Application and method of target gene for regulating dendrobium flower type change by AP3-3 transcription factor

Abstract

The invention relates to the technical field of dendrobium nobile flower type molecular mechanism regulation and control, in particular to application and a method for regulating and controlling a target gene of dendrobium nobile flower type change through an Ap3-3 transcription factor, and the method comprises the following steps: S1, obtaining a binding site of the transcription factor DoAP3-3 in a dendrobium officinale genome through DNA affinity purification sequencing (DAP-seq); s2, analyzing differential expression genes of the dendrobium officinale wild type plant and the DoAP3-3 overexpression plant through transcriptome sequencing (RNA-se q); s3, performing intersection analysis on the binding site associated gene identified in the step S1 and the differential expression gene in the step S2, and screening out a target gene directly regulated and controlled by DoAP3-3; key target genes such as MADS6, ERECTA and the like are identified through a DAP-seq and RNA-seq combined technology, and a foundation is laid for preliminarily analyzing a regulation mechanism between the floral organ characteristic gene DoAP3-3 and the target gene thereof and exploring a highly-specific and diversified flower form forming mechanism in orchid plants.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular mechanism regulation of Dendrobium flower shape, and in particular to an application and method of a target gene for regulating the change of Dendrobium flower shape by an Ap3-3 transcription factor. Background Art

[0002] Dendrobium plants are an important group in the Orchidaceae family. Their floral organ morphological diversity (such as differently shaped lip petals and unusual flowers) has extremely high ornamental value and evolutionary research significance. Traditional studies believe that flower type differentiation is mainly regulated by MADS-box transcription factors, among which class B genes (AP3 / PI) play a core role in the development of petals and stamens. However, due to the high degree of specialization of organs such as the lip petals, the AP3 homologous genes of orchid plants may have unique functional differentiation. For example, the DoAP3-3 gene of Dendrobium officinale has been shown to be involved in the formation of the lip petals, but the downstream target gene network directly regulated by it and the molecular mechanism for the formation of flower type specificity remain unclear.

[0003] Currently, the analysis of transcription factor regulatory networks primarily relies on chromatin immunoprecipitation sequencing (ChIP-seq). However, the lack of specific antibodies and stable genetic transformation systems in non-model plants such as Dendrobium has limited its application. DNA affinity purification sequencing (DAP-seq), an emerging in vitro binding site detection technology, has been applied in Arabidopsis and rice, but its feasibility in orchids and its combination with RNA-seq for target gene analysis have not yet been reported.

[0004] Regarding flower pattern regulation mechanisms, it is known that the ERECTA gene influences petal size by regulating cell division, and the AS2 gene is involved in establishing petal polarity. However, evidence remains lacking as to whether these genes are directly regulated by the AP3-3 transcription factor. Furthermore, the molecular basis for the morphological variation of the peculiar flower organs and lip petals in Dendrobium remains unexplained. Therefore, developing transcription factor target gene identification techniques suitable for Dendrobium, deciphering the key pathways involved in AP3-3 flower pattern regulation, and establishing efficient genetic transformation systems to support functional studies remain urgent technical bottlenecks. Summary of the Invention

[0005] The purpose of the present invention is to provide an application and method of the target gene of the Ap3-3 transcription factor in regulating the change of Dendrobium flower shape. By combining DAP-seq and RNA-seq technology, key target genes such as MADS6 and ERECTA were identified, which laid the foundation for the preliminary analysis of the regulatory mechanism between the floral organ characteristic gene DoAP3-3 and its target genes, and explored the formation mechanism of highly specialized and diversified floral morphology in orchid plants.

[0006] In order to achieve the above technical objectives and the above technical effects, the present invention is implemented through the following technical solutions:

[0007] An application of an AP3-3 transcription factor to a target gene for regulating flower shape changes in Dendrobium, wherein the target gene is selected from at least one of MADS6, NAC029, NAC054, WOX3, AS2, ERECTA, and AG, and the sequences are shown in SEQ ID NOs. 1-7, respectively;

[0008] Its promoter sequence includes:

[0009] LOC110101756(MADS6);

[0010] LOC110105798(NAC029);

[0011] LOC110106875(NAC054);

[0012] LOC110111262(WOX3);

[0013] LOC110111071(AS2);

[0014] LOC110094200(ERECTA);

[0015] LOC110098137(AG);

[0016] The application includes changing the morphology, size or symmetry of Dendrobium flower organs by gene editing or expression regulation of the target gene.

[0017] In another aspect, the present invention provides a method for identifying target genes of Ap3-3 transcription factors that regulate changes in flower shape in Dendrobium officinale, comprising the following steps:

[0018] S1: DNA affinity purification sequencing (DAP-seq) was used to obtain the binding sites of the transcription factor DoAP3-3 in the Dendrobium officinale genome;

[0019] S2: Transcriptome sequencing (RNA-seq) was used to analyze the differentially expressed genes between wild-type Dendrobium officinale plants and DoAP3-3 overexpressing plants;

[0020] S3: Intersection analysis of the binding site-associated genes identified in step S1 and the differentially expressed genes in step S2 was performed to screen out target genes directly regulated by DoAP3-3;

[0021] The target gene is selected from at least one of MADS6, NAC029, NAC054, WOX3, AS2, ERECTA, and AG.

[0022] On the other hand, the present invention provides a method for regulating the development of Dendrobium officinale flower shape, by overexpressing the transcription factor DoAP3-3 to regulate the expression level of the above-mentioned target gene, thereby changing the morphology, size or symmetry of the floral organ.

[0023] Beneficial effects of the present invention:

[0024] The present invention uses a combination of DAP-seq and RNA-seq to effectively identify the target genes of the Ap3-3 transcription factor that regulate changes in Dendrobium flower shape. Traditional transcription factor target gene research usually relies on ChIP-seq technology, but ChIP-seq is greatly limited in Dendrobium due to the lack of specific antibodies and stable transformation systems. The present invention uses DAP-seq technology to identify the binding sites of DoAP3-3 in the Dendrobium officinale genome using an in vitro system, overcoming the limitations of in vivo experiments. After performing RNA-seq differential expression analysis on wild-type Dendrobium and DoAP3-3 overexpressing plants, the target genes directly regulated by DoAP3-3 were identified. This not only improves the accuracy and efficiency of target gene identification, but also verifies its feasibility for the first time in orchid plants, establishing a technical platform for studying the molecular mechanisms of floral organ development in the Dendrobium genus.

[0025] The present invention reveals the regulatory mechanism of the DoAP3-3 transcription factor in the development of Dendrobium flower patterns, enhancing the understanding of the molecular basis for the formation of complex flower patterns in orchids. Through DAP-seq analysis, it was found that DoAP3-3 mainly binds to the gene promoter region and identified multiple key target genes related to flower development, including MADS6, NAC029, WOX3, ERECTA, etc. These genes play an important role in the formation and development of floral organs. MADS6 regulates floral organ determinacy, ERECTA affects petal cell division and size, WOX3 participates in sepal morphogenesis, and AS2 is an important factor in the establishment of petal polarity. The above target gene regulatory network reflects the core role of DoAP3-3 in the multi-level regulation of floral organ morphology and development. Combined with the direct interaction between DoAP3-3 and MADS6 verified by yeast one-hybrid experiments, it supports its mechanism of regulating target gene expression through direct binding, revealing the biological function of DoAP3-3 in flower pattern development.

[0026] The present invention provides an implementable molecular tool for the genetic improvement of Dendrobium flower type. Through gene editing technology or transgenic expression regulation means, the key target genes accurately identified in the present invention, such as MADS6, ERECTA, AS2, etc., can be used to efficiently change the morphology, size and symmetry of Dendrobium flower organs, thereby achieving precise regulation and design of flower type. The significant phenotypic differences in flower type exhibited by plants overexpressing DoAP3-3 verified the effectiveness of the regulatory pathway. It provides clear molecular targets and theoretical basis for the flower type breeding of non-model plants, especially orchids, and expands the breeding and application prospects of orchid flower type diversity.

[0027] Of course, any product implementing the present invention does not necessarily need to achieve all of the advantages described above at the same time. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0029] Figure 1 These are the morphological diagrams of floral organs of WT and DoAP3-3 overexpressing plants; ①③⑤ are sepals; ②⑥ are petals; ④ is the lip; ⑦ is the gynandrostem.

[0030] Figure 2 Schematic diagram of the DoAP3-3 binding peak distribution detected by DAP-seq; A: DoAP3-3 binding peak distribution within the whole genome; B: DoAP3-3 binding peak distribution within the promoter region.

[0031] Figure 3 Schematic diagram of the distribution of peak functional elements; A: Statistical diagram of the distribution of peak functional elements within the genome; B: Statistical diagram of the distribution of peak functional elements within the promoter region.

[0032] Figure 4 This is a schematic diagram of the predicted transcription factors of target genes related to flower development;

[0033] Figure 5 This is a schematic diagram of GO enrichment analysis of the top 30 pathways in target genes related to flower development;

[0034] Figure 6 Schematic diagram of differentially expressed gene analysis; A: Analysis of differentially expressed genes between WT and OE plants; B: Cluster heat map analysis of differentially expressed genes related to flower development.

[0035] Figure 7Schematic diagram of direct target genes of transcription factor DoAP3-3 in regulating floral organ and flower type development identified by DAP-seq and RNA-seq; A: Venn diagram; B: Cluster heat map of 30 target genes.

[0036] Figure 8 This is an analysis chart of target gene expression levels related to floral organ and flower shape development;

[0037] Figure 9 This is the DAP-seq binding peak diagram of target genes related to floral organ and flower type development;

[0038] Figure 10 This is a schematic diagram of the analysis of cis-acting elements of seven key downstream target genes that regulate flower pattern changes;

[0039] Figure 11 It is a schematic diagram of candidate target gene motif analysis;

[0040] Figure 12 This is a schematic diagram of motif enrichment analysis of candidate target genes related to flower type development;

[0041] Figure 13 This is a schematic diagram of yeast one-hybrid interaction verification. DETAILED DESCRIPTION

[0042] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts shall fall within the scope of protection of the present invention.

[0043] Example 1

[0044] The present embodiment describes an application of an AP3-3 transcription factor to a target gene for regulating changes in flower shape of Dendrobium, wherein the target gene is selected from at least one of MADS6, NAC029, NAC054, WOX3, AS2, ERECTA, and AG.

[0045] The application includes changing the morphology, size or symmetry of Dendrobium flower organs by gene editing or expression regulation of the target gene.

[0046] In another aspect, the present invention provides a method for identifying target genes of Ap3-3 transcription factors that regulate changes in flower shape in Dendrobium, comprising the following steps:

[0047] S1: DNA affinity purification sequencing (DAP-seq) was used to obtain the binding sites of the transcription factor DoAP3-3 in the Dendrobium officinale genome;

[0048] S2: Transcriptome sequencing (RNA-seq) was used to analyze the differentially expressed genes between wild-type Dendrobium officinale plants and DoAP3-3 overexpressing plants;

[0049] S3: Intersection analysis of the binding site-associated genes identified in step S1 and the differentially expressed genes in step S2 was performed to screen out target genes directly regulated by DoAP3-3;

[0050] The target gene is selected from at least one of MADS6, NAC029, NAC054, WOX3, AS2, ERECTA, and AG.

[0051] On the other hand, the present invention provides a method for regulating the development of Dendrobium officinale flower shape, by overexpressing the transcription factor DoAP3-3 to regulate the expression level of the above-mentioned target gene, thereby changing the morphology, size or symmetry of the floral organ.

[0052] Example 2

[0053] Materials and Methods

[0054] Material

[0055] Wild-type (WT) and DoAP3-3-overexpressing (OE) D. officinale plants were obtained from previous studies in our laboratory. Fresh leaves of D. officinale were collected from the Dendrobium cultivation base of Yunnan Academy of Forestry and Grassland Sciences.

[0056] method

[0057] DNA affinity purification and sequencing

[0058] DNA was extracted from the leaves of Dendrobium officinale and fragmented. Target fragments were screened using magnetic beads and a library was constructed using a library construction kit (NEXTflex Rapid DNA-Seq Ki). A halo-tag in vitro expression plasmid for the transcription factor DoAP3-3 was constructed and protein expression was performed using a wheat germ system. The purified transcription factor DoAP3-3 and affinity tag fusion protein were co-incubated with the genomic DNA library. The target protein and DNA complex were extracted and purified using Halo Tag-specific magnetic beads, PCR amplified, and analyzed using Qubit TM The quality of the DNA library was tested using the dsDNA-HS Assay Kit, and sequencing was performed by Wuhan Aijibaike Biotechnology Co., Ltd.

[0059] DAP-seq sequencing data analysis

[0060] Raw data were quality-controlled using Fastqc (version 0.11.5) and filtered using Trimmomatic (version 0.36). Clean reads were aligned to the Dendrobium catenatum (GCF_001605985.2) reference genome using BWA (version 0.7.15-r1140). MACS (version 2.1.1.20160309) software was used to identify peaks enriched for the transcription factor DoAP3-3 relative to the control genome. Significantly enriched peaks were screened using a q value < 0.001 and a fold change greater than 2. Gene oncogenes and KEGG (Knowledge of Genes and Genomes) analyses were performed on genes associated with these peaks. Enrichment motifs for DoAP3-3 target genes were analyzed using Homer, MEME, and MEME-ChIP software. Binding peaks were visualized using IGV software. Red peaks represent replicate 1, blue peaks represent replicate 2, and green peaks represent the negative control (input). The exon-intron structure and orientation (arrows) of each gene are shown at the bottom of the panel. Red arrows indicate binding within 2000 bp above and below the target gene TSS. [0-50] represents the strength of binding, as reflected by peak height.

[0061] Transcriptome sequencing

[0062] RNA from mixed flower buds at different stages (from flower bud to fully open) was extracted from wild-type Dendrobium officinale plants (WT) and DoAP3-3 overexpressing plants (OE), and RNA sequencing was performed using the Illumina high-throughput sequencing platform. The sequencing was completed by Beijing Qingke Biotechnology Co., Ltd.

[0063] Transcriptome sequencing data analysis

[0064] The raw data from the sequencing was filtered using Fastqc, and the clean reads were aligned to the reference genome of Dendrobium catenatum (GCF_001605985.2) using Hist. Gene expression differentials between the sequencing results of DoAP3-3-overexpressing and WT plants of Dendrobium officinale were analyzed using DEGseq software. Differentially expressed genes were screened using a FoldChange ≥ 2 and an FDR < 0.01. The differentially expressed genes were then compared with six major public databases, including NR, eggNOG, GO, KEGG, Swissprot, and Pfam. Annotation information from each database was obtained to identify differentially expressed genes related to floral organ development.

[0065] Bioinformatics analysis of promoter cis-acting elements

[0066] The plant cis-element database Plant CARE was used to predict and analyze the cis-acting elements of the promoter sequences of key candidate target genes, and the visualization analysis was performed using TBtools software.

[0067] Yeast one-hybrid interaction experiments

[0068] Using the cDNA of the DoAP3-3 overexpressing plant of Dendrobium officinale as a template, the DoAP3-3 coding sequence was amplified and connected to the pGADT7 vector (Clontech, USA) to obtain pGADT7-DoAP3-3 as prey. Using the MADS6 gene DNA of Dendrobium officinale as a template, the specific DNA sequence of the target gene promoter was amplified and cloned into the pHIS2 vector (Clontech, USA) to obtain pHIS2-MADS6 as bait. The MADS6 gene constructed into the pHIS2 vector and the DoAP3-3 gene constructed into pGADT7 were transformed into the yeast strain Y187 by co-transfection. Three single colonies were randomly picked from the transformation reaction plates, and the OD 600 =0.002, dilution (10 0 , 10 -1 , 10 -2 ) were spotted onto the corresponding defective plates without histidine but with different concentrations (0, 2.5, 5, 10, 20, 30, 40, 50, 75, 100 mmol / L) of HIS3 protein competitive inhibitor 3-AT (3-amino-1, 2, 4-triazole), and cultured at 30°C for 3 days to screen the appropriate concentration and eliminate the self-activation phenomenon. The single colonies successfully verified in the experimental group (pHIS2-MADS6 + pGADT7-DoAP3-3), the negative control group (pHIS2-MADS6 + pGADT7), and the positive control group (pGAD53m + pHIS2-p53) were resuspended in 2 mL of ddH2O water and the OD 600 =0.002, the concentration was set to 3 gradients of 10 0 , 10 -1 , 10 -2 , aspirate 10 μL and spot it on SD-TL (-trp, -leu), SD-TLH (-trp, -leu, -his), and SD-TLH+40mmol / L 3AT culture medium (refer to Clontech's PT3024-1 / Yeast Protocols Handbook), with 3 spots on each plate, and culture at a constant temperature of 30℃ for 3 to 5 days.

[0069] Results and Analysis

[0070] Comparative analysis of floral organ morphology between wild-type Dendrobium officinale plants and DoAP3-3 overexpressing plants

[0071] Differences in flower shape between wild-type Dendrobium officinale plants and DoAP3-3 overexpressing plants Figure 1 As shown, the flower morphology differs only in the distribution of petals and sepals, and in the size and color of the lip; other morphological changes remain unchanged. The two petals of the overexpressing flowers are rotated counterclockwise by more than 90 degrees, almost overlapping the sepals, and the petals and sepals are either mosaic or fused. The lip of the overexpressing flowers is larger, and the proximal protuberance on the lip abdomen is flatter, while the purple spot on the distal axis has disappeared.

[0072] DAP-seq sequencing data analysis

[0073] DNA was extracted from Dendrobium officinale leaves and sequenced using DAP-Seq to obtain raw DAP-seq data. The clean reads were aligned to the genome sequence of Dendrobium catenatum (GCF_001605985.2) with a 99.72% alignment rate. Peaks binding to the transcription factor DoAP3-3 were also identified. All transcription factor binding peaks were summarized and categorized, yielding a total of 175,468 peaks across the genome with an average peak length of 399.43 bp. The distribution of functional elements within the peaks was statistically analyzed, revealing that 6,579 peaks were located in the promoter region, accounting for 3.75% of the total peaks ( Figure 2 ), 56.85% of the peaks were located in the intergenic regions, 3.75% of the peaks were located in the promoter regions, and the 5′-untranslated regions and 3′-untranslated regions were the least distributed ( Figure 3 A). 90.74% of the peaks located in the promoter region are bound to the promoter ( Figure 3 B) This indicates that DoAP3-3 is a transcription factor with DNA binding ability and gene regulatory activity.

[0074] DAP-seq analysis of potential downstream target genes of the transcription factor DoAP3-3 in floral development

[0075] A total of 5768 potential target genes were screened within the promoter range, corresponding to 6579 peaks, of which 82 potential target genes related to floral organ and flower type development were found, corresponding to 94 peaks. These potential target genes include transcription factor family genes and functional protein genes, such as MYB-related, MADS-MIKC, AP2 / ERF-ERF, B3, B3-ARF, C2C2-YABBY, HB-BELL, MADS-M-type, NAC and other transcription factor family genes ( Figure 4); TAA1, DCR, ERECTA and other functional protein genes.

[0076] Functional annotation of genes related to flower development detected by DAP-seq was performed, and the top 30 pathways with P < 0.05 were screened for enrichment analysis. The results showed that the enriched GO terms in Dendrobium officinale were mainly biological processes, including reproductive structure development, floral organ development, floral whorl development, and other biological processes ( Figure 5 ).

[0077] Transcriptome sequencing analysis

[0078] RNA was extracted from reproductive-stage flower buds of wild-type Dendrobium officinale plants and DoAP3-3-overexpressing plants. RNA sequencing was performed using the Illumina high-throughput sequencing platform, generating 15.88 Gb of data with a Q30 base percentage of 95.98% or higher. Clean reads from each sample were aligned to the reference genome, with alignment efficiencies ranging from 87.82% to 91.43%.

[0079] Transcriptome sequencing analysis identifies differentially expressed genes regulated by the transcription factor DoAP3-3 in floral development

[0080] To identify genes regulated by DoAP3-3 in D. officinale, we conducted comparative transcriptome analysis of flower buds of wild-type (WT) and DoAP3-3 overexpressing (OE) D. officinale plants by transcriptome sequencing. A total of 8101 differentially expressed genes were found, of which 3845 genes were down-regulated and 4256 genes were up-regulated ( Figure 6 A). Further screening of these differentially expressed genes revealed 86 differentially expressed genes related to flower development, of which 32 genes were up-regulated and 54 genes were down-regulated ( Figure 6 B) The expression levels of genes such as AS2, MADS16, NAC054, DL, LFL1, and GI in the overexpression plants were lower than those in wild-type plants. The expression levels of genes such as WOX3, FT, FZP, GATA22, and ENDO2 in the overexpression plants were higher than those in wild-type plants.

[0081] DAP-seq combined with RNA-seq analysis of potential direct target genes of transcription factor DoAP3-3 in regulating floral organ and flower type development

[0082] The 82 target genes related to flower development identified in DAP-seq were compared with the 85 DEGs from RNA-seq analysis of overexpression plants and wild-type plants, and 30 common genes were found ( Figure 7A). In DoAP3-3 overexpressing plants, 19 genes were positively regulated target genes and 11 were negatively regulated target genes ( Figure 7 B). These target genes can be divided into three categories according to their functions: the first category is related to floral induction during the floral development stage, among which NAC035, FT, LFL1, LHY, ZHD4, ZTL, AOD3, and RVE8 are involved in regulating flowering time; the second category is related to floral organ primordium formation, i.e. floral organ development, among which MADS6 affects floral meristem-determined floral organ development, floral organ formation, and floral whorl morphogenesis; FZP and BAM1 determine floral meristem identity and specify floral organ identity; AG and CYP40 participate in the control of organ identity during early floral development and play a crucial role in maintaining floral meristem formation. The third category plays a role in the determination of floral tissues. DL determines carpel identity and floral meristem, ATH1 controls the formation of the boundary between floral organs and stems, affecting floral organ shedding, TAA1 and FHA2 affect floral organ and stamen development, LUG regulates floral organ development, and JMJ706 participates in floral morphogenesis. The third category participates in the maturation of floral organs during the floral development stage, namely flower type development, among which NAC054 and NAC029 affect flower development, WOX3 participates in sepal formation, AS2 affects petal development, ERECTA regulates flower morphology and inflorescence structure, and DCR affects trichome morphogenesis. In DoAP3-3 overexpressing plants, the expression levels of WOX3, NAC029, MADS6, ERECTA, and AG were all higher than those in the wild type, while the expression levels of AS2 and NAC054 were lower ( Figure 8 , Figure 9 ), combined with the floral phenotypic changes of overexpressed plants and their gene functions, these genes are very likely to be the key downstream target genes of the transcription factor DoAP3-3.

[0083] Analysis of key candidate target genes regulating floral pattern by transcription factor DoAP3-3

[0084] This study selected seven target genes closely related to flower development from the identified direct target genes, namely MADS6, NAC029, NAC054, WOX3, AS2, ERECTA, and AG, as key candidate target genes. Cis-acting element analysis of these seven target genes showed that in addition to containing multiple transcription initiation core elements TATA-box and CAAT-box, the cis-acting elements distributed in the promoter regions of these seven genes are related to plant hormone response, light response, plant growth and development, and biotic and abiotic stress responses ( Figure 10 ).

[0085] To gain a deeper understanding of the DNA binding properties of DoAP3-3, this study used MEME and MEME-CHIP software to perform motif analysis and motif enrichment analysis on the promoters and peak overlap sequences of seven candidate target genes. Motif analysis found that except for AG, the remaining target genes can bind to motif1, motif2, and motif3 ( Figure 11 ). Motif enrichment analysis showed that the GA / CT-rich ( Figure 12 ).

[0086] To further verify the direct role of DoAP3-3 in regulating flower pattern development, this study selected promoter regions that showed enriched binding sites in DAP-seq analysis ( Figure 9 , Figure 11 ) was further verified by yeast one-hybrid experiments to verify whether there is an interaction between DoAP3-3 and MADS6 to regulate flower shape changes. Figure 13 It can be seen that the positive control (pGAD53m+pHIS2-p53) can grow normally on SD-TL, SD-TLH, and SD-TLH+40mmol / L 3AT plates; the negative control group (pHIS2-MADS6+pGADT7) can grow normally on SD-TL and SD-TLH plates, but does not grow on SD-TLH+40mmol / L3AT plates; the experimental group (pHIS2-MADS6+pGADT7-DoAP3-3) can grow normally on SD-TL and SD-TLH plates, but grows weakly on SD-TLH+40mmol / L 3AT plates, indicating that DoAP3-3 interacts with the promoter of MADS6 in yeast.

[0087] DAP-seq, as an in vitro method for detecting transcription factor binding sites, successfully transfers in vivo binding experiments to in vitro, greatly improving the efficiency of DNA binding site discovery and making it applicable to the study of transcription factors in non-model plants. Building on previous research by our group, we generated flowers from plants overexpressing the gene in Dendrobium officinale using the in-vase flowering technique and conducted transcriptome sequencing analysis. Combined DAP-seq and RNA-seq analysis successfully identified seven key candidate target genes that directly bind to the transcription factor DoAP3-3 and regulate floral morphological development: MADS6, NAC029, NAC054, WOX3, AS2, ERECTA, and AG. These genes may play important roles in regulating floral morphological development through specific regulation or interactions with other genes. This study provides a preliminary understanding of the regulatory mechanism between the floral organ identity gene DoAP3-3 and its target genes, laying the foundation for understanding the mechanisms of highly specialized and diverse floral morphogenesis in orchids.

[0088] The preferred embodiments of the present invention disclosed above are intended only to help illustrate the present invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the present invention to the specific embodiments described. Obviously, many modifications and variations are possible based on the content of this specification. These embodiments are selected and described in detail in this specification to better explain the principles and practical applications of the present invention, thereby enabling those skilled in the art to better understand and utilize the present invention. The present invention is limited only by the claims and their full scope and equivalents.

Claims

1. An application of an AP3-3 transcription factor to regulate target genes for changes in flower shape in Dendrobium, characterized in that: The target gene is selected from at least one of MADS6, NAC029, NAC054, WOX3, AS2, ERECTA, and AG; the sequences are shown in SEQ ID NOs. 1-7; and the application includes regulating the target gene by gene editing or expression to change the morphology, size, or symmetry of Dendrobium flower organs.

2. A method for identifying target genes of Ap3-3 transcription factors that regulate changes in flower shape in Dendrobium officinale, characterized by: The following steps are involved: S1: The binding sites of transcription factor DoAP3-3 in the genome of Dendrobium officinale were obtained by DNA affinity purification and sequencing; S2: Transcriptome sequencing was used to analyze the differentially expressed genes between wild-type Dendrobium officinale plants and DoAP3-3 overexpressing plants; S3: Intersection analysis of the binding site-associated genes identified in step S1 and the differentially expressed genes in step S2 was performed to screen out target genes directly regulated by DoAP3-3; The target gene is selected from at least one of MADS6, NAC029, NAC054, WOX3, AS2, ERECTA, and AG.

3. A method for regulating the flower shape development of Dendrobium officinale, characterized in that: The expression level of the target gene according to claim 1 is regulated by overexpressing the transcription factor DoAP3-3, thereby changing the morphology, size or symmetry of the floral organ.

4. Application of the method as claimed in claim 2 in cultivating new varieties of Dendrobium officinale.

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