CN108893470A - A kind of non-coding RNA OsiR and application thereof of anti-oxidant and high temperature resistant stress - Google Patents

A kind of non-coding RNA OsiR and application thereof of anti-oxidant and high temperature resistant stress Download PDF

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CN108893470A
CN108893470A CN201810666398.0A CN201810666398A CN108893470A CN 108893470 A CN108893470 A CN 108893470A CN 201810666398 A CN201810666398 A CN 201810666398A CN 108893470 A CN108893470 A CN 108893470A
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osir
coding rna
high temperature
resistance
strain
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CN108893470B (en
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林敏�
陈明
张维
周正富
田烨
高丽华
战嵛华
陆伟
柯秀彬
燕永亮
王劲
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Biotechnology Research Institute of CAAS
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli

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Abstract

The present invention has found a kind of non-coding RNA sequence that host's oxidative stress resistance and high temperature stress resistance can be improved from extreme bacterial strain abnormal cocci.The present invention constructs the recombinant vector containing the gene, by it in prokaryotic host cell expression in escherichia coli.It is demonstrated experimentally that significantly enhancing the resistance to oxidative stress and high temperature stress after the non-coding RNA is expressed in prokaryotic host cell, having a wide range of applications potentiality in terms of engineered strain transformation.

Description

A kind of non-coding RNA OsiR and application thereof of anti-oxidant and high temperature resistant stress
Technical field
The invention belongs to gene engineering technology fields, and in particular to a kind of non-coding RNA of anti-oxidant and high temperature resistant stress OsiR gene and application thereof.
Background technique
Bacterium non-coding RNA (non-conding RNA, ncRNA), is some sizes in 40-500 nucleotide range The relatively small non-coding RNA molecule of molecular weight, be usually located at area between protein coding gene, with stem-loop formed answer Miscellaneous higher structure, most of termination minor structures having independent of rho factor.In recent years the study found that non-coding in bacterium RNA can be used as the post-transcriptional control factor, and by the expression of number of mechanisms controlling gene, important work is played in vital movement With.
Summary of the invention
Present invention finds a kind of non-coding RNA new gene, expression is induced by oxidative stress, participates in the anti-oxidant of bacterium And high temperature resistant stress procedure, it is named as osiR gene, which is 251bp, Genbank ID：MF964218.Non-coding The DNA sequence dna of RNA osiR gene is SEQ ID NO:1, the RNA sequence such as SEQ ID NO of transcription:Shown in 2.
Present invention discover that the rna gene has a wide range of applications potentiality in terms of engineered strain transformation, oxygen can be specifically responded Stress signal, hence it is evident that improve the oxidation resistance of bacterial strain, and the high temperature stress resistance of host strain can be significantly improved.
Specific invention work is as follows：
1. the recombinant strain of building expression osiR gene
1) digestion is carried out by the sequence dna fragment of the PCR complete osiR gene cloned, is inserted into expression vector In pRADZ3, construction recombination plasmid pRADZ3-OsiR.
2) the recombinant plasmid pRADZ3-OsiR for importing osiR gene is transferred to recipient E. coli Trans10, obtains engineering Bacterial strain Trans-OsiR (detailed in Example 1)；
2, the stress resistance analysis of osiR genetic recombination engineered strain Trans-OsiR is expressed
1) H of various concentration is utilized2O2It is impacted, studies the oxidative stress resistance of recombinant strain Trans-OsiR (being detailed in example 2)
2) 48 DEG C of shaking table cultures are utilized, the high temperature stress resistance of research recombinant strain Trans-OsiR (is detailed in example 3)
The experimental results showed that：Non-coding RNA osiR gene can significantly improve host cell and resist oxidative stress and high temperature The ability of stress has a good application prospect in terms of improving bacterial strain stress resistance.
Sequence table information
SEQ ID NO.1：The DNA sequence dna of non-coding RNA osiR gene.
SEQ ID NO.2：RNA sequence after non-coding RNA osiR genetic transcription.
Detailed description of the invention：
Fig. 1 recombinant expression carrier pRADZ3-OsiR constructs schematic diagram；
Fig. 2 recombinant expression carrier pRADZ3-OsiR double digestion proof diagram.M1:Trans 15K maker；M2:Trans 100bp maker；1:The non-digestion of pRADZ3-OsiR；2:PRADZ3-OsiR double digestion；
Fig. 3 expresses the oxidative stress resistance figure of the engineering bacteria Trans-OsiR of OsiR.Trans-OsiR is expression osiR Recombinant bacterial strain；Trans-Z3 is the control strain containing empty carrier；
Fig. 4 expresses the high temperature stress resistance figure of the engineering bacteria Trans-OsiR of OsiR.Trans-OsiR is expression osiR Recombinant bacterial strain；Trans-Z3 is the control strain containing empty carrier.
Specific embodiment
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are only used for illustrating this The method of invention, rather than limit the scope of the invention.It is all that specific experiment condition is not specified, it is according to art technology Normal condition known to personnel.
Shuttle plasmid pRADZ3：This laboratory saves；
Escherichia coli Trans10：For Beijing Quan Shi King Company commercial product.
Embodiment 1 expresses the building of the Escherichia coli recombinant strain of osiR gene
One, experimental material
Escherichia coli Trans10：For Beijing Quan Shi King Company commercial product.
Pcr template DNA：Deinococcus radiodurans genomic DNA
Two, experimental method
1. according to the Deinococcus radiodurans genome design 1 announced to PCR specific primer：
OsiR-F：5'CGGACTAGTGACATGTATTCGTCTGACC 3’
OsiR-R：5'CGCCATATGCCTGGCCGAGCTCACCGG 3’
2. amplifying objective gene sequence from Deinococcus radiodurans genomic DNA by PCR method.
Reaction condition：95 DEG C of 10min, [95 DEG C of 30sec, 58 DEG C of 30sec, 72 DEG C of 0.5min] 35 circulations, 72 DEG C 10min。
3.PCR product carries out double digestion after glue recycles, using SpeI and NdeI enzyme.Obtain the osiR containing cohesive end Gene is connected on pRADZ3 carrier, is constructed colibacillus high expression vector pRADZ3-OsiR (Fig. 1).The expression vector is turned Change Escherichia coli Trans10, the recombinant strain of building expression osiR gene.Positive colony bacterial strain is chosen, Plasmid DNA is extracted Different recons is screened, carries out double digestion and sequence verification using SpeI and NdeI enzyme.
Three, experimental result
Using round pcr successful clone Deinococcus radiodurans osiR gene, the recombination of expression osiR is successfully constructed Colibacillus engineering strain.Through PCR, digestion, sequence verification insetion sequence is correct, is Trans-OsiR by the Strain Designation.Contain There is the control strain of pRADZ3 empty plasmid to be named as Trans-Z3.
Four, experiment conclusion
Complete the building of the recombination bacillus coli engineered strain of expression osiR.
The oxidative stress Analysis of Resistance of the expression of embodiment 2 osiR genetic recombination engineered strain Trans-OsiR
One, experimental material
Recombinant strain：The Trans-osiR bacterial strain for the expression osiR gene that embodiment 1 obtains
Control strain：Trans-Z3 bacterial strain described in embodiment 1 containing empty plasmid.
Two, experimental method
1. culture
It picks them separately single colonie on solid plate to be inoculated in the LB liquid medium containing Amp, 37 DEG C of constant-temperature table cultures Overnight
2. oxidative stress (H2O2Impact)
By seed liquor switching in new LB culture medium, culture to OD600About 0.6 or so.Respectively take 1mL thallus that final concentration is added For 10,15,20mM H2O210min is impacted,
3. result viewing, with blank
Each processing carries out multiple proportions gradient dilution to 10-5.The sample of not oxidised processing is blank control.Draw each place The 8 μ L point of dilution gradient sample of reason is on LB solid plate, the oxidative stress resistance of 37 DEG C of culture observation bacterial strains.
Three, experimental result
As shown in Figure 3：
10mM H2O2Under oxidative stress, recombinant bacterial strain Trans-OsiR is compared with control strain Trans-Z3, survival rate Improve 10 times.
The H of 15mM and 20mM concentration2O2Under oxidative stress, recombinant bacterial strain Trans-OsiR and control strain Trans-Z3 phase 2 orders of magnitude are improved than, survival rate, about 100 times.
Four, experiment conclusion
H2O2Impact experiment is aoxidized the results show that the expression of non-coding RNA osiR significantly improves host e. coli Oxidative stress resistance.
The high temperature stress Analysis of Resistance of the expression of embodiment 3 osiR genetic recombination engineered strain Trans-OsiR
One, experimental material
Recombinant strain：The Trans-OsiR bacterial strain for the expression osiR gene that embodiment 1 obtains
Control strain：Trans-Z3 bacterial strain described in embodiment 1 containing empty plasmid.
Two, experimental method
1. culture
It picks them separately single colonie on solid plate to be inoculated in the LB liquid medium containing Amp, 37 DEG C of constant-temperature table cultures Overnight.
2.48 DEG C high temperature stress
By seed liquor switching in new LB culture medium, culture to OD600About 0.8 or so.5mL bacterium solution is respectively taken to be placed in 48 DEG C Shaking table impacts 4h.
3. result viewing, with blank
Each processing carries out multiple proportions gradient dilution to 10-5.The sample of not oxidised processing is blank control.Draw each place The 8 μ L point of dilution gradient sample of reason is on LB solid plate, the high temperature stress resistance of 37 DEG C of culture observation bacterial strains.
Three, experimental result
As shown in Figure 4：
Under 48 DEG C of high temperature stress, compared with control strain Trans-Z3, survival rate improves recombinant bacterial strain Trans-OsiR 2 orders of magnitude, about 100 times.
Four, experiment conclusion
48 DEG C of high temperature impact experimental results show that the expression of non-coding RNA osiR significantly improves host e. coli High temperature stress resistance.
Sequence table
<110>Biological Technology institute, Chinese Academy of Agricultural Sciences
<120>A kind of non-coding RNA OsiR and application thereof of anti-oxidant and high temperature resistant stress
<160> 2
<170> PatentIn version 3.1
<210> 1
<211> 251
<212> DNA
<213>Deinococcus radiodurans（Deinococcus radiodurans）
<400> 1
atgtattcgt ctgacctcga cctcaccacc aaagccctca tgcgcgaaca ggcccttcac 60
gagccgcagg ccatgacctc atgggaagct catgaccgcc gcgaaagccg cagcgtgcgc 120
cgcgccctga actggctgga acgccgcctg cgccggagcg cttgaggccg catcatttgc 180
ccacgcagaa gttgcgaaac acagcgtcca ccacatcttc ctgcacgtcc tgaccggtga 240
gctcggccag g 251
<210> 2
<211> 532
<212> RNA
<213>Deinococcus radiodurans（Deinococcus radiodurans）
<400> 2
auguauucgu cugaccucga ccucaccacc aaagcccuca ugcgcgaaca ggcccuucac 60
gagccgcagg ccaugaccuc augggaagcu caugaccgcc gcgaaagccg cagcgugcgc 120
cgcgcccuga acuggcugga acgccgccug cgccggagcg cuugaggccg caucauuugc 180
ccacgcagaa guugcgaaac acagcgucca ccacaucuuc cugcacgucc ugaccgguga 240
gcucggccag g 251

Claims (3)

1.SEQ ID NO:Application of the gene of the non-coding RNA of sequence shown in 2 in terms of improving engineered strain resistance.
2. application described in claim 1, the raising engineered strain resistance are that the non-coding RNA specifically responds the oxygen side of body Compel signal, hence it is evident that improve the oxidation resistance of bacterial strain.
3. application described in claim 1, the raising engineered strain resistance are the height that the non-coding RNA improves bacterial strain Warm stress resistance.
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CN103382475A (en) * 2012-05-03 2013-11-06 中国科学院植物研究所 DNA fragment related to drought tolerance and alkali resistance of plant and application thereof
CN102807991A (en) * 2012-08-15 2012-12-05 中国农业科学院生物技术研究所 Application of deinococcus radiodurans R1 trkB genes to cultivation of salt-tolerant plants
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