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Protein for catalytic synthesis of amyrin, and preparation method and application for protein

A kind of technology of amyrin and protein, applied in the direction of biochemical equipment and method, botany equipment and method, application, etc., can solve the problem of not separating multifunctional OSC enzyme, etc.

Active Publication Date: 2020-09-29
GUANGZHOU UNIVERSITY OF CHINESE MEDICINE
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although some multifunctional enzymes have been obtained in other plants, such as a multifunctional enzyme cloned from tomato, the main product is β-amyrin, in addition to various by-products such as α-amyrin and δ-amyrin (Zhonghua Wang, Plant Physiology, 2011), but so far no multifunctional OSC enzyme that can simultaneously catalyze the synthesis of α-amyrin, β-amyrin and δ-amyrin has been isolated

Method used

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  • Protein for catalytic synthesis of amyrin, and preparation method and application for protein
  • Protein for catalytic synthesis of amyrin, and preparation method and application for protein
  • Protein for catalytic synthesis of amyrin, and preparation method and application for protein

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Embodiment 1

[0048] The embodiment of the present invention provides a method for preparing a protein used to catalyze the synthesis of myringin, which includes the following steps.

[0049] (1) Acquisition of ItOSC2 coding gene

[0050] Iris (Iris tectorum Maxim) leaf total RNA was used as a template, and two primer pairs whose sequences were shown in Table 1 were used for RT-PCR amplification respectively. Specifically, the primers include a sequence of the candidate gene and a homology arm sequence on the pESC-URA or pDEST1-GFP vector, the lowercase letters indicate the homologous sequence of the vector, and the uppercase letters indicate the sequence of the gene. Specifically include the following steps.

[0051] Table 1 Primer Sequence

[0052]

[0053] In Table 1, F is an upstream primer, and R is a downstream primer.

[0054] 1. Extraction of total RNA

[0055] Apply the Rapid Universal Plant RNA Extraction Kit 3.0 (product number: 0416-50) of Beijing Huayueyang Biotechnology...

Embodiment 2

[0075] Obtaining and expressing ItOSC2 tobacco expressing plants.

[0076] 1. Construction of tobacco expression vector pDEST1-GFP-ItOSC2

[0077] pDEST1-GFP was double digested with restriction enzymes FastDigest Mlu I and FastDigest Xho I (Thermo Fisher Scientific). The digestion system is: 3 μL 10× FastDigest Buffer, 3 μL FastDigestMlu I (FD0564), 3 μL FastDigest Xho I (FD0694), 2.4 μg pDEST1-GFP, plus ddH 2 Add O to 30 μL of the system, and flick to mix well; the reaction conditions are: 37°C, 1h. After the enzyme digestion was completed, it was detected by 1% agarose gel electrophoresis (100V, 30min), and the enzyme-digested vector was recovered by cutting the gel.

[0078] The recovered fragment of ItOSC2 (Example 1) was homologously ligated with the double-digested product. The ligation system is: 2 μL pDEST1-GFP double enzyme digestion product, 3 μL gel recovery product, 2 μL 5×CEⅡ Buffer, 1 μL Exnase Ⅱ, add ddH 2 Supplement O to 10 μL of the system, gently pipette...

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Abstract

The invention discloses a protein for catalytic synthesis of amyrin, and a preparation method and an application for the protein, and relates to the technical field of biomolecules. The protein has anamino acid sequence as shown in SEQ ID No. 2, or has a sequence which has 90% or above of sequence homology with the sequence as shown in the SEQ ID No. 2 and has the same protein function as the sequence as shown in the SEQ ID No. 2. According to the protein, 2,3-squalene oxide is used as a substrate; alpha-amyrin, beta-amyrin and delta-amyrin can be catalytically synthesized simultaneously; thealpha-amyrin is mainly synthesized; and a new direction is provided for research of multifunctional OSC enzymes.

Description

technical field [0001] The invention relates to the technical field of biomolecules, in particular to a protein used to catalyze the synthesis of amyrin, its preparation method and application. Background technique [0002] Triterpenoids are one of the most abundant natural products in nature, and they are also important plant secondary metabolites. They mainly exist in ferns, fungi, monocotyledon and dicotyledon in the form of free, glycoside or ester. Plants and animals contain a small amount (Yang Yang et al., Organic Chemistry, 2013). According to different molecular structures, triterpenes can be divided into chain triterpenes, monocyclic triterpenes, bicyclic triterpenes, tricyclic triterpenes, tetracyclic triterpenes and pentacyclic triterpenes (Li Jiaomei, Chinese Herbal Medicine, 2014), Common are tetracyclic triterpenes and pentacyclic triterpenes. [0003] Triterpenoids have a variety of important biological activities, such as anti-virus, hypoglycemic, disease ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/90C12N15/61C12N15/81C12N1/19C12N15/84A01H5/00A01H6/82C12P33/00
CPCC12N9/90C12N15/81C12N15/8243C12P33/00C12Y504/99007
Inventor 段礼新刘中秋吴世丹黄柳芳季爱加梁锦才
Owner GUANGZHOU UNIVERSITY OF CHINESE MEDICINE
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