Gene encoding protein having trehalose synthesis-promoting activity and use thereof
A protein and trehalose technology, applied in genetic engineering, plant genetic improvement, application, etc., can solve the problems of poor low-temperature preservation of baker's yeast, and achieve the effect of eliminating thorny problems and reducing weight
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Embodiment 1
[0101] Example 1 Cloning of a gene (nonScTSL1) encoding a protein having trehalose synthesis-promoting activity
[0102] As a result of searching using the comparison database described in Japanese Patent Application Laid-Open No. 2004-283169, the nonScTSL1 gene (SEQ ID NO: 1) encoding a protein having trehalose synthesis-promoting activity of S. cerevisiae was found. According to the obtained nucleotide sequence information, the primers nonScTSL1_for (sequence number 3) / nonScTSL1_rv (sequence number 4) used to amplify the full-length gene were respectively designed, and the strain Saccharomyces pastorianus Weihenstepan34 / 70 strain (referred to as "W34 / The chromosomal DNA of 70 strains") was used as a template for PCR to obtain a DNA fragment including the full-length gene of nonScTSL1.
[0103] The nonScTSL1 gene fragment obtained above was inserted into pCR2.1-TOPO vector (manufactured by Invitrogen) by TA cloning. The nucleotide sequence of the nonScTSL1 gene was analyzed...
Embodiment 2
[0104] Example 2 Expression analysis of nonScTSL1 gene in beer trial brewing
[0105] Brewery yeast Saccharomyces pastorianus W34 / 70 strain was used for trial brewing, and the mRNA extracted from the fermenting yeast cell was detected by Saccharomyces DNA microarray.
[0106] Wort extract concentration 12.69%
[0107] Wort volume 70L
[0108] Dissolved oxygen concentration in wort 8.6ppm
[0109] Fermentation temperature 15°C
[0110] The amount of yeast added 12.8×10 6 cells / mL
[0111] The fermented liquid was sampled over time to observe the changes in the amount of yeast proliferation (Figure 1) and the apparent concentration of the extract (Figure 2). At the same time, the yeast cells were sampled to prepare mRNA, and the prepared mRNA was labeled with biotin to be hybridized with the brewer's yeast DNA microarray. Signal detection was performed using the GeneChip Operating System (GCOS; GeneChip Operating Software 1.0, manufactured by Affymetrix Corporation), and t...
Embodiment 3
[0112] Example 3 Construction of nonScTSL1 high expression strain
[0113] The nonScTSL1 / pCR2.1-TOPO obtained by the method described in Example 1 was digested with restriction enzymes SacI and NotI to prepare a DNA fragment containing the nonScTSL1 gene. The fragment was connected to pYCGPYNot treated with restriction enzymes SacI and NotI to construct nonScTSL1 high expression vector nonScTSL1 / pYCGPYNot. pYCGPYNot is a YCp-type yeast expression vector, and the introduced gene is highly expressed through the promoter of pyruvate kinase gene PYK1. Yeast selectable markers include the aminoglycoside antibiotic (Geneticin) resistance gene G418 r , selectable markers for E. coli include the ampicillin resistance gene Amp r .
[0114] Using the high expression vector obtained by the above method, adopt the method described in Japanese Patent Application Laid-Open 07-303475 to transform the AJL4004 strain, adopt the YPD plate medium (1% yeast extract, 2 % polypeptone, 2% glucos...
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