Genetic manipulation system based on Haloarcula hispanica and pyrF gene and its application

A gene, halophilic archaea technology, applied in the field of genetic operating system, can solve the problems of lack of transformation, high-frequency homologous recombination, hindering theoretical research and genetic engineering development of halophilic archaea, etc., to increase the pressure of selection , easy to filter effects

Active Publication Date: 2012-09-19
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 Haloarcula hispanica and pyrF gene and its application
  • Genetic manipulation system based on Haloarcula hispanica and pyrF gene and its application
  • Genetic manipulation system based on Haloarcula hispanica and pyrF gene and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1. Obtaining of orotidine-5'-monophosphate decarboxylase PyrF and its coding gene

[0061] The genome of Haloarcula hispanica CGMCC 1.2049 was sequenced and its structure was studied. As a result, one 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:

[0062] P1: 5′TAT GAATTC GAGCGGGCTTCTACCTGC3′( EcoRI )

[0063] P2: 5′GGC GGTACC TTAGCGGAACTGATTCAG3′( KpnI )

[0064] Using the genome of Haloarcula hispanica CGMCC 1.2049 as a template, PCR amplification was carried out using primer pairs P1 and P2 as upstream and downstream primers, and a PCR product with a length of 954 bp was obtained.

[0065] The above PCR amplification procedure is: 94°C for 3min pre-denaturation; then 94°C for 30s, 57°C for 30s, 72°C for 60s for 30 cycles; 72°C for another 7 minutes of extension. The amplification system is 25μl.

[0066] The PCR amplified product ...

Embodiment 2

[0067] Example 2. Functional identification of pyrF gene

[0068] Each liter of AS-168 medium is prepared as follows: 5.0g acid hydrolyzed 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 0.36mg MnCl 2 ·4H 2 O is dissolved in distilled water, and the volume is adjusted to 1 liter with distilled water, and the pH is 7.1-7.2.

[0069] Acid hydrolyzed casein was purchased from Bacto Difco, catalog number 223120. Yeast extract was purchased from OXOID, the product catalog number is LP0021.

[0070] 1. RT-PCR detects the transcription of pyrF gene in Har.hispanica CGMCC 1.2049.

[0071] 1) Culture conditions: Pick a single colony of Har.hispanica CGMCC 1.2049 and place it on an AS-168 medium 37°C shaker at 200 rpm for 4 days.

[0072] 2) RNA extraction: take a sterile 1.5ml EP tube to collect 1.5ml bacterial solution, centrifuge at 4℃, 12000rpm for 1min, and suck up the supernatant with a pipe...

Embodiment 3

[0111] Example 3. Construction of an integrated plasmid vector pHAR based on the pyrF gene

[0112] The pUCm-PyrF (containing the full length of pyrF and containing its own promoter) in Example 1 was double-cut with restriction enzymes EcoRI and KpnI and digested to recover the target gene pyrF fragment; the vector pUBP was used with restriction enzyme EcoRI Double enzyme digestion with KpnI, recover the large vector fragment, ligate the large vector fragment and the target gene pyrF fragment with T4 DNA ligase 16℃ overnight, the construction process is as follows figure 1 Shown. Then the ligation product was transformed into E. coli JM109 competent by heat shock transformation method, and screened on a plate containing ampicillin resistance. The obtained positive clones were quickly detected and the plasmids were extracted for sequencing. The result was EcoRI and KpnI in the vector pUBP The gene shown at nucleotide 477-1430 in SEQ ID NO: 2 was inserted between the sites along th...

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Abstract

The invention discloses a genetic manipulation system based on Haloarcula hispanica 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 Haloarcula hispanica. 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 Salina spp. and pyrF gene and its application. Background technique [0002] In 1977, American Woese discovered the third form of life on earth-archaea, which led to the three-domain theory of life, that is, life is composed of Archaea, Bacteria and eukaryotic 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.); but they are similar to bacteria in terms of central metabolism (such as productivity). Therefore, studying archaea is not only of great significance for clarifying the basic laws of life movement, revealing the origin of life and species evolution, but also helps to understand some important biological processes in more complex eukaryotes. From the perspective of bi...

Claims

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Application Information

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