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Biological sweet protein monellin gene

A technology of sweetness and protein, applied in the direction of plant peptides, plant products, botanical equipment and methods, etc., can solve the problem of no research reports on sweet protein genes, so as to improve the utilization value, nutritional value and eating quality, and improve Effect of protein content and sweetness

Inactive Publication Date: 2012-05-23
SOUTHWEST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Although there are many studies on sweet protein and its gene at home and abroad, so far, in the field of sericulture, there is no research report on the sweet protein gene that takes into account the preference of mulberry codons

Method used

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  • Biological sweet protein monellin gene

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Cloning of the monellin gene:

[0029] (1) According to the amino acid sequence of the monellin protein reported by Kondo K etc. (Kondo, K., Y.Miura, et al. High-level expression of a sweet protein, monellin, in the food yeast Candida utilis.NatBiotechnol, 1997, 15 (5): 453-7), according to the codon preference of Pichia pastoris and Morus alba, a 294bp sweet protein monellin gene was synthesized;

[0030] (2) Artificially synthesize the sequence onto the PUC57-T cloning vector;

[0031] (3) After obtaining positive clones, send them to GenScript for sequencing.

Embodiment 2

[0033] Recombination of monellin gene in Pichia pastoris GS115:

[0034] (1) The T clone containing the target fragment and the pPIC9K empty plasmid were double digested with EcorI and NotI respectively, and the target fragments were recovered respectively, connected with T4 DNA ligase and transformed into E. coli DH5α competent cells, and obtained by screening pPIC9K containing -cloning of monellin, and then send the cultured bacterial liquid to GenScript for sequencing to prove that the monellin gene has been connected to the expression vector pPIC9K;

[0035] (2) Extract the plasmid with the correct sequence for linearization and dephosphorylation, and cultivate fresh yeast GS115 at the same time to prepare competent; then transform the plasmid (pPIC9K expression vector with monellin gene) treated in the previous step into yeast by electric shock transformation method GS115;

[0036] (3) The colonies grown in the MD medium were planted on the YPD plate containing G418 to s...

Embodiment 3

[0039] Blind test of target protein sweetness:

[0040] (1) control sample: mass ratio 10% sucrose aqueous solution

[0041] (2) Preparation of series concentration experimental samples:

[0042] First weigh 1.0g of the target protein sample and dilute it to 100ml with distilled water to form a 1.0% sample solution; measure 20ml, 12.5ml, 10.0ml, 5ml, 4ml, 3.3ml, 2.5ml of the 1.0% sample solution into a 100ml volumetric flask , diluted to 100ml, the solution concentrations are 0.2%, 0.125%, 0.1%, 0.05%, 0.04%, 0.033%, 0.025% respectively;

[0043](3) Sample tasting comparison: An evaluation team composed of at least 5 people tastes the samples, and judges which of the series concentration samples has the same or similar sweetness as the 10% sucrose solution, so as to calculate the sweetness of the samples. The formula is: Sweetness = 10% / sample concentration. The blind test results of the sweetness of the target protein are as follows:

[0044] Table 1 Relationship between ...

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Abstract

The invention discloses a biological sweet protein monellin gene, which is obtained by preference synthesis of pichiapastoris and mulberry codon according to a reported single-chain monellin amino acid sequence. When the gene is imported into the pichiapastoris to perform expression, blind test experiments prove that an expression product of the gene has sweetness. The gene takes the preference of the mulberry codon into full consideration, can be used as a target gene of mulberry transgenic study so that mulberry leaves have better mouthfeel, and can be used as a tea making raw material and put into a medicament to become beverage and medicaments of people. Meanwhile, once the gene is successfully transformed to the fruiting mulberry, the gene can greatly improve the protein content and sweetness of the mulberry, and improve the nutritional value and edible quality of the mulberry.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a biological sweet protein monellin gene. Background technique [0002] Monellin, derived from the fruit of the West African plant Dioscoreophyllum Cumminsii, is a high-sweet, low-calorie protein sweetener. The food made from it is suitable for patients with diabetes and cardiovascular disease, and it can also prevent dental caries in children. Therefore, this sweet protein is expected to be developed as a substitute for nutritious carbohydrates in the food industry. Promising food additive. [0003] Currently, the U.S. Food and Drug Administration (FDA) approves monellin as a "generally recognized as safe" (GRAS) food additive. There are many foreign companies producing sweet protein, which can be used as an additive or auxiliary material in food, beverage, health care products, medicine and cosmetics. However, because it is composed of protein, its stability is not as good as th...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12N15/81C07K14/415A01H5/00
Inventor 余茂德金筱耘王茜龄李镇刚李军黎其友张琼予
Owner SOUTHWEST UNIV
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