Cytochrome P450 enzyme gene for taking part in sanguinarine and chelerythrine synthesis in macleaya cordata and application of cytochrome P450 enzyme gene

A technology of chelerythrine and cytochrome, which is applied in application, genetic engineering, plant genetic improvement, etc.

Active Publication Date: 2016-11-16
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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  • Cytochrome P450 enzyme gene for taking part in sanguinarine and chelerythrine synthesis in macleaya cordata and application of cytochrome P450 enzyme gene
  • Cytochrome P450 enzyme gene for taking part in sanguinarine and chelerythrine synthesis in macleaya cordata and application of cytochrome P450 enzyme gene
  • Cytochrome P450 enzyme gene for taking part in sanguinarine and chelerythrine synthesis in macleaya cordata and application of cytochrome P450 enzyme gene

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Effect test

Embodiment 1

[0024] Example 1 Mining and acquisition of genes involved in the synthesis of sanguinarine and chelerythrine

[0025] 1 Gene mining

[0026] 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 cytochrome P450 enzyme 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 De Novo. A total of 14 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 out 14 can...

Embodiment 2

[0036] Example 2 Using the yeast expression system to verify the function of the candidate gene for the synthesis of sanguinarine and chelerythrine, and compare the transformation efficiency of related genes in Boluohui and Poppy and Poppy

[0037] Mc2661, Mc9485, Mc217, Mc11229, Mc11218, Mc10725 and the coenzyme CPR gene 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 6 shown. Using the yeast expression system to compare the transformation efficiency of Mc11229 and Mc11218 in Boluohui, PsP6H in poppy and EcP6H in poppy ( Image 6 ).

[0038] The CPR19967 gene (SEQ ID No.6) is a coenzyme gene of the P450 gene in the oxidation substrate, and the addition of CPR19967 is b...

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Abstract

The invention discloses cytochrome P450 enzyme gene for taking part in sanguinarine and chelerythrine synthesis in macleaya cordata and application of the cytochrome P450 enzyme gene. For the first time, five cytochrome P450 enzyme genes taking part in sanguinarine and chelerythrine synthesis are found in macleaya cordata genome and comprise gene Mc9485, gene Mc2661, gene Mc217, gene MC11229 and gene Mc11218. A brewer's yeast system verifies that the steps of reaction are respectively verified by upstream precursor feeding and synthesis of sanguinarine and chelerythrine intermediate is achieved. According to the cytochrome P450 enzyme gene, the conversion efficiencies of the same function genes in the macleaya cordata, the papaver somniferum and the eschscholzia californica are compared by the brewer's yeast heterogeneous expression system, and the conversion efficiency of the macleaya cordata is obviously higher than those of the papaver somniferum and the eschscholzia californica. According to the cytochrome P450 enzyme gene, a molecular mechanism of the sanguinarine synthesis in the macleaya cordata is further revealed. Thus, a theoretical basis and a molecular assistant breeding target are provided for the breeding of the macleaya cordata with high sanguinarine and chelerythrine content. Meanwhile, valuable experience is provided for in-vitro artificial synthesis of the sanguinarine and the chelerythrine.

Description

technical field [0001] The invention relates to the fields of genetic engineering and molecular biology, in particular to a cytochrome P450 enzyme 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 fr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N15/54C12N9/02C12N9/10C07K14/415C12P17/18C12P17/16
CPCC07K14/415C12N9/0071C12N9/0073C12N9/1007C12P17/16C12P17/18C12Y114/13055C12Y114/13071C12Y114/21005C12Y201/01147
Inventor 曾建国黄鹏黄三文尚轶
Owner MICOLTA BIORESOURCE INC LTD
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