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Red monascus alpha-amylase gene as well as preparation method and application thereof

A kind of Monascus rubrum, amylase technology, applied in its preparation, genetic engineering technology and fermentation of mold, Monascus rubrum α-amylase gene field, can solve the problem of lack of α-amylase high-efficiency expression gene and the like

Active Publication Date: 2018-05-15
JIANGXI SCI & TECH NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] The present invention aims at the technical defects of the prior art, and provides a Monascus ruberus α-amylase gene, its preparation method and application, so as to solve the lack of a high-efficiency expression of α-amylase for genetic engineering in the prior art genetic technical issues

Method used

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  • Red monascus alpha-amylase gene as well as preparation method and application thereof
  • Red monascus alpha-amylase gene as well as preparation method and application thereof
  • Red monascus alpha-amylase gene as well as preparation method and application thereof

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Embodiment 1

[0057] 1. Determination of the target gene

[0058] This section describes the finding and cloning of effective amylase genes from the 13 α-amylase genes predicted by the Monascus ruberum NRRL1597 genome-wide database.

[0059] In the Monascus ruber database (https: / / genome.jgi.doe.gov / Monru1 / Monru1.home.html), the protein numbers (Protein ID) of the predicted 13 α-amylase genes are: P324551, P379161, P411620 , P435885, P63242, P440333, P454978, P460054, P464710, P469192, P469571, P501041, P472279. Through experiments, it was found that the expression of Aspergillus oryzae α-amylase A gene in Monascus can significantly promote starch degradation and increase the yield of Monascus pigment. Therefore with the protein (AOamyA) sequence of Aspergillus oryzae α-amylase A gene, and above-mentioned 13 kinds of α-amylases, construct phylogenetic tree, pattern is as follows figure 1 shown. Among them, the homology relationship between protein number P 440333 and AOamyA is 79%. Acco...

Embodiment 2

[0114] A Monascus ruberus CICC41233α-amylase gene, the DNA sequence of which is shown in SEQ ID NO:1.

[0115] The amino acid sequence of the protein encoded by the α-amylase gene of Monascus ruberus is shown in SEQ ID NO:3.

[0116] The preparation method of the above-mentioned Monascus ruber α-amylase gene comprises the following steps: extracting the total DNA of the Monascus ruber strain as a template, and carrying out PCR amplification with a pair of primers whose sequences are SEQ ID No:4 and SEQ ID No:5 , the amplified product is the Monascus ruber α-amylase gene.

[0117] The method for constructing a high-yielding strain of Monascus pigment by applying the above-mentioned Monascus ruber α-amylase gene comprises the following steps:

[0118] 1) Take the Monascus rubra α-amylase gene and the pNeo0380 vector, respectively digest the two with restriction endonucleases HindIII and Sac I, and connect the digested products with T4DNA ligase to obtain a binary plasmid Expre...

Embodiment 3

[0125] A Monascus ruberus CICC41233α-amylase gene, the DNA sequence of which is shown in SEQ ID NO:1.

[0126] The method for constructing a high-yielding strain of Monascus pigment by the above-mentioned Monascus ruber α-amylase gene comprises the following steps:

[0127] 1) Take the Monascus α-amylase gene and the pNeo0380 vector, respectively digest the two with restriction endonucleases Hind III and Sac I, and connect the digested products with T4DNA ligase to obtain a binary plasmid Expression vector pNeo0380-440333;

[0128] 2) The binary plasmid expression vector pNeo0380-440333 was mediated by Agrobacterium tumefaciens EHA105, transformed into a Monascus ruber strain, and positive clones were screened to obtain the high-yielding strain of Monascus pigment.

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Abstract

The invention provides a red monascus alpha-amylase gene as well as a preparation method and application thereof. According to the technical scheme, an amylase protein expression gene highly homologous with the monascus alpha-amylase A is obtained through the screening in the red monascus NRRL1597 whole genome; a PCR amplification method is designed around the sequence feature. On the basis, the red monascus CICC41233 is used as an experiment strain; PCR amplification is performed to obtain a target gene; then, the target gene is used for building a dual plasmid expression vector pNeo0380-440333; then, through agrobacterium tumefaciens EHA105 mediation, the vector is transferred into the parent red monascus to obtain a red monascus high-yield strain. The strain can obviously promote the degradation of starch rice, so that the yield of the red monascus can be improved. On the basis, a special fermentation method for red monascus production is designed around the biological characteristics of the recombination strain; the production yield of the red monascus is obviously higher than that of a wild strain; meanwhile, the proportion of ethanol-soluble ingredients in the red monascus isimproved.

Description

technical field [0001] The invention relates to the technical field of industrial microorganisms, and further relates to genetic engineering technology and mold fermentation technology, in particular to a Monascus ruberus α-amylase gene, its preparation method and application. Background technique [0002] Monascus pigment is a natural pigment fermented by microorganism Monasus spp. with rice as raw material. It has a history of more than one thousand years in my country. As a food additive, monascus pigment is widely used in food processing and cosmetics manufacturing and other fields; because it also has a wide range of biological activities such as regulating blood lipids, lowering blood pressure, preventing vascular sclerosis, anti-diabetes, inhibiting obesity, anti-inflammation, anti-allergic, anti- Peroxidation, anti-cancer, anti-bacterial, anti-fungal, etc., its application in the development of probiotics and health care products and the medical field has also attrac...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/57C12N9/30C12N15/80C12P1/02C12R1/66
CPCC12N9/242C12P1/02C12Y302/01001C12N1/145C12R2001/66
Inventor 龙传南曾斌谢韶斌张冬生曾旭谢坚梁玉梅刘梦梦王杰
Owner JIANGXI SCI & TECH NORMAL UNIV
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