CN1177038C - Halophilic and alkalophilic bacillus strain and its special plasmid and application - Google Patents
Halophilic and alkalophilic bacillus strain and its special plasmid and applicationInfo
- Publication number
- CN1177038C CN1177038C CNB021007144A CN02100714A CN1177038C CN 1177038 C CN1177038 C CN 1177038C CN B021007144 A CNB021007144 A CN B021007144A CN 02100714 A CN02100714 A CN 02100714A CN 1177038 C CN1177038 C CN 1177038C
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- plasmid
- liking
- alkali
- bacterium
- pnb101
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Abstract
The present invention relates to a halophilic and alkalophilic bacillus, special plasmid thereof, and an application thereof. The present invention provides an extremely halophilic and alkalophilic archaeobacterium which is Natronobacterium sp. AS-7091 CGMCC No. 0673 having plasmid pNB101 with the sequence 1. The present invention has the technical scheme that plasmid pNB101 and derivation plasmid and a recombined carrier thereof supplied by Natronobacterium sp. As-7091 and a mutant or variant thereof can be transformed into the extremely halophilic and alkalophilic archaeobacterium, particularly the halophilic and alkalophilic bacillus and neutral extremely halophilic archaeobacterium having close relationship with the extremely halophilic and alkalophilic archaeobacterium; the present invention is used for researching and developing the microbiological resources.
Description
Technical field
The present invention relates to a strain has a liking for salt alkali bacillus and contained proprietary plasmid and application, particularly a strain extreme halotolerant and has a liking for the ancient bacterium of alkali and proprietary plasmid and application.
Background technology
According to the criteria for classification of present highest level, tellurian life can be divided into three major types, is called territory (domains), i.e. bacterium (Bacteria) territory, ancient bacterium (Archaea) territory and eukaryote (Eukarya) territory.The understanding of people to life quintessence has been deepened in the discovery of ancient bacterium, and therefore becomes the focus of life science fundamental research.Ancient bacterium lives in extreme environment mostly, therefore is again the important object of extreme environment Microbial resources exploitation.
Extremely halophilic archaea (Extremely Halophilic Archaea) can the suitableeest growth in the hypersaline environment of 2-5M NaCl, is main extreme halotolerant environmental microorganism.Extremely halophilic archaea is of a great variety, comprises Halobacterium, Halococcus, Haloarcula, Haloferax, Halorubrum, Halobaculum, Halogeometricum, Natrialba, Natronobacterium, Natronococcus, Natronomonas, Natrinema, 13 genus such as Natronorubrum.Halobacterium wherein, Haloferax etc. are the neutral pole terminal halophilic archaea, Natronobacterium, Natronococcus etc. have a liking for the ancient bacterium of alkali for extreme halotolerant.Extremely halophilic archaea has DEVELOPMENT PROSPECT, in a large number can be as in this quasi-microorganism, having for the extreme enzyme of exploitation, biologically active factors, the precursor of degradable plastics, have the bacteria rhodopsin of development biological computation machine chip potentiality etc., and it is had a liking for salt and has a liking for the alkali Study on Mechanism and will provide possible theoretical foundation to the contrary crop of research and development novel anti.
The research and development of extremely halophilic archaea needs corresponding genetic operating system, but its genetic operating system is also quite deficient at present, the genetic operating system that extreme halotolerant is had a liking for the ancient bacterium of alkali does not appear in the newspapers in the world as yet, and the several systems that made up in the neutral pole terminal halophilic archaea (J Bacteriol 1990,172:756-761; J Biol Chem 1992 267:5829-5843) can only be used for limited several genus, and this has limited the theoretical investigation and the genetically engineered exploitation of this class important microbe to a great extent.Therefore it is significant that novel strain type, the especially extreme halotolerant of researching and developing extremely halophilic archaea had a liking for the new genetic operating system of the ancient bacterium of alkali.Moreover the extreme microorganism genetic operating system is not only research extreme environment microbial genetics, and the first-selected system of expressing extreme enzyme and extreme microorganism biologically active factors, and this system also has other valuable feature.The host bacterium of having a liking for the ancient bacterium genetic operating system of alkali as extreme halotolerant is that extreme halotolerant is had a liking for the ancient bacterium of alkali, its culture environment can't grow common bacteria because of high salt and high pH value, therefore available open-air salt pond its gene engineering product of fermentative production reaches the purpose of saving cost and developing the saltings.
PUC pUC is the principal mode of microorganism hereditary operating system.1), the method for physics such as the gene (DNA) that is used to transform must be by transforming such as electricity, and mineral ion is handled, the protoplastis fusion or chemistry imports in this organism gene transformation to a kind of microorganism success must must meet the following conditions:.2), enter biological intravital foreign gene (DNA) and must be able to be replicated (self-replicating or duplicate as the part of host genome).By from desiring to be separated its wild plasmid the host transformed, make up replicating plasmid vector by gene recombination then, after being cloned on the above-mentioned plasmid vector, just can realize foreign DNA purpose well with plasmid replication.3), use one or more selective markers, the conversion physical efficiency is selected from the non-transformed cell group, but the antibiotics resistance gene of activity expression in the target host microorganism by separating clone or design, and chain with foreign DNA place plasmid, can realize orthoselection.
Summary of the invention
One of purpose of the present invention provides the new extreme halotolerant of a strain and has a liking for the ancient bacterium of alkali.
It is to have a liking for salt alkali bacillus (Natronobacterium sp.AS-7091) CGMCC № 0673 that extreme halotolerant provided by the invention is had a liking for the ancient bacterium of alkali.
Have a liking for salt alkali bacillus (Natronobacterium sp.AS-7091), be deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center on 01 11st, 2002, it abbreviates CGMCC as, and deposit number is CGMCC № 0673.
Of the present inventionly have a liking for salt alkali bacillus AS-7091 and in Inner Mongol alkalescence salt lake, separate and obtain, have the suitableeest growth in the alkaline environment of the high salt of 2-5M NaCl and pH8-11, Gram-negative, characteristic such as shaft-like.This bacterium can be at saline and alkaline substratum (every liter of medium component: casamino acids 7.5g, yeast extract 10g, Trisodium Citrate 3g, MgSO
47H
2O 0.1g, KCl 2g, NaCl 200g, Na
2CO
38g, divalence Fe and Mn trace PH9.5) are gone up growth, and bacterium colony is rounded, neat in edge, 7-10 days bacterium colonies of 37 ℃ of growths are red.
Another object of the present invention provides a kind of proprietary plasmid of having a liking for salt alkali bacillus (Natronobacteriumsp.AS-7091) that derives from, this plasmid can be used to transform halophilic archaea, and what especially extreme halotolerant was had a liking for the ancient bacterium of alkali has a liking for salt alkali bacillus (Natronobacterium).
Plasmid provided by the present invention is to derive from the plasmid pNB101 that has a liking for salt alkali bacillus (Natronobacterium sp.AS-7091), and it has the nucleotide sequence of sequence 1, is made up of 2535 Nucleotide, and its chain type is double-stranded, and the topology shape is a ring-type.
Plasmid pNB101 comprises restricted zymogram as shown in Figure 1, and common restriction endonuclease sites comprises Dra I, Hinc II, Hind III, Not I, Sac I, each one of Sal I etc., each three of two of MseI etc. and Pst I and XhoI etc.More than the part of these restriction enzyme sites can be used for DNA reorganization etc.
Plasmid pNB101 does not contain Apa I, BamH I, Bgl II, Cla I, EcoR I, EcoR V, Kpn I, NcoI, Nde I, Pvu II, Sac II, Sma I, Sph I, common restriction enzyme sites such as Xba I, so the part of these restriction enzyme sites can be in order to be designed to the multiple clone site of derivative vector.
Genes encoding at plasmid pNB101 is read frame (Open Reading Frame, orf) in, the dna sequence dna of 1-1167 in the sequence 1, the coded albumen and the neutral halophilic archaea plasmid pHSB2 (J.Bacteriol.1993 of function fragment OrfA of the required trans element of plasmid replication promptly encodes, 175:1081-1086), pGRB (NucleicAcids Res.1990,18:3408), pHSB1 (Can.J.Microbiol.1989,35:86-91) and the homology in the plasmid replication associated protein correlation function district of pHGN1 (Nucleic Acids Res.1989:17:10501) etc. up to 30-40%.The dna sequence dna of 1168-1894 in the sequence 1, coded albumen and certain cyclin of eukaryote (CDC2L5) kinases (Proc.Natl.Acad.Sci.U.S.A.1992, the 89:579-583 of function fragment OrfB of the required trans element of plasmid replication promptly encodes; Biochem.Biophys.Res.Commun.2000,279:832-837) etc. the homology of relevant range is more than 30%.The dna sequence dna of 1168-1894 is the function fragment of the required cis element of coding plasmid replication in the sequence 1.
Certainly; by plasmid pNB101 transform gained with have a liking for bacterium that salt alkalophilic bacillus and intestinal bacteria are representative, contain the recombinant vectors of plasmid pNB101 and transform gained to have a liking for the bacterium that salt alkalophilic bacillus and intestinal bacteria are representative, all should be subjected to protection of the present invention.
Have a liking for the ancient bacterium of alkali because plasmid pNB101 provides at extreme halotolerant, especially have a liking for and duplicate necessary trans and cis element in the saline and alkaline Bacillaceae (Natronobacterium).Therefore can conclude that what it provided in assurance duplicates under the complete prerequisite of required element, can transform the recombinant vectors or the plasmid of deriving that extreme halotolerant is had a liking for the ancient bacterium of alkali to wherein introducing other clear and definite function DNA fragment of character, can constituting.PNB101 provides a plurality of sites that the function DNA fragment makes up the recombinant vectors or the plasmid of deriving of introducing, as Sac I site, it is single restriction enzyme site in plasmid pNB101, and after being positioned at the end of Orf B just, enzyme is cut not destroy any of pNB101 plasmid and its conversion and have a liking in the ancient bacterium of alkali at extreme halotolerant and is duplicated required element herein.Therefore be introduced in the extreme ancient bacterium effectively resistance herein and select gene, can be built into and be applicable to that extreme halotolerant has a liking for the genetic operating system of alkali bacterium.The resistant maker gene that can introduce the escherichia coli plasmid replicon on this basis and be suitable in intestinal bacteria, screening also, can constitute intestinal bacteria-extreme halotolerant and have a liking for the ancient bacterium shuttle vectors of alkali, conveniently in intestinal bacteria, carry out genetic manipulation and have a liking in the ancient bacterium of alkali carrying out genetic analysis and applied research at extreme halotolerant.
By having a liking for plasmid pNB101 and derive plasmid and the recombinant vectors that salt alkali bacillus (Natronobacterium sp.AS-7091) and mutant thereof or varient provide, can transform extreme halotolerant and have a liking for the ancient bacterium of alkali, especially have a liking for saline and alkaline Bacillaceae (Natronobacterium) and have a liking for the nearer neutral pole terminal halophilic archaea of the ancient bacterium relationship of alkali, be used to research and develop this quasi-microorganism resource with extreme halotolerant.
The present invention will be further described below in conjunction with specific embodiment.
Description of drawings
Fig. 1 is the restriction enzyme site collection of illustrative plates of plasmid pNB101 of the present invention
Fig. 2 is a recombinant vectors pNB102 structure collection of illustrative plates
Fig. 3 is a recombinant vectors pNB103 structure collection of illustrative plates
Embodiment
Embodiment 1, based on the recombinant vectors pNB102 of plasmid pNB101
Vulkamycin. PA-93 resistant gene (Nov with the Natrinema altunense sp source
R, J Bacteriol 1991,173:642-648) the Sac I site of introducing pNB101 as the resistance selectable marker gene of having a liking at extreme halotolerant in the ancient bacterium of alkali bacterium, and obtains ammonia benzyl cyanomycin resistant gene (Amp from pBR322
R) and escherichia coli plasmid replicon, add the multiple clone site of synthetic, can constitute applied intestinal bacteria-extreme halotolerant and have a liking for the ancient bacterium shuttle vectors pNB102 (as shown in Figure 2) of alkali.
Because pNB102 contains from pNB101 complete having a liking in the ancient bacterium of alkali at extreme halotolerant and duplicates required trans and cis element, promptly the pNB101 plasmid is cut the dna sequence dna pNB101 ori of acquisition by Sac I enzyme; Contain and in intestinal bacteria, duplicate required trans and cis element, be i.e. escherichia coli plasmid replicon ColE1 ori; And contain intestinal bacteria and at extreme halotolerant and have a liking for the resistance selectable marker gene Amp of screening transformant in the ancient bacterium of alkali
RAnd Nov
R, and be used for cloning site such as the Kpn I that foreign gene inserts, Apa I, EcoR V, Sma I, Xba I etc.Therefore pNB102 is that an effective intestinal bacteria-extreme halotolerant is had a liking for the ancient bacterium shuttle vectors of alkali, but transformed into escherichia coli and extreme halotolerant are had a liking for the ancient bacterium of alkali, and clonal expression foreign gene therein.
Embodiment 2, based on the recombinant vectors pNB103 of plasmid pNB101
Nervinolin (mevinolin) resistant gene (Mev with the Natrinema altunense sp source
R, J Biol Chem 1992,267:5829-5834) the Sac I site of introducing pNB101 as the resistance selectable marker gene of having a liking at extreme halotolerant in the ancient bacterium of alkali bacterium, and obtains ammonia benzyl cyanomycin resistant gene (Amp from pBR322
R) and escherichia coli plasmid replicon, add the multiple clone site of synthetic, can constitute applied intestinal bacteria-extreme halotolerant and have a liking for the ancient bacterium shuttle vectors pNB103 (as shown in Figure 3) of alkali.
Because pNB102 contains from pNB101 complete having a liking in the ancient bacterium of alkali at extreme halotolerant and duplicates required trans and cis element, promptly the pNB101 plasmid is cut the dna sequence dna pNB101 ori of acquisition by Sac I enzyme; Contain and in intestinal bacteria, duplicate required trans and cis element, be i.e. escherichia coli plasmid replicon ColE1 ori; And contain intestinal bacteria and at extreme halotolerant and have a liking for the resistance selectable marker gene Amp of screening transformant in the ancient bacterium of alkali
RAnd Mev
R, and be used for cloning site such as the Kpn I that foreign gene inserts, Cla I, EcoR I etc.Therefore pNB103 is that an effective intestinal bacteria-extreme halotolerant is had a liking for the ancient bacterium shuttle vectors of alkali, but transformed into escherichia coli and extreme halotolerant are had a liking for the ancient bacterium of alkali, and clonal expression foreign gene therein.
Sequence table
<160>1
<210>1
<211>2535
<212>DNA
＜213〉have a liking for salt alkali bacillus (Natronobacterium sp.AS-7091)
<400>1
atgaacaacc?gtccagacgc?gagagagggc?gtcagccgcc?gggtttcgga?actcgacccg 60
tccgcccttc?ccgacggcac?cgcgttccgc?ccgatctccg?tcaccgaggg?caccggcctg 120
cgcgacgagg?ccgtgctggt?ccgcaccgag?aacgggcacc?gggcaccgct?ggacacgttc 180
gagtcggtgc?cagatggcgc?agaactgcag?gcacggtccg?tctaccgggt?gctgaacgca 240
tggcgggact?ggttcgatgg?ctaccggaac?gcgcacatcg?agtacgagga?cccggacggg 300
aaaaccgtac?gcacaccgct?tgagaacagc?tatcagccgg?agtacgggaa?acgctactac 360
gcgaagatca?aggactggga?gcgtggcata?gaacgggcgt?atcaggcccc?gacgatggtg 420
atggtgacac?tttcagcatc?gagcgagaac?gcgaagggtg?ggcgacggtg?cccggccgat 480
cacatgcgcg?acatcgccag?aggctggaac?agcgcgagaa?aggcgctaca?ccgcgtactt 540
cgacgtttcg?agtgggagta?cgcgaaggtc?tgggagcctc?accagagcgg?ctacggtcac 600
atgcacgtcg?cggtcgccgt?cgacgacccg?gccgacgaga?tcgagggcga?gacgttccgg 660
ccggtggtcc?ggtcgcacgt?cgagaacgtg?gaaccggcag?gatcggccgc?acacggcctg 720
aacgccgtgg?gcatgggcga?tacggtgagc?gtgaaccgag?aggtggagaa?cctcggttcg 780
tacatcagcg?agtacatcgg?aatcttcggg?gaagaaccca?ccgagcggcc?ggtttccgaa 840
cagatgttct?acgcgacgtg?ctgggcgaca?gggacgcgcc?gggtggactt?cagcaacgga 900
gcgcacgaga?tcatggcgat?ggagcagttc?cggcgagaga?cgggcttacg?gcccgaggat 960
cgtgggggcg?acacgtttga?ccagtggcgt?gacggcgcga?ccggcgagac?ggacgcagac 1020
ggctcgtcag?gcggttcgtg?gtcggtggac?agtatctgca?ccgtcaccaa?cggcacgccg 1080
acgtacagcg?acccgacagc?gggcggccag?aggctcacgc?ccatcgacgg?ccgggcgggt 1140
gttgatccgc?cagcccatcg?agactgaccg?gcccacagcc?caccgtcccc?ggttgtacac 1200
acccccttta?ggggggtgtg?tacagacagt?cagaggagtc?tatatataag?acgcgcgtag 1260
ccggtacaga?gcttctaccg?gccggtttcg?tcgtttccgt?cactgtccac?gcggcgaaac 1320
ctcgtgcaac?acgtagtaca?gagtctctac?cgtctggttt?cggcccttct?gtccggggtg 1380
ttgcaagtgc?tgttgtggtg?gctttacacg?ggtttctgga?cggccggtac?aggctctgca 1440
aagctttaaa?ccggccgtat?acgatgatac?ggtcacatgg?cgaagaagaa?cgtaagcgcg 1500
agggtgccac?cacagatagc?tgacgggata?gacgagtacg?cggaagcgtg?gaatctgaac 1560
cgtacagagg?ctatgacggc?acttctcgga?ttcgctgtac?agaacgcacc?agatccccga 1620
gaggtagacg?ttgatgccga?cgtgaaccca?gcggaacggg?tgtatacgct?ggagttagag 1680
cctgagaacg?ccgaacacgt?cgaagattct?gacgtgtctg?tcgagcaggc?catcaacaac 1740
gtgctgagcg?tctacagcgg?gtgatctgga?gcggctaaca?gagaggtaga?gagtctgact 1800
aaagcgtata?cgctggatag?cgggtagtac?agcgtcccta?cgtaggaatt?aagtgggcgg 1860
gggagcgatg?gactggtaag?caaagctacg?caacgtgatc?gaagtccggc?agaaggggcc 1920
ggacacgacc?gctcagggcc?gtgccatcgg?ccctgaacgc?ggcgaggaac?ttgtggcagg 1980
gttccgtgtc?ctacccttct?gcgtccgtcg?gcaaaatcgt?ttcgaccaag?ccagacggcc 2040
ggtataccat?gctgaaagtg?ccgagtacgg?tactttcgga?gatgttactc?agagtaacag 2100
ggaacagccc?acagcccacc?gcgagattcc?tcgagcggtc?ccgatgcgtc?tgcagggtcc 2160
tcgagtagca?ccagagcatc?tgcaggccga?tccatccggc?aggcggtccc?atcgtgtgcg 2220
gtcccagcat?cgcgcgagtg?ccggatgggt?gaaagcccgg?aacggccgcg?ttagcggccg 2280
caggcccgga?acacagggag?cgcagcgacc?gtcctcgttc?cggcgagcga?gcgcgttccg 2340
gtacgtgggc?cggtcgagga?ggcccgaggc?gcgagcggca?gctccacgag?gagcgccgag 2400
ccacccggct?gacgacgacc?ggcgggggcg?gtcgcggtga?gctcgagtcc?agggcgtgac 2460
gactgggacg?gccgggtaga?tcgagccgcc?gcaggcggcg?ataggccggg?tgacggttct 2520
acttctcggc?cctcc 2535
Claims (7)
1, salt alkali bacillus (Natronobacterium sp.AS-7091) CGMCC № 0673 is had a liking in a strain.
2, a kind of plasmid pNB101 that has a liking for salt alkali bacillus (Natronobacterium sp.AS-7091) that derives from, it has the nucleotide sequence of sequence 1.
3, transform the bacterium of gained by plasmid pNB101.
4, bacterium according to claim 3 is characterized in that: described bacterium is intestinal bacteria or has a liking for the salt alkali bacillus.
7, the recombinant vectors that contains plasmid pNB101.
8, transform the bacterium of gained by the recombinant vectors that contains plasmid pNB101.
9, bacterium according to claim 8 is characterized in that: described bacterium is intestinal bacteria or has a liking for the salt alkali bacillus.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB021007144A CN1177038C (en) | 2002-01-18 | 2002-01-18 | Halophilic and alkalophilic bacillus strain and its special plasmid and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB021007144A CN1177038C (en) | 2002-01-18 | 2002-01-18 | Halophilic and alkalophilic bacillus strain and its special plasmid and application |
Publications (2)
| Publication Number | Publication Date |
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| CN1432644A CN1432644A (en) | 2003-07-30 |
| CN1177038C true CN1177038C (en) | 2004-11-24 |
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| Application Number | Title | Priority Date | Filing Date |
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| CNB021007144A Expired - Fee Related CN1177038C (en) | 2002-01-18 | 2002-01-18 | Halophilic and alkalophilic bacillus strain and its special plasmid and application |
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Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100422333C (en) * | 2004-06-25 | 2008-10-01 | 中国科学院微生物研究所 | Superhalophilic antient bacteria plasmid and its derivative plasmid carrier |
| CN100347302C (en) * | 2004-08-11 | 2007-11-07 | 中国科学院微生物研究所 | Nikkomycin biological synthesis regulating gene and its coded protein and engineering bacterium for expression |
| CN1308449C (en) * | 2005-08-05 | 2007-04-04 | 中国科学院微生物研究所 | Carrier and engineering bacteria for expressing extremehalophile purple membrane |
| CN104195062B (en) * | 2014-06-06 | 2016-08-24 | 迪丽拜尔·托乎提 | A kind of halophilic archaea and application thereof |
| CN106479928B (en) * | 2016-11-02 | 2019-10-11 | 中国科学院微生物研究所 | The indigenous plasmid of one plant of resistance to resistance to high COD salt water meningitidis strains and the source bacterial strain with high salt |
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2002
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