R2R3-MYB transcription factor for specifically regulating and controlling accumulation of anthocyanin in spotted region of dendrobium nobile flower and application of R2R3-MYB transcription factor

Abstract

The invention discloses an R2R3-MYB transcription factor for specifically regulating and controlling anthocyanin accumulation in a spotted region of a dendrobium nobile flower and application of the R2R3-MYB transcription factor, the R2R3-MYB transcription factor comprises an R2R3-MYB gene-DnMYB1, and the application of the R2R3-MYB transcription factor comprises the following steps: carrying out targeted regulation and control through a transgenic technology, and carrying out transgenic breeding; in the phalaenopsis and white flower gardening variety, the heterologous transient overexpression of the target gene can significantly promote the accumulation of anthocyanin in petals of the phalaenopsis and white flower gardening variety; in the wild type dendrobium nobile, the accumulation of the anthocyanin in the floral quilt of the wild type dendrobium nobile can be obviously reduced through targeted silencing of the target gene. The gene plays an important role in the aspect of anthocyanin biosynthesis, can enrich a transgenic material library in which specific pigments are accumulated in a spot region of the orchid, and provides powerful support for molecular breeding for cultivating a diversity coloring mode of the orchid in the future.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to an R2R3-MYB transcription factor that specifically regulates the accumulation of anthocyanins in the perianth patch region of Dendrobium nobile and its application. Background Technology

[0002] Most orchids exhibit a bilaterally symmetrical floral structure, consisting of two whorled floral organs: the outermost whorled contains three sepals—one dorsal sepal and two lateral sepals; the second whorled contains three petals, two of which are morphologically similar to the sepals, while the third is a highly specialized lip. Because the sepals and petals are so similar in morphology that they are difficult to distinguish, they are often collectively referred to as the "tepals." The diversity of tepal coloration patterns, especially the unique coloration pattern on the lip, is not only a key characteristic of orchids' adaptation to pollination and evolution but also an important manifestation of their ornamental value. Therefore, genetically improving orchid flower color based on market demand and public preference is one of the main goals of breeders.

[0003] Currently, orchid breeding primarily relies on interspecific hybridization. However, traditional breeding methods are often accompanied by high levels of randomness and uncertainty, typically requiring significant time and effort, and multiple generations of selection to obtain the target variety. In recent years, with the rapid development of science and technology, targeted molecular breeding technology based on transgenic technology has been widely applied in the fields of crops and horticulture. This technology mainly controls specific phenotypic traits through gene overexpression or gene knockout, thereby allowing plants to acquire or lose the corresponding traits, ultimately cultivating ideal varieties. Naturally occurring homeomorphic mutants of Dendrobium nobile are ideal materials for studying perianth coloration patterns. Therefore, utilizing these mutants to obtain key genes controlling specific phenotypic traits is a prerequisite and important resource for molecular breeding.

[0004] Anthocyanins are natural pigments found in plant tissues that produce pink, red, blue, and purple colors. The anthocyanin biosynthetic pathway and its key structural genes have been extensively studied. The expression of these genes is mainly regulated by the MYB-bHLH-WD40 protein complex, with MYB playing a major regulatory role. Although many studies have reported on MYB transcription factors regulating flower color in orchids, the current research findings are still insufficient compared to the vast number of orchid species, the rich color variations, and the differences and specificities of homologous genes among species. This makes it difficult to meet the broad needs of future molecular breeding improvements for orchid flower color.

[0005] Therefore, supplementing and expanding the resources of R2R3-MYB transcription factors that promote anthocyanin synthesis in different orchid species remains an important foundation and prerequisite for future molecular improvement of flower color in orchid ornamental flowers.

[0006] In view of this, the present invention is hereby proposed. Summary of the Invention

[0007] The purpose of this invention is to provide an R2R3-MYB transcription factor that specifically regulates anthocyanin accumulation in the perianth patch area of ​​Dendrobium nobile and its application, so as to expand and enrich the transcription factor library for controlling perianth coloration patterns in orchids.

[0008] The objective of this invention is achieved through the following technical solution:

[0009] A specific R2R3-MYB transcription factor that regulates anthocyanin accumulation in the perianth patches of Dendrobium nobile, comprising the R2R3-MYB gene—DnMYB1, with its CDS sequence as follows:

[0010] .

[0011] The applications of the R2R3-MYB transcription factor that specifically regulates anthocyanin accumulation in the perianth patch region of Dendrobium nobile include one or more of the following applications:

[0012] The R2R3-MYB gene—DnMYB1 is targeted and regulated through transgenic technology for transgenic breeding;

[0013] The R2R3-MYB gene—DnMYB1, when heterologously and transiently overexpressed in white Phalaenopsis orchid cultivars, significantly promoted the accumulation of anthocyanins in the petals of white Phalaenopsis orchid cultivars.

[0014] The R2R3-MYB gene—DnMYB1, by targeting and silencing the target gene, can significantly reduce the accumulation of anthocyanins in the perianth of wild-type Dendrobium nobile.

[0015] Compared with existing technologies, the application of the R2R3-MYB transcription factor in this invention, which specifically regulates anthocyanin accumulation in the perianth patches of *Dendrobium nobile*, demonstrates its function. The function of the R2R3-MYB transcription factor was verified in the petals of the white-flowered Phalaenopsis sogo Yukidian 'V3' orchid using heterologous transient overexpression technology, showing that it promotes anthocyanin accumulation in the petals of this white-flowered Phalaenopsis cultivar. Simultaneously, silencing this gene in the perianth of wild-type *Dendrobium nobile* using VIGS technology significantly inhibited anthocyanin accumulation. Therefore, the R2R3-MYB gene enriches the genetic resource pool of specific pigment accumulation in orchid perianth patches, providing important theoretical basis and genetic resources for creating new orchid varieties with diverse perianth patterns using molecular breeding methods.

[0016] WIPO ST.26 Standard Serial List:

[0017] 1. CDS sequence of R2R3-MYB gene—DnMYB1:

[0018] . Detailed Implementation

[0019] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them, and do not constitute a limitation on the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the protection scope of the present invention.

[0020] First, the following explanations are provided for the terms that may be used in this article:

[0021] The terms “including,” “contains,” “comprising,” “having,” or other similar semantic descriptions shall be interpreted as non-exclusive inclusion.

[0022] The term "composed of" excludes any technical features not expressly listed. When used in a claim, it closes the claim to exclude all technical features other than those expressly listed, except for associated conventional impurities. If the term appears only in a clause of a claim, it limits the claim to the elements expressly listed in that clause; elements recited in other clauses are not excluded from the overall claim.

[0023] The technical solution provided by this invention will be described in detail below. Contents not described in detail in the embodiments of this invention are prior art known to those skilled in the art. Where specific conditions are not specified in the embodiments of this invention, they shall be performed according to conventional conditions in the art or conditions recommended by the manufacturer. Reagents or instruments used in the embodiments of this invention whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0024] The present invention relates to an R2R3-MYB transcription factor that specifically regulates anthocyanin accumulation in the perianth patch region of Dendrobium nobile, including the R2R3-MYB gene—DnMYB1, whose CDS sequence is as follows:

[0025] .

[0026] The aforementioned applications of the R2R3-MYB transcription factor, which specifically regulates anthocyanin accumulation in the perianth patches of Dendrobium nobile, include one or more of the following applications:

[0027] The R2R3-MYB gene—DnMYB1 is targeted and regulated through transgenic technology for transgenic breeding;

[0028] The R2R3-MYB gene—DnMYB1, when heterologously and transiently overexpressed in white Phalaenopsis orchid cultivars, significantly promoted the accumulation of anthocyanins in the petals of white Phalaenopsis orchid cultivars.

[0029] The R2R3-MYB gene—DnMYB1, by targeting and silencing the target gene, can significantly reduce the accumulation of anthocyanins in the perianth of wild-type Dendrobium nobile.

[0030] In summary, the technical solution of this invention specifically involves isolating and identifying an R2R3-MYB gene, named DnMYB1, that is specifically expressed in the perianth patch region of *Dendrobium nobile*. While the R2R3-MYB gene, which has been shown to promote anthocyanin accumulation, plays a similar function in different plants, its CDS sequence is not identical. Therefore, isolating and identifying an R2R3-MYB gene that specifically regulates anthocyanin accumulation in the perianth patch region of *Dendrobium nobile* can enrich the gene library for orchid molecular breeding, providing support for the future cultivation of new orchid varieties with diverse perianth patterns.

[0031] To more clearly demonstrate the technical solution and its effects provided by the present invention, the embodiments of the present invention will be described in detail below with reference to specific examples.

[0032] The following sections will use the heterologous transient overexpression of the DnMYB1 gene in the white-flowered Phalaenopsis orchid cultivar P. sogo Yukidian 'V3' and the silencing of the two genes in the perianth of wild-type Dendrobium nobile as examples to illustrate the technical scheme for transgenic breeding using the R2R3-MYB gene provided by this invention, thereby verifying the feasibility of this breeding path.

[0033] Example:

[0034] The specific process by which the R2R3-MYB gene's function in regulating anthocyanin accumulation, as described in this invention, was confirmed in both white-flowered Phalaenopsis orchid cultivars and wild-type Dendrobium nobile is as follows:

[0035] I. Heterologous transient overexpression of the DnMYB1 gene in white Phalaenopsis orchids

[0036] 1) Transcriptome sequencing was performed on the spotted and non-spotted areas of the lip petal of wild-type Dendrobium nobile, the spotted and non-spotted areas of the petal petal-lipped mutant, and the spotted and non-spotted areas of the lateral sepals of the lateral sepal-lipped mutant to identify the R2R3-MYB gene DnMYB1 specifically expressed in the spotted area.

[0037] 2) Total RNA was extracted from the perianth patch region of Dendrobium nobile and reverse transcribed to synthesize cDNA. The full-length CDS sequence was amplified and cloned using specific primers for DnMYB1, and verified by sequencing to ensure sequence integrity and accuracy.

[0038] 3) The pCAMBIA1302 vector was double-digested at the BamHI and PstI sites to linearize the vector. Based on the sequence at the end of the linearized vector, specific primers containing homologous arms were designed to amplify DnMYB1;

[0039] 4) The target fragment containing homologous arms was ligated to the linearized vector using a seamless cloning method. The ligation product was transformed into E. coli DH5α and plated on LB plates containing 50 μg / mL kanamycin. Positive single clones were selected for sequencing verification to ensure the accuracy of the overexpression vector.

[0040] 5) Transform the correctly sequenced overexpression vector into Agrobacterium GV3101, plate it on LB agar plates containing 50 μg / mL kanamycin, invert the plates and incubate them at 28℃ for 2-3 days. Select single colonies and inoculate them into 10 mL of LB liquid medium (50 mg / L kanamycin + 25 mg / L rifampin), and incubate them in a shaker at 28℃ and 200 rpm for 16 h. Extract the plasmid for sequencing identification.

[0041] 6) Adjust the OD600 value of the correctly sequenced bacterial culture to 0.75. Cover the centrifuge tube with aluminum foil to block light, incubate at 28°C for 3 hours, and add 100 μM acetylsuccinone;

[0042] 7) Using a medical syringe, draw 300 μL of injection solution. Select a healthy, 2 cm long, pale yellow-green flower bud of the Phalaenopsis sogo Yukidian 'V3' white-flowered horticultural variety. Carefully peel open the flower bud by hand. Use the syringe needle to make small incisions on the median sepal, lateral sepals, and petals. After removing the needle, hold the syringe nozzle against the incision and inject the overexpression injection solution into it at a uniform speed.

[0043] 8) Place the infected plants in a greenhouse under natural light conditions for cultivation. When the flower buds are fully open and show obvious color phenotypes, take photos and collect samples for subsequent pigment content determination.

[0044] II. Silencing the DnMYB1 gene in the perianth of wild-type Dendrobium nobile using virus-induced gene silencing (VIGS) technology

[0045] 1) Total RNA was extracted from the perianth patch region of Dendrobium nobile and reverse transcribed to synthesize cDNA. The full-length CDS sequence was amplified and cloned using specific primers for DnMYB1, and verified by sequencing to ensure sequence integrity and accuracy.

[0046] 2) Double digestion of the pTRV2-GFP vector at the EcoRI and BamHI sites was performed to linearize the vector. Based on the sequence at the end of the linearized vector and the non-conserved domain sequence (186 bp in length) in the CDS sequence of the Dendrobium nobile DnMYB1 gene, specific primers containing homologous arms were designed to amplify the target fragment.

[0047] 3) The DnMYB1 target fragment containing homologous arms was ligated to the linearized pTRV2-GFP vector using a seamless cloning method to construct the VIGS silencing vector. The ligation product was transformed into E. coli DH5α, plated on LB plates containing 50 μg / mL kanamycin, and positive single clones were selected for sequencing verification to ensure the accuracy of the silencing vector.

[0048] 4) Transform the correctly sequenced silencing vector into Agrobacterium GV3101, plate it on LB agar containing 50 μg / mL kanamycin, invert it and incubate it in a 28℃ incubator for 2-3 days. Select a single colony and inoculate it into 10 mL of LB liquid medium (50 mg / L kanamycin + 25 mg / L rifampin), place it in a shaker at 28℃ and 200 rpm for 16 h, and extract the plasmid for sequencing identification.

[0049] 5) Resuspend the cells of pTRV1, pTRV2-GFP, and the correctly sequenced bacterial culture in infection buffer and adjust the bacterial concentration to OD600 = 0.75. Mix pTRV1 with equal volumes of pTRV2-GFP and pTRV2-DnMYB1-GFP bacterial cultures, respectively. Cover the centrifuge tubes with aluminum foil to protect them from light. After standing at room temperature for 3 h, add 100 μM acetylsuccinone.

[0050] 6) Select wild-type Dendrobium nobile flower buds (1-1.5 cm in length, with a slightly colored base on the lip and the rest being entirely green), and use a sterile syringe to inject VIGS bacterial solution into the flower bud tissue and the adjacent flower stalk. The injection volume per plant should be controlled at 150 μL.

[0051] 7) Place the infected plants in a greenhouse under normal light conditions to grow. When the perianth matures, take photos and collect plant samples for subsequent pigment content determination.

[0052] As can be seen from the above implementation scheme, the present invention can enrich the library of transgenic materials that promote anthocyanin accumulation in the perianth patch area of ​​Dendrobium nobile, and the present invention can be used to target and regulate the perianth patch traits of Dendrobium nobile through transgenic technology.

[0053] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. An R2R3-MYB transcription factor that specifically regulates anthocyanin accumulation in the perianth patch region of Dendrobium nobile, characterized in that, Including the R2R3-MYB gene—DnMYB1, its CDS sequence: .

2. The application of the R2R3-MYB transcription factor as described in claim 1, which specifically regulates anthocyanin accumulation in the perianth patch region of Dendrobium nobile, characterized in that, Includes one or more of the following applications: The R2R3-MYB gene—DnMYB1 is targeted and regulated through transgenic technology for transgenic breeding; The R2R3-MYB gene—DnMYB1, when heterologously and transiently overexpressed in white Phalaenopsis orchid cultivars, significantly promoted the accumulation of anthocyanins in the petals of white Phalaenopsis orchid cultivars. The R2R3-MYB gene—DnMYB1, by targeting and silencing the target gene, can significantly reduce the accumulation of anthocyanins in the perianth of wild-type Dendrobium nobile.