Function and application of novel triterpene synthase

A triterpene compound and protein technology, applied in the function and application field of triterpene synthase, can solve the problem of unclear function of OSC gene

Active Publication Date: 2012-01-18
INST OF BOTANY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Seven gene sequences encoding full-length OSCs enzymes were found in rice, and the biochemical functions of four OSC genes were identified, they were cycloartenol synthase gene (0s02g0471...

Method used

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  • Function and application of novel triterpene synthase
  • Function and application of novel triterpene synthase
  • Function and application of novel triterpene synthase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1、0

[0049] Example 1, the acquisition of OsOSC8 gene

[0050] According to the rice genome sequence, the primers of OsOSC8 were designed: 5'-end primer: 5'-ATGTGGAAGCTCAAGATTGCCGAG-3'; 3'-end primer: 5'-TCAAGTTGGCGCTGTTGTACTTGC-3'. The japonica rice variety Zhonghua No. 11 (Oryza sativa L.spp. Japonica, Prospect of Rice Flower Breeding, Li Meifang, Crop Journal 03:17-18, 1991, publicly available from the Institute of Botany, Chinese Academy of Sciences.) Flower total RNA as template , under the guidance of the above-mentioned primer pair, RT-PCR amplifies the cDNA sequence of OsOSC8, and the specific method includes the following steps:

[0051] 1. Extraction of total RNA: Extract the total RNA of seedlings for 2 weeks by Trizol method (reagents used are purchased from Invitrogen Company), the specific method is as follows: collect 100 mg of rice material, immediately grind it in liquid nitrogen, add 1 mL of Trizol reagent, and mix well After that, put it at 25 °C for 5 minutes; ...

Embodiment 2

[0057] Example 2, Obtaining rice transfected with 0s0SC8 and functional research of 0s0SC8

[0058] 1. Transform 0s0SC8 rice to obtain

[0059] 1. Construction of 0s0SC8 expression vector

[0060] 1) pCR8-OsOSC8 was completely digested with restriction endonuclease Mlu I. The digestion system is: 5 μg plasmid, 2.5 μL 10× digestion buffer, 1 μL Mlu I, plus ddH 2 O Supplement the reaction system to 50 μL. The enzyme digestion reaction conditions are: enzyme digestion at 37°C for 1 hour.

[0061] 2) Separate the digested products by agarose electrophoresis, recover the linearized products, and dissolve them in 20 μL ddH 2 O middle.

[0062] 3) The linearized product obtained in step 2) is subjected to LR reaction. Add the following components in the 5 μl reaction system: 0.5 μL terminal carrier pH7WG2D, 1 (Karimi, M., Inzé, D. and Depicker, A. (2002) GATEWAYvectors for Agrobacterium-mediated plant transformation, Trends in Plant Science; 7 (5 ): 193-5, the public can obtai...

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Abstract

The invention discloses a function and application of a novel triterpene synthase, and also provides application of 0s0SC8 protein in preparation of a triterpenoid compound, which is characterized in that the amino acid sequence of the 0s0SC8 protein is a sequence 2 in a sequence table. The experiment of the invention proves that: the biochemical function of a 0s0SC8 gene in rice is the synthesisof (3S, 13S)-malabarica-17,21-dien-3beta,14-diol, thus proving that the 0s0SC8 gene encodes (3S, 13S)-malabarica-17,21-dien-3beta,14-diol synthase.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the function and application of a novel triterpene synthase. Background technique [0002] Triterpenoids, including free triterpenes and triterpenoid saponins, are widely present in higher plants. Triterpenoids are synthesized through the isoprene pathway. DAPP) first forms geranium diphosphate (GPP) under the action of vanilla diphosphate synthase (GPS), and then converts it into farnesyl diphosphate (FPP) by farnesyl diphosphate synthase (FPS), and then converts it into farnes diphosphate (FPP) by squalene synthase. Squalene is synthesized under the action of Squalene synthase (SQS), and then converted into 2,3-oxidosqualene (2,3-oxidosqualene) catalyzed by squalene epoxidase (SQE). 2,3-Oxysqualene is cyclized by oxysqualene cyclases (OSCs) to form precursors of triterpenoids, such as β-amyrin. At present, dozens of genes encoding OSCs enzymes have been isolated from different p...

Claims

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Application Information

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IPC IPC(8): C12P15/00C12N9/00C12N15/52C12N15/63
Inventor 漆小泉薛哲勇刘丹段礼新
Owner INST OF BOTANY CHINESE ACAD OF SCI
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