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Application of peony PsMYB1 gene in changing color and flower color of plant mottles

A peony and gene technology, applied in the field of plant biology, can solve the problems of MYB1 gene research that has not been reported, and achieve the effect of bright color of petals, increased expression level, and increased total anthocyanin content

Active Publication Date: 2022-08-02
YANGZHOU UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The above results provide a theoretical basis for the study of the molecular mechanism of the formation of peony variegation traits, but the research on the regulation of MYB1 gene in the formation of peony variegation has not been reported.

Method used

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  • Application of peony PsMYB1 gene in changing color and flower color of plant mottles
  • Application of peony PsMYB1 gene in changing color and flower color of plant mottles
  • Application of peony PsMYB1 gene in changing color and flower color of plant mottles

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1 Cloning of the full-length cDNA sequence of the peony PsMYB1 gene

[0053] 1. Obtaining the cDNA sequence of the 3′ end of the PsMYB1 gene: The peony variety 'Haihuang' (purchased from Heze, Shandong) was selected as the material. First, according to the change of petal shape during plant development, it was divided into 4 stages, namely dew color. stage, loose valve stage, early blooming stage and blooming stage ( figure 1 ), the mixed samples of peony petals from four periods were used as materials, and the MiniBEST Plant RNA Extraction Kit (TaKaRa) was used to extract total RNA. The first strand of cDNA was produced by reverse transcription using 3'full RACE Core Set Ver.2.0 (TaKaRa). The reverse transcription system was: 1μL RNA, 1μL 3'-RACE Adaptor, 1μL dNTP Mixture (10mM each), 2μL 5×M -MLV Buffer, 0.25μL RNase Inhibitor, 0.25μL Reverse Transcriptase M-MLV (RNase H - ), 4.5μL RNase Free ddH 2 O; reverse transcription program: reaction at 42°C for 60mi...

Embodiment 2

[0055] Example 2 Homology alignment and phylogenetic tree analysis between the predicted amino acid sequence of the peony PsMYB1 gene and the MYB protein related to anthocyanin biosynthesis

[0056] The amino acid sequence SEQ ID NO.2 of the peony 'sea yellow' PsMYB1 gene and the amino acid sequence of the MYB gene related to the anthocyanin biosynthesis pathway in other plants were saved as TXT files, and then loaded into DNAMAN5.2.2 software for the same process. source alignment, such as image 3 As shown, the peony PsMYB1 protein and other MYB proteins related to anthocyanin biosynthesis both contain R2 and R3 conserved domains at the N-terminus. Save the amino acid sequence SEQ ID NO.2 of the peony 'sea yellow' PsMYB1 gene and the amino acid sequence of the R2R3-MYB gene related to the anthocyanin biosynthesis pathway in other plants as TXT files, and then load them into MEGA 7.0 software, The phylogenetic tree was constructed by the neighbor joining method, and Bootstra...

Embodiment 3

[0057] Example 3 Expression of peony PsMYB1 gene silencing vector in peony petals

[0058] 1. Construction of peony PsMYB1 gene silencing vector: Design primers containing enzyme cleavage sites Xba I and BamH I for amplifying PsMYB1-specific sequences (upstream primer: 5'-AAGGTTACCGAATTCTCTAGAACGGATACCAGGCCGGAG-3' (SEQ ID NO.6), downstream Primer: 5'-CGTGAGCTCGGTACCGGATCCATGGGTAGACCACCTTGCTGTG-3' (SEQ ID NO. 7)). PCR amplification system: 12.5 μL 2×Phanta Flash Master Mix (Vazyme), 1 μL ForwardPrimer, 1 μL Reverse Primer, 2 μL cDNA template containing PsMYB1 gene in Example 1, 8.5 μL ddH 2 O. Reaction program: pre-denaturation at 98°C for 30s; denaturation at 98°C for 10s, annealing at 60°C for 5s, extension at 72°C for 5s, a total of 35 cycles; extension at 72°C for 1 min. After the reaction, the PCR reaction solution was analyzed by agarose gel electrophoresis, and the PsMYB1-specific fragment containing the restriction site was recovered by TSP601-DNA gel recovery kit (Ts...

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Abstract

The invention discloses application of a peony PsMYB1 gene in changing color and flower color of plant mottles. A constructed PsMYB1 gene silencing vector is transformed into peony petals for expression, after the petals grow for 7 days, the phenotype of PsMYB1 transgenic peony flower spots is observed, and it is found that the color of the flower spots is obviously light, the total anthocyanin content in the flower spots is obviously reduced compared with a wild type, and the expression level of anthocyanin biosynthesis related genes in the flower spots is obviously reduced; and a new peony germplasm with light-color piebald is created. A constructed PsMYB1 gene overexpression vector is transformed into tobacco for expression, after the plant blooms, the petal phenotype of the PsMYB1 transgenic tobacco is observed, and it is found that the color of petals is obviously red, the total anthocyanin content in the petals is obviously increased compared with that of wild type petals, and the expression level of anthocyanin biosynthesis related genes in the petals is obviously increased; and a new tobacco germplasm with bright petals is created.

Description

technical field [0001] The invention belongs to the field of plant biotechnology, and in particular relates to the application of the peony PsMYB1 gene in changing the variegation and flower color of plants. Background technique [0002] Petal stains are commonly found in ornamental plants, and they are distributed on floral organs in a fixed form and location, such as the petals or calyx of Pansy (Viola × wittrockiana), Oncidium hybridum (Oncidium hybridum), and Paeoniarockii (Paeoniarockii) There are dark spots at the base (Shang Xiao, Wang Jian, Li Qin, Gong Sheng, Sun Haiyan, Zhang Xuanbing. Research progress on the molecular mechanism of variegation formation in plants. Chinese Journal of Horticulture, 2014, 41:1485-1494). In angiosperms, variegation is also an important visual signal of plants to pollinators, which promotes the specific interaction between plants and pollinators, and plays a very important role in plant reproduction and dispersal (Davies KM, Albert NW)...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C07K14/415C12N15/11C12N15/84C12N15/66A01H5/02A01H6/00A01H6/82C12Q1/6895
CPCC07K14/415C12N15/825C12N15/8218C12N15/8205C12N15/66C12Q1/6895C12Q2600/13C12Q2600/158
Inventor 陶俊栾雨婷钱祎赵大球汤寓涵孙静
Owner YANGZHOU UNIV
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