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Transmethylase gene participating in synthesis of sanguinarine and chelerythrine in macleaya cordata and application of transmethylase gene

A technology of methyltransferase and chelerythrine, applied in the field of genetic engineering and molecular biology

Active Publication Date: 2016-11-09
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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  • Transmethylase gene participating in synthesis of sanguinarine and chelerythrine in macleaya cordata and application of transmethylase gene
  • Transmethylase gene participating in synthesis of sanguinarine and chelerythrine in macleaya cordata and application of transmethylase gene
  • Transmethylase gene participating in synthesis of sanguinarine and chelerythrine in macleaya cordata and application of transmethylase gene

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Effect test

Embodiment 1

[0023] Example 1 Mining and acquisition of genes involved in the synthesis of sanguinarine and chelerythrine

[0024] 1 Gene mining

[0025] 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 methyltransferase gene sequences of sanguinarine and chelerythrine published in NCBI in other species (poppy, poppy) as reference genes, and performed BLAST comparison on the whole genome sequence of Boluohui DeNovo. 11 candidate genes were obtained. 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 to screen 11 candidate genes for gene...

Embodiment 2

[0034] Example 2 Using Yeast Expression System to Verify the Function of Synthetic Candidate Genes of Sanguinarine and Chelidonine

[0035] The genes Mc8567, Mc833, Mc830, Mc769, Mc2833 and Mc9487 were respectively constructed on the expression vector pYES2 (Invitrogen), and transformed into yeast. Induce yeast to express protein, then carry out precursor feeding to collect yeast. After cleavage, the compound was extracted with methanol. After the samples were prepared, they were detected by UPLC-Q-TOF. The results are as follows Figure 3-Figure 7 shown.

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Abstract

The invention discloses a transmethylase gene participating in synthesis of sanguinarine and chelerythrine in macleaya cordata and application of the transmethylase gene. Six transmethylase genes participating in synthesis of sanguinarine and chelerythrine are found in a macleaya genome for the first time and comprise the Mc2833 gene, the Mc769 gene, the Mc8567 gene, the Mc833 gene, the Mc830 gene and the Mc9487 gene. A saccharomyces cerevisiae system is utilized for verifying, several steps of reactions are verified through upstream precursor feeding, and synthesis of sanguinarine and chelerythrine intermediates can be achieved. The invention further discloses a molecular mechanism of sanguinarine in synthesis of macleaya cordata, and theoretical bases and molecular assisted breeding objectives are provided for sanguinarine and chelerythrine content macleaya cordata breeding; meanwhile, valuable 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 methyltransferase 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 op...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/81C12N1/19
CPCC12N9/1007C12N15/81C12N2800/102C12Y201/01116C12Y201/01117C12Y201/01122C12Y201/01128C12Y201/0114
Inventor 曾建国黄鹏卿志星
Owner MICOLTA BIORESOURCE INC LTD
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