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Mutant of SgCS gene of momordica grosvenori and application of gene

A technology of Luo Han Guo and mogroside, applied in the direction of genetic engineering, plant gene improvement, application, etc., can solve the problems of inability to form active protein and slow research

Active Publication Date: 2014-09-03
GUILIN GFS MONK FRUIT CORP
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0007] The research on the molecular biology of Luo Han Guo is more common in the study of genetic diversity and kinship through the method of molecular markers. Compared with other medicinal plants, the research is slightly slower. Currently, there are few reports on the functional genes of Luo Han Guo. Although GENEBANK Sequence structure of Mongolic cucurbitacienol synthase (SgCS) and Mongolic arjool synthase (SgCAS) can already be retrieved in the Chinese Academy of Sciences. When the nuclear host is transformed, it is found that the gene cannot form an active protein after being transferred into the host. Therefore, it is necessary to improve and optimize the gene sequence through certain molecular biological function improvement or molecular evolution methods to overcome mogroside. Key difficulties in the V biosynthetic pathway

Method used

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  • Mutant of SgCS gene of momordica grosvenori and application of gene
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  • Mutant of SgCS gene of momordica grosvenori and application of gene

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Experimental program
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Effect test

Embodiment 1

[0019] Embodiment 1, the bioinformatics analysis of SgCS gene gene and the acquisition of mutant sequence

[0020]Using the SgCS-ORF sequence as the analysis material, the online software Protparam (http: / / web.expasy.org / protparam / ) of ExPAEy Proteomics Server was used to predict the physical and chemical properties of the protein encoded by the SgCS gene, and the online software Interproscan (http: / / www.ebi.ac.uk / Tools / pfa / iprscan / ) to predict the conserved domain of the protein, and use the SOPMA online software (http: / / www.ibcp.fr / predict.html) to perform secondary Structural prediction, through the online software SWISS MODEL (http: / / swissmodel.expasy.org / ) for tertiary structure prediction, through SignalP4.1Server (http: / / www.cbs.dtu.dk / services / SignalP / ) , PSORT (http: / / wolfpsort.org / ) and TMHMM Server v.2.0 software (http: / / www.cbs.dtu.dk / services / TMHMM / ) to predict the signal peptide, subcellular localization and translocation of the protein The location of the memb...

Embodiment 2

[0029] Example 2, Utilizing Yeast to Express SgCS Gene to Produce Cucurbitadienol

[0030] Yeast expression vector pYES2 and yeast strain IVF were purchased from Invitrogen; yeast extract, peptone, and agar powder were purchased from Sigma; other conventional drugs were imported or domestic analytical reagents.

[0031] YPD solid medium formula: 1% yeast extract, 1% peptone, 2% glucose, 2% agar powder

[0032]Yeast extraction buffer: Weigh HEPES1.192g, 75mM EDTA (ethylenediaminetetraacetic acid), 0.1% (v / v) Triton X-100 (polyethylene glycol octylphenyl ether), adjust the pH to 7.4, set Dissolve in 100mL.

[0033] SD solid medium: 0.67% yeast amino acid-free medium, 0.077% Ura deletion amino acid mixture (Clotech), 2% galactose

[0034] SDG liquid medium: 0.67% yeast amino acid-free medium, 0.077% Ura missing amino acid mixture, 2% galactose

[0035] 1. Construction of yeast expression vector pYES2-SgCS

[0036] Using the positive clone containing the mutated SgCS gene as a...

Embodiment 3

[0061] Example 3, Construction of SgCS gene plant overexpression vector and Agrobacterium tumefaciens-mediated transformation of Arabidopsis thaliana

[0062] Wild-type Arabidopsis thaliana (Columbia type) was selected as the material for genetic transformation. Arabidopsis seeds were first soaked and sterilized in 70-75% alcohol for 15 minutes, then washed twice in absolute ethanol, and then sterilized. Dry on filter paper for about 1 minute. Vernalize in refrigerator at 4°C for 4-7 days, plant on vermiculite nutrient soil (1:1 ratio) and cultivate in greenhouse until flowering. pBI121 plant expression vector (Clontech Company).

[0063] 1. The plant overexpression vector construction of SgCS gene;

[0064] 1) Select XbaI and SalI as restriction sites for upstream and downstream primers to construct plant expression vectors. Use the Primer premier5.0 software to design primers containing restriction sites, and the upstream and downstream primers are as follows:

[0065] S...

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Abstract

The invention relates to mutant protein of a SgCS gene of momordica grosvenori, a nucleotide sequence coded by the gene, and application of the gene to production of mogroside V in a yeast host or a plant host.

Description

technical field [0001] The invention relates to a mutant of the SgCS gene derived from Luo Han Guo and its use in the production of mogroside V. Background technique [0002] Luo Han Guo (Siraitia grosvenorii) contains a variety of saponins, and it has been reported that more than 20 types of cucurbitane-type tetracyclic triterpene-type saponins have been isolated from Luo Han Guo, among which mogroside IV, glycoside V and simonoside I are the most reported so far. The three components with the highest sweetness in glycosides are 392, 425 and 563 times sweeter than sucrose, respectively. Among all sweet glycosides, mogroside V has the highest content, accounting for about 20% of the total saponin content; at the same time, as the main active ingredient, mogroside V has antitussive, expectorant, antispasmodic activities, anticancer, anti-inflammatory, Hypoglycemic and other effects, making it one of the few new sweeteners with therapeutic functions excavated from traditional...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/90C12N15/61C12N15/81C12P33/00C12N15/84A01H5/00
CPCC12N9/90C12P33/00C12P33/20C12Y504/99033
Inventor 马小军赵欢唐其
Owner GUILIN GFS MONK FRUIT CORP
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