CN103911318B - The red bacterium in radiation hardness desert and application thereof - Google Patents
The red bacterium in radiation hardness desert and application thereof Download PDFInfo
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- CN103911318B CN103911318B CN201410083784.9A CN201410083784A CN103911318B CN 103911318 B CN103911318 B CN 103911318B CN 201410083784 A CN201410083784 A CN 201410083784A CN 103911318 B CN103911318 B CN 103911318B
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Abstract
The present inventor's separation and Culture from the red building tree rhizosphere soil at Taklimakan Desert of Xinjiang edge goes out a new bacterial strain, confirm that this Pseudomonas is in red bacterium, is named as the red bacterium in desert, Rhodobacter through qualification? shamoensis? W402.The present invention finds that this bacterium has radiation hardness function, can be used as the function stem that stressful environmental is repaired, and can build genetically modified organism, cultivates degeneration-resistant border biology.
Description
Technical field
The invention belongs to microbial technology field, relate to the red bacterium in a kind of new desert.The invention still further relates to the application of this bacterium as radiation hardness function stem aspect.
Background technology
Along with the development of nuclear power technology, nuclear leakage in various degree works the mischief to environment and human health, causes global concern.Therefore, screening has the bacterial strain of excellent radiation tolerance, and study the repair ability of its DNA damage, the reparation for radiation environment has positive meaning.
Therefore, new can having important practical significance by radiation-resistant microorganism is found.
The more Anti-radiation Microbes of current R and D mostly is the gram-positive microorganism that Deinococcus belongs to, as D.radiodurans, D.gobiensis, D.radiophilus, D.radiopugnans, D.proteolyticus, D.geothermalis and D.murrayi etc.
But the rare report of Gram-negative bacteria that Rhodobacter belongs to has bacterial strain and the radiation-resisting functional of radiation resistance.
Summary of the invention
The object of the invention is to isolate the novel species microorganism with tolerance uv-radiation ability.
The present inventor's separation and Culture from the red building tree rhizosphere soil at Taklimakan Desert of Xinjiang edge goes out a kind of new bacterial classification, confirm that this bacterial classification belongs to red bacterium through qualification, be named as the red bacterium in radiation hardness desert, Rhodobactershamoensis[sha.mo.en ' sis.N.L.masc.adj.].
This bacterial classification is in China General Microbiological DSMZ of the depositary institution preservation that State Intellectual Property Office specifies, and preservation date is on February 13rd, 2014, and preservation registration number is CGMCCNo.8817.
The present invention finds that this bacterium has radiation hardness function.
The culture condition of the described red bacterium in radiation hardness desert is:
(1) substratum: adopt LB nutrient agar.Concrete formula is: Tryptones 10g/L, yeast extract 5g/L, sodium-chlor 10g/L
(2)pH7.0；
(3) culture temperature: 30 DEG C.
The red bacterium in radiation hardness desert of the present invention (R.shamoensisW402) has following character:
1. morphological specificity
Visible under an electron microscope, be rod-shaped bacterium (Fig. 4).
2. the feature on various substratum:
On LB nutrient agar, bacterium colony presents lemon yellow, convex, surface wettability, neat in edge, without mucilage secretion (Fig. 3).
On TGY substratum, growing way is slow.
3. physiological and biochemical property
This bacterial strain is strict aerobic growth, Gram-negative, without mobility, and non-autotrophic bacteria strain.L-LEU arylamine enzyme positive, acid phosphatase enzyme positive, naphthols-AS-BI-phosphoric acid hydrolysis enzyme positive, alpha-gluconase activity enzyme positive.
4. utilization of carbon source
Can with glucose, lactose, semi-lactosi, fructose, L-rhamnosyl, ALANINE, D-MANNOSE for carbon source, but can not with L-trehalose, sucrose, D-cellobiose, D-melibiose and alpha-cylodextrin etc. for carbon source.
5. other character
Optimum growth temperature 30 DEG C.Cell walls lipid acid contains SummedFeature8 (C
18:1ω 7cand/orC
18:1ω 6c), C
18:0, 11-MethylC
18:1ω 7c, C
17:0, C
10:03-OH, C
16:0and C
12:03-OH, wherein with SummedFeature8 (C
18:1ω 7cand/orC
18:1ω 6c), C
18:0and 11-MethylC
18:1ω 7c is main.
The 16SrDNA of the red bacterium in radiation hardness desert that the present invention finds and the significant difference (similarity <96%) of other kind belonged to together:
Be 95.833% with the similarity of RhodobactercapsulatusATCC11166;
Be 95.681% with the similarity of Rhodobactersphaeroides2.4.1;
Be 95.529% with the similarity of HaematobactermissouriensisH1892;
Be 95.309% with the similarity of RhodobactermegalophilusJA194;
Be 95.202% with the similarity of RhodobacterveldkampiiATCC35703;
Be 94.337% with the similarity of RhodobacterazotoformansKA25;
Be 93.486% etc. with the similarity of RhodobacteraestuariiJA296.
In addition, reference culture is belonged to red bacterium---the contrast experiment of RhodobactercapsulatusATCC11166 shows, and there were significant differences (table 1) for the Microbiological Characteristics of the red bacterium in radiation hardness desert that the present invention finds and reference culture.
Red bacterium is compared with the Microbiological Characteristics of R.capsulatusATCC11166 in table 1 radiation hardness desert
Note: "+" represents positive, "-" represents negative, and " ± " represents is variable in different strains
Red bacterium has good ultraviolet (UV) tolerance in radiation hardness desert of the present invention, under equal conditions contrast with intestinal bacteria (E.coliK12) bacterial strain, result shows, the red bacterium in radiation hardness desert of the present invention is under identical ultraviolet irradiating dose, and its survival rate is higher than control strain more than 2 times (Fig. 2);
The red bacterium in desert of the present invention is the Gram-negative bacteria that Rhodobacter belongs to, and this genus never has report to have the bacterial strain of radiation hardness resistance.By the research to its radiation hardness mechanism, the understanding of people for radiation tolerance mechanism of production can be deepened further, build new genetically modified organism, make it obtain other good character.
Therefore, the red bacterium in radiation hardness desert of the present invention, can directly as the genetic donor of the genetically modified organism of uv-resistant radiation, and the uv-resistant radiation gene that it is excellent proceeds in other organism, makes it can obtain excellent degeneration-resistant proterties; Meanwhile, it also may as new engineering strain, by accepting other excellent genes external thus obtaining more good character; In addition, carried out the improvement of proterties by traditional selection by mutation means, the bacterial strain of more multiple characters can also be obtained.To the reparation molecule mechanism of its DNA damage of research, promote that new DNA technique is at the development important in inhibiting of environment protection, biological restoration, human health etc.
Accompanying drawing explanation
The red bacterium in Fig. 1 radiation hardness desert and other red bacterium belong to the evolutionary analysis (based on 16SrDNA sequential analysis) between member;
The red bacterium in Fig. 2 radiation hardness desert and control strain (E.coliK12) contrast the tolerance of uviolizing, in figure, the survival curve of the red bacterium in curve representation radiation hardness desert that right side is longer, the survival curve of the curve representation control strain that left side is shorter;
The colonial morphology of the red bacterium in Fig. 3 radiation hardness desert;
The scanning electron microscopic picture of the red bacterium in Fig. 4 radiation hardness desert.
Biomaterial preservation information:
Title: the red bacterium in radiation hardness desert (Rhodobactershamoensis)
Deposit number: CGMCCNo.8817
Depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center
The preservation time: on February 13rd, 2014.
Address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City
Embodiment
The separation and Culture of the red bacterium in embodiment 1 radiation hardness desert
Sand is gathered in red building tree rhizosphere soil in Taklimakan Desert of Xinjiang edge.Take soil sample 1.0g, put into the triangular flask containing 99mL sterilized water, put shaking table vibration 20min and make that soil is dispersed becomes soil supension (10
-2).Therefrom draw 100 μ L soil supensions with 100 μ L liquid-transfering guns, inject the centrifuge tube dividing in advance and 900 μ L sterilized waters are housed, pressure-vaccum 3 times, shaken well (10
-3).Then use same method, being configured to extent of dilution is 10
-4, 10
-5soil bacteria suspension.The bacteria suspension 200 μ L getting suitable concn coats on LB nutrient agar flat board, and upset flat board is placed in 30 DEG C of incubators and cultivates 3 days, can obtain the red bacterium in radiation hardness desert of the present invention.
LB nutrient agar: 10 ‰ sodium-chlor; 5 ‰ yeast extracts; 10 ‰ Tryptoness; 15 ‰ agar.
Culture temperature is 30 DEG C.
The ultraviolet resistance test of the red bacterium in embodiment 2 radiation hardness desert
1, test method
Be inoculated in LB liquid nutrient medium by radiation hardness desert of the present invention Rhodobacter strain, after shaking table is cultured to growth logarithmic phase, 4 DEG C, the centrifugal 10min of 5000rpm collects thalline; Thalline is resuspended in equal-volume 0.1M potassiumphosphate Buffer (pH7.0) (K
2hPO
427.816g, KH
2pO
410.608g, is dissolved in 1L distilled water, 121 DEG C of sterilizing 20min), 4 DEG C afterwards, the centrifugal 10min of 5000rpm collects thalline, is again resuspended in by thalline in equal-volume 0.1M potassiumphosphate Buffer (pH7.0).
Bacterium liquid is placed in culture dish, stirs with the iron wire of sterilizing, make thalline even.Culture dish is placed in 0 ~ 3600J/m respectively
2uv irradiating under various dose; Get postradiation bacterium liquid dilution spread LB nutrient agar dull and stereotyped, counting after cultivating in incubator.Be effectively dull and stereotyped with colony number 30-300 on substratum, statistics.Simultaneously with intestinal bacteria (E.coliK12) bacterial strain for contrast.Test organisms and contrast bacterium have respectively carried out 3 groups of parallel tests.
2, result
Experimental result shows, at different dosages, the survival rate of the red bacterium in radiation hardness desert of the present invention is all significantly higher than control strain (table 2, table 3).
The survival condition of table 2 radiation hardness desert red bacterium (R.shamoensisW402) uviolizing
| Treatment dosage (J/m 2) | 0 | 720 | 1440 | 2160 | 2880 | 3600 |
| Survival rate (%) | 100.00 | 101.25 | 18.29 | 0.7 | 0.045 | 0.00325 |
| Survival rate standard deviation | 0 | 0 | 0.09 | 0.0016 | 8.2E-06 | 1.52E-05 |
The survival condition of table 3 intestinal bacteria (EscherichiacoliK12) uviolizing
| Treatment dosage (J/m 2) | 0 | 120 | 240 | 360 | 480 | 600 | 720 |
| Survival rate (%) | 100.00 | 61.23 | 12.25 | 2.80 | 0.41 | 0.01 | 1.7E-04 |
| Survival rate standard deviation | 0.027 | 0.067 | 0.01 | 0.0026 | 0.00037 | 1.7E-05 | 1.41E-07 |
The extraction of embodiment 3 radiation hardness desert Rhodobacter strain 16SrDNA
Present invention also offers the genome extraction in the red bacterium in radiation hardness desert (R.shamoensisW402), the amplification of 16SrDNA and sequence measurement, specifically carry out according to following operation:
1. Extraction Methods of Genome
The genome of the red bacterium in radiation hardness desert of the present invention uses
the bacterial genomes DNA extraction kit (centrifugal cylindricality) of company is extracted, and refers to its specification sheets.
2.PCR amplification method
The design of the universal primer sequence that the pcr amplification of the 16SrDNA of the red bacterium in radiation hardness desert of the present invention is used is with reference to the article of WilliamG.Weisburg (1991), and primer is synthesized by Beijing Qing Ke biotech company.The test kit used that increases is bought in precious biotechnology (Dalian) company limited.
Forward primer F27 (5 '-AGAGTTTGATCATGGCTCAG-3 '),
Reverse primer R1492 (5 '-TACGGTTACCTTGTTACGACTT-3 '),
Table 4PCR amplification system
| Composition | Add-on |
| ExTaq archaeal dna polymerase | 0.5μL |
| dNTPs | 5μL |
| F27 primer | 1μL |
| R1492 primer | 1μL |
| ExTaq Buffer(10×) | 5μL |
| Template | 1μL |
| ddH 2O | Be settled to 50 μ L |
PCR response procedures
By the 16SrDNA sequence obtained that increases, be connected to
on the pSURE-T carrier of company, precious biotechnology (Dalian) company limited is entrusted to check order for insertion sequence.
Claims (2)
1. deposit number be CGMCCNo.8817 the red bacterium in radiation hardness desert (
rhodobactershamoensis).
2. the red bacterium in desert according to claim 1 is as the application of radiation hardness function stem.
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| CN103911318B true CN103911318B (en) | 2016-01-13 |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101245330A (en) * | 2008-02-03 | 2008-08-20 | 中国农业科学院生物技术研究所 | Gobi abnormal cocci |
| CN101481672A (en) * | 2009-01-22 | 2009-07-15 | 广东省微生物研究所 | Radiation resistant microbacterium and use thereof |
| CN101696414A (en) * | 2009-11-10 | 2010-04-21 | 中国农业科学院生物技术研究所 | Gene capable of improving radiation resistance of organisms and application thereof |
| CN103911317A (en) * | 2014-03-07 | 2014-07-09 | 中国农业科学院生物技术研究所 | Bacteria capable of resisting glyphosate, and applications thereof |
-
2014
- 2014-03-07 CN CN201410083784.9A patent/CN103911318B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101245330A (en) * | 2008-02-03 | 2008-08-20 | 中国农业科学院生物技术研究所 | Gobi abnormal cocci |
| CN101481672A (en) * | 2009-01-22 | 2009-07-15 | 广东省微生物研究所 | Radiation resistant microbacterium and use thereof |
| CN101696414A (en) * | 2009-11-10 | 2010-04-21 | 中国农业科学院生物技术研究所 | Gene capable of improving radiation resistance of organisms and application thereof |
| CN103911317A (en) * | 2014-03-07 | 2014-07-09 | 中国农业科学院生物技术研究所 | Bacteria capable of resisting glyphosate, and applications thereof |
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