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Agilawood sesquiterpenoid synthase protein ASS4 and encoding gene and application thereof

A sesquiterpene and enzyme protein technology, applied in agarwood sesquiterpene synthase protein ASS4 and its encoding gene and application field, can solve the problem of lack of white wood incense formation technology, undisclosed, difficult large-scale, industrialized artificial production Agarwood and other issues, to achieve important economic value and application prospects, reduce damage

Inactive Publication Date: 2013-10-16
INST OF MEDICINAL PLANT DEV CHINESE ACADEMY OF MEDICAL SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

One of the main technical bottlenecks causing this situation is the lack of stable and efficient Agarwood Agarwood technology, and it is difficult to carry out large-scale and industrialized artificial production of Agarwood medicinal materials in a targeted manner.
In order to accelerate and increase the amount of Agarwood aroma formation, a large number of researches on artificial aroma formation technology have been carried out at home and abroad since the 1930s, but the induction formation mechanism of agarwood has not been revealed, resulting in no breakthrough in stable and efficient aroma formation technology ( Zhang Zheng, Yang Yun, Wei Jianhe, Meng Hui, Sui Chun, Chen Huaiqiong. Research progress on the mechanism of aroma formation of A. argentina and the hypothesis of defense response-induced aroma formation. Chinese Herbal Medicine, 2010, 41(1): 19-21.)

Method used

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  • Agilawood sesquiterpenoid synthase protein ASS4 and encoding gene and application thereof
  • Agilawood sesquiterpenoid synthase protein ASS4 and encoding gene and application thereof
  • Agilawood sesquiterpenoid synthase protein ASS4 and encoding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Cloning of embodiment 1ASS4

[0061] 1.1 Extraction and detection of total RNA of A.

[0062] The stems of A. argentina after injury treatment were taken, ground into a fine powder with liquid nitrogen, and 0.5 g of the fine powder was weighed, and the total RNA of A. arbusae was extracted according to the method of the Total RNA Purification Kit. Specific steps are as follows:

[0063] (1) Take 500 mg of Akiras sinensis powder, add 600 μl of Lysis Solution and mix the sample evenly.

[0064] (2) After mixing evenly, centrifuge at 12000rpm for 2min, and transfer the supernatant to a new centrifuge tube.

[0065] (3) Add 70% ethanol equal to the volume of the supernatant (about 600 μl), shake well, and vortex to mix.

[0066] (4) Put the spin column into a centrifuge tube and add the above liquid, centrifuge at 12000rpm for 1min, and discard the waste liquid in the collection tube.

[0067] (5) Add 400μl of washing solution containing DNAseI, centrifuge at 12000rpm f...

Embodiment 2

[0132] Example 2 Functional Verification of Agarwood Sesquiterpene Synthase Gene ASS4

[0133] 2.1 Construction of Prokaryotic Recombinant Expression Vector of Arboria sesquiterpene Synthase Gene

[0134] 2.1.1 Plasmid extraction by alkaline lysis

[0135] Plasmids were extracted using the high-purity plasmid small-scale extraction kit from TIANGEN Company, and the specific operations were as follows:

[0136] (1) Add 500μl of equilibrium solution to the adsorption column, centrifuge at 12000rpm for 1min, and discard the waste solution.

[0137] (2) Take 15ml of the bacterial solution and add it to a centrifuge tube, centrifuge at 12000rpm for 1min, and remove the supernatant.

[0138] (3) Add 500 μl solution Ⅰ to mediate and suspend the bacterial cell pellet.

[0139] (4) Add 500 μl of solution II, turn over 6 to 8 times to fully lyse the bacteria.

[0140] (5) Add 700 μl of solution III, turn it upside down 6 to 8 times, mix well, a white flocculent precipitate appears, ...

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Abstract

The invention provides agilawood sesquiterpenoid synthase protein ASS4 and an encoding gene ASS4 thereof. The protein comprises the following components: (1), protein formed by amino acid sequence shown in SEQ ID NO.2; (2), protein derived from the protein in (1), having the same function as the protein in (1), and formed in the way that one or more amino acids in the amino acid sequence shown in SEQ ID NO.2 are replaced and deleted or added. The invention further provides a preparation method and application of the agilawood sesquiterpenoid synthase protein ASS4. The agilawood sesquiterpenoid synthase protein ASS4, provided by the invention, can be used for catalyzing agilawood sesquiterpenoid externally, the gene ASS4 provided by the invention can be converted into prokaryotic cell, yeast or more advanced eukaryocyte, so as to have corresponding agilawood sesquiterpenoid synthase activity, and the mass production of agilawood sesquiterpenoid can be realized, and the damage to the resource of rare and endangered South China medical aquilaria sinensis is reduced, therefore, the agilawood sesquiterpenoid synthase protein ASS4 has significant economic value and application prospect.

Description

technical field [0001] The invention belongs to the technical field of biological genetic engineering, and in particular relates to an agarwood sesquiterpene synthase protein ASS4 and its coding gene and application. Background technique [0002] my country's traditional and precious medicinal material agarwood comes from the rare and endangered plant Aquilaria sinensis (Lour.) Gilg (Editorial Committee of Pharmacopoeia of the People's Republic of China. 2010. Pharmacopoeia of the People's Republic of China (Part I). Beijing: China Medical Science and Technology Press.). Agarwood has been used as medicine for thousands of years in traditional Chinese medicine, Tibetan medicine and Indian traditional medicine. Hong Kong is also named for its rich history of producing and exporting agarwood. Due to the high value of agarwood (currently, the international transaction price of super-grade agarwood blocks is as high as $10,000 / kg) and the huge demand in the international market,...

Claims

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Application Information

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IPC IPC(8): C12N9/88C12N15/60C12N15/63C12N1/15C12N1/19C12N1/21C12P5/00
Inventor 张争魏建和梁良韩晓敏徐艳红杨云高志晖刘洋洋孟慧陈旭玉
Owner INST OF MEDICINAL PLANT DEV CHINESE ACADEMY OF MEDICAL SCI
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