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Flavonoid-3,5'-hydroxylase gene cloned from moth orchid, its coded sequence and application

A flavonoid, hydroxylase technology, applied in the fields of molecular biology and genetic engineering

Inactive Publication Date: 2008-04-23
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Before the publication of the invention, there was no public report on the Phalaenopsis flavonoid-3'5'-hydroxylase gene sequence mentioned in this patent application, its protein sequence or its application in transgenic plants

Method used

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  • Flavonoid-3,5'-hydroxylase gene cloned from moth orchid, its coded sequence and application
  • Flavonoid-3,5'-hydroxylase gene cloned from moth orchid, its coded sequence and application
  • Flavonoid-3,5'-hydroxylase gene cloned from moth orchid, its coded sequence and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Cloning of Phalaenopsis flavonoid-3’5’-hydroxylase gene

[0037] 1 Phalaenopsis hybrida (Phalaenopsis hybrida) material was collected from the Institute of Vegetable Horticulture, Shanghai Academy of Agricultural Sciences, the variety is Formosa rosa, and the flower colors are purple, white, and yellow. Plant materials are grown under normal conditions in the greenhouse (L / D, 16h / 8h; 25-28°C).

[0038] 2 RNA extraction. Take about 100 mg of fresh material and grind it fully with liquid nitrogen. Add 1mL TriBlue reagent, shake vigorously for 15s, and leave at room temperature for 5min. Add 0.2 mL of chloroform to remove protein, transfer the supernatant to a new centrifuge tube, add an equal volume of isopropanol, mix well, and place at room temperature for 10 minutes. Wash the precipitate with 1 mL of 75% ethanol prepared by DEPC and repeat once. Dry at room temperature for 5-10 minutes, dissolve in 20μL DEPC water, measure OD value, and detect by electrophoresis.

[0039] 3...

Embodiment 2

[0043] Analysis of copy number of Phalaenopsis flavonoid-3’5’-hydroxylase gene

[0044] The Phalaenopsis genome was extracted by CTAB method, and 10 μg DNA was digested with three commercially available enzymes, Bgl II, EcoR V and Hind III, and digested overnight at 37°C. The digested products were separated by electrophoresis using 0.8% agarose gel. Transfer DNA and fix it on nylon membrane. Probe hybridization confirms that it is but a copy. (see picture 1)

Embodiment 3

[0046] Phalaenopsis flavonoid-3'5'-hydroxylase gene eukaryotic expression in petunia plants and phenotype identification of transgenic plants.

[0047] 1 Construction of expression vector. First amplify the complete coding reading frame, and introduce restriction endonuclease recognition sites on the forward and reverse primers (depending on the selected vector) to construct the expression vector. Using the amplified product obtained in Example 1 as a template, after PCR amplification, the Phalaenopsis flavonoid-3'5'-hydroxylase gene was forward cloned into the vector p2301.

[0048] 2 Transfer to Agrobacterium. Prepare Agrobacterium competent, and use heat shock transformation method to introduce plasmid into Agrobacterium.

[0049] 3 Agrobacterium leaf disc method to transform petunia.

[0050] (1) Cut petunia leaves into 0.8×0.8cm (1cm) squares, put them into MSO solution containing Agrobacterium, shaker at 190rpm for 10min.

[0051] (2) Dry the leaves and place them on MS1 ​​c...

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Abstract

The present invention belongs to the field of molecular biology and gene technology, and is especially the nucleotide coding sequence of flavoid-3', 5'-hydroxylase gene expressed in butterfly orchid, and its application in changing flower color. The present invention relates to the recombinant expression vector including the said gene, the transgenic plant, nucleotide primer sequence for obtaining the gene, the oligonucleotide sequence probe for detecting endogenous expression mode and method of analyzing and identifying the expression mode of the endogenous gene in butterfly orchid. Transforming the gene into petunia obtains transgenic plant with differentially expressed flavoid-3', 5'-hydroxylase gene and altered flower color.

Description

Technical field [0001] The present invention belongs to the technical fields of molecular biology and genetic engineering, and specifically relates to a nucleotide coding sequence of a flavonoid-3'5'-hydroxylase gene expressed in Phalaenopsis, a recombinant expression vector containing the above gene, and a transgene Plants, and methods for analyzing and identifying the expression pattern of this endogenous gene in Phalaenopsis. Background technique [0002] The color of flowers in nature is ever-changing, which is inseparable from its complex metabolic pathways and diverse metabolites. As early as 1664, Robert Boyle studied the effect of pH on plant color. Flower color is mainly determined by flavonoids, carotenoids and alkaloids. Among them, flavonoid pigments are a type of vacuole, which mainly exist in vacuoles. The accumulation of different pigments of flavonoids in the vacuoles produces various colors. At the same time, the difference in the composition and concentration of...

Claims

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Application Information

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IPC IPC(8): C12N15/52C12N9/00C12N15/82C12Q1/68
Inventor 明凤王敬文刘启昆石金磊
Owner FUDAN UNIV
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