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Preparation method of sulfite-resistant recombinant saccharomyces uvarum

A grape juice yeast, sulfite resistant technology, applied in the field of genetic engineering

Pending Publication Date: 2022-02-08
SOUTHWEST FORESTRY UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, Met4p can play an important role in regulating cell homeostasis when Saccharomyces cerevisiae is subjected to sulfur stress, but the mechanism of sulfite resistance of this gene in grape juice yeast has not been reported yet

Method used

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  • Preparation method of sulfite-resistant recombinant saccharomyces uvarum
  • Preparation method of sulfite-resistant recombinant saccharomyces uvarum
  • Preparation method of sulfite-resistant recombinant saccharomyces uvarum

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Construction of MET4 gene expression vector

[0024] The small fragment of Pgem-T-MET4 digested by NcoI and BglII and purified was constructed into pCAMBIA1301 which was also digested by these two enzymes, and constructed according to the method introduced by Sambrook, Fritsch and Manitis (Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press, 1989). Then use polymerase chain reaction (Polymerase Chain Reaction, PCR) to verify the expression vector, and send the vector to Shanghai Sangong for sequencing to check whether the vector construction is successful.

[0025] genetic transformation

[0026] Transfer pCAMBIA1301-MET4 into Escherichia coli DH5α by electroporation, and spread on LB (1% yeast extract, 1% tryptone, 2% agar and 0.5% NaCl) containing 10 mg / mL hygromycin (HYG) ) plates and incubated overnight at 37°C. The large transformed colony was picked into 3 mL LB liquid medium (1% yeast extrac...

Embodiment 2

[0036] PCR analysis

[0037] DNA samples were prepared according to the method of Nardi et al. (2010). Ten candidate colonies were randomly selected from the MET4 transformants of S. uvarum strain A9 for PCR analysis. PCR reaction mixture (25 μL) includes 0.2 μL of 5 U / μL Taq enzyme, 0.5 μL of 10 mM dNTP, 1 μL of 10 μM primer, 2 μL of DNA template, 2.5 μL of 10×PCR buffer (including mg 2+ ) and 17.8 μL of dH 2 O.

[0038] The primers used in PCR are

[0039] HYG-F: 5'-TGCTGCTCCATACAAGCCAA-3';

[0040] HYG-R: 5'-ACCGCAAGGAATCGGTCAAT-3'.

[0041] The PCR reaction was carried out according to the following procedure: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s; annealing at 56°C for 30 s, cycled 35 times; extension at 72°C for 60 s, and finally a final extension at 72°C for 8 min, determined according to the method of Liu Xiaozhen et al. (2018) Yield of PCR product.

[0042] RNA extraction and cDNA synthesis

[0043] After culturing in liquid YPD for...

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Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to a preparation method of sulfite-resistant recombinant saccharomyces uvarum, MET4 gene with a base sequence as shown in SEQ ID No.1 is transferred into saccharomyces uvarum, and the obtained recombinant saccharomyces uvarum can resist sulfite to 40 mM which is 50% higher than 20 mM of an initial strain. According to the method, the expression quantity of the HOM3 gene can be up-regulated by overexpressing the MET4 gene by the saccharomyces uvarum, so that more sulfite in a culture medium, namely sulfite in a living environment of the saccharomyces uvarum, can be consumed, the concentration of the sulfite in the environment is reduced, and the transformant can continuously survive or ferment under the condition that the content of the sulfite is relatively high, and thus, the transformantcan further resist sulfite. Besides, the saccharomyces uvarum overexpresses the MET4 gene, up-regulation of a fermentation stress reaction gene and a heat shock protein gene can be triggered, then the sulfite resistance of the saccharomyces uvarum is further improved, and continuous fermentation of the saccharomyces uvarum in a sulfite environment is facilitated.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a sulfite-resistant recombinant grape juice yeast and a preparation method thereof. Background technique [0002] Wine quality is partly determined by the yeast species or strain characteristics of the fermentation process. In addition to the common Saccharomyces cerevisiae, grape juice yeast (Saccharomyces uvarum) is also often used in winemaking because of its low-temperature fermentation ability and ability to produce special aromas. As we all know, sulfite is a widely used preservative, which is toxic to many microorganisms. During the fermentation process, under the action of yeast, it can also make wine produce a special flavor. At present, there is no compound that can completely replace this kind of additives. However, in Saccharomyces cerevisiae cells, sulfite will not only damage the cell structure, but also combine with some enzymes or metabol...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/81C12N15/65C12G1/022C12R1/85
CPCC07K14/395C12N15/81C12N15/65C12G1/0203C12G2200/11Y02A50/30
Inventor 张汉尧张智铭魏卓赵文娟刘小珍
Owner SOUTHWEST FORESTRY UNIVERSITY
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