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Cladosporium endogenic fungus capable of producing veralkol

A technology of resveratrol and endophytic fungi, applied in the field of bioengineering, can solve the problems of numerous extraction steps, complex extract components, limited application and the like

Inactive Publication Date: 2007-04-18
林忠平
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, although the vast majority of plants contain stilbene synthase substrates, they lack the stilbene synthase gene
[0005] At present, commercial resveratrol mainly uses grape skins, grape seeds and knotweed with relatively high content as raw materials, using methanol, ethanol, ethyl acetate, etc. as extraction solvents, and is separated by high performance liquid chromatography, silica gel column chromatography, etc. High content of resveratrol can be obtained through purification, but the resveratrol existing in natural plants is trace amount after all, and there are many extraction steps and complex components of the extract, which must be separated and purified to obtain the finished product. Issues such as limited and small production capacity
However, the hazards of catalysts and reagents in the chemical synthesis process to the environment and human body greatly limit its application.

Method used

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  • Cladosporium endogenic fungus capable of producing veralkol
  • Cladosporium endogenic fungus capable of producing veralkol
  • Cladosporium endogenic fungus capable of producing veralkol

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0120] Embodiment 1: the isolation of endophytic fungus in creeper

[0121] Rinse the newly collected creeper stem section with tap water, drain the water, cut it into small pieces of about 2cm, and then carry out the following surface disinfection according to the conventional aseptic operation: soak in 75% alcohol for 1 minute, soak in 0.1% mercuric chloride for 30 minutes, The phloem was taken and cultured on a PDA plate. The fungus grown 4-7 days after inoculation was picked with an inoculation needle to pick up mycelia and transferred to CYM slant medium for purification, and stored at 4°C for later use.

Embodiment 2

[0123] Embodiment 2: the cloning of stilbene synthase (sts) gene in creeper endophytic fungus

[0124] The following primers (synthesized by Shanghai Bioengineering Co., Ltd.) were designed with reference to the stilbene synthase gene (sta) of Ivy ivy:

[0125] STS1 (upstream primer): 5'>CGG GAT CCG CCA TGG CTT CAG TTG AGAAAT TTA G<3',

[0126] Contains BamHI site

[0127] STS2 (downstream primer): 5'>GTG AGC TCG AAG GGTAAA CCA TTC TCT TTT AT<3',

[0128]Contains SacI site

[0129] Using the genome of endophytic fungi as a template, add 2 μl of DNA template, 10×PCR buffer (MgCl 2 ) 5ul, 2.5mM dNTP mix 4ul, each primer (10μM) 2.5ul, Taq DNA polymerase (5U / ul) 0.5ul, ddH 2 O 34ul. The PCR reaction conditions were: pre-denaturation at 94°C for 5 min; 30 cycles of denaturation at 94°C for 30 s, annealing at 56°C for 45 s, and extension at 72°C for 90 s; extension at 72°C for 10 min, and incubation at 10°C for 1 min. The PCR product was run on an ag...

Embodiment 3

[0179] Example 3: 18S Identification of Fungi

[0180] The following primers were designed according to the 18S conserved sequence of eukaryotic rDNA:

[0181] 18S upstream: 5'-AGCGAAACTG CGAATGGC-3';

[0182] Downstream of 18S: 5'-CATCCTTGGC AAATGCTTTC-3';

[0183] Add to the 50μl PCR reaction system: 2μl DNA template, 10×PCRbuffer (MgCl 2 ) 5ul, 2.5mMdNTP mix 4ul, primers (10uM) 2.5ul each, Taq DNA polymerase (5U / ul) 0.5ul, ddH 2 O 34ul. The PCR reaction conditions were: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 60 s, 30 cycles; extension at 72°C for 10 min, and incubation at 10°C for 1 min. The PCR product was run on an agarose recovery gel, the target band was excised and recovered, ligated with the pMD-18 vector and sequenced.

[0184] The sequencing results were compared by BLAST, and the endophytic fungi were identified as: Ascomycota, Dothideomycetes et., Mycosphaerellaceae, Cladodporium sp...

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Abstract

This invention has clone Stilbene synthase gene (sts) from Parthenocissi Tricuspidatae endogeny eumycete, the coding sequence similarity between Stilbene synthase gene and Parthenocissi Tricuspidatae is 95.25%,this eumycete belongs to Cladosporium sp by 18SrDNA series assessment. High performance liquid chromatogram and mass chromatographic analysis indicate that this eumycete can also composite resveratrol by culture in vitro. The analysis about non- coding part of endogeny eumycete sts gene indicates that: it is very different from regulatory element of sts gene which is in plants. Importing exogenous gene(such as VHb,ipt and iaaM and so on ) in endogeny eumycete can promote endogeny eumycete growing.

Description

1. Technical field: [0001] The present invention relates to isolating a Cladosporium caulis from the endophytic fungus of creeper, the fungus contains the key stilbene synthase gene (sts gene) in the resveratrol synthesis pathway with a coding region of 1179bp. The invention also utilizes genetic engineering technology to genetically improve the bacteria, so that the metabolism and growth speed of the bacteria under the oxygen-limited condition are obviously improved compared with the non-transgenic bacteria. The invention belongs to the technical field of biological engineering. 2. Background technology: [0002] Resveratrol (Res) is a stilbene compound, the chemical name is 3,4',5-trihydroxy-1,2-stilbene (3,4',5-trihydrolystilbene), and the molecular formula is C 14 h 12 o 3 , with a relative molecular weight of 228.25, with cis and trans structures, and the trans structure is more stable. Resveratrol often exists in the form of glycosides in combination with glucose, ...

Claims

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Application Information

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IPC IPC(8): C12N1/14C12N15/31C12N15/52C12N15/29A61K36/06C12N1/15A01N63/04
Inventor 林忠平王晓丽胡鸢雷刘树君
Owner 林忠平
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