CN101245330A - Gobi abnormal cocci - Google Patents
Gobi abnormal cocci Download PDFInfo
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- CN101245330A CN101245330A CNA2008100575592A CN200810057559A CN101245330A CN 101245330 A CN101245330 A CN 101245330A CN A2008100575592 A CNA2008100575592 A CN A2008100575592A CN 200810057559 A CN200810057559 A CN 200810057559A CN 101245330 A CN101245330 A CN 101245330A
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Abstract
The invention provides a bacterial strain with excellent radiation tolerance. A new bacterial strain-Deinococcus gobiensis is obtained by separation and culture from the yellow sand of desert surface. The Deinococcus gobiensis can be used as a functional bacterial strain for repairing radiation environment, constructing a transgenic creature and culturing a radiation tolerant creature.
Description
Technical field
The invention belongs to microbial technology field, relate to a kind of abnormal cocci with radiation tolerance.
Background technology
In recent years, radiation has very big influence to environment, and the especially development of nuclear power technology has caused nuclear leakage problem in various degree.Therefore, the bacterial strain that screening has good radiation tolerance has positive meaning for the reparation of radiation environment.Simultaneously,, can further deepen the understanding of people, make up new genetically modified organism, make it obtain other good character for the radiation tolerance mechanism of production by research to its radiation hardness mechanism.
Summary of the invention
The purpose of this invention is to provide a kind of bacterial classification with good radiation tolerance.
The present invention's separation and Culture from top layer, the Gobi desert yellow ground soil in somewhere, Xinjiang has obtained a new abnormal cocci, belongs to the new bacterial classification of a kind of microorganism.
This bacterium called after Gobi abnormal cocci, Deinococcus gobiensis[gobi.en ' sis.N.L.masc.adj.].
This bacterial classification is in the common micro-organisms center preservation of China Committee for Culture Collection of Microorganisms of specified depositary institution of State Intellectual Property Office, and preservation date is on January 25th, 2008, and preservation registration number is CGMCCNo.2358.
The culture condition of described Gobi abnormal cocci is:
(1) substratum: adopt the TGY nutrient agar.Concrete prescription is: Tryptones 10g/L, yeast extract 5g/L, glucose 1g/L
(2)pH7.0；
(3) culture temperature: 30 ℃.
1. morphological specificity
Under electron microscope, as seen,, mostly be bigeminy, a small amount of tetrad, accidental monomer for spherical gram positive bacterium.
2. the feature on various substratum:
On the TGY nutrient agar, brick-red, convex that bacterium colony presents, surface wettability, neat in edge, no exocytosis thing.
On the LB nutrient agar, can not grow.
3. physiological and biochemical property
This bacterial strain is strict aerobic growth, Gram-positive, and the catalase positive, oxidase negative, the urase positive does not produce H
2S, gelatine liquefication is fast, can hydrolyzed starch and casein, can reduce nitrate.
4. utilization of carbon source
Can be carbon source with glucose, sucrose, lactose, fructose, D-raffinose, L-aspartic acid, L-Histidine, but can not be carbon source with L-pectinose, maltose, D-trehalose, D-wood sugar, L-rhamnosyl, L-tryptophane and L-arginine etc.
5. other character
30 ℃ of optimum growth temperatures.The cell walls sugar is measured and is contained glucose and a small amount of ribose.Cell walls lipid acid contains laurostearic acid, tridecylic acid, TETRADECONIC ACID, pentadecanoic acid, palmitic acid, 16 carbon monoenoic acids, margaric acid, 17 carbon monoenoic acids, stearic acid, oleic acid, linolic acid and epoxidation linolenic acid, wherein based on palmitic acid, 16 carbon monoenoic acids, margaric acid, 17 carbon monoenoic acid and oleic acid.
The 16S rDNA of the Gobi abnormal cocci that the present invention finds and the significant difference (similarity＜95%) of other kind of generic:
With the similarity of Deinococcus hohokamensis KR245 be 93.273%;
With the similarity of Deinococcus gradis be 93.182%;
With the similarity of Deinococcus deserti VCD115 be 93.182%;
With the similarity of Deinococcus navajonenensis KR-39 be 93.182%;
With the similarity of Deinococcus radiodurans KR-39 be 93.066%;
With the similarity of Deinococcus indicus Wt/laT be 93.052%;
With the similarity of Deinococcus hopiensis KR125 be 92.238%;
With the similarity of Deinococcus sonorensis KR136 be 91.603%;
With the similarity of Deinococcus yunnanensis 7 be 91.454% or the like.
In addition, through belonging to reference culture with abnormal cocci---the contrast experiment of radiation hardness abnormal cocci R1 (D.radioduransR1) confirms that there were significant differences (seeing Table 1) for the Gobi abnormal cocci that the present invention finds and the Microbiological Characteristics of this bacterial strain.
The Microbiological Characteristics of table 1 Gobi abnormal cocci and radiation hardness abnormal cocci R1 relatively
Annotate: "+" expression is positive, and "-" expression is negative, and " W " expression is weak positive, and ND represents not have related data
Gobi abnormal cocci of the present invention has ultraviolet ray and gamma rays tolerance preferably, under equal conditions compare with radiation hardness abnormal cocci (D.radiodurans) R1 bacterial strain, find that Gobi abnormal cocci of the present invention is at same ultraviolet irradiation amount (254nm, 567Jm
2About) under, its survival rate is higher than control strain more than 2 times (referring to Fig. 4); And under the gamma rays dose irradiation of 10kGy, its survival rate is higher than control strain more than 3 times (referring to Fig. 5).
Gobi abnormal cocci of the present invention, the function stem of not only can be directly repairing as radiation environment, can also be as the genetic donor of new genetically modified organism, the radiation tolerance gene that it is good changes in other organism, makes it can obtain good degeneration-resistant proterties; Simultaneously, it also may be as new engineering strain, thereby obtains more good proterties by accepting external other excellent genes; In addition, carry out the improvement of proterties, can also obtain the more bacterial strain of multiple characters by traditional selection by mutation means.
Description of drawings
The colonial morphology of Fig. 1 Gobi abnormal cocci;
The scanning electron microscope picture of Fig. 2 Gobi abnormal cocci;
Fig. 3 Gobi abnormal cocci and other abnormal cocci belong to the evolutionary analysis (based on 16S rDNA sequential analysis) between the member;
Fig. 4 Gobi abnormal cocci and control strain D.radiodurans R1 are to the tolerance contrast of uviolizing, and among the figure, blue curve is represented the survival curve of the unusual coccus in Gobi desert, and red curve is represented the survival curve of control strain;
Fig. 5 Gobi abnormal cocci and control strain D.radiodurans R1 are to the tolerance contrast of gamma rays irradiation, and among the figure, blue curve is represented the survival curve of the unusual coccus in Gobi desert, and red curve is represented the survival curve of control strain.
Embodiment
The isolation cultivation method of embodiment 1 Gobi abnormal cocci
On the Gobi desert in somewhere, Xinjiang, gather yellow ground soil.Soil sampling 2cm
3About, use the gamma radiation irradiation of 10k Gy.Changed in the 20ml TGY substratum under 30 ℃, 200rpm enrichment culture afterwards over to 112 hours, each is coated on the TGY nutrient agar flat board with 200 μ L with the enrichment culture thing, the upset flat board places 30 ℃ of incubators to cultivate several days, can obtain Gobi abnormal cocci of the present invention.
TGY nutrient agar: glucose, 1 ‰; Yeast extract, 5 ‰; Tryptones, 10 ‰; Agar, 15 ‰.
Culture temperature is 30 ℃.
The gamma rays resistance test of embodiment 2 Gobi abnormal coccis
1, test method
Gobi abnormal cocci bacterium liquid of the present invention is passed through 5000rpm, 10min, 4 ℃ of centrifugal collection thalline; Thalline is resuspended in equal-volume 0.1M potassiumphosphate Buffer (pH 7.0), reuses 5000rpm, 10min, 4 ℃ of centrifugal collection thalline afterwards, so wash thalline 2 times; Thalline is resuspended in equal-volume 0.1M potassiumphosphate Buffer (pH 7.0) once more the most at last.It is packed as 4ml bacteria suspension/15ml centrifuge tube, and respectively with the gamma rays irradiation of 0-15kGy dosage, the dilution coating is counted on TGY nutrient agar flat board, statistics after no longer changing to number for centrifuge tube.
Be contrast with radiation hardness abnormal cocci (D.radiodurans) R1 bacterial strain simultaneously.
Test organisms and contrast bacterium have respectively carried out 5 groups of parallel tests.
2, result
See Table 2, table 3.Gobi abnormal cocci of the present invention respectively 5,10, under the gamma rays dose irradiation of 15kGy, its survival rate all be higher than control strain more than 3 times (referring to mistake! Do not find Reference source.)。
The survival condition of table 2Deinococcus gobiensis gamma rays irradiation
| Treatment dosage (kGy) | 0 | 5 | 10 | 15 |
| Survival rate | 100.00％ | 85.87％ | 21.63％ | 4.17％ |
| The standard deviation of survival rate | 42.46％ | 11.30％ | 1.43％ | 0.40％ |
The survival condition of table 3 Deinococcus radiodurans R1 gamma rays irradiation
| Treatment dosage (kGy) | 0 | 5 | 10 | 15 |
| Survival rate | 100.00 ％ | 25.76 ％ | 5.93％ | 1.00％ |
| The standard deviation of survival rate | 13.41％ | 3.22％ | 0.60％ | 0.12％ |
The ultraviolet resistance test of embodiment 3 Gobi abnormal coccis
1, test method
Gobi abnormal cocci bacterium liquid of the present invention is passed through 5000rpm, 10min, 4 ℃ of centrifugal collection thalline; Thalline is resuspended in equal-volume 0.1M potassiumphosphate Buffer (pH 7.0), reuses 5000rpm, 10min, 4 ℃ of centrifugal collection thalline afterwards, so wash thalline 2 times; Thalline is resuspended in equal-volume 0.1M potassiumphosphate Buffer (pH 7.0) once more the most at last.Be placed in the culture dish that stirs with iron wire, through 0～648.7J/m
-2The uv irradiating of various dose, the dilution coating is counted on TGY nutrient agar flat board, statistics after no longer changing to number.
Be contrast with radiation hardness abnormal cocci (D.radiodurans) R1 bacterial strain simultaneously.
Test organisms and contrast bacterium have respectively carried out 4 groups of parallel tests.
2, result
See Table 4, table 5.Gobi abnormal cocci of the present invention is under same 9 various dose ultraviolet irradiation amounts, and survival rate all is significantly higher than control strain (referring to mistake! Do not find Reference source.)。
The survival condition of table 4Deinococcus gobiensis uviolizing
| Treatment dosage (Jm -2) | 0 | 81.1 | 162. 2 | 243. 2 | 324. 3 | 405. 4 | 486. 5 | 567. 7 | 648. 6 |
| Survival rate (%) | 100. 00 | 104. 60 | 111. 49 | 108. 91 | 102. 01 | 108. 62 | 112. 07 | 73.6 8 | 81.7 0 |
| The variance of survival rate (%) | 7.33 | 6.70 | 1.53 | 2.49 | 3.07 | 7.47 | 4.60 | 7.28 | 11.0 5 |
The survival condition of table 5Deinococcus radiodurans R1 uviolizing
| Treatment dosage (Jm -2) | 0 | 81.1 | 162.2 | 243.2 | 324. 3 | 405. 4 | 486. 5 | 567. 7 | 648. 6 |
| Survival rate (%) | 100. 00 | 101. 60 | 115.7 1 | 124.3 6 | 99.0 4 | 52.5 6 | 42.6 3 | 14.3 9 | 6.28 |
| The variance of survival rate (%) | 4.81 | 5.98 | 5.34 | 13.03 | 26.9 2 | 0.43 | 2.99 | 2.48 | 0.17 |
The extracting method of embodiment 4 Gobi abnormal cocci bacterial strain 16S rDNA
The present invention also provides the genome extraction of this Gobi abnormal cocci (Deinococcus gobiensis), amplification and the sequence measurement of 16SrDNA, specifically carries out according to following operation:
1. genome extracting method
The genome of Gobi abnormal cocci of the present invention uses
The bacterial genomes of company
DNA extraction test kit (centrifugal cylindricality) extracts, and sees its specification sheets for details.
2.PCR amplification method
The design of the universal primer sequence that the pcr amplification of the 16S rDNA of Gobi abnormal cocci of the present invention is used is with reference to the article of William G.Weisburg (1991), and primer is held up biotech company of section by Beijing and synthesized.The used test kit that increases is bought in precious biotechnology (Dalian) company limited.
Forward primer F27 (5 '-AGAGTTTGATCATGGCTCAG-3 '),
Reverse primer R1492 (5 '-TACGGTTACCTTGTTACGACTT-3 '),
The pcr amplification system is:
The PCR response procedures
16S rDNA sequence with amplification obtains is connected in
On the pSURE-T carrier of company, entrust precious biotechnology (Dalian) company limited to check order for insertion sequence, the accession number on the GenBank database is EU427464.
Claims (5)
1. Gobi abnormal cocci (Deinococcus gobiensis) CGMCC 2358.
2. the described Gobi abnormal cocci of claim 1, the GenBank accession number of the sequence of its 16S rDNA part is EU427464.
3. claim 1 or 2 described Gobi abnormal coccis are as the application of the function stem aspect of radiation environment reparation.
4. claim 1 or the 2 described Gobi abnormal coccis application aspect the structure genetically modified organism.
5. claim 1 or 2 described Gobi abnormal coccis are improved the application of plant trait aspect in selection by mutation.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101696414B (en) * | 2009-11-10 | 2012-01-25 | 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 |
| CN102080089B (en) * | 2009-11-26 | 2012-09-05 | 中国农业科学院生物技术研究所 | Gene of enhancing stress resistance such as drought resistance and the like of plant and application thereof |
| CN103911318A (en) * | 2014-03-07 | 2014-07-09 | 中国农业科学院生物技术研究所 | Radiation resistant Rhodobacter shamoensis W402, and applications thereof |
| CN109182310A (en) * | 2018-09-26 | 2019-01-11 | 中国农业科学院生物技术研究所 | A kind of application of radiation hardness Gobi abnormal cocci keratinase gene |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1200099C (en) * | 2003-04-09 | 2005-05-04 | 浙江大学 | Method for increasing yield of superoxide dismutase of radioresistant coccus by radiation induction |
-
2008
- 2008-02-03 CN CN2008100575592A patent/CN101245330B/en active Active
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101696414B (en) * | 2009-11-10 | 2012-01-25 | 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 |
| CN102080089B (en) * | 2009-11-26 | 2012-09-05 | 中国农业科学院生物技术研究所 | Gene of enhancing stress resistance such as drought resistance and the like of plant and application thereof |
| CN103911318A (en) * | 2014-03-07 | 2014-07-09 | 中国农业科学院生物技术研究所 | Radiation resistant Rhodobacter shamoensis W402, and applications thereof |
| CN103911318B (en) * | 2014-03-07 | 2016-01-13 | 中国农业科学院生物技术研究所 | The red bacterium in radiation hardness desert and application thereof |
| CN109182310A (en) * | 2018-09-26 | 2019-01-11 | 中国农业科学院生物技术研究所 | A kind of application of radiation hardness Gobi abnormal cocci keratinase gene |
| CN109182310B (en) * | 2018-09-26 | 2021-10-08 | 中国农业科学院生物技术研究所 | Application of radiation-resistant Gobi deinococcus keratin gene |
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