High-throughput screening system for breeding high-nucleic-acid yeast and application

A nucleic acid yeast, high-throughput technology, applied in the field of microorganisms, to achieve the effect of broad market prospects, huge economic value, and avoiding the problem of transgenic

Active Publication Date: 2018-11-27
QILU UNIV OF TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the search found that: Utilizing the high efficiency of RNA polymerase I in rDNA gene transcription, the rDNA gene promoter guides the expression of the reporter gene yeGFP, and constructs a regulatory disturbance that can cause changes in rRNA synthesis through induction, indirectly reflecting rRNA content The changed high-throughput large-scale screening system and related articles or patents applied to the screening of high-nucleic acid yeast have not yet been reported

Method used

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  • High-throughput screening system for breeding high-nucleic-acid yeast and application
  • High-throughput screening system for breeding high-nucleic-acid yeast and application
  • High-throughput screening system for breeding high-nucleic-acid yeast and application

Examples

Experimental program
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Effect test

Embodiment 1

[0040] Embodiment 1: Construction of reporter plasmid YEp-Hyg B-yeGFP

[0041] 1. Construction of plasmid YEp-Hyg B containing hygromycin B resistance gene

[0042] (1) Construction of hygromycin B resistance gene expression cassette

[0043] Using the plasmid YEp-CH as a template, using the primer Sal I-pJ-TEF1-F

[0044] (5'-CATTTCCCCGAAAAGTGCCACCTGACGTCGACATGGAGGCCCAGAATACC-3') and pJ-TEF1-Nco I-R (5'-CCTCCATGGCAGTATAGCGACCAGCATTC-3') amplified about 1500bp hygromycin B gene expression cassette Sal with Sal I and Nco I restriction sites For I-TEF1p-Hyg B-TEF1t-NcoI, PCR amplification conditions were pre-denaturation at 95°C for 3 min, denaturation at 95°C for 45 s, annealing at 52°C for 15 s, extension at 72°C for 1.5 min, 30 cycles, and final extension at 72°C for 5 min.

[0045] (2) Construction of plasmid YEp-Hyg B containing hygromycin B resistance gene

[0046] Use Sal I and Nco I to digest plasmid YEplac195 and hygromycin B gene expression cassette Sal I-TEF1p-HygB...

Embodiment 2

[0068] Example 2: Construction of Saccharomyces cerevisiae host cells containing reporter plasmids

[0069] 1. Transformation of Saccharomyces cerevisiae host cells with reporter plasmid

[0070] The control plasmid YEp-Hyg B and the reporter plasmid YEp-Hyg B-yeGFP were transformed into the yeast strain CGMCC No.9084 (named Y08 in the present invention) with a relatively high nucleic acid content after preliminary screening, and the transformation method used was PEG- LiAc-mediated Saccharomyces cerevisiae transformation method, using YPD plates containing 200mg / L hygromycin B to screen transformants, select transformants, extract plasmids from yeast, and then use primers Sac I-rDNAp-F and URA3-Xho I-R PCR amplification was carried out. The PCR amplification conditions were pre-denaturation at 95°C for 3 min, denaturation at 95°C for 45 s, annealing at 52°C for 15 s, extension at 72°C for 1.5 min, 30 cycles, and final extension at 72°C for 5 min. A band of about 1400bp was a...

Embodiment 3

[0073] Example 3: Utilizing a new high-throughput screening system to screen high-nucleic acid yeast engineered bacteria

[0074] 1. Using ARTP to mutagenize the host cells containing the reporter plasmid

[0075] (1) Strain activation: Saccharomyces cerevisiae industrial strain Y082 expressing a high-throughput screening system was inoculated onto a YPD plate containing 200 mg / L hygromycin B, cultured upside down at 30°C for 2 to 3 days, and then inoculated to a plate containing 200 mg / L In the YPD liquid of hygromycin B, shake culture for 12 to 24 hours, transfer the bacterial liquid to fresh YPD containing 200mg / L hygromycin B to control the bacterial concentration OD 600 ≈0.1, shake culture at 30°C for several hours, control the final concentration of bacteria OD 600 Within ≈1.0, it was used as the starting strain for ARTP mutagenesis.

[0076] (2) ARTP mutagenesis: Take 1 mL of the bacterial liquid, suspend it in a 1.5 mL EP tube, centrifuge at 8000 r / min for 2 min, and...

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Abstract

The invention relates to a high-throughput screening system for breeding high-nucleic-acid yeast. The high-throughput screening system comprises reporter plasmid Yep-Hyg B-yeGFP and a host cell containing the Yep-Hyg B-yeGFP, wherein the reporter plasmid Yep-Hyg B-yeGFP is annular and sequentially comprises a YEplac195 plasmid skeleton, a yeast enhanced green fluorescent protein gene yeGFP expression box and a hygromycin B resistance gene expression box from 3' to 5'; the original strain of the host cell is a saccharomyces cerevisiae industrial strain whose nucleic acid content is confirmed tobe higher than a normal value through screening. The invention further discloses application of the screening system in high-nucleic-acid yeast engineering bacterium breeding. The screening system has the advantages that the defect that traditional high-nucleic-acid yeast breeding cannot directly reflect RNA content can be overcome, the instability of the plasmid in the yeast can be utilized to easily eliminate the plasmid through continuous passage, and the system is significant to the application of the high-nucleic-acid yeast in food industry.

Description

technical field [0001] The invention relates to the technical field of microorganisms, in particular to a high-throughput screening system for breeding high-nucleic acid Saccharomyces cerevisiae and its application. Background technique [0002] Ribonucleic acid (RNA) not only performs biological functions in cells, but also is an important new food raw material, food additive and pharmaceutical intermediate. RNA and its degradation products, or corresponding derivatives are active ingredients of many drugs [Wang Nan, Cai Xiaxia, Li Yong. Research progress on exogenous nucleotides and immune function. Food Science, 2016,37(5):278-282 .], which has a positive effect on the health care efficacy and immunity improvement of humans and animals. In recent years, 5'-inosinic acid (5'-IMP) and 5'-guanylic acid (5'-GMP) prepared by RNA catalyzed by multiple enzymatic transformations are called disodium nucleotides, which have With its unique "umami" taste and rich in nutrition, it ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/81C12N15/65C12Q1/04G01N21/64C12R1/865
CPCC12N15/65C12N15/81C12Q1/04G01N21/64
Inventor 鲍晓明徐丽丽曾杜文邱晨曦易勇张继祥吴倩杜显雨
Owner QILU UNIV OF TECH
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