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Genetic manipulation system based on Hfx. mediterranei and pyrF gene and its application

A Mediterranean halophilic bacteria and gene technology, applied in the field of genetic operating system, can solve the problems of lack of transformation, high-frequency homologous recombination, weak selection pressure of antibiotics, etc., and achieve the effect of increasing selection pressure and easy screening.

Active Publication Date: 2014-01-01
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although some progress has been made in molecular genetics in recent years, such as the establishment of the protoplast transformation method of extreme halophilic archaea mediated by Polyethylene glycol (PEG, polyethylene glycol), gene knockout, gene complementation and site-directed mutation and other technologies in extreme halophilic archaea Application in Haloarchaea, but only limited to some species, not universally applicable, thus limiting in-depth study of other strains
In addition, novobiocin, anisomycin, movinolin, and thiostrepton are generally used for the selection of resistance to extreme halophilic archaea, but these resistance selection marker genes are mostly derived from the bacteria's own genes It is obtained by cloning after mutation and applied to the transformation of the integrated plasmid vector system, which is prone to high-frequency homologous recombination of the endogenous gene corresponding to the bacteria, resulting in the generation of false positive transformants
Moreover, the selection pressure of these antibiotics is weak, and transformants are often not obtained
This situation has greatly hindered the in-depth and systematic theoretical research and genetic engineering development of halophilic archaea.

Method used

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  • Genetic manipulation system based on Hfx. mediterranei and pyrF gene and its application
  • Genetic manipulation system based on Hfx. mediterranei and pyrF gene and its application
  • Genetic manipulation system based on Hfx. mediterranei and pyrF gene and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1, the acquisition of orotidine-5'-monophosphate decarboxylase PyrF and its coding gene

[0062] The genome of Haloferax mediterranei CGMCC 1.2087 was sequenced and its structure was studied. As a result, an orf in the genome was annotated as pyrF. Design a pair of primers P1 / P2 for PCR amplification of the pyrF coding gene and its promoter sequence. The primer sequences are as follows:

[0063] P1: 5′GGC GAATTC GTTTTCTTGTGTGCGGTT3′( EcoRI )

[0064] P2: 5′GTT GGTACC TTACCGGTACTGGTTCAG3′( KpnI )

[0065] Using the genome of Haloferax mediterranei CGMCC 1.2087 as a template, PCR amplification was performed with primer pair P1 and P2 as upstream and downstream primers, and a PCR product with a length of 897 bp was obtained.

[0066] The above-mentioned PCR amplification program is: pre-denaturation at 94°C for 3 minutes; then 30 cycles at 94°C for 30s, 57°C for 30s, and 72°C for 60s; and extension at 72°C for 7 minutes. The amplification system is 25 μl...

Embodiment 2

[0068] Example 2. Identification of pyrF gene function and construction of Hfx.mediterraneiΔpyrF

[0069] Each liter of AS-168 medium was prepared as follows: 5.0g casamino acids, 5.0g yeast extract, 1.0g sodium glutamate, 3.0g sodium citrate , 200g NaCl, 20g MgSO 4 ·7H 2 O, 2.0g KCl, 0.36g FeSO 4 ·7H 2 O and 0.36 mg MnCl 2 4H 2 Dissolve O in distilled water and make up to 1 L with distilled water, pH 7.1-7.2.

[0070] Acid hydrolyzed casein was purchased from Bacto Difco, catalog number 223120. Yeast extract was purchased from OXOID, catalog number LP0021.

[0071] 1.RT-PCR detection of pyrF gene transcription in Hfx.mediterranei CGMCC 1.2087.

[0072] 1) Culture conditions: A single colony of Haloferax mediterranei CGMCC 1.2087 was picked and placed in AS-168 medium on a shaker at 37° C. at 200 rpm for 4 days.

[0073] 2) RNA extraction: Take a sterilized 1.5ml EP tube to collect 1.5ml of bacterial solution, centrifuge at 12000rpm at 4°C for 1min, and suck up the su...

Embodiment 3

[0114] Example 3, construction of integrated plasmid vector pHFX based on pyrF gene

[0115] In Example 1, pUCm-PyrF (containing the full length of pyrF and containing its own promoter) was double-digested with restriction endonuclease EcoRI and KpnI and gel-cut to recover the pyrF fragment of the target gene; Digest with KpnI, recover the large fragment of the vector, connect the large fragment of the vector with the pyrF fragment of the target gene with T4DNA ligase at 16°C overnight, the construction process is as follows figure 1 shown. Then the heat shock transformation method was used to transform the ligation product into Escherichia coli JM109 competent, screened on a plate containing ampicillin resistance, and quickly detected the positive clones obtained, and extracted the plasmid for sequencing. The gene shown in nucleotides 512-1408 in SEQ ID NO: 2 was inserted between the sites along the direction from EcoRI to KpnI, indicating that the constructed recombinant ve...

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Abstract

The invention discloses a genetic manipulation system based on Hfx. mediterranei and pyrF genes and its application. The genetic manipulation system comprises recombinant halophilic archaea and a recombinant plasmid carrier. The recombinant halophilic archaea is prepared by defunctionalizing an encoding gene of a protein sequence of SEQ ID NO:1 in initial halophilic archaea. The recombinant plasmid carrier at least contains a replication origin required by replication of Escherichia coli, an expression cassette for screening resistance selectable marker genes of Escherichia coli transformants, an expression cassette of the encoding gene of the protein sequence of SEQ ID NO:1, and polycloning sites for allowing exogenous genes to insert. The genetic manipulation system can be used as a high-efficiency gene knockout system, and can carry out extensive genetic function research and metabolic pathway illumination on Hfx. mediterranei. According to the experiment, the frequency of positive recombinants obtained from genetic transformation by using the inventive system is greatly improved.

Description

technical field [0001] The invention relates to a genetic operating system based on Mediterranean halobacteria and pyrF gene and its application. Background technique [0002] In 1977, the American Woese discovered the third life form on the earth-archaea, which led to the proposal of the three-domain theory of life, that is, life is composed of Archaea, Bacteria and eukaryotes. The biological domain (Eucarya) is composed. The archaeal domain includes Crenarchaeota, Euryarchaeota, Korarchaeota and Nanoarchaeota. [0003] Archaea are similar to eukaryotes in the transmission of genetic information (such as DNA replication, transcription, translation, etc.); while they are close to bacteria in terms of central metabolism (such as energy production). Therefore, the study of archaea is not only of great significance for elucidating the basic laws of life movement, revealing the origin of life and species evolution, but also helps to understand some important biological process...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/63C12N1/19C12N1/21C12N7/01C12N9/88C12N5/10C12N15/09C12N1/20C12N15/60C12N1/15
Inventor 向华刘海龙
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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