Degenerate primers for identifying hybrid isoprenoid-producing bacteria and application thereof

A technology of isoprenoid and degenerate primers, applied in the biological field, can solve problems such as blindness, time-consuming, and tediousness, and achieve the effects of strong purpose, avoiding tediousness, and improving work efficiency

Active Publication Date: 2017-02-01
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] In order to solve the technical problems of cumbersome, time-consuming, blindness and easy to miss screening in the process of fermentation of a large number of strains and purification and identification of fermentation products in the identification of traditional heterozygous isoprenoid compound-producing bacteria, the present invention provides a The degenerate primers used to identify heterozygous isoprenoid compound-producing bacteria adopt the following technical scheme:

Method used

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  • Degenerate primers for identifying hybrid isoprenoid-producing bacteria and application thereof
  • Degenerate primers for identifying hybrid isoprenoid-producing bacteria and application thereof
  • Degenerate primers for identifying hybrid isoprenoid-producing bacteria and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Design of degenerate primer pairs

[0040]Due to the phenomenon of degeneracy of amino acid codons, that is, the amino acid sequence of the final protein expressed by different DNA sequences may be the same, so comparing the published protein sequences of prenyltransferases, primer pair 1 was based on the conservative Design degenerate primers for region motif I and motif II. For primer pair 2, the upstream and downstream primers were designed based on the conserved regions of prenyltransferase, motif I and motif V, and for primer pair 3, the upstream and downstream primers were designed based on the conserved regions of prenyltransferase, motif I and motif IV.

[0041] The protein sequences of isopentenyltransferases from different microorganisms were collected from NCBI, Streptomycessp.SN-5936-dimethylallyltryptophan synthase (GenBank number: BAJ07990.1), Streptomyces coelicolor A3(2) SCO7467 (GenBank number: CAC36059. 1), Streptomyces lividans TK24 (GenBan...

Embodiment 2

[0049] Example 2 Extraction of microbial DNA

[0050] Five heterozygous isoprenoid-producing strains were taken: Streptomyces sp.neau-D50, Streptomyces scoelicolor A3(2), Streptomyces lividans TK24, Streptomyces ambofaciens ATCC23877, Salinispora arenicola CNS-205, and their respective genomes were extracted.

[0051] The extraction method of microbial DNA is as follows: (1) add 5 ml of SET buffer solution to the centrifuge tube containing microbial cells, pipette and mix to resuspend the bacteria, then add 100 μl lysozyme solution (50 mg / ml) to a final concentration of 1mg / ml, incubate in a 37°C water bath for 30-60min until the bacterial suspension is viscous and transparent; (2) Slowly add 140μl proteinase K solution (20mg / ml) to the bacterial suspension to a final concentration of 0.5mg / ml, Mix by inverting, then add 600 μl of 10% SDS solution to a final concentration of 1%, mix by inverting, then incubate in a water bath at 55°C for 2 hours, and mix by inverting slowly ev...

Embodiment 3

[0052] Example 3 Verification of Primer Validity and Accuracy

[0053] The three sets of degenerate primers designed in Example 1 were used to perform PCR on each genome, and then detected by electrophoresis. The PCR product was recovered, and the PCR product was sent to Bao Biological Engineering (Dalian) Co., Ltd. for sequencing. PCR reaction system: 10 μl of 10× amplification buffer, 200 μmol / L of each dNTP mixture, 10-100 pmol of each primer, 0.1-2 μg of template DNA, 1.5 U of Taq DNA polymerase, Mg 2+ 1.5mol / L, add deionized water to 100μL. The amplification reaction conditions are: 94°C for 10 min; 94°C for 1 min; 53°C for 30 s; 72°C for 1 min.

[0054] As a result, it was found that the PCR product of primer pair 1 had a single band at 357bp (such as figure 2 shown), indicating that the primers can indeed be used to amplify the isopentenyl transferase gene from the heterozygous isoprenoid compound-producing bacteria. The sequencing results of the PCR products were ...

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Abstract

The invention discloses degenerate primers for identifying hybrid isoprenoid-producing bacteria and application thereof, belonging to the technical field of biology. The sequences of the degenerate primers are disclosed as SEQ ID NO.1-SEQ ID NO.2. The invention also discloses a method for identifying hybrid isoprenoid-producing bacteria by using the degenerate primers. A direct PCR (polymerase chain reaction) method is utilized to identify hybrid isoprenoid-producing bacteria, thereby avoiding the defects of high complexity, time consuming, blindness, high leakage possibility and the like in the strain fermentation, fermentation product purification and identification and other processes in the traditional identification method, greatly enhancing the work efficiency, and having higher purposiveness. The degenerate primers have important meanings in discovering hybrid isoprenoid microbial natural product drugs and exploring novel prenyltransferase.

Description

technical field [0001] The invention relates to a degenerate primer and its application for identifying heterozygous isoprenoid compound-producing bacteria, belonging to the field of biotechnology. Background technique [0002] Isoprenoids are a class of compounds with the most abundant structural diversity in nature, and their basic skeleton is composed of isoprene (C5) units. According to the number of C5 contained, they can be divided into monoterpenes , sesquiterpenes, triterpenes and tetraterpenes. The diversity of isoprenoid compound structures determines the diversity of its biological functions, which can be used as antibiotics, hormones, insecticides, fungicides, anticancer drugs, etc. in the fields of agriculture and medicine. Fungi and plants are the main sources of isoprenoid compounds. Recent studies have shown that prokaryotes can also produce isoprenoid compounds, especially actinomycetes, an important source of microbial natural product drugs, whose isopreno...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/465
CPCC12Q1/686C12Q1/689C12Q2565/125
Inventor 张继向文胜王相晶申一波潘通
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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