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A mutant of Luo Han Guo sgcas gene and its application

A kind of Luo Han Guo, genetic engineering technology, applied to the mutant of Luo Han Guo SgCAS gene and its application field, which can solve the problems of slow research and inability to produce active protein, etc.

Active Publication Date: 2016-03-16
GUILIN GFS MONK FRUIT CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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 produce active protein after it is 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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  • A mutant of Luo Han Guo sgcas gene and its application
  • A mutant of Luo Han Guo sgcas gene and its application
  • A mutant of Luo Han Guo sgcas gene and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1, Bioinformatics analysis of SgCAS gene and acquisition of mutant sequence

[0020]Using the SgCAS-ORF sequence as the analysis material, the online software Protparam (http: / / web.expasy.org / protparam / ) of ExPAEyProteomicsServer was used to predict the physical and chemical properties of the protein encoded by the SgCAS 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 analyze the secondary structure of the protein encoded by SgCAS Prediction, the prediction of the tertiary structure is carried out through the online software SWISSMODEL (http: / / swissmodel.expasy.org / ), through SignalP4.1Server (http: / / www.cbs.dtu.dk / services / SignalP / ), PSORT ( http: / / wolfpsort.org / ) and TMHMMServerv.2.0 software (http: / / www.cbs.dtu.dk / services / TMHMM / ) to predict the signal peptide, subcellular localization and transmembrane ...

Embodiment 2

[0055] Example 2, using yeast to express the SgCAS mutant gene to produce cycloachool

[0056] 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.

[0057] YPD solid medium formula: 1% yeast extract, 1% peptone, 2% glucose, 2% agar powder

[0058] Yeast extraction buffer: weigh HEPES1.192g, 75mMEDTA (ethylenediaminetetraacetic acid), 0.1% (v / v) TritonX-100 (polyethylene glycol octylphenyl ether), adjust the pH to 7.4, and dissolve in 100mL.

[0059] SD solid medium: 0.67% yeast amino acid-free medium, 0.077% Ura deletion amino acid mixture (Clotech), 2% galactose

[0060] SDG liquid medium: 0.67% yeast amino acid-free medium, 0.077% Ura missing amino acid mixture, 2% galactose

[0061] 1. Construction of yeast expression vector pYES2-SgCAS

[0062] Using the positive clone containing the SgCAS mutant gene ...

Embodiment 3

[0099] Example 3, the construction of SgCAS plant RNA interference vector and the effect of transforming tobacco

[0100] In this section, Nicotiana tabacum was selected as the material for genetic transformation, plant interference vector pBI121 (purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd.), and Agrobacterium GV3101 (Invitrogen Company). Restriction enzymes BamHI, PacI, SalI and AscI were purchased from NEB (Beijing) Co., Ltd.

[0101] Tobacco Genetic Transformation Medium

[0102] Configuration of MSB0 basic medium (MS+B5 basic medium):

[0103] Macroelements (10X Mother Solution I):

[0104] Component Concentration

[0105] KNO319.0g / L

[0106] NH4NO316.50g / L

[0107] KH2PO41.7g / L

[0108] MgSO4·7H2O3.7g / L

[0109] CaCl2 2H2O4.4g / L

[0110] Trace elements (1000X mother liquor II):

[0111] Component Concentration

[0112] KI0.83g / L

[0113] H3BO36.2g / L

[0114] ZnSO4·7H2O8.6g / L

[0115] Na2MnO4·2H2O0.25g / L

[0116] CuSO4·5H2O0.025g / L

[...

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Abstract

The invention relates to a mutant protein from an SgCAS gene of momordica grosvenori, a nucleotide sequence encoding the mutant protein and application of the gene in production of Ring Joe alcohol or mogroside V in yeast hosts or plant hosts.

Description

technical field [0001] The invention relates to a mutant of the SgCAS gene derived from Luo Han Guo and its use in the production of mogroside V. Background technique [0002] Luo Han Guo (Siraitigrosvenorii) contains a variety of saponins, and it has been reported that more than 20 types of cucurbitane-type tetracyclic triterpene saponins have been isolated from Luo Han Guo, among which mogroside IV, glycoside V and simonoside I are the sweetest ingredients reported so far. The three components with the highest sweetness among 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 fro...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/90C12N15/61C12N1/19C12P33/00A01H5/00
CPCC12N9/90C12P33/00C12P33/20C12Y504/99008
Inventor 马小军赵欢唐其
Owner GUILIN GFS MONK FRUIT CORP