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Construction method and application of monascus purpureus YJX-8 cDNA library

A technology of YJX-8 and Monascus purpureus, applied in the field of microorganisms

Pending Publication Date: 2022-06-07
BEIJING TECHNOLOGY AND BUSINESS UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, so far, the regulatory mechanism of ester synthases derived from Monascus purple to efficiently catalyze the synthesis of ethyl caproate and ethyl octanoate has not been reported.

Method used

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  • Construction method and application of monascus purpureus YJX-8 cDNA library
  • Construction method and application of monascus purpureus YJX-8 cDNA library

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Experimental program
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Effect test

Embodiment 1

[0017] Example 1 Construction of Monascus purpureus YJX-8 cDNA library

[0018] 1. Monascus purpureus YJX-8 culture

[0019] Monascus purpureus YJX-8 was inoculated into PDA slant medium, and cultured at 30°C for 72h, so that the mycelium covered the medium. The spores were washed with sterile water, and the spore suspension was incubated at 30°C for 5 days at 180 rpm.

[0020] 2. Monascus purpureus YJX-8 total RNA extraction

[0021] The hyphae were rinsed with DEPC water, dried with filter paper, quickly placed in a pre-cooled mortar, and fully ground into powder by adding liquid nitrogen.

[0022] 3. First-strand synthesis of Monascus purpureus YJX-8 cDNA

[0023] Using Monascus purpureus YJX-8 total RNA as template, according to Gold yeast two-hybrid system (Clontech company) instructions to synthesize the first strand of cDNA.

[0024] Add 2μL of RNA, 2μL of Oligo-dT Primer, ddH to the centrifuge tube 2 O 1 μL, the reaction conditions are 72 °C, 2 min; ice bath for...

Embodiment 2

[0036] Example 2 Screening of Monascus purpureus YJX-8 ester synthase LIP05 interacting protein

[0037] 1. Construction of pGBKT7-LIP05 bait vector

[0038] The primers were designed with the LIP05 sequence after removing the signal peptide as the template. The primer sequences are as follows:

[0039] LIP05-F: 5'-ATGGAGGCC GAATTC CTCCCCCCTAACACCC-3';

[0040] LIP05-R: 5’-GATCCCCGG GAATTC TCACGATGAAGCAGC-3'.

[0041] The plasmid containing the LIP05 gene was used as a template for PCR amplification to obtain the target gene fragment, and the bait vector pGBKT7 was digested with EcoR I to obtain the target vector fragment. After the fragment was recovered and purified by gel, it was ligated and transformed, and the recombinant vector pGBKT7-LIP05 was successfully obtained. .

[0042] 2. Decoy vector pGBKT7-LIP05 toxicity and transcriptional autoactivation activity detection

[0043] Take 1 μL of pGBKT7-LIP05 and pGBKT7 plasmids to transform Y2HGold yeast strains, respe...

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Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to a construction method of a monascus purpureus YJX-8 cDNA library and application of the library to screening of ester synthase LIP05 interaction protein. According to the construction method of the monascus purpureus YJX-8 cDNA library disclosed by the invention, LIP05 interaction proteins, namely cell nucleic acid binding proteins and eukaryotic translation initiation factors, are obtained by screening the library constructed according to the method, and expression of the LIP05 can be regulated and controlled on transcription and translation levels. The cDNA library constructed by the invention has an application prospect and a practical value for screening interaction proteins.

Description

technical field [0001] The invention belongs to the technical field of microorganisms, and in particular relates to a method for constructing a Monascus purpureus YJX-8 cDNA library and an application for screening an ester synthase LIP05 interaction protein using the library. Background technique [0002] The main substances of liquor are water and ethanol, and a small amount of flavor substances such as esters, acids and ketones are contained, which determine the flavor of the product. Among them, ethyl hexanoate and ethyl caprylate are important characterization substances for the quality of Luzhou-flavor liquor, and the content of ethyl hexanoate and other ester substances in the wine body as an important measure of product quality has reached a broad consensus in the industry. During the brewing process of liquor, the ester production cycle is long, and the amount of ester synthesis is low and unstable, resulting in a low yield of high-quality Luzhou-flavor liquor, whic...

Claims

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Application Information

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IPC IPC(8): C12Q1/6806C12N15/10C40B50/06C12N15/81C12N15/52C12Q1/6897C12R1/865
CPCC12Q1/6806C12N15/1096C40B50/06C12N15/81C12N9/93C12Y601/02C12Q1/6897C12Q2521/107C12Q2521/327C12Q2531/113C12Q2565/201Y02A50/30
Inventor 李秀婷杜秉昊李微微庞泽敏
Owner BEIJING TECHNOLOGY AND BUSINESS UNIVERSITY
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