Phagl6-2 gene and its application in controlling plant floral organ curvature

Abstract

The application discloses a PhAGL6-2 gene and application thereof in controlling curling of plant flower organs. The nucleotide sequence of the PhAGL6-2 gene is shown as SEQ ID NO. 1. Through research on the function of the PhAGL6-2 gene of Phalaenopsis, it is found for the first time that the gene specifically regulates curling / bending of petals, and has no obvious influence on the shape of labellum, thereby providing a new gene resource for directional improvement of the flower organ shape of orchid plants.

Description

Technical Field

[0001] This invention belongs to the fields of molecular biology and biotechnology, specifically relating to the PhAGL6-2 gene, a member of the MADS-box transcription factor family of Paphiopedilum B, and its application in controlling the curling of plant floral organs. Background Technology

[0002] The morphology of floral organs (including size, shape, and degree of curling) is an important economic trait in ornamental plants. Orchids are renowned for their highly specialized floral organ structures, with the morphological diversity of the labellum being particularly prominent. Paphiopedilum plants possess unique hooded labels, the formation of which is closely related to the curling / bending of floral organs. However, the molecular mechanisms regulating floral organ curling, especially the key genes and their functions in orchids, currently lack in-depth research.

[0003] AGL6 (AGAMOUS-LIKE 6) is an important member of the MADS-box transcription factor family and plays a crucial role in floral organ identity determination. In the orchid "P-code" model, AGL6-2 is thought to be primarily involved in labellial identity determination. However, whether AGL6-2 has additional functions in regulating sepal and petal morphology remains unclear. Summary of the Invention

[0004] The purpose of this invention is to provide the PhAGL6-2 gene of Paphiopedilum and its application in regulating the curling of sepals and petals in plants.

[0005] This invention, through transcriptome sequencing and qRT-PCR analysis, revealed that the class B MADS-box family transcription factor PhAGL6-2 was specifically and highly expressed in the lip-like petals of both the wild-type and trilip mutants of *Paphiopedilum henri*, and its expression level was highly correlated with the presence of the lip-like structure. Figure 1 ).

[0006] This invention demonstrates through virus-induced gene silencing (VIGS) experiments that silencing PhAGL6-2 in *Paphiopedilum henri* results in stable and significant dorsal axial bending in the lower region of the petals and the edge of the median sepal, while the overall morphology of the lip remains largely unchanged. Figure 4 In Dendrobium Chao Praya Smile, heterologous silencing of PhAGL6-2 also resulted in significant dorsal axial bending of the median sepals, lateral sepals, and petals, while the morphology of the lip remained largely unchanged. Figure 3 The above results indicate that PhAGL6-2 is a key regulatory factor that specifically controls the curling / bending morphology of sepals and petals.

[0007] This invention provides cytological evidence that PhAGL6-2 drives the curling of sepals and petals by regulating the proliferation balance of epidermal cells on the axial (inner) and dorsal (outer) sides (i.e., creating an asymmetry in the number of inner and outer epidermal cells). Silencing PhAGL6-2 resulted in curled sepals and petals with a significantly higher number of inner epidermal cells than outer epidermal cells, while the cell area showed no significant difference. Figure 4 ).

[0008] This invention further demonstrates through heterologous overexpression experiments that heterologous expression of PhAGL6-2 in Arabidopsis thaliana results in transgenic plants exhibiting significant upward curling of rosette leaves, smaller petals, and a slight bending at the apex. Figure 2 This indicates that PhAGL6-2 has a conserved function in inducing organ curling.

[0009] Based on the above findings, the first objective of this invention is to provide the PhAGL6-2 gene, which controls the curling of plant sepals and petals, and whose encoded amino acid sequence is shown in SEQ ID NO.2.

[0010] Preferably, the nucleotide sequence of the PhAGL6-2 gene is shown in SEQ ID NO.1.

[0011] A second objective of this invention is to provide the application of the PhAGL6-2 gene in controlling the curling of plant sepals and petals.

[0012] Preferably, the PhAGL6-2 gene is silenced in plants to induce dorsal axis bending of sepals and / or petals, thereby altering the degree of bending.

[0013] Preferably, overexpression in plants causes the rosette leaves of the transgenic plants to curl upwards significantly, and the petals to become smaller with a slight bend at the tip.

[0014] Preferably, the plant is Paphiopedilum henri, Dendrobium nobile, or Arabidopsis thaliana.

[0015] A third objective of this invention is to provide a method for controlling the curling of sepals and petals in plants by overexpressing or silencing the PhAGL6-2 gene in plants.

[0016] Preferredly, silencing the PhAGL6-2 gene in *Paphiopedilum henri* resulted in a stable and significant dorsal axial bending in the lower region of the petals and the edge of the median sepal, while the overall morphology of the lip remained largely unchanged.

[0017] Preferably, PhAGL6-2 is silenced in Dendrobium nobile, resulting in significant dorsal axial bending of the median sepals, lateral sepals and petals, while the morphology of the lip remains basically unchanged.

[0018] Preferably, heterologous overexpression of PhAGL6-2 in Arabidopsis thaliana resulted in the rosette leaves of the transgenic plants curling upwards significantly, and the petals becoming smaller with a slight bend at the tip.

[0019] Preferably, the sequence shown in SEQ ID NO.3 is used as a specific fragment, and the pTRV2-PhAGL6-2 silencing vector is constructed by cloning it into the pTRV2 vector using the ClonExpress II OneStep Cloning Kit. The vector is then transformed into Agrobacterium to obtain the transformation solution, which is then transformed into flowering plants.

[0020] Preferably, the flowering plant can be any type of flowering plant, such as Paphiopedilum henri or Dendrobium nobile.

[0021] This invention, through studying the function of the PhAGL6-2 gene in Paphiopedilum, has for the first time discovered that this gene specifically regulates the curling / bending morphology of petals, while having no significant effect on the morphology of the lip, providing a new gene resource for the targeted improvement of the floral organ morphology of orchids. Attached Figure Description

[0022] Figure 1 This study analyzes the expression characteristics of PhAGL6-1 and PhAGL6-2 in mutants of different floral organs and lip morphology.

[0023] (A–B) Expression levels of PhAGL6-1 and PhAGL6-2 in different floral organs of wild-type and trilip mutants. Actin (BMK_Unigene_064712) was used as an internal reference gene. Sampling organs included the median sepal, synepithelial sepal, petals / lip-like petals, lip, and column. Data are expressed as mean ± standard deviation (mean ± SD) (n = 3). Different letters indicate significant differences under Duncan's multiple comparison test (P < 0.05). (C) Floral morphology of Paphiopedilum 'Pacific Shamrock' wild-type (CK) and three lip morphology mutants (T1–T3). CK: Normal lip with a distinct hood structure; T1: Lip with a double hood structure; T2: The hood structure is significantly lighter (approximately 2–3 times lighter than normal); T3: The hood structure is completely absent, and the lip is flattened. Scale bar = 1 cm. (D) Expression levels of PhAGL6-2 in different floral organs (median sepal, synepithelial sepal, petals, and lip) of CK and T1–T3 plants. Actin (BMK_Unigene_064712) was used as an internal reference gene. Data are expressed as mean ± standard deviation (n = 3). Different letters indicate significant differences under Duncan's multiple comparison test (P < 0.05).

[0024] Figure 2This is a phenotypic analysis of heterologous overexpression of PhAGL6-2 in Arabidopsis thaliana.

[0025] (A–C) Rosette leaves and floral morphology of wild-type (WT) Arabidopsis thaliana plants. (D–F) Phenotypic characteristics of the 35S::PhAGL6-2 transgenic plant, characterized by distinctly upward-curving rosette leaves and petals that are significantly smaller than those of the WT plant with a slight curve at the tip. Scale bar: A, D = 1 cm; B, C, E, F = 1 mm.

[0026] Figure 3 This study investigated the effects of heterologous silencing of PhAGL6-2 on floral organ morphology in Dendrobium nobile. Treatment with pTRV2-PhAGL6-2 resulted in dorsal-axis bending of the sepals and petals, while the lip remained unchanged.

[0027] (A) Morphological characteristics of flowers in the control treatment (pTRV2-Empty), showing front, back, and side views in sequence, as well as separated floral organs, including lateral sepals (LS), median sepals (DS), petals (P), and lip (L). (B) Three representative flowers (1st–3rd) from the same inflorescence after pTRV2-PhAGL6-2 treatment, showing a consistent dorsoaxial bending phenotype of floral organs. Scale bar = 0.5 cm.

[0028] Figure 4 This study investigated the effects of silencing PhAGL6-2 on floral organ morphology and cellular characteristics in *Paphiopedilum henri*. Silencing caused dorsoaxial bending of the petals and median sepal, while the lip remained unchanged. Cytological analysis revealed a significantly higher number of inner epidermal cells than outer epidermal cells in the bent areas.

[0029] (A, B) Overall floral morphology of plants in the control treatment (pTRV2-Empty) and the PhAGL6-2 silencing treatment (pTRV2-PhAGL6-2), with three independent VIGS flowers shown for each treatment. (C) Front and back views of the control flower and isolated floral organs, including the median sepal (DS), petals (P), and lip (L), showing their axial and dorsal morphology, respectively. (D) Representative flower (V1) of the PhAGL6-2 silencing plant, showing the dorsal bending phenotype of the median sepal and petal base region. Scale bar = 1 cm. (E) SEM images and quantitative analysis of the petal epidermis of the control and PhAGL6-2 silencing plants. The left image shows the epidermal cell count, and the right image shows the cell area count. Error bars represent mean ± SD, and asterisks indicate significant differences (*P < 0.01, Student's t-test). Scale bar = 50 μm. (F) Relative expression levels of PhAGL6-2 in different floral organs of *Paphiopedilum henri*, normalized by Actin (BMK_Unigene_064712). RNA was obtained from 3 independent biological replicates. Data are presented as mean ± SD (*P < 0.05, Student's t-test). Detailed Implementation

[0030] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the embodiments described in this specification are merely illustrative of the invention and are not intended to limit the invention.

[0031] Example 1: Cloning and Expression Analysis of the PhAGL6-2 Gene

[0032] Total RNA was extracted from *Paphiopedilum henneruis* flower buds and reverse transcribed into cDNA. Based on the sequence information obtained from transcriptome sequencing, specific primers (PhAGL6-2-ORF-F; 5'-3': ATGGGAAGAGGAAGAGTTGA and PhAGL6-2-ORF-R; 5'-3': TCAAAACAGACCATCCCAACA) were designed, and the complete coding region (CDS) of the PhAGL6-2 gene was obtained by PCR amplification. Sequencing results showed that the nucleotide sequence of the PhAGL6-2 gene is shown in SEQ ID NO.1, and the encoded protein sequence is shown in SEQ ID NO.2.

[0033] Example 2: Analysis of PhAGL6-2 expression by transcriptome sequencing and qRT-PCR

[0034] (1) Total RNA extraction and transcriptome sequencing

[0035] Wild-type *Paphiopedilum henri* and its naturally occurring trilipped mutant were used as materials. At stage 4 of flower development, the lip and petals of the wild-type were collected, as well as the lip-like petals and lip of the trilipped mutant, with three biological replicates for each. Simultaneously, the sepals, synepithelial plates, petals / lip-like petals, lip, and column of both the wild-type and trilipped mutant were collected for qRT-PCR analysis. Total RNA was extracted using a polysaccharide-polyphenol plant total RNA extraction kit. RNA concentration and purity were determined using Nanodrop 2000, and RNA integrity was detected by agarose gel electrophoresis. High-quality RNA samples (total RNA ≥ 1 μg, concentration ≥ 35 ng / μL, OD260 / 280 ≥ 1.8) were selected for transcriptome sequencing using the Illumina NovaSeq platform. After quality control, the raw data were assembled using Trinity software, and gene expression abundance was expressed as FPKM values. Differentially expressed genes were analyzed using FDR < 0.01 and FC ≥ 2 as screening criteria.

[0036] (2) qRT-PCR verification

[0037] Mesial sepals, synepithelial sepals, petals / lip-like petals, lip, and column of *Paphiopedilum henri* wild-type and trilip mutant were collected for qRT-PCR analysis. cDNA was synthesized according to the instructions of the Total Gold Reverse Transcription Kit. qRT-PCR reactions were performed using PerfectStart® Green qPCR SuperMix. The qRT-PCR primers for PhAGL6-2 were: PhAGL6-2-QF: 5'-AACCGTCAAGTAACCTTTTCG-3'; PhAGL6-2-QR: 5'-TTGTAATGTCGGGGCTCC-3'. Actin was used as an internal reference gene. Relative expression levels were calculated using the 2^−ΔΔCT method.

[0038] Transcriptome differential expression analysis revealed several genes significantly upregulated in the lip and lip-like petals in the comparison groups of petals versus lip petals and petals versus lip-like petals. Among them, the transcript abundance of PhAGL6-2 in lip-like petals and lip petals was approximately 5-fold and 8-fold higher than that in petals, respectively. qRT-PCR analysis showed that PhAGL6-2 exhibited highly specific expression in the lip and lip-like petals of wild-type and trilip mutants, while its expression was extremely low in sepals and column. The expression level of PhAGL6-2 homologs was positively correlated with the inward curvature of the lip, and PhAGL6-2 expression was highly correlated with the hood-like structure of the lip and lip-like petals of *Paphiopedilum*. Figure 1 ).

[0039] Example 3: Functional analysis of heterologous overexpression of PhAGL6-2 in Arabidopsis thaliana

[0040] (1) Construction of overexpression vector

[0041] EcoRI and XbaRI were selected as restriction enzyme sites based on the multiple cloning site region of the vector. Primers with homologous arms were designed using the In-Fusion Cloning function in SnapGene (version: v6.0.2).

[0042] C17-PhAGL6-2-F: gcttgatatcgaattcATGGGAAGAGGAAGAGTTGAGCT; C17-PhAGL6-2-R: cagcgaattatctagaAACAGACCATCCCAACATGAAATTGT. The target fragment containing the PhAGL6-2 plasmid was amplified using the 2 × Phanta Flash Master Mix high-fidelity enzyme from Novizan as a template.

[0043] The pGreen 35S-GFP vector was double-digested with Takara fast digester to obtain a linear vector. The target fragment was then ligated to the linear vector using Chloride homologous recombinase. Maintaining a molar ratio of linear vector to target fragment between 1:1 and 1:10 yielded the recombinant plasmid 35S::PhAGL6-2. Sequencing verified the correct recombinant plasmid, which was then transformed into Agrobacterium GV3101 competent cells.

[0044] The pGreen 35S-GFP vector was kindly provided by a collaborating professor; the reference is: Yu H, Ito T, Wellmer F, Meyerowitz EM (2004) Repression of AGAMOUS-LIKE 24 is a crucial step in promoting flower development. Nat Genet 36: 157. The applicant also holds this vector and guarantees its availability to the public for 20 years from the date of application.

[0045] (2) Transformation of Arabidopsis thaliana by inflorescence infection method

[0046] The wild-type Arabidopsis thaliana (Col-0) was transformed using the inflorescence infection method: Agrobacterium carrying the recombinant plasmid was cultured to an OD600 of 0.6-0.8, and the bacterial cells were collected by centrifugation and resuspended in transformation osmotic buffer (5% sucrose, 0.015% Silwet L-77). Arabidopsis inflorescences were immersed in the bacterial solution for 1 minute and cultured in the dark for 24 h, after which normal growth resumed. The infection was repeated once a week later. After seed harvesting, transgenic positive plants were obtained through Kan resistance selection.

[0047] (3) Phenotypic observation

[0048] Transgenic plants were cultured in an artificial climate chamber (22℃±2℃, 16 h light / 8 h dark). Phenotypic observation was performed on T2 generation homozygous plants (35S::PhAGL6-2). Heterologous expression of PhAGL6-2 in Arabidopsis thaliana resulted in transgenic plants with significantly upward curling of rosette leaves, smaller petals, and slight bending at the tips. Figure 2 This indicates that PhAGL6-2 has a conserved function in inducing organ curling.

[0049] Example 4: Functional Analysis of the VIGS Technology-Silent PhAGL6-2

[0050] (1) Carrier construction

[0051] To construct a viral-induced gene silencing vector for PhAGL6-2, a specific fragment of approximately 200 bp was obtained from the low-conservation C-terminal region of the PhAGL6-2 protein, as shown in SEQ ID NO.3, based on BLAST alignment results. This fragment can form a perfectly matched 21 nt siRNA with the AGL6-2 homolog in Dendrobium 'Chao Praya Smile', while minimizing the risk of non-specific matching with other MADS-box genes. The target fragment was cloned into the pTRV2 vector using the ClonExpress II OneStep Cloning Kit (Vazyme) to construct the pTRV2-PhAGL6-2 silencing vector.

[0052] (2) Transformation of Agrobacterium

[0053] The constructed pTRV2-PhAGL6-2, empty pTRV2 (control), and helper pTRV1 were transformed into Agrobacterium (EHA105). Agrobacterium was cultured at 28°C until the OD600 reached 0.4–0.6. After centrifugation, the cells were collected and resuspended in KC medium containing 100 µM acetylsyleugenone, and incubated at room temperature for 30 min. Before infection, pTRV1 was mixed with each pTRV2 construct at a 1:1 (v / v) ratio to obtain the transformed bacterial solution.

[0054] (3) Injection of inflorescences and leaves to transform flowering plants

[0055] Transformed bacterial culture was injected into the base of newly emerging inflorescences and the abaxial surface of the uppermost 2-3 leaves of *Paphiopedilum henri* or *Dendrobium Chao PrayaSmile* using a needle-free 1mL syringe. After inoculation, the plants were placed in darkness for 24 h, followed by culture under normal greenhouse conditions. Phenotypic analysis was performed on *Paphiopedilum henri* and *Dendrobium Chao PrayaSmile* plants 60-80 days post-infection, with a supplementary infection at 30-40 days. All experiments were independently replicated in triplicate. Gene silencing efficiency was verified by qRT-PCR detection of PhAGL6-2 transcription levels in *Paphiopedilum henri*.

[0056] Heterologous silencing of PhAGL6-2 in Dendrobium nobile also resulted in significant dorsal-axis bending of the median sepals, lateral sepals, and petals, while the morphology of the lip remained largely unchanged. Figure 3 The above results indicate that PhAGL6-2 is a key regulatory factor that specifically controls the curling / bending morphology of sepals and petals.

[0057] Heterologous silencing of PhAGL6-2 in *Sophora henleyana* yielded the following results: Figure 4 As shown, compared with the control, the floral organs of the silenced PhAGL6-2 plants exhibited significant phenotypes: both lateral petals and the median sepal showed obvious dorsal-axis bending, abnormal protrusions appeared on the surface of the lip, and the overall morphology of the lip did not change significantly. Figure 4 This result indicates that PhAGL6-2 is essential for maintaining the flat morphology of floral organs and normal lip development. The inner and outer epidermal cells of the curved petals of the control and PhAGL6-2-silenced plants obtained in Example 2 were observed using scanning electron microscopy (SEM). Figure 4 As shown, there was no significant difference in the number of inner and outer epidermal cells in the control petals. However, in petals that underwent dorsal bending after PhAGL6-2 silencing, the number of inner (axial) epidermal cells was significantly greater than that of outer (dorsal) epidermal cells, but there was no significant difference in the area of ​​inner and outer epidermal cells. This indicates that PhAGL6-2 controls the bending morphology of floral organs by regulating the asymmetric proliferation of inner and outer epidermal cells.

[0058] SEQ ID NO.1 ATGGGAAGAGGAAGAGTTGAGCTGAAGCGCATCGAGAACAAGATCAACCGTCAAGTAACCTTTTCGAAGCGCAGGAATGGGATCATGAAGAAGGCCTACGAGCTCTCCGTACTCTGCGACGCCGAGATCGCACTCATCATCTTCTCTAGCCGCGGCAAGCTCTTTGAGTTCGGGAGCCCCGACATTACAAAAACGCTGGAGCGCTATAAGCGGTGCACGTTTACACCACAGACTATTGATCCTACAGATCACGAAACACTGAACTGGTACCAGGAATTATCAAAGCTCAAAGCAAAGTATGAATCTCTACAGCGTTCCCAAAGGCATCTGCTCGGGGAAGATCTAGAAATGTTGAGTTTGAAGGAATTACAACAGTTGGAACGTCAACTTGAATCATCTCTATCACAAGCAAGACAAAAAAGGACTCAGATGATGTTGGAACAGATGGAAGAGCTAAAGAAGAAAGAGCGTCATCTCGGTGATATTAACAAGCAGCTAAAGCATAAGCTCGGGGCAGACGGTGGATCCATGAGAGCTATCACAAGTTCCTTGCGACCTGGTGCTGCAGCCAGTACTGAGACATTCCCTGCACATTCCAACGCTATGGACGCTGAACCCACTCTTCAAATAGGGTACCATCAGTATACCCCTTCTGAAGCAAGTATTTTGAGGAACGGTGGGTGTGCCGGGAACAATTTCATGTTGGGATGGTCTGTTTGA

[0059] SEQ ID NO.2 MGRGRVELKRIENKINRQVTFSKRRNGIMKKAYELSVLCDAEIALIIFSSRGKLFEFGSPDITKTLERYKRCTFTPQTIDPTDHETLNWYQELSKLKAKYESLQRSQRHLLGEDLEMLSLKELQQLERQLESSLSQARQKRTQMMLEQMEELKKKERHLGDINKQLKHKLGADGGSMRAI

[0060] SEQ ID NO.3 ATGGGAAGAGGAAGAGTTGAGCTGAAGCGCATCGAGAACAAGATCAACCGTCAAGTAACCTTTTCGAAGCGCAGGAATGGGATCATGAAGAAGGCCTACGAGCTCTCCGTACTCTGCGACGCCGAGATCGCACTCATCATCTTCTCTAGCCGCGGCAAGCTCTTTGAGTTCGGGAGCCCCGACATTACAAAAACGCTGGAGCGCTATAAGCGGTGCACGTTTACACCACAGACTATTGATCCTACAGATCACGAAACACTGAACTGGTACCAGGAATTATCAAAGCTCAAAGCAAAGTAT。

Claims

1. The PhAGL6-2 gene, which controls the curling of plant sepals and petals, is characterized by, The encoded amino acid sequence is shown in SEQ ID NO.

2.

2. The PhAGL6-2 gene according to claim 1, characterized in that, The nucleotide sequence of the PhAGL6-2 gene is shown in SEQ ID NO.

1.

3. The application of the PhAGL6-2 gene as described in claim 1 or 2 in controlling the curling of plant sepals and petals.

4. The application according to claim 3, characterized in that, Silencing the PhAGL6-2 gene in plants can induce dorsal-axis bending of sepals and / or petals, altering the degree of bending; or overexpression in plants can cause the rosette leaves of transgenic plants to curl upwards significantly, and the petals to become smaller and have curved tips.

5. The application according to claim 3, characterized in that, The plants mentioned are Paphiopedilum henri, Dendrobium nobile, or Arabidopsis thaliana.

6. A method for controlling the curling of plant sepals and petals, characterized in that, It is to overexpress or silence the PhAGL6-2 gene as described in claim 1 or 2 in plants.

7. The method according to claim 6, characterized in that, The PhAGL6-2 gene was silenced in *Paphiopedilum henri*, resulting in a stable and significant dorsal axial bending in the lower region of the petals and the edge of the median sepal, while the overall morphology of the lip remained unchanged.

8. The method according to claim 6, characterized in that, PhAGL6-2 was silenced in Dendrobium nobile, resulting in significant dorsal axial bending of the median sepals, lateral sepals, and petals, while the morphology of the lip remained largely unchanged.

9. The method according to claim 6, characterized in that, Heterologous overexpression of the PhAGL6-2 gene in Arabidopsis thaliana resulted in transgenic plants with rosette leaves that curled upwards, smaller petals, and curved tips.

10. The method according to claim 6, characterized in that, The sequence shown in SEQ ID NO.3 is used as a specific fragment and cloned into the pTRV2 vector using the ClonExpress II One Step Cloning Kit to construct the pTRV2-PhAGL6-2 silencing vector. The vector is then transformed into Agrobacterium to obtain the transformation solution, which is then transformed into flowering plants. Preferably, the flowering plants are Paphiopedilum henri or Dendrobium nobile.

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