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Novel campanula flavonoid 3',5'-hydroxylase gene and use of same

An amino acid and base sequence technology, applied in genetic engineering, oxidoreductases, enzymes, etc., can solve unpredictable and complex problems

Inactive Publication Date: 2014-12-24
SUNTORY HLDG LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0016] As described above, although delphinidin can be produced by expressing the F3'5'H gene in different kinds of plants, it is difficult to predict which promoter will be used for the F3'5'H gene from which plant in the target plant The expression is good. In order to efficiently produce delphinidin, that is, the delphinidin content in the total amount of anthocyanins is 80% or more, preferably 85% or more, more preferably 90% or more, and still more preferably 95% or more. Repeated trials and complexities are required. experiments, etc.

Method used

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  • Novel campanula flavonoid 3',5'-hydroxylase gene and use of same
  • Novel campanula flavonoid 3',5'-hydroxylase gene and use of same
  • Novel campanula flavonoid 3',5'-hydroxylase gene and use of same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] [Example 1: Separate isolation of Bellflower F3'5'H cDNA]

[0067] Based on the translated sequence of Campanula medium F3'5'HcDNA (accession number D14590) registered in the DNA database GenBank, two primers CamF1 (5'-GTGAAGCCACCATGTCTATAG-3', sequence number 3) and CamR1 (5' - GCATTTGCCTAGACAGTGTAAG-3', SEQ ID NO: 4). RNA was extracted from petals of commercially available bellflower buds using RNeasy Mini Plant Kit (QIAGEN), and 1st strand DNA was synthesized using RT-PCR kit. Using this 1st strand DNA as a template, PCR was performed using the primers described above. The resulting DNA fragment was cloned into pCR-TOPO II. Inside, the nucleotide sequence of clone #4 (pSPB2561) was determined by analysis with a DNA sequencer (SEQ ID NO: 1). The F3'5'H registered as accession number D14590 consists of 523 residues compared to the F3'5'H encoded by clone #4 consisting of 521 residues relative to the F3'5'H registered as D14590 Showing 96% sequence identity.

reference example 1

[0068] [Reference Example 1: Introduction of pSPB130 into the Chinese rose variety "Lengshui" (constitutive expression of pansy F3'5'H gene and torenia 5AT gene)]

[0069] The binary vector pSPB130 (constitutively expressing the F3'5'H gene of pansy and the anthocyanin 5-acyltransferase gene of torenia in plants) described in Patent Document 3 was introduced into Agrobacterium ( Agrobacterium tumefaciens) Agl0. This transformed Agrobacterium was introduced into the lavender rose variety "Lengshui" by the method described in Patent Document 3, and 164 transformants were obtained. When the anthocyanin pigments in petals were analyzed, delphinidin accumulation was confirmed in 51 of 164 transformants, and the delphinidin content rate was the highest at 76.1% (36.6% on average).

[0070] Analysis values ​​of representative transformants are shown in Table 1 below.

[0071] 【Table 1】

[0072]

[0073] Del: delphinidin, Cya: anthocyanin, Pel: anthocyanin

[0074] Delphinidin ...

Embodiment 2

[0075] [Example 2: Introduction of pSPB2564 into the rose variety "Lengshui" (expression from the F3'5'H gene of Bellflower under the control of the E12 35S promoter)]

[0076] The expression cassette in which the bellflower F3'5'H gene cDNA obtained in Example 1 was inserted between the El2 35S promoter and the nopaline synthase terminator (nos terminator) described in Patent Document 3 was inserted into a binary vector pBinPLUS (van Engelen et al. Transgenic Research 4, 288-290, 1995), and the resulting plasmid was designated as pSPB2564. pSPB2564 was introduced into Agrobacterium tumefaciens Agl0. Using this transformed Agrobacterium, the lavender rose variety "Lengshui" was transformed by the method described in Patent Document 3, and 55 transformants were obtained. When the anthocyanin pigments in the petals were analyzed, delphinidin accumulation was confirmed in 48 of the 55 transformants, and the delphinidin content was the highest at 97.3% (average 61.22%).

[0077]...

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Abstract

Provided are a novel campanula flavonoid 3',5'-hydroxylase gene and a plasmid that contains the gene under the control of a cauliflower mosaic virus 35S promoter.

Description

【Technical field】 [0001] The present invention relates to the technique of introducing foreign genes into plant cells so as to carry out genetic manipulation. In more detail, the present invention relates to a novel bellflower flavonoid 3',5'-hydroxylase gene and a plasmid comprising the gene under the control of the cauliflower mosaic virus 35S promoter, developed by the Agrobacterium method using the plasmid The invention relates to a method for producing a horticultural variety rose plant which can significantly increase delphinidin content in petals through transformation. 【Background technique】 [0002] Methods for improving plant varieties include (i) mating and hybridization of stamens and pistils, (ii) natural or artificial mutations, (iii) genetic recombination, and the like. Among them, when gene recombination technology is used, it is possible to impart a new shape to the plant by expressing a useful gene in the target plant without being bound by the genetic con...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09A01H5/00
CPCC12N9/0073C12N15/825C12N15/8205C12Y114/13088A01H5/00
Inventor 田中良和胜元幸久
Owner SUNTORY HLDG LTD
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