CN100560712C - A kind of new Pasteur staphylococcus LF-2 and application thereof - Google Patents
A kind of new Pasteur staphylococcus LF-2 and application thereof Download PDFInfo
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- CN100560712C CN100560712C CNB2007100172493A CN200710017249A CN100560712C CN 100560712 C CN100560712 C CN 100560712C CN B2007100172493 A CNB2007100172493 A CN B2007100172493A CN 200710017249 A CN200710017249 A CN 200710017249A CN 100560712 C CN100560712 C CN 100560712C
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Abstract
The invention discloses a kind of new microorganism Pasteur staphylococcus (Staphylococcus pasteuri) LF-2, be preserved in Chinese typical culture collection center (being called for short CCTCC) on November 19th, 2006, its preserving number is CCTCC NO:M 206127.This Pasteur staphylococcus LF-2, energy is the degrading plant toxin effectively, toxic alkaloid 2 particularly to extracting, separate from glacier whin over-ground part, 2,6, the toxicity of 6-tetramethylpiperidone (be called for short TMPD) can thoroughly solve the murder by poisoning of glacier whin, for active effect is played in the biology control and the range ecology research of grassland poisonous weeds.
Description
Technical field
The present invention relates to new bacterial strain and the application of this bacterial strain in preparation degrading plant toxin medicine of Pasteur staphylococcus (Staphylococcus pasteuri).
Background technology
Loco weed (Locoweed) is the general name that pulse family Astragalus and whin belong to poisonous plants, also is the most serious class poisonous weeds of harm pasture animal husbandry in the world wide.In China, loco weed is distributed widely in the vast grassland in northwest, North China and southwest, and hazard area reaches 1,100 ten thousand hm
2In recent years, both at home and abroad in harm, toxic ingredient, the toxicity mechanism of loco weed, prevent and kill off and the research of aspect such as utilization has obtained impressive progress, but also do not control the effect method that loco weed spreads so far, cause loco weed poisoning problem to be on the rise, become the great trouble of pasture animal husbandry.
Glacier whin (Oxytropis.glacialis) is a kind of of loco weed, also is one of Qinghai-Tibet peculiar grass seeds, mainly is distributed in the Ali area, Tibet, spreads all over the Cuoqin, changes then Geji, day soil, Ka Er, counties such as Pulan.It has very strong resistance, and it is vigorous to grow in desert, and rate of propagation forms advantage group easily apparently higher than other plant of locality.It can cause that chronic poisoning takes place multiple animal, make its occur with the central nervous system function disorder be feature toxicity symptom (Yang Xiaoping. Clinical Symptoms of Toxicosis in Animals by Crazyweed. Wuhan University Of Science and Engineering's journal, 2002,15 (3): 25-28; Xi Linqiao, Ma Liping. present Research and the progress of meadow poisonous weeds whin. grassland and lawn, 2003, (3): 19-22), serious generation death.According to investigation statistics in 1996, actual grassland area 9.30 ten thousand hm that herd in 11 townshiies, Cuoqin County
2, the glacier whin endangers serious 5.40 ten thousand hm that have
2, reach 250,000 (only) because of poisoning causes livestock death in 15 years of 1978～1993 years, about 2,205 ten thousand yuan of direct economic loss, average annual dead about 1.7 ten thousand (only), annual financial loss 150～2,500,000 yuan; There are 166.67 ten thousand hm the Gaize County
2Glacier, meadow whin hazard ratio is more serious, 1987～nineteen ninety-five livestock be poisoned to death 21.28 ten thousand (only), direct economic loss reaches 2,562 ten thousand yuan, in 9 years, the livestock that is poisoned to death every year about 2.4 ten thousand (only), financial loss reaches many ten thousand yuan of 35O; Glacier, Geji County whin spreads heavier grassland 48.87 ten thousand hm
2, be poisoned to death more than the livestock 7500 (only) financial loss 120～1,500,000 yuan every year.Till nineteen ninety-five, the live-stock inventory that three animal husbandry counties, Ali east are poisoned to death because of the glacier whin is more than 530,000, loss surpass 6,172 ten thousand yuan (Li Qinfan, Wang Jianhua. the ecotope characteristics of glacier whin growth. domestic animal ecology, 2004,25 (1): 42-44; Wang Jianhua, Zhang Zhi's perseverance, high superstar. Ali area, Tibet animal " Herba oxytropis glabrae " toxonosis report of survey. animal toxicology, 1998,13 (1-2): 22-27; Zhang Zhi's perseverance, high superstar, Shen Songdong is about the report to Ali area, Tibet " Herba oxytropis glabrae " investigation. animal toxicology, 1998,13 (1-2): 11-13); In addition, the not lethal livestock of glacier whin poisoning that searches for food, its performance and quality significantly descend, can not drive or pack, fur is with low quality, weight loss, the underproduction of milk meat, fecundity weaken, and monster, miscarriage and stillborn foetus etc. occur, and its loss surpasses snow disaster or epidemic disease calamity.And drought-resistant, the cold resistance of glacier whin, with good forage competition meadow, cause large quantities of good forage output to reduce and deterioration of grasslands.In the livestock glacier whin recurrent area of poisoning, the herdsman dare not herd, cause these grasslands be not fully utilized (Wu Da, take charge of red beautiful, Wang Jianhua. Study on Locoweed. Practaculture Science, 2003,20 (2): 43-47).This shows that the glacier whin is very serious to the harm of southern Tibet area domestic animal, bring very big loss for Tibet livestock industry.
At present both at home and abroad to preventing and kill off of glacier whin mainly take manually to excavate, chemistry goes out and removes and the method for biocontrol.Though the glacier whin is poisonous weeds, but it belongs to leguminous plants, comprehensive nutrition, if can rationally utilize, turn waste into wealth, will obtain good economic benefits, this respect Chinese scholars has been carried out many useful explorations, mainly comprises reasonable rotation grazing, intermittently feeds and methods such as daily ration collocation, detoxification utilization.Wherein, aspect the detoxification of poisonous plants, its method has physics detoxification, chemical detoxication, immunoprophylaxis and microbiological deterioration etc.In recent years, using microbe degraded toxic substance is the focus of studying both at home and abroad always, but the research aspect the microbiological deterioration plant poison is less, but also obtained certain achievement, be separated to multiple microorganism strain more than 70 of the sinigrin of can degrading in the Zhou Bincong rapeseed cake, through identifying that these bacterium are white torulopsis (Torulopsis candida), zhizopchin (Rhizopus chinensis), Mucor racemosus (Mucor racemosus), aspergillus oryzae (Aspergillus oryzae), determine that by fermentation test its virus elimination rate is 80% (Zhou Bin, separation screening takes off the research of sinigrin toxin bacterial strain in the rapeseed cake, Yunnan University's journal, 1996,18 (2): 122-125).Tan Beiying etc. are from the ox cud of Weizhou Island, Guangxi; isolate 4 strain anaerobic bacteriums; through being accredited as lactobacillus (Lactobacillus) (2 strain); streptococcus bovis (Streptococcus bovis) and clostridium sporogenes (Clostridium sporogenes); above-mentioned bacterial strains has degrading activity to mimosine and dihydroxy-pyridine toxoid; these detoxification bacterium live in the cud; can protect host animal to avoid the murder by poisoning (Tan Beiying of Leucaena leucocephala (L.); Wang Xuming; Wang does; the degrade rumen bacteria of deleterious mimosine and dihydroxy-pyridine compound. the microorganism journal; 1994; 34 (5): 379-384); its research method can be used for the research of glacier whin, in the hope of thoroughly solving the murder by poisoning problem of glacier whin, makes the abundant nutrition of animal use glacier whin and does not poison.
2,2,6,6-tetramethylpiperidone (2,2,6,6-tetramethyl-4-piperidone, TMPD) be a kind of main toxic alkaloid (Tan Yuanyou, Wang Jianhua, the Li Qinfan etc. that extract, separate from glacier whin over-ground part, the evaluation of glacier, Ali area, Tibet whin toxic alkaloid, herding and animal doctor, 2002,34 (5): 12-13).At present, still not having effective ways reduces the TMPD toxicity in the whin of glacier.If can using microbe degraded TMPD, will be to the murder by poisoning of thorough solution glacier whin, for active effect is played in the biology control and the range ecology research of grassland poisonous weeds.
Summary of the invention
At above-mentioned problems of the prior art and defective, the object of the invention is to provide a kind of new Pasteur staphylococcus LF-2 bacterial strain, this bacterial strain can effectively be degraded, 2,6, (toxicity that is called for short TMPD thoroughly solves the murder by poisoning of glacier whin, for active effect is played in the biology control and the range ecology research of grassland poisonous weeds to the 6-tetramethylpiperidone.
For achieving the above object, technical scheme of the present invention is as follows:
A kind of new Pasteur staphylococcus LF-2 (Staphylococcus pasteuri LF-2), its preserving number is CCTCC NO:M 206127.
Be preserved in Chinese typical culture collection center (be called for short CCTCC) on November 19th, 2006, its preserving number is CCTCC NO:M 206127, the depositary institution address: Chinese Wuhan Wuhan University.
It is the application of Pasteur staphylococcus LF-2 (Staphylococcus pasteuri) in the degrading plant toxin that the present invention also has a purpose.
The present invention also have a purpose be Pasteur staphylococcus LF-2 (Staphylococcus pasteuri) at degrading plant toxin 2,2,6, the application in the 6-tetramethylpiperidone.
It is the application of Pasteur staphylococcus LF-2 (Staphylococcus pasteuri) in preparation degrading plant toxin medicine that the present invention also has a purpose.
It is that Pasteur staphylococcus LF-2 (Staphylococcus pasteuri) is in preparation degrading plant toxin 2,2,6, the application in the 6-tetramethylpiperidone that the present invention also has a purpose.
This strain TMPD degradation bacteria all comes from the ambient soil of ground loco weed growths such as Tibet, Inner Mongol, Qinghai, gets through manually separating, screening, and the specific embodiment on limit is seen below in concrete separation, screening.
It below is the feature description of Pasteur staphylococcus of the present invention (Staphylococcus pasteuri) LF-2.
1, morphological feature
The thalline of LF-2 bacterial strain is spherical, 0.8～1.0 μ m, and its form under transmission electron microscope is seen Fig. 1, its form under scanning electron microscope is seen Fig. 2; Gram-positive, single, paired and irregular heap shape exists, no pod membrane, no gemma, no mobility; Bacterium colony is rounded, and smooth surface is moistening, protuberance, and neat in edge, canescence, translucent; Glucose fermentation produces not aerogenesis of acid, and amphimicrobian, edwardsiella hoshinae, M.R are tested positive, and V.P tests negative, hydrolyzed starch, and reduction nitrate, the catalase positive, oxidase negative, urease-positive is grown in 5%NaCl and 7%NaCl.
2. physiological and biochemical property
Concrete physiological and biochemical property sees Table 1.
The physiological and biochemical property of table 1.LF-2 bacterial strain
| Experimental project | The result | Experimental project | The result |
| Carbohydrate produces acid | The V-P test | - | |
| Glucose | + | The starch hydrolysis | + |
| Lactose | + | Nitrate reduction | + |
| Sucrose | + | Catalase | + |
| D-N.F,USP MANNITOL | - | Oxydase | - |
| The carbohydrate aerogenesis | - | Urase | + |
| Aerobic | + | Thrombin coagulase | - |
| Anaerobism | + | The 5%NaCl growth | + |
| Indoles produces | - | The 7%NaCl growth | + |
| The M.R test | + |
Annotate: "+" expression growth or reacting positive; "-" expression is not grown or reaction negative.
3,16S rDNA sequence homology analysis
Get LF-2 bacterial strain list colony inoculation in the 10mlLB liquid nutrient medium, after cultivating, 37 ℃ of overnight shakings get the centrifugal supernatant that goes of 1.5ml bacterium liquid, prepare pcr template DNA according to the extracting method shown in the bacterial genomes DNA extraction test kit of U-Gene company ,-20 ℃ of preservations are standby.
The primer that is used for the PCR reaction of 16S rDNA is a pair of universal primer.Forward primer BSF8/20:5 '-AGAGT TTGAT CCTGG CTCAG-3 '; Reverse primer BSR1541/20:5 '-AAGGAGGTGA TCCAG CCGCA-3 '.
PCR reaction system (50 μ L) is: Taqmix25 μ L, primer BSF8/20 (10 μ mol/L) 2 μ L, primer BSR1541/20 (10 μ mol/L) 2 μ L, template DNA 1 μ L, redistilled water 20 μ L.The PCR reaction conditions is: 94 ℃ of pre-sex change 2min; 94 ℃ of 1min, 56 ℃ of 1min, 72 ℃ of 2min circulate 29 times; 72 ℃ of 10min.Through 1% agarose gel electrophoresis, the image that gel imaging system shows down as shown in Figure 3, M1 is Dl2000, LF-2 is the PCR product, M2 is 200bp Ladder.The PCR product reclaims test kit recovery purifying with the gel of U-Gene company.Order-checking is finished by the rich inferior biotechnology in Shanghai limited liability company, and order-checking is primer BSF8/20 with primer.
The GenBank accession number of LF-2 bacterial strain 16SrDNA is EF127830, and the 16SrDNA sequence is shown in sequence in the sequence table.
The 16S rDNA sequence of LF-2 bacterial strain is carried out sequence homology analysis by the Blast retrieval system of NCBI, utilize DNAStar software building systematic evolution tree, see Fig. 4.Draw by Fig. 4, the genetic evolution distance of LF-2 bacterial strain is nearest with Staphylococcus, Pasteur staphylococcus (Staphylococcuspasteuri) is in the branch of a minimum together, reach 99.80% with the homology of known bacterial strain Staphylococcus pasteuri (AJ717376), illustrate that this bacterium belongs to the Pasteur staphylococcus on the Molecular Phylogeny taxonomy.Through 16S rDNA sequence homology analysis, according to principle of classification and the combining form, physiological and biochemical property of homology in phylogeny, with reference to " uncle's outstanding bacteriology handbook (the 8th edition) ", determine that finally the LF-2 bacterial strain is Pasteur staphylococcus (Staphylococcus pasteuri).This bacterium can be sole carbon source with TMPD, the toxicity of degradable TMPD.
The component of described LB liquid nutrient medium and proportioning are: peptone 10g, yeast extract 5g, NaCl 10g, distilled water 1L, pH are 7.0～7.2,121 ℃ of autoclaving 15min.
The main toxin T MPD of TMPD degradation bacteria degradable of the present invention glacier whin is for the murder by poisoning that thoroughly solves the glacier whin lays the foundation.
Description of drawings
Fig. 1 is the transmission electron microscope photo of LF-2 bacterial strain;
Fig. 2 is the stereoscan photograph of LF-2 bacterial strain;
Fig. 3 is 1% agarose gel electrophoresis figure of LF-2 bacterial strain;
Fig. 4 utilizes DNAStar software building systematic evolution tree;
Fig. 5 is different incubation time TMPD degradation rate curves;
Embodiment
Providing the separation screening way of Pasteur staphylococcus LF-2 and Pasteur staphylococcus LF-2 thereof below in conjunction with accompanying drawing and contriver tests at degraded TMPD and further specifies beneficial effect of the present invention.
Embodiment 1: the separation of acid-producing Klebsiella bacterium LF-1-1, screening method
1, soil sample: the ambient soil that picks up from ground loco weed growths such as Tibet, Inner Mongol, Qinghai;
2, the separation screening of bacterial strain: the soil inoculation of getting 4 sources in 20g～30g loco weed growing environment respectively is in ordinary broth, after cultivating 48h, 37 ℃ of expansion bacterium on plain agar, carry out streak inoculation, cultivate 24h for 37 ℃, choose single colony inoculation in the ordinary broth that contains 50mg/L TMPD, cultivate 48h for 37 ℃, preserve bacterial classification; Previous step preserved the bacterial classification inoculation that gets off in the ordinary broth that contains 100mg/L TMPD, put 37 ℃ and cultivate 48h; Repeat above process, each cultivation all increases TMPD content, reduces the content of organotrophy composition in the nutrient solution, preserves bacterial classification.Through 5 cultivations, when TMPD content was increased to 300mg/L in the substratum, 4 ℃ of preservation bacterial classifications were standby; With the bacterial classification inoculation of above-mentioned preservation in ordinary broth, cultivating 48h for 37 ℃ activates and expands bacterium, the centrifugal 15min of 7500r/min, abandoning supernatant, sedimentary thalline washes 3 times with the ditalimfos phthalate buffer, make the bacteria suspension of 20mL at last with the ditalimfos phthalate buffer, be inoculated in respectively and contain on 150mg/L TMPD substratum and the no nutritional medium, behind 37 ℃ of cultivation 48h, choose well-grown bacterial strain on the TMPD substratum, line is inoculated in TMPD substratum and no nutritional medium respectively again, repeat to cultivate, choose purely, obtain a strain degradation bacteria, called after LF-2 bacterial strain.
The component of described broth medium and proportioning are: extractum carnis 5g, peptone 10g, NaCl 5g, KH
2PO
11g, distilled water 1L, pH are 7.4～7.6,121 ℃ of autoclaving 20min.
The component of described plain agar substratum and proportioning are: extractum carnis 5g, peptone 10g, NaCl 5g, KH
2PO
41.0g, agar 20g, distilled water 1L, pH 7.4～7.6,121 ℃ of autoclaving 20min.
The component of described no nutritional medium and proportioning are: NaCl 1.0g, K
2HPO
41.0g, NH
4Cl1.0g, MgSO
47H
2O 0.4g, FeCl
30.01g agar 20g is dissolved in the 1000mL distilled water, pH is 7.2～7.4,121 ℃ of sterilization 30min.
Described TMPD substratum: according to different needs, in no nutritional medium, add TMPD, regulate 7.2～7.4,121 ℃ of sterilizations of pH 30min.
Test example 1: the degraded TMPD effect test of Pasteur staphylococcus LF-2
Adopt the ultraviolet-visible spectrophotometer method to measure the degradation rate of this bacterium to TMPD.Be inoculated in this degradation bacteria in the ordinary broth respectively, cultivating 48h for 37 ℃ activates and expands bacterium, the centrifugal 15min of 7500r/min, abandoning supernatant, sedimentary thalline washes 3 times with the ditalimfos phthalate buffer, makes the bacteria suspension of 20mL at last with the ditalimfos phthalate buffer, be inoculated in respectively and contain in the 150mg/L TMPD nutrient solution, measured the absorbancy of nutrient solution every 3 days, measurement result is brought regression curve into, calculates its concentration.The degradation rate of concrete TMPD sees Table 2.As can be seen from Table 2, cultivated through 30 days, the TMPD degradation rate of LF-2 bacterial strain is 71.24%.Different incubation time TMPD degradation rate curves are seen Fig. 5, as can be seen from Figure 5 each bacterial classification after inoculation in the 15d degradation speed the fastest, cultivate 18d after, degradation speed reduces, and illustrates that three strain degradation bacteria inoculation TMPD nutrient solution cultivates after 15 days, TMPD can well be degraded.
Sequence table
＜110〉Xibei Univ. of Agricultural ﹠ Forest Science ﹠ Technology
＜120〉a kind of new microorganism Pasteur staphylococcus LF-2 and application thereof
<160>1
<210>1
<211>1492
<212>DNA
＜213〉Pasteur staphylococcus (Staphylococcus pasteuri)
<400>1
ggcccggggg gcatgctata catgcagtcg agcgaacaga taaggagctt gctcctttga 60
cgttagcggc ggacgggtga gtaacacgtg gataacctac ctataagact gggataactt 120
cgggaaaccg gagctaatac cggataacat attgaaccgc atggttcaat agtgaaaggc 180
ggctttgctg tcacttatag atggatccgc gccgtattag ctagttggta aggtaacggc 240
ttaccaaggc aacgatacgt agccgacctg agagggtgat cggccacact ggaactgaga 300
cacggtccag actcctacgg gaggcagcag tagggaatct tccgcaatgg gcgaaagcct 360
gacggagcaa cgccgcgtga gtgatgaagg tcttcggatc gtaaaactct gttatcaggg 420
aagaacaaat gtgtaagtaa ctgtgcacat cttgacggta cctgatcaga aagccacggc 480
taactacgtg ccagcagccg cggtaatacg taggtggcaa gcgttatccg gaattattgg 540
gcgtaaagcg cgcgtaggcg gttttttaag tctgatgtga aagcccacgg ctcaaccgtg 600
gagggtcatt ggaaactgga aaacttgagt gcagaagagg aaagtggaat tccatgtgta 660
gcggtgaaat gcgcagagat atggaggaac accagtggcg aaggcgactt tctggtctgt 720
aactgacgct gatgtgcgaa agcgtgggga tcaaacagga ttagataccc tggtagtgca 780
cgccgtaaac gatgagtgct aagtgttagg gggtttccgc cccttagtgc tgcagctaac 840
gcattaagca ctccgcctgg ggagtacgac cgcaaggttg aaactcaaag gaattgacgg 900
ggacccgcac aagcggtgga gcatgtggtt taattcgaag caacgcgaag aaccttacca 960
aatcttgaca tcctttgacc gctctagaga tagagtcttc cccttcgggg gacaaagtga 1020
caggtggtgc atggttgtcg tcagctcgtg tcgtgagatg ttgggttaag tcccgcaacg 1080
agcgcaaccc ttaagcttag ttgccatcat taagttgggc actctaagtt gactgccggt 1140
gacaaaccgg aggaaggtgg ggatgacgtc aaatcatcat gccccttatg atttgggcta 1200
cacacgtgct acaatggaca atacaaaggg cagctaaacc gcgaggtcaa gcaaatccca 1260
taaagttgtt ctcagttcgg attgtagtct gcaactcgac tacatgaagc tggaatcgct 1320
agtaatcgta gatcagcatg ctacggtgaa tacgttcccg ggtcttgtac acaccgcccg 1380
tcacaccacg agagtttgta acacccgaag ccggtggagt aaccatttat ggagctagcc 1440
gtcgaaggtg ggacaaatga ttggggtgaa gtcgtaacaa gtagccatag gc1492
Claims (3)
1. Pasteur staphylococcus (Staphylococcus pasteuri) LF-2, its preserving number is CCTCCNO:M 206127.
2. the described Pasteur staphylococcus of claim 1 LF-2 is at degrading plant toxin 2,2,6, the application in the 6-tetramethylpiperidone.
3. the described Pasteur staphylococcus of claim 1 LF-2 is in preparation degrading plant toxin 2,2,6, the application in the 6-tetramethylpiperidone medicine.
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