Method for constructing monascus strain capable of achieving high yield of acid protease

A technology of acid protease and monascus, which is applied in the direction of microorganism-based methods, botany equipment and methods, biochemical equipment and methods, etc., can solve the problems of non-directional stability and large working range of natural breeding methods, and achieve Strong stability, good market application prospects, and the effect of genetically stable transformants

Inactive Publication Date: 2015-11-11
LUZHOU LAOJIAO GRP CO LTD +2
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In order to obtain Monascus strains with high acid protease production, common methods include natural selection, mutation breeding, and gene recombination breeding; Disadvantages of low rear stability

Method used

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  • Method for constructing monascus strain capable of achieving high yield of acid protease
  • Method for constructing monascus strain capable of achieving high yield of acid protease
  • Method for constructing monascus strain capable of achieving high yield of acid protease

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Embodiment 1: Cloning of Monascus acid protease gene

[0031] 1. Monascus RNA extraction and cDNA double-strand synthesis:

[0032] Scrape off the Monascus hyphae cultured on malt agar medium for 12 days with a sterile spoon, grind with liquid nitrogen and extract its RNA with OMEGA’s fungal RNA extraction kit; take 500ng of the extracted Monascus RNA and use reverse transcription reagents The cassette synthesizes the first strand and the second strand, and the synthesized cDNA double strands are stored at -20°C.

[0033] 2. Primer design and gene amplification:

[0034] According to the Monascus purple acid protease gene published in GenBank Asp (AB090877.1) sequence (SEQIDNO.1) and features of the multiple cloning site of plasmid pBC-Hygro, designed with software PrimerPremier5.0 Asp Gene primers. The primer sequences are:

[0035] Asp -ORF-F:5'-TCCCCCGGGATGGTCGTCTTTCAGCAAGATCAC-3'

[0036] Asp -ORF-R: 5'-TCCCCCGGGTTATGCCTGAGGGGCAAATCCGA-3'

[0037] Using the...

Embodiment 2

[0038] Example 2 Acid protease gene Monascus expression vector pBC-Hygro- Asp The build:

[0039] The sequence of Example 1 is correct Asp Fragment and vector pBC-Hygro were used separately Sma Carry out enzyme digestion with I restriction endonuclease, use gel extraction kit to recover and purify the digested Asp and linear pBC-Hygro vector, then use T4DNA ligase to Asp The fragment is cloned into the pBC-Hygro vector; the recombinant expression vector pBC-Hygro- Asp The construction map is as figure 2 shown.

[0040] Use pBC-Hygro empty vector and recombinant expression vector pBC-Hygro- Asp Electrophoresis together, the results show that the recombined plasmid is about 1200bp longer than the empty plasmid vector, as shown in image 3 as shown, image 3 Middle M: ​​DL23130DNAMarker, 1: vector pBC-Hygro- Asp , 2: pBC-Hygro vector; primers for recombinant plasmid Asp -ORF-F and Asp -ORF-R was amplified by PCR to obtain a specific band of about 1200bp, such as Fi...

Embodiment 3

[0041] Example 3 Agrobacterium-mediated homologous transformation of Monascus

[0042] 1. Mediated transformation:

[0043] The carrier pBC-Hygro- Asp The recombinant plasmid was transformed into Agrobacterium tumefaciens. Pick a single colony of recombinant Agrobacterium from the YEB plate (containing 34 μg / mL chloramphenicol, 50 μg / mL rifampicin) and inoculate it in YEB liquid medium (containing 34 μg / mL chloramphenicol, 50 μg / mL rifampicin), Shake the bacteria at 28°C and 200r / min in the dark for 2 days.

[0044] 2. PCR verification:

[0045] Take the bacterial liquid cultivated in step 1 for PCR verification to determine whether the recombinant vector has been transferred into Agrobacterium. Add the inducer of Agrobacterium tumefaciens to the recombinant Agrobacterium bacterium cultured in step 1, and shake the bacteria at 200r / min for 4h; the inducer is 200μmol / mL acetosyringone solution; -80 (v / v) of 0.85% saline (m / v) eluted Monascus spores from solid culture.

[...

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Abstract

The invention discloses a method for constructing a monascus strain capable of achieving high yield of acid protease. The method is characterized by firstly amplifying an Asp fragment of an acid protease gene in monascus from a monascus genome, then connecting the Asp fragment obtained through amplification to an empty vector to construct a recombinant expression vector of acid protease in monascus, transforming the recombinant expression vector of acid protease in monascus into agrobacterium tumefaciens to obtain recombinant agrobacterium tumefaciens and then guiding the recombinant agrobacterium tumefaciens into monascus by utilizing an agrobacterium tumefaciens-mediated method, thus obtaining the monascus strain capable of achieving high yield of acid protease. The monascus transformant strain capable of achieving high yield of acid protease, which is obtained by a gene recombination method, has the characteristics of high-efficiency expression, accuracy in processing and genetic stability of the transformant and can achieve passage stability under non-selective pressure. The expression quantity of the acid protease gene in the monascus strain capable of achieving high yield of acid protease is 3.30 times the expression quantities of wild type genes, so that high yield of acid protease can be achieved.

Description

technical field [0001] The invention belongs to the technical field of biological breeding, and in particular relates to a method for constructing a Monascus strain with high acid protease production. Background technique [0002] Monascus is a functional strain in the fermentation industry, which contains acid protease in its body Asp gene, which expresses the acid protease Asp , acid protease Asp It has the following effects on liquor brewing: [0003] 1. Promote the growth of microorganisms: [0004] The development of microorganisms requires protein to provide nitrogen and energy for them. The acidity of the fermented grains in the cellar and the acidity in the cellar at the initial stage of fermentation are relatively high. At this time, the acidic protease in Aspergillus can strengthen the decomposition of protein into amino acids, enriching the amino acids in the fermented grains content, which is conducive to the growth of microorganisms. [0005] 2. Improve t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/80C12N15/57C12N1/15C12R1/645
Inventor 邱思佳沈才洪霍丹群唐玉明候长军林锋
Owner LUZHOU LAOJIAO GRP CO LTD
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