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Mylia taylori sesquiterpene synthetase MTb and gene sequence thereof

A sesquiterpene synthase, calyx moss technology, applied in genetic engineering, plant genetic improvement, lyase and other directions, can solve the problems of little research on moss plants and rarity of lichenaceae plants.

Active Publication Date: 2019-06-07
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the special growth environment of the small calyx moss, the plants belonging to the family Caricaceae are very rare, so there are few studies on this kind of moss plants, and there is no relevant literature on the sesquiterpene synthase in the small calyx moss

Method used

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  • Mylia taylori sesquiterpene synthetase MTb and gene sequence thereof
  • Mylia taylori sesquiterpene synthetase MTb and gene sequence thereof
  • Mylia taylori sesquiterpene synthetase MTb and gene sequence thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1. Cloning and expression of the WQ011 / pESC-LEU-MTb bacterial strain Sesquiterpene synthase gene MT-29066

[0021] Total RNA was extracted from Lily calyx by RNAprep pure Plant Kit. The first-strand cDNA was synthesized using the Clontech SMARTer PCRcDNA Synthesis Kit, the first-strand cDNA was amplified by PCR to synthesize the second-strand cDNA, and then the double-strand DNA was amplified by secondary PCR and used for SMRTbell library construction, and PacBio ISO-Seq was used platform for sequencing.

[0022] According to the MT-29066 gene sequence SEQ ID NO.2 obtained by sequencing, primers were designed to amplify the target gene. The primer sequences are as follows:

[0023] Primer MT-29066-F: 5'CGC GGATCC ATGGGGATCGAGACGAGACC 3' (SEQ ID NO. 3)

[0024] Primer MT-29066-R: 5'CCG CTCGAG TCAGACCTTAAAGCGAATGTTTG 3' (SEQ ID NO. 4)

[0025] Using the cDNA sequence obtained by reverse transcription as a template, the MT-29066 gene sequence was amplified b...

Embodiment 2

[0047] Example 2. Induced fermentation

[0048] (1) The transformant WQ011 / pESC-LEU-MTb was transferred to 50 mL of synthetic medium (with out Leu), 180 r / min, 30° C., and cultured on a shaking table for 30 h.

[0049] (2) At 30h, 10g / L galactose was added to induce expression to 90-120h, and at 36h and 72h, 10g / L ethanol was added as a supplementary carbon source.

[0050] (3) The fermentation broth was extracted with 3 mL of n-hexane for 15 min to detect by GC-MS.

Embodiment 3

[0051] Embodiment 3.GC-MS detects fermentation product

[0052] (1) GC-MS detection method

[0053] Chromatographic column: TG-5MS; Ion source; EI, 70eV; Injection volume: 1uL; Injection temperature: 200°C; Detector temperature: 280°C; Column temperature: 240°C; / min Raise the temperature to 280°C and maintain it for 5min.

[0054] (2) The output was calculated by the external standard method. Accurately prepare 0.02, 0.04, 0.06, 0.08 and 1.0 mg / L standard substances respectively, draw a standard curve with the peak area as the ordinate and the concentration as the abscissa.

[0055] The results show that: if image 3 As shown, the target peak in the GC-MS spectrum is compared with the reference mass spectrum data in the NIST14 database, and it is found that the chromatographic peak of the n-hexane extract of the WQ011 / pESC-LEU-MTb strain fermentation broth at a retention time of 17.73min is Piperaceae orange Solanol (Cubedol), 50mL shake flask output is 0.14mg / L. The chr...

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Abstract

The invention discloses mylia taylori sesquiterpene synthetase MTb and a gene sequence thereof. The overall length of the gene is 1206bp, 401 amino acids are coded, the molecular weight of pheron is 46.6KDa, pESC-LEU yeast protein expression plasmid is used as a carrier, and saccharomyces cerevisia Saccharomyces cerevisiae WQ011 is used as a host, so that the heterologous expression of the mylia taylori sesquiterpene synthetase MTb is realized, and a recombinant strain WQ011 / pESC-LEU-MTb can generate cubedol(Cubedol) and cadinol (Cadinol) at the same time. Based on 50m of a shake flask, the cubedol is 0.14mg / L, and the cadinol is 0.24mg / L. The obtained gene has great significance on research of a moss plant sesquiterpene compound.

Description

technical field [0001] The invention belongs to the field of enzyme genetic engineering and enzyme engineering, and specifically relates to a sesquiterpene synthase MTb derived from Myliataylori and a gene sequence MT-29066. Background technique [0002] Terpenoids are natural products with the most complex chemical structure and conformation. There are about 80,000 terpenoids discovered so far, accounting for one-third of natural products, and they are widely distributed in plants and microorganisms. The most basic structural units required for the biosynthesis of terpenoids are Dimethylallyl diphosphate, DMAPP and Isopentenyl diphosphate, IPP comes from the mevalonate pathway and deoxyxylulose phosphate ester pathway. Different amounts of DMAPP and IPP are polymerized end-to-end and then catalyzed by a special terpene synthase to form a monoterpene (C 10 ) sesquiterpene (C 15 ) and diterpenes (C 20 ) and other natural products. Sesquiterpenoids have the most structure...

Claims

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Application Information

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IPC IPC(8): C12N9/88C12N15/60C12N15/81C12N1/19C12R1/865
Inventor 乔建军闫晓光梁冬梅李伟国
Owner TIANJIN UNIV