Myb transcription factor for regulating color formation of cymbidium and application thereof

Abstract

This invention belongs to the field of plant genetic engineering, specifically relating to a MYB transcription factor that regulates the color formation of Cymbidium goeringii. CeMYB104 And its applications. The MYB transcription factor CeMYB104 The nucleotide sequence is shown in SEQ ID NO.1. Transformation into white Phalaenopsis orchids using a transient expression method showed that the MYB transcription factor... CeMYB104 CeMYB104 CeMYB104 CeMYB104 CeMY This invention can induce the accumulation of anthocyanins in the tepals of white Phalaenopsis orchids, turning them purplish-red. This invention provides guidance for the breeding and in-depth research of orchid varieties with different flower colors.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering, specifically relating to a MYB transcription factor CeMYB104 that regulates color formation in Cymbidium ensifolium and its applications. Background Technology

[0002] Cymbidium ensifolium, also known as the Four Seasons Orchid, is a perennial herbaceous plant belonging to the genus Cymbidium in the family Orchidaceae. It is a large group within the Cymbidium genus, mainly distributed in Fujian, Zhejiang, Anhui, Jiangxi, Sichuan, and Yunnan provinces of my country, and in Southeast Asian countries, as well as India and Japan. Cymbidium ensifolium has a long history of cultivation in my country, with numerous varieties and highly valued for its ornamental qualities, including rich foliage and plant variegation, and unique and varied flower colors.

[0003] Flower color is one of the important ornamental traits of orchids, influenced by various factors such as epidermal cell shape and environmental factors. The most important factor is the type and content of pigments. Pigments, as secondary metabolites of plants, are widely distributed in the cells of flowers, leaves, and other tissues, giving plants a variety of colors. Flower color is one of the main ornamental traits of Cymbidium ensifolium. To further enhance the horticultural value of Cymbidium ensifolium, flower color improvement can be a new goal in its variety development. Currently, the breeding of colored Cymbidium ensifolium varieties still mainly relies on screening from wild species, which is extremely inefficient and progresses very slowly. Furthermore, with the increasing destruction of wild Cymbidium ensifolium resources, the breeding of new colored varieties will become even more difficult. Using genetic engineering for targeted improvement of flower color is the most cutting-edge technology in the field of flower color breeding, but current research on Cymbidium ensifolium flower color is limited, and its molecular mechanisms are unclear, urgently requiring systematic research.

[0004] The MYB gene family is one of the largest transcription factor families, among which the R2R3-MYB transcription factor can alter flower color by influencing the formation of flavonoids. MYB has been identified as a major determinant of the MBW complex in anthocyanin biosynthesis, and its spatiotemporal expression pattern determines the morphology and localization of anthocyanins. Although research on MYB transcription factors in orchids has increased in recent years, it is still far from sufficient, and further research is needed to supplement and expand the resources of MYB transcription factors in orchids that can promote anthocyanin synthesis. Summary of the Invention

[0005] The purpose of this invention is to provide a MYB transcription factor that regulates the flower color formation of Cymbidium ensifolium. The transcription factor CeMYB104, which regulates the flower color formation of Cymbidium ensifolium, was cloned from Cymbidium ensifolium, and the application of this transcription factor was clarified.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A MYB transcription factor CeMYB104 that regulates the formation of blue pigment in cymbidium orchids, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0007] A primer pair for amplifying the aforementioned MYB transcription factor CeMYB104, the nucleotide sequence of which is as follows: CeMYB104-F: 5'-aacacgggggactcttgaccatggATGAAGAGGAAGCCATTCTGTGAC-3', CeMYB104-R: 5'-tgaaaagttcttctcctttactagtTTAAAAATTCATCCAAATATCTGTCTCCAATG-3'.

[0008] An expression vector containing the aforementioned MYB transcription factor CeMYB104.

[0009] A recombinant bacterium containing the aforementioned MYB transcription factor CeMYB104.

[0010] The above-mentioned application of the MYB transcription factor CeMYB104 in increasing the anthocyanin content in the perianth segments of orchid plants, wherein the orchid plant is Phalaenopsis.

[0011] The above-mentioned application of the MYB transcription factor CeMYB104 in altering the coloration of the perianth segments of an orchid, wherein the orchid is Phalaenopsis.

[0012] The significant advantages of this invention are: Flower color is essentially formed by the accumulation of pigments on the perianth segments. Anthocyanins are important coloring substances widely found in plants. In this invention, the function of the MYB transcription factor CeMYB104 was verified in white Phalaenopsis orchids using transient overexpression technology. It was found that CeMYB104 can induce the accumulation of anthocyanins in the perianth segments, turning the perianth segments of white Phalaenopsis orchids purplish-red. This invention provides strong support for future molecular breeding of orchids with red flowers. Attached Figure Description

[0013] Figure 1 The results show the color change of white Phalaenopsis orchid petals after transient overexpression of CeMYB104. A, B, and C represent 0, 3, and 6 days after injection of pCAMBIA1302-CeMYB104 Agrobacterium, respectively; D, E, and F represent 0, 3, and 6 days after injection of pCAMBIA1302 Agrobacterium, respectively. Detailed Implementation

[0014] To make the content of this invention easier to understand, the technical solution of this invention will be further described below with reference to specific embodiments, but this invention is not limited thereto.

[0015] This invention screened and identified a MYB transcription factor that regulates flower color formation in Cymbidium ensifolium, named CeMYB104, from the genome of Cymbidium ensifolium. Its CDS sequence is as follows (SEQ ID NO.1): 5'--3'.

[0016] Example 1 Orchid perianth segments were collected, and total RNA was extracted from the samples using the OMEGA Total RNA Extraction Kit according to the manufacturer's instructions. The RNA was then processed using the Yeasen Hifair II 1st Strand cDNA Synthesis assay. Total RNA was reverse transcribed using Kit (11119ES60) to obtain cDNA. Using the reverse-transcribed cDNA as a template, PCR amplification was performed using primers containing the homologous arm sequence from the pCAMBIA1302 plasmid vector to obtain the CeMYB104 sequence containing the homologous arm sequence. After gel purification, the PCR product was homologously recombined into the NcoI and SpeI restriction sites of the pCAMBIA1302 plasmid vector using Yeasen's one-step rapid cloning kit (10911ES20) to obtain the recombinant expression vector pCAMBIA1302-CeMYB104 containing CeMYB104. The recombinant expression vector pCAMBIA1302-CeMYB104 was transformed into E. coli DH5α competent cells (Weidi Corporation), and bacterial culture PCR was performed for verification. Bacterial cultures containing the correct bands were selected for sequencing analysis. The primer pairs containing the homologous arm sequences of the pCAMBIA1302 plasmid vector are as follows: CeMYB104-F: 5'-aacacgggggactcttgaccatggATGAAGAGGAAGCCATTCTGTGAC-3', CeMYB104-R: 5'-tgaaaagttcttctcctttactagtTTAAAAATTCATCCAAATATCTGTCTCCAATG-3'.

[0017] The correctly sequenced positive clone plasmid pCAMBIA1302-CeMYB104 and the empty vector plasmid pCAMBIA1302 were transformed into GV3101 Agrobacterium competent cells, respectively, and positive transformants were screened by PCR. 100 μL of Agrobacterium containing the empty vector pCAMBIA1302 and the empty vector pCAMBIA1302-CeMYB104 were respectively placed in 10 mL of LB broth containing kanamycin and rifampin antibiotics, and cultured at 28°C and 180 rpm for approximately 24 h until the bacterial culture reached OD. 600 The value is 0.6 to 0.8. The formulation of the LB liquid culture medium containing kanamycin and rifampicin antibiotics is: 10 μL 50 mg / mL kanamycin antibiotic + 10 μL 25 mg / mL rifampicin antibiotic + 10 mL LB liquid culture medium.

[0018] Centrifuge the cultured Agrobacterium tumefaciens at 5000 rpm for 5 minutes at room temperature, removing as much supernatant as possible; add invasion staining buffer to the bacterial pellet to reduce OD. 600Adjust the concentration to approximately 0.6–0.8, then incubate at 27°C for about 2 hours. After incubation, inject the solution into the perianth segments of the white Phalaenopsis orchid buds that are about to bloom. The infection buffer is prepared as follows: dissolve 1.9524 g of MES in 10 mL of sterile water to obtain an MES solution; dissolve 0.196 g of acetylsalicylic acid (AS) in 10 mL of dimethyl sulfoxide (DMSO) to obtain an AS solution; dissolve 2.03 g of magnesium chloride in 10 mL of sterile water to obtain a MgCl2 solution; take 100 mL of MS medium, add 1 mL of MES solution, 1 mL of MgCl2 solution, and 100 μL of AS solution, and mix well to obtain the infection buffer. After injection, incubate in an artificial climate chamber under the following conditions: 24°C, 16 hours of light, 8 hours of darkness, and 70% humidity. Observe phenotypic changes and record photographs after 0, 3, and 6 days of incubation.

[0019] like Figure 1 As shown, after injection of Agrobacterium tumefaciens pCAMBIA1302-CeMYB104, the petals of white Phalaenopsis orchids turned purplish-red. This result indicates that CeMYB104 can alter the flower color of orchids.

Claims

1. Overexpression of MYB transcription factor CeMYB104 Its application in increasing the anthocyanin content in the perianth segments of orchid plants is characterized by: The MYB transcription factor CeMYB104 The nucleotide sequence is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that: The orchid mentioned is the Phalaenopsis orchid.

3. Overexpression of MYB transcription factor CeMYB104 The application of changing the white Phalaenopsis orchid petals to a purplish-red color is characterized by: The MYB transcription factor CeMYB104 The nucleotide sequence is shown in SEQ ID NO.1.

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