Gene expression analysis of flavonoid synthesis pathway structure in GbMYB1 transgenic Arabidopsis

Key structural gene expression of the flavonoid synthesis pathway differed significantly in

GbMYB1 transgenic Arabidopsis compared to wild type (Figure 19). In the phenylpropanoid metabolic pathway, the expression levels of key enzyme genes AtPAL1,

AtPAL2 and AtPAL4 were significantly up-regulated, and only the expression level of

AtPAL3 showed a down-regulation pattern; the expression level of AtC4H gene was up- regulated; the 4CL gene involved in p-coumaroyl CoA synthesis, except for the down- regulation of At4CL3 gene expression level, the other four 4CL gene levels were significantly up-regulated, with At4CL5 having the most significant change. In addition, two acetyl CoA carboxylase (ACC) genes catalyzing the carboxylation of acetyl CoA to malonyl CoA were up-regulated. Some coumaroyl CoA was catalyzed to form naringenin into the flavonoid synthesis pathway, in which AtCHS and AtCHSL3 gene expression levels changed significantly and AtCHSL2 gene expression levels were down-regulated; in CHI gene, AtCHI did not change significantly, AtCHIL and AtFAP2 genes were induced to be up-regulated, and AtFAP1 and AtFAP3 gene expression levels were down- regulated.

In addition, AtF3H gene involved in dinydrokaempferol synthesis was up-regulated; Ath31601941 'H gene involved in dihydroquercetin synthesis showed a tendency to be down-regulated.

In the flavonol synthesis pathway, dihydroflavonols are catalyzed to form flavonols, and their key enzyme gene FLS, all four FLS genes are up-regulated except AtFLS5 expression levels, which do not change significantly. Some dihydroflavonols are involved in anthocyanin synthesis, of which both AtDFR and AtANS genes are down-regulated, and the final gene of this synthesis pathway, flavonoid glycosyltransferase (UFGT), is up- regulated, and AtUF3GT gene, which converts anthocyanins into anthocyanins, is up- regulated. Two other UDP glycosyltransferase (UGT) genes, AtRT and AtUGT73C6, were up- and down-regulated, respectively. O — methyltransferases (OMT) are a class of modifying enzymes that further methylate the products of flavonoid synthesis to generate diverse flavonoids, and the expression levels of all three OMT genes were upregulated in transgenic plants. 9.2 Gene expression analysis of flavonoid synthesis pathway structure in GbMYB39 transgenic Arabidopsis

The flavonoid content of GbMYB39 transgenic Arabidopsis was significantly increased compared with wild type, and the expression levels of structural genes in the synthetic pathway were significantly different (Figure 20). In transgenic plants, AtPAL1 and AtPAL2 expression levels were significantly up-regulated, AtPAL3 and AtPAL4 expression levels were down-regulated; AtC4H gene expression levels were up-regulated; 4CL gene, except At4CL1 gene expression levels did not change significantly, the other four 4CL gene expression levels were significantly up-regulated. Unlike GbMYB1 transgenic plants, expression levels of both AtACC1 and AtACC2 were down-regulated in GbMYB39 transgenic plants. In addition, three CHS genes, AtCHS expression was up-regulated, and AtCHSIL2 and AtCHSIL3 showed a small down-regulation, among the CHI genes,

AtCHI gene levels were significantly up-regulated, and other genes were consistent with the trend in GbMYB1 transgenic plants. AtF3H gene involved in dihydrokaempferol synthesis was up-regulated; AtF3 'H gene involved in dinydroquercetin synthesis showed a significant down-regulation trend. Flavonol synthesis pathway key enzyme genes FLS,

AtFLS1 did not change significantly, AtFLS2 and AtFLS6 expression was up-regulated,

AtFLS3 and AtFLS5 expression was down-regulated.

Both AtDFR and AtANS genes involved in anthocyanin synthesis were also dowfggo1941 regulated. Among the four UGTs, AtRT, AtUF3GT and AtUGT73C6 gene expression levels did not change significantly, and AtUFGT gene was up-regulated. In addition, all three OMT gene expressions were upregulated, with AtOMT1 gene expression levels being significantly upregulated. 9.3 Gene expression analysis of flavonoid synthesis pathway structure in GbMYB41 transgenic Arabidopsis

Expression levels of key structural genes in the flavonoid synthesis pathway were also regulated in GbMYB41 transgenic Arabidopsis (Figure 21). PAL gene, AtPAL1, AtPAL2 and AtPAL4 expression levels were significantly up-regulated, AtPAL3 was down- regulated; AtC4H gene expression level was up-regulated; 4CL gene, At4CL2 gene level was not significantly changed, At4CL3 was down-regulated, At4CL1, At4CL5 and At4CL8 were up-regulated. Unlike GbMYB1 and GbMYB39 transgenic plants, AtACC1 expression was down-regulated and AtACC2 expression was up-regulated in GbMYB41 transgenic plants. CHS gene, AtCHS expression was up-regulated, AtCHSL2 and

AtCHSL3 expression was down-regulated; in CHI gene, AtFAP2 was not significantly changed, AtCHI and AtCHIL genes were induced and up-regulated, AtFAP1 and AtFAP3 gene expression levels were down-regulated; AtF3H gene expression was up-regulated;

AtF3 'H gene expression was not significantly changed; in FLS gene, AtFLS1 and AtFLS3 expression was down-regulated, AtFLS2, AtFLS5 and AtFLS6 expression was up- regulated; in anthocyanin synthesis pathway, AtDFR and AtANS genes were down- regulated; UGT gene, AtRT and AtUF3GT expression levels were up-regulated, AtUFGT and AtUGT73C6 expression levels were not significantly changed; OMT gene expression levels were up-regulated.

The above description is only a preferred specific implementation manner of the present invention, but the scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

SEQUENCE LISTING LU601941 <ST26SequenceListing dtdVersion="V1_3" fileName="Application of miR858a-GbMYBs regulating the biosynthesis of flavonoids in Ginkgo biloba L.xml" softwareName="WIPO

Sequence" softwareVersion="2.3.0" productionDate="2025-06-05"> <ApplicantFileReference>Yangtze University</ApplicantFileReference> <ApplicantName languageCode="en">Yangtze University</ApplicantName> <ApplicantNameLatin>Yangtze University</ApplicantNameLatin> <InventorName languageCode="en">Jiabao Ye</InventorName> <InventorNameLatin>Yejiabao</InventorNameLatin> <InventionTitle languageCode="zh">Application of miR858a-GbMYBs regulating the biosynthesis of flavonoids in Ginkgo biloba L.</InventionTitle> <SequenceTotalQuantity>4</SequenceTotalQuantity> <SequenceData sequencelDNumber="1"> <INSDSeq> <INSDSeq_length>250</INSDSeq_length> <INSDSeq_moltype>DNA</INSDSeq_moltype> <INSDSeq_division>PAT</INSDSeq_division> <INSDSeq_feature-table> <INSDFeature> <INSDFeature_key>source</INSDFeature_key> <INSDFeature_location>1..250</INSDFeature_location> <INSDFeature_quals> <INSDQualifier> <INSDQualifier_name>mol_type</INSDQualifier_name> <INSDQualifier_value>other DNA</INSDQualifier_value> </INSDQualifier> <INSDQualifier id="q2"> <INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>Ginkgo biloba</INSDQualifier_value> LU601941 </INSDQualifier> </INSDFeature_quals> </INSDFeature> </INSDSeq_feature-table> <INSDSeq_sequence>accccttgattaaactgatcatecgtegagecttttcctgagctatattgggattttgggage cttttcctgatgtgagaccgccagtaattcttaatttcgttgtctgticggectggceatctictctgcaatcaaagaccatctgcac atgccaccaaaacatgatcaaataacaggtaaaaattgcccaaaatttatttagcgcggataggtggcectttcatgtagaa atgagaggcaaagtcgtca</INSDSeq_sequence> </INSDSeq> </SequenceData> <SequenceData sequencelDNumber="2"> <INSDSeq> <INSDSeq_length>819</INSDSeq_length> <INSDSeq_moltype>DNA</INSDSeq_moltype> <INSDSeq_division>PAT</INSDSeq_division> <INSDSeq_feature-table> <INSDFeature> <INSDFeature_key>source</INSDFeature_key> <INSDFeature_location>1..819</INSDFeature_location> <INSDFeature_quals> <INSDQualifier> <INSDQualifier_name>mol_type</INSDQualifier_name> <INSDQualifier_value>other DNA</INSDQualifier_value> </INSDQualifier> <INSDQualifier id="q12"> <INSDQualifier_name>organism</INSDQualifier_name> <INSDQualifier_value>Ginkgo biloba</INSDQualifier_value>

</INSDQualifier> LU601941 </INSDFeature_quals> </INSDFeature> </INSDSeq_feature-table> <INSDSeq_sequence>atgggtcggtctccttgetgtgaaaaagctcataccaacaaaggggcettggaccaaaga ggaagacgacaggcttattgcacacattcaggctcatggagaaggatgctggaggtccctccccaaggcetgcagggcetg ctgcgttgtgggaagagttgcaggctccggtggataaactatctccgtcccgacctcaagegtgggaatttttcagaagaa gaagacgaactcatcatcaagctccacgcecctcttagggaacaagtggtctctcatcgcaggaagattgeccggtegeac agataacgaaataaaaaattattggaacacccatataaaaagaaaattgattagcaggggcgtagacccacagtcgcea tcaacccatccagaccggctatcagtcggccaagctaattgcageggattctccacagatttttgcaticcagecggaatacg ccggagatggcagacttcttcgaatacaatgacatgatgagtacatcagtggagcagcagcagacggcagcetccagcta attcggatggtcagggcagtggaatcgaagaccatccggacctcaacctcgaattgtgtatcagtttgecctetggecegg ctagcataagcagagccaccacagccgattcaaaggaggtttccagtccaaatagtgeccgggtcgaaggcagacgcetg tgtgctgtcacatgggattgcaaatcaatggaggaggcetgetgtgaggataatagatgctcacatgaagaccatgtggcetg cgacggctggccattttaattacaggcaactgcgggtataa</INSDSeq_sequence> </INSDSeq> </SequenceData> <SequenceData sequencelDNumber="3"> <INSDSeq> <INSDSeq_length>1182</INSDSeq_length> <INSDSeq_moltype>DNA</INSDSeq_moltype> <INSDSeq_division>PAT</INSDSeq_division> <INSDSeq_feature-table> <INSDFeature> <INSDFeature_key>source</INSDFeature_key> <INSDFeature_location>1..1182</INSDFeature_location> <INSDFeature_quals> <INSDQualifier> <INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value> LU601941 </INSDQualifier> <INSDQualifier id="q14"> <INSDQualifier_name>organism</INSDQualifier_name> <INSDQualifier_value>Ginkgo biloba</INSDQualifier_value> </INSDQualifier> </INSDFeature_quals> </INSDFeature> </INSDSeq_feature-table> <INSDSeq_sequence>atgggtcgagctcegtgcetgtgccaaggttggattgaataggggecgatggactgctga agaagatgacattttagctagatatattgaagcccatggcgaaggttgctggcgagcacttcccaaaaatgcaggattget gcggtgcggaaagagttgcaggtigagatggatcaactatcticgticggatgtgaagegtggcaacatttgcgaagaag aagaggagcttatcatcaaactccacaatcttctgggcaatcggtggtcattgattgcaggcecgtatgectggtcgaacgga caatgagataaaaaactactggaatactcatctaagccggaagctcgtagacagaggaatcaatccecgtgacccacaa gccattagatgctaaagtcatgcctaagccctccgeatgtatcagcaatgcggcgaagcetgaagaaaacagacgagtgt ggtattgttgatcaagtgtctggaataacccgacgagatgtggttttgcagaagcccgagaaagaagagaatccaagatc tcgagtaagccagaaaactgtggaaaaaaacgcagaggattccactgttaaaggcaaaaggaagaggacttcgttaa aattggcttccagcagtgaaggccgaggtcagaagctgaaaatgttgggegaccttaaattaactgaacaaccttcctcca cagtttcagtgcagtcgagctgtgaatcatgttccgacttcgattccccteccecectggaggecatgacatctaatectagtigttct gatcaggctcagttgtittgtgaaaaagcticacagagtgcccaggtagaggcaaaggaaaatgaagattacaaggtac aagatgcaggaattgcggtagcattacaggaagatccaagcagttcatttactgttaattcggattctctgtgggataattcgt tttcgtcgcagatactttcttcagacatggacttgtttggatcattagaaacggaagctctgticgagtattgtitacctaatgecgg atgcgaattcaaatgcagagagcatggagatgtctggcgatccagacgaactgtggtctttctttcagtctgcaaatgcagg aggaggagactcaacttatcccaactccttggcatggattcttcctcecgegteeccececateccggetga</INSDSeq_seq uence> </INSDSeq> </SequenceData> <SequenceData sequencelDNumber="4"> <INSDSeq> <INSDSeq_length>1383</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype> LU601941 <INSDSeq_division>PAT</INSDSeq_division> <INSDSeq_feature-table> <INSDFeature> <INSDFeature_key>source</INSDFeature_key> <INSDFeature_location>1..1383</INSDFeature_location> <INSDFeature_quals> <INSDQualifier> <INSDQualifier_name>mol_type</INSDQualifier_name> <INSDQualifier_value>other DNA</INSDQualifier_value> </INSDQualifier> <INSDQualifier id="q16"> <INSDQualifier_name>organism</INSDQualifier_name> <INSDQualifier_value>Ginkgo biloba</INSDQualifier_value> </INSDQualifier> </INSDFeature_quals> </INSDFeature> </INSDSeq_feature-table> <INSDSeq_sequence>atgagctttaacgtgttccaaggacaagagggctcaactttactccttggaaaccgattca tagatgattctgtgagtttgccattcgcgattggagaaataaaagtgaagtcggatcacatgatgggacgttccecttgttget caaagcatagtgttaacaggggtgcttggactgcggttgaggacagtttactgagaaaatatattgaaacccacggagaa ggtggttggagatctcticctaagaaagcagggctacaacgatgtggaaagagctgcaggttgagatggctgaattatctc cgccccaacatcaagcatggtaatatttctgctgatgaagaagagcttataatacgaatgcacggactccttgggaacag gtggtcgttaattgcaggaagagtgccaggccgaacggacaacgaaatcaagaattactggaacactcatttgggtaag aaggttgcagctctcaagggtgatgataccaaaacgcataaaaatgttigtgcagaatagcccgactcttgcettgtaatatga attctagtgatgatatgccatcgagticaattagtaaactgaagaaaacaaatatggaccgaagtcgatcatcagcattigt aatccctaatgtacacaatttgaaatatatagatacctcaaatcgcttctcctctcticaaggaaccgttcatgtgatggatggg ggaaaaagccttcattgtaatcctaaagctataggtccgagtttgcaaggaaatagtcccatggatgacaaaatagactca aacccagtgaaggcaaacttgatctctactgaactgcattctacggtgtccctagtaaaactccatgatgtcgaacctcacg cctcagacgtcctggagaatgatgataatctctatctcagtaacacagtctcagaggatgatgagctattecttgatactgatt 601941 gctcaatggaggaaagtccagaaatattaaacttgttaaagcatcataataacgtaaatacagaagatggctctaacatc actcctgcatcgacacccgatgaccatatgttagaccagatgaatggcaagaatataaticaggaacagagaagccaa atccttgatgtcctcaacttctttgaagttggagaagcagagaaagaggtgtgctgcaatattcatgagtgggggcaggatc ggcattacttgaaaggttcgtcttcgegtttaatggatgacagactaattcaatggccggatgatcatgaggatatgcagcetg cagggaggaaatggttttggaattgctagtcctcatctcgattatgcaggtttgacagatggtgcaacgtgggaagceatctgt ttggtatgaagagtaa</INSDSeq_sequence> </INSDSeq> </SequenceData> </ST26SequenceListing>

Claims (6)

CLAIMS LU601941

1. An application of miR858a-GbMYBs regulating the biosynthesis of flavonoids in Ginkgo biloba L., comprising screening and identification of Gb-miR858a-GbMYBs regulatory components.

2. The application according to claim 1, a precursor sequence of Gb-miR858a is shown as SEQ ID NO. 1; the GbMYBs comprise GbMYB1, GbMYB39 and GbMYB41, with corresponding nucleotide sequences shown as SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4 respectively.

3. The application according to claim 2, wherein the GbMYBs positively regulates biosynthesis of flavonoids in G. biloba.

4. The application according to claim 3, wherein the Gb-miR858a is involved in the biosynthesis of flavonoids in G. biloba by targeting the GbMYBs.

5. The application according to claim 4, wherein content of flavonoids in G. biloba and expression level of GbMYBs in G. biloba are increased by exogenous hormone treatment.

6. The application according to claim 5, wherein the exogenous hormone is MeJA or

ABA.