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Isoarborinol synthesis related protein, and coding gene and application thereof

A technology for encoding genes and proteins, which is applied to the protein related to the synthesis of terpene alcohols in isocaper, and its encoding genes and application fields, can solve the problem of not being isolated from any organism, and achieve the effect of far-reaching application value.

Inactive Publication Date: 2010-01-27
INST OF BOTANY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] Isocapryl alcohols are widely found in higher plants, among which the content of gramineous plants such as Imperatacylindrica (Imperatacylindrica) stems and leaves is more, isocapryl alcohol methyl ester (cylindrin) is sugarcane (Saccharum officinarum), Budi coconut (Buti

Method used

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  • Isoarborinol synthesis related protein, and coding gene and application thereof
  • Isoarborinol synthesis related protein, and coding gene and application thereof
  • Isoarborinol synthesis related protein, and coding gene and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0043] Embodiment 1, the acquisition of OsOSC12 gene

[0044] According to the blastn search in the NCBI database of the oxidative squalene cyclase (OSC) gene AsCS1 (GenBank Accession Number: AJ311790) and AsbAS (GenBank Accession Number: AJ311789) sequences from oat, 12 homologs were found in the rice genome For the higher full-length cDNA sequence, the following primers were designed according to the nucleotide sequence of one of the genes (OsOSC12):

[0045] 5' end primer: 5'-ATGTGGAGGCTGAAGGTGGC-3';

[0046] 3' end primer: 5'-TCATGCACGCCTCGTGGAG-3'.

[0047] The japonica rice variety Zhonghua No. 11 (Oryza sativa L.spp.japonica) seedling total RNA was used as a template, and the above primers were used for RT-PCR amplification. The specific steps are as follows:

[0048] 1. Extraction of total RNA

[0049] Use the Trizol method (the reagents used were purchased from Invitrogen) to extract the total RNA of 2-week-old seedlings. The specific method is: collect 100 mg of r...

Embodiment 2

[0059] Example 2, the acquisition and identification of OsOSC12 yeast transformed cell line

[0060] 1. Construction of yeast expression vector pPICZ-OSC12

[0061] 1) The pTE-OsOSC12 and pPICZA vectors (Invitrogen) were completely digested with restriction endonuclease Not I, respectively. Enzyme digestion system: 5 μg pPICZA, 2.5 μL 10× digestion buffer, 1 μL Not I, add ddH 2 O Supplement the reaction system to 50 μL. The enzyme digestion reaction conditions are: enzyme digestion at 37°C for 1 hour.

[0062] 2) Separation of the digested products by agarose electrophoresis, recovery of about 2.2kb fragment containing OsOSC12 and about 3kb fragment of pPICZA vector, respectively, dissolved in 20μL ddH 2 O middle.

[0063] 3) The two fragments obtained in step 2) are subjected to a ligation reaction. The ligation reaction system is: 2 μL T 4 DNA ligase, 2 μL 10× ligase buffer, 3 μL recovered DNA fragment containing OsOSC12 gene, 1 μL pPICZA vector fragment. The ligation...

Embodiment 3

[0078] Embodiment 3, cultivating OsOSC12 yeast transformed cells to produce isocaperene alcohol

[0079] 1. Culture of OsOSC12 yeast transformed cells

[0080] Add 25ml of MGY medium (1.34% yeast nitrogen source, 1% glycerol, 0.00004% biotin) to a 250ml culture flask, inoculate a single OsOSC12 yeast transformed cell, and culture with shaking at 28-30°C and 250-300rpm until logarithmic growth is achieved Period (OD 600 =2-6), about 16-18 hours. Collect the cells by centrifugation (1500-3000g, 5min), discard the supernatant, and resuspend to OD with MM medium (1.34% yeast nitrogen source, 0.00004% biotin, 0.5% methanol) 600 =1.0, about 100-200ml. Transfer the culture to a 1L long-necked culture bottle, cover it with 2 layers of sterile gauze and cultivate it for 72 hours, and add 100% methanol every 24 hours to keep the final concentration of methanol at 0.5%. The test set 100 repetitions.

[0081] Cultivate the empty carrier cells, and the operation is the same as above. ...

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Abstract

The invention discloses an isoarborinol synthesis related protein, and a coding gene and application thereof. The isoarborinol synthesis related protein provided by the invention is a protein of (a) or (b): (a), a protein consisting of amino acid sequences shown in a sequence 2 in a sequence table; and (b), a protein which is derived from the sequence 2, obtained by replacing and/or deleting and/or adding one or more amino acid residues of the amino acid sequences in the sequence 2, and related to the isoarborinol synthesis. The OsOSC12 protein and the coding gene thereof cloned from japonica rice are gene sequences which obtain isoarborinol synthetase for the first time. The coding gene of the isoarborinol synthesis related protein is introduced into yeast cells, and yeast converted cells of the gene are obtained and then are cultured to prepare isoarborinol. The isoarborinol synthesis related protein and the coding gene thereof have important application value and profound significance.

Description

technical field [0001] The invention relates to a protein related to the synthesis of isocapryl terpene alcohol, its coding gene and application. Background technique [0002] Sterols and saponins have important physiological activities and biological functions. Sterol is a substance with cyclopentane perhydrophenanthrene as the skeleton (also known as steroid nucleus), which can be divided into animal sterols, plant sterols and fungal sterols. Animal sterols are mainly cholesterol. Phytosterols mainly include Sitosterol, Stigmasterol and Campestanol. Bacteroids mainly include ergosterol. Phytosterols can inhibit the body's absorption of cholesterol, promote the degradation and metabolism of cholesterol, inhibit the biochemical synthesis of cholesterol, and have the functions of inhibiting cardiovascular disease, anti-cancer, anti-tumor, anti-inflammatory, improving immunity, regulating growth, and anti-virus. Sterols also have good antioxidant properties and can be used...

Claims

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Application Information

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IPC IPC(8): C12N9/00C12N15/52C12N15/11C12N15/81C12N1/19C12P33/00C12R1/84C12R1/865
Inventor 漆小泉薛哲勇段礼新
Owner INST OF BOTANY CHINESE ACAD OF SCI
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