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DNA fragment with promoter function and application

A promoter and functional technology, applied in the field of DNA fragments, to achieve the effect of strong specific expression activity

Active Publication Date: 2015-05-20
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Non-patent literature Marcus Schallme et al. (Developments in the use of Bacillus species for industrial production. Canadian Journal of Microbiology [J]. 2004,50:1-17.) reported the use of the α-amylase promoter of Bacillus amyloliquefaciens Used to express extracellular α-amylase with obvious thermostability and used in saccharification, the disadvantage is that the activity of α-amylase promoter needs to be further improved

Method used

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  • DNA fragment with promoter function and application
  • DNA fragment with promoter function and application
  • DNA fragment with promoter function and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] culture of bacteria

[0035] Take out the glycerol tube of Bacillus amyloliquefaciens (-80°C) and streak it on the LB solid plate at 37°C for 20-24h, pick a single colony in 10mL LB medium with 1% final concentration of glucose, and culture at 37°C, 200rpm until OD 6008.5-10 (U2000 full-wavelength spectrophotometer, Hitachi, Japan).

Embodiment 2

[0037] Bacterial total RNA extraction, transcriptome library construction and sequencing

[0038] Collect 1mL cells in Example 1 and centrifuge quickly at 10000g for 1min to extract bacterial total RNA, such as figure 1 . For the specific extraction method, please refer to the Cell Bacterial Total RNA Extraction Kit of Tiangen Biochemical Technology Co., Ltd. The samples used for RNA-Seq sequencing library preparation were tested by Agilent Technologies 2100 Bioanalyzer, and the mixed DNA molecules were treated with DNaseI (RNase Free), and the vast majority of total RNA was removed with Ribo-Zero (Gram-Positive Bacteria) kit (USA) rRNA, purified mRNA. The mRNA was first broken into fragments of appropriate size, using the fragmented mRNA as a template, reverse transcriptase and random primers were added to synthesize double-stranded cDNA, and then the synthesized cDNA was purified with the kit QIAquick PCR Purification Kit (Qiagen). Fill in the cohesive ends of the cDNA, a...

Embodiment 3

[0040] Screen and clone promoter fragments

[0041] The RNA-Seq sequencing data was used to analyze the transcript structure of Bacillus amyloliquefaciens and the whole genome to analyze the gene containing the transcription start position. Through RPKM quantification, a highly expressed gene was screened. Its sequence is shown in SEQ ID NO 1. Using the genomic DNA of the Bacillus amyloliquefaciens XH7 strain as a template, amplify a 300bp DNA fragment, namely the Pscut01 promoter fragment, with artificially synthesized primers SEQ ID NO 3 and SEQ ID NO 4, such as figure 2 , consistent with the size of the target product. Introduce the restriction site EcoRI, KpnI.

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Abstract

The invention discloses a DNA fragment with a promoter function and an application. The DNA fragment is any one of the following sequences: (a) a nucleotide sequence shown in SEQ ID NO: 1 or a complementary sequence of the nucleotide sequence; (b) a nucleotide sequence having a promoter function identical to SEQ ID NO: 1 or a complementary sequence of the nucleotide sequence obtained by substituting, deleting or adding one or more nucleotides on the nucleotide sequence shown in the SEQ ID NO: 1; and (c) sequences of one or more ribosome bind sites added in the nucleotide sequence shown in the SEQ ID NO: 1. The DNA has a promoter function and is high in expression activity, and high expression of exogenous genes can be realized under a condition that inducers are not needed, and the DNA fragment is applied to expression of heatproof beta-galactosidase and transglutaminase. An effective tool is particularly provided for expression of bacillus amyloliquefaciens.

Description

technical field [0001] The invention relates to a DNA segment, in particular to a DNA segment with promoter function and its application. Background technique [0002] Bacillus amyloliquefaciens is one of the important industrial microbial production strains widely used in agriculture, medicine and food industry. At present, there are still many limitations in the efficient production of high-value-added recombinant enzymes in Bacillus amyloliquefaciens. Among them, the lack of efficient transcription promoters, controllable promoters, and characteristic promoters are the main limiting factors that limit the high-efficiency expression of genes. Promoter is an important component of gene expression, and it is the place where RNA polymerase binds to start transcription and synthesis of mRNA. The binding efficiency of promoter and RNA polymerase is the key to affect the expression of enzyme gene. Different from Escherichia coli, studies have shown that Bacillus subtilis is ric...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/75C12N15/56C12N15/54C12N1/21C12R1/07
Inventor 潘力廖瑜玲王斌刘欣
Owner SOUTH CHINA UNIV OF TECH
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