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Flavoprotein oxidase genes of macleaya cordata in synthesis of sanguinarine and chelerythrine and application of flavoprotein oxidase genes

A technique for chelerythrine and flavoprotein, applied in the field of flavoprotein oxidase gene

Active Publication Date: 2016-10-26
MICOLTA BIORESOURCE INC LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Related synthetic genes have been cloned from opium poppy (Papaver somniferum L.), poppy (Eschscholzia californica Cham.) and other plants, but no gene has been cloned from Boluohui

Method used

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  • Flavoprotein oxidase genes of macleaya cordata in synthesis of sanguinarine and chelerythrine and application of flavoprotein oxidase genes
  • Flavoprotein oxidase genes of macleaya cordata in synthesis of sanguinarine and chelerythrine and application of flavoprotein oxidase genes
  • Flavoprotein oxidase genes of macleaya cordata in synthesis of sanguinarine and chelerythrine and application of flavoprotein oxidase genes

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Embodiment 1

[0016] Example 1 Mining and acquisition of genes involved in the synthesis of sanguinarine and chelerythrine

[0017] 1 Gene mining

[0018] Since the synthetic pathways of sanguinarine and chelerythrine in plants such as poppy and poppy are known ( figure 1 ), and related functional genes have also been cloned. We used the flavoprotein oxidase-related gene sequences of other species (poppy, poppy) that have been published in NCBI as reference genes, and performed BLAST on the whole genome sequence of Boluohui De Novo A total of 14 candidate genes were obtained by comparison. And because the contents of sanguinarine and chelerythrine are extremely high in pods, the contents in stems are extremely low. Precursor compounds pro-opiine and allocryptopine are found in extremely high levels in roots and very low levels in stems. Taking this as a clue, we analyzed the transcriptome data (RNA-Seq) of Boluohui roots, stems, leaves, flowers and fruits, and screened out 14 candidate ...

Embodiment 2

[0026] Example 2 Using the yeast expression system to verify the function of the candidate gene for sanguinarine and chelerythrine synthesis, and compare the transformation efficiency of related genes in Boluohui and poppy

[0027] The Mc20113, Mc6407, Mc6408, Mc6426, and PsDBOX genes were respectively constructed on the expression vector pYES2 (Invitrogen), and transformed into yeast. Yeast is induced to express protein, and then the precursor is fed to collect the yeast. After lysis, the compound is extracted with methanol. After the sample is prepared, it is detected by UPLC-Q-TOF. The results are as follows Figure 3-Figure 4shown. Using yeast expression system to compare the transformation efficiency of Mc6407, Mc6408 in Boluohui and PsDBOX in poppy ( Figure 4 ).

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Abstract

The invention discloses flavoprotein oxidase genes of macleaya cordata in synthesis of sanguinarine and chelerythrine and application of the flavoprotein oxidase genes. Three flavoprotein oxidase genes joining in synthesis of sanguinarine and chelerythrine are found in a macleaya cordata genome for the first time, including genes Mc20113, Mc6407 and Mc6408. Steps of reaction are respectively verified by using a brewer's yeast system through upstream precursor feeding, and synthesis of sanguinarine and chelerythrine and midbodies can be achieved. The conversion efficiency of same functional genes of macleaya cordata and opium poppy is further compared by using a brewer's yeast heterologous expression system, and the result shows that the conversion rate of the gene of macleaya cordata is remarkably higher than that of opium poppy. The invention further discloses a molecular mechanism of synthesis of sanguinarine in macleaya cordata, and a theoretic basis and molecular assistant breeding targets are provided for breeding of macleaya cordata with high contents of sanguinarine and chelerythrine; and meanwhile, precious experience is provided for in-vitro artificial synthesis of sanguinarine and chelerythrine.

Description

technical field [0001] The invention relates to the fields of genetic engineering and molecular biology, in particular to a flavoprotein oxidase gene involved in the synthesis of sanguinarine and chelerythrine in Boluohui and its application. Background technique [0002] Sanguinarine and chelerythrine are mainly distributed in sanguinaria canadensis L., opium poppy (Papaver somniferum L.), poppy (Eschscholzia californica Cham.), celandine (Chelidonium majus), purple cordyces ( Corydalis edulis Maxim) and Bo Luohui (Macleaya cordata (Willd.) R.Br.). For the last two decades, sanguinarine has been used in Europe as an additive to replace antibiotics to promote animal growth. At present, Boluohui is the main source plant of sanguinarine in the world, and it is listed as an animal feed additive by the European Food Safety Authority. The synthetic pathways of sanguinarine and chelerythrine have been elucidated in previous studies. Related synthetic genes have been cloned from...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N9/02
CPCC12N9/0004C12Y121/03003
Inventor 曾建国黄鹏刘秀斌
Owner MICOLTA BIORESOURCE INC LTD
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