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Application of benzylisoquinoline alkaloid and derivative synthetic gene Ac6OMT thereof

A technology of benzylisoquinoline and alkaloids, applied in the fields of plant genetic improvement, application, genetic engineering, etc.

Active Publication Date: 2021-11-23
INST OF MEDICINAL PLANT DEV CHINESE ACADEMY OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the research on the biosynthetic pathway of benzylisoquinoline alkaloids and their derivatives of Aristolochia northerna is still blank. Therefore, the synthesis of benzylisoquinoline alkaloids and their derivatives There is also less research on the gene of aristolochic acid

Method used

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  • Application of benzylisoquinoline alkaloid and derivative synthetic gene Ac6OMT thereof
  • Application of benzylisoquinoline alkaloid and derivative synthetic gene Ac6OMT thereof
  • Application of benzylisoquinoline alkaloid and derivative synthetic gene Ac6OMT thereof

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0025] Determination of Aristolochic Acid Content in the Root, Stem, Leaf and Flower of Aristolochia militaris in Example 1

[0026] 1) Sample pretreatment

[0027] The four tissues of rhizome, leaf and flower were taken out from the -80°C refrigerator, and ground into fine powder using liquid nitrogen. Weigh 0.1 g of powder into a centrifuge tube, add 2 ml of 70% methanol for shaking, infiltrate at room temperature for 15 min, and then sonicate for 30 min. After centrifugation, the supernatant was collected, and the remaining precipitate was added to 2ml of 70% methanol for shaking, and ultrasonicated for 30min. The supernatant was collected after centrifugation and combined with the previously collected supernatant. After filtering through a 0.22μm filter membrane, wait for loading;

[0028] 2) UPLC detection of aristolochic acid A, B, C, D content

[0029] Use ACQUITYUPLC BEH C18 column (2.1X100nm, 1.7μm; Waters); detection wavelength: 254nm; column temperature: 30°C; f...

Embodiment 2

[0030] Example 2 Screening and Phylogenetic Analysis of Ac6OMT Gene Based on Aristolochia northernis Genome and Transcriptome Data

[0031] The OMT protein sequence of Arabidopsis thaliana was extracted from the Tair database, and Aristolochia miraculouse genome data were compared by BLASTP to identify AcOMTs of Aristolochia miraculouse. The 6 OMT protein sequences (Ps6OMT, Tf6OMT, Cj6OMT, Ps4'OMT1, Ps4'OMT2, Cj4'OMT) and AcOMTs of other species related to the synthesis of benzylisoquinoline alkaloids were searched in the Uniprot database and passed through MUSCLE comparison, construction of NJ phylogenetic tree, bootstrap selection repeated 1000 times. Such as figure 2 , focusing on 7 AcOMTs clustered together with 6 OMTs with known functions, AcOMT1-7. According to the transcriptome data, the expression FPKM values ​​of these 7 AcOMTs in different tissues were extracted, and the heat map was drawn ( image 3 )Compare. It was found that in different tissues, the expressi...

Embodiment 3

[0032] Example 3 Cloning of Aristolochia miraculouse Ac6OMT gene

[0033] According to the open reading frame of the Ac6OMT gene sequence, primers were designed to amplify the full length of the gene, and the cDNA of Aristolochia militaris was used as a template to perform PCR amplification to obtain the nucleotide sequence of the Ac6OMT gene, such as SEQ ID No.1, and the full length of the gene was 1056bp. After translating the nucleotide sequence, the amino acid sequence of Ac6OMT was deduced, including 351 amino acid residues, such as SEQ ID No.2. Ac6OMT gene full-length amplification primers are shown in Table 1:

[0034] Table 1

[0035]

[0036]

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Abstract

The invention discloses an application of an Ac6OMT gene in production of benzylisoquinoline alkaloid and a derivative thereof. The nucleotide sequence of the Ac6OMT gene is as shown in SEQ ID NO. 1; and the synthesized amino acid sequence is as shown in SEQ ID NO. 2. The invention further discloses application of the Ac6OMT gene in breeding transgenic plant varieties which are synthesized or cannot be synthesized with the benzylisoquinoline alkaloid and the derivative thereof, and records a method for regulating organisms to synthesize the benzylisoquinoline alkaloid and the derivative thereof. The Ac6OMT coding gene in a synthetic pathway of the benzylisoquinoline alkaloid and derivatives thereof is subjected to functional identification, and the gene can be applied to biosynthesis and molecular breeding of the aristolochia contorta benzylisoquinoline alkaloid.

Description

technical field [0001] The invention relates to the technical field of synthesis of benzylisoquinoline alkaloids and derivatives, and more specifically relates to the application of a synthetic gene Ac6OMT of benzylisoquinoline alkaloids and derivatives thereof. Background technique [0002] Aristolochic acid, a benzylisoquinoline alkaloid derivative, has attracted much attention because of its severe nephrotoxicity and carcinogenicity, and at the same time, the use of Chinese herbal medicines containing aristolochic acid is also restricted. Since 2005, the Pharmacopoeia has successively deleted aristolochic acid medicinal materials such as aristolochia, qingmuxiang, tianxianteng, fangji, and guanmutong. In the 2020 edition of "Chinese Pharmacopoeia", only aristolochic acid medicinal materials remain. . However, most of these aristolochic acid medicinal materials have been used for a long time and have significant curative effects, and no suitable alternatives have been fou...

Claims

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Application Information

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IPC IPC(8): C12N15/54C12N9/10C12N15/82A01H5/00A01H6/00
CPCC12N9/1007C12N15/8243C12Y201/01128Y02A50/30
Inventor 卢善发崔馨云李彩丽
Owner INST OF MEDICINAL PLANT DEV CHINESE ACADEMY OF MEDICAL SCI
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