CN1266278C - Clone procaryotic biological salt-resistant related gene method - Google Patents
Clone procaryotic biological salt-resistant related gene method Download PDFInfo
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- CN1266278C CN1266278C CN 200410084812 CN200410084812A CN1266278C CN 1266278 C CN1266278 C CN 1266278C CN 200410084812 CN200410084812 CN 200410084812 CN 200410084812 A CN200410084812 A CN 200410084812A CN 1266278 C CN1266278 C CN 1266278C
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Abstract
The present invention provides a method for cloning salt-resistant related genes of prokaryotic organisms, which relates to gene engineering. The method for cloning salt-resistant related genes of prokaryotic organisms comprises the following steps: extracting total DNA from salt-resistant pseudomonas or other salt-resistant prokaryotic organisms; incompletely enzyme-cutting the total DNA; carrying out gel electrophoresis to the enzyme-cut total DNA; finding enzyme-cutting segment areas to cut out gel and recover DNA segments; completely enzyme-cutting the total DNA into linear; co-culturing the recovered DNA segments and linear carriers; adding ligase to form recombinant plasmid; treating the recombinant plasmid by CaCl2 used for conversion to obtain competent recipient bacteria; coating transformant solution on a high-salt LB agar culture medium; selecting transformants; extracting the recombinant plasmid; inserting the segments; measuring sequence; carrying out homology comparison to judge the character of genes; drawing the growth curve of the transformants and a comparison growth curve and comparing the growth state differences thereof to verify that the genes are salt-resistant related genes for the second time.
Description
Technical field
The present invention relates to a kind of genetically engineered, especially a kind of fast method of clone procaryotic biological salt-resistant related gene.
Background technology
At present the conventional way of a known of clone is to search by bioinformation from certain prokaryotic organism, learn the sequence of other biological this gene after, follow two technological lines again and carry out clone gene:
1. the oligonucleotide dna probe that synthesizes this gene, with radio isotope or this molecular probe of on-radiation chemical substance mark, Southern hybridization is carried out in this probe and this biological gene group library that makes up in advance, angled out the dna fragmentation that contains goal gene, this sequencing fragment; Structure contains this segmental recombinant expression, transformed acceptor cell, detect this dna fragmentation and transcribe new albumen that the back transformant obtains and biological function thereof (referring to document 1:Sambrook J, Fritsch E F, Maniantis T:Molecular Cloning:A Laboratory Mannual (2
Dd) .Cold Spring HarborLaboratory Press, 1989. (Jin Dongyan, Li Mengfeng etc. translate. molecular cloning experiment guide (second edition). and Beijing: Science Press, 1996)).
2. lack nucleotide primer according to two ends sequences Design and synthetic 3 ' end and 5 ' end oligomerization of known, total DNA with this biology is that template is carried out pcr amplification, reclaim amplified production and construction recombination plasmid, order-checking, transformed acceptor cell, detect this dna fragmentation transcribe new albumen that the back transformant obtains and biological function thereof (referring to document 2: Wu Naihu writes. Principles of Gene Engineering. Beijing: Science Press, 1998).
Method 1 needs to make up in advance this biological genomic library, and chemosynthesis radioactivity then or on-radiation dna probe carry out a large amount of Southern hybridization again, and expensive, time-consuming, experimental period is long again;
Though method 2 ratio method 1 are simple,, many cover loop parameter, the especially temperature of " annealing " are set in the PCR reaction then because therefore different biological gene order and incomplete same often need synthesize how right primer.Like this, method 2 generally all to carry out repeatedly grope repeatedly just may clone goal gene, even may come to an end with the failure of an experiment.
In addition, the gene that the strategy of above-mentioned two kinds of clone genes all can only be cloned known (being that forefathers cloned), gene that can't cloned sequence the unknown with certain function.
Summary of the invention
The object of the present invention is to provide a kind of not only method of quick but also economic clone procaryotic biological salt-resistant related gene.
Concrete steps of the present invention are as follows:
1 with extremely salt-tolerant pseudomonas or other salt tolerant prokaryotic organism, comprise that various salt tolerant bacteriums and blue-green algae are seeded in the substratum that contains NaCl0.1～0.9mol/L, and temperature is 15～32 ℃, shake with the speed of 100～150rpm to be cultured to the exponential growth middle and later periods; If the salt tolerant blue-green algae should be cultivated under illumination;
2 conventional alkaline denaturations extract extremely salt-tolerant pseudomonas or the procaryotic total DNA of other salt tolerant, detect purity, its OD through UV spectrum
260/ OD
280Ratio answers 〉=1.8.The molecular weight that detects the DNA that extracts with 1% agarose gel electrophoresis whether 〉=21kb, as if OD
260/ OD
280Ratio 〉=1.8 illustrate that gained DNA is purer, can enter next step: if OD
260/ OD
280Ratio＜1.8 illustrate that gained DNA purity is not enough, should be further purified, until OD
260/ OD
280Ratio 〉=1.8;
3, under aseptic condition, above-mentioned total DNA is cut at 37 ℃ of incomplete enzymes that carried out 60～100 minutes with restriction endonuclease (E.coRI) or other restriction endonuclease that produces sticky end, total DNA after enzyme is cut is through 1% agarose gel electrophoresis of brominated second pyridine, finding molecular weight under ultraviolet lamp is the endonuclease bamhi zone of 1～3kb, cut out the gel that contains above-mentioned this endonuclease bamhi, reclaim dna fragmentation wherein;
4, under aseptic condition with E.coRI at artificial constructed expression vector, as the multiple clone site of pUC18 that its complete degestion is linear, dephosphorylation is handled, the dna fragmentation and the linear carrier that reclaim are educated altogether, add ligase enzyme and connect 10～24 hours, form recombinant plasmid, transform and use CaCl at 16 ℃
2Handle the competence recipient bacterium that obtains;
5, transformant solution is spread upon high salt and containing on the LB nutrient agar of Amp-Xgal-IPTG respectively, cultivate after 48～60 hours, selecting to grow on high salt nutrient agar is containing the transformant that bacterium colony is white in color on the Amp-Xgal-IPTG substratum again;
6, select recombinant plasmid in the salt tolerant transformant at least in extracting above-mentioned 10, cut out wherein insertion fragment, order-checking with E.coRI;
Each that 7, will predict inserts fragment sequence and the information in the biometric database and carries out gene and proteinic homology relatively, the preliminary character of judging the insertion gene;
8, detect the product of exogenous gene expression in the transformant and the new function that transformant obtains, judge the character of this gene;
9, each transformant and contrast are seeded in respectively contain in the liquid LB substratum of high salt that NaCl is 0.9～1.0mol/L, cultured continuously in the shaking table of 37 ℃ of constant temperature, 100～150rpm, this moment, contrast only can slowly be carried out cell fission, each salt tolerant transformant is faster growing then, sampling detect wherein contain the bacterium number, draw the growth curve of transformant and contrast respectively, relatively the difference of both growth conditions verifies once more that whereby this gene is a salt-resistant related gene.
In above-mentioned steps 3, can adopt gel to reclaim test kit and reclaim dna fragmentation in the gel that contains endonuclease bamhi; Total DNA after enzyme is cut is through 1% agarose gel electrophoresis of brominated second pyridine (regulation that the existing custom of making an appointment of its content becomes, promptly about 1 ‰).
In step 4, said dephosphorylation is handled and can be undertaken by the method for above-mentioned document 1.The dna fragmentation and the linear carrier that reclaim are educated altogether, and generally at least 1 times of the amount of carrier is to dna fragmentation; Conversion CaCl
2Handle the competence recipient bacterium that obtains and all use intestinal bacteria usually.
In step 5, be preferably in and respectively get 100 μ L transformant solution under the aseptic condition and evenly spread upon high salt (contained NaCl≤1.1mol/L) and containing on the LB nutrient agar of Amp-Xgal-IPTG with the aseptic glass rod of trilateral respectively, cultivate after 48～60 hours, selecting routinely to grow on high salt nutrient agar is containing the transformant that bacterium colony is white in color on the Amp-Xgal-IPTG substratum again.
In step 7, said biometric database is GenBank or other databases.
In step 9, said contrast is the intestinal bacteria of handling without genetics, is seeded in respectively to contain in the liquid LB substratum of high salt that NaCl is 0.9～1.0mol/L, and cultured continuously is 48～72 hours in the shaking table of 37 ℃ of constant temperature, 100～150rpm.Every sampling in 4～8 hours detect wherein contain the bacterium number.
Compare with existing conventional gene clone method, outstanding advantage of the present invention is:
1. simple and direct.Since do not need to set up loaded down with trivial details experimental arrangements such as genomic library, so step of the present invention is less, easier;
2. economical.Because experimental technique is easier, and need not the medicine of various costlinesses, therefore expense of the present invention is lower;
3. can clone unknown gene.Because the present invention is not a target to clone which gene, but design obtains salt-resistant related gene through screening, therefore will may obtain the unknown gene relevant with this biology salt tolerant;
4. can clone a plurality of genes simultaneously.Because the present invention has made up numerous recombinant plasmids that comprise range gene at random, through high salt conditional filtering, obtains a plurality of genes relevant with this biology salt tolerant with cloning simultaneously;
5. improve the gene that can be used for cloning other kind slightly.As long as change targetedly aspect the genescreen condition simultaneously in conjunction with follow-up gene identification, can be cloned the gene of other kind.
Embodiment
Following examples will be in conjunction with the various salt tolerant prokaryotic organism of clone, and the invention will be further described comprising the salt-resistant related gene of various salt tolerant bacteriums and blue-green algae.
Embodiment 1: its salt-resistant related gene of clone from the pseudomonas of extremely salt-tolerant the steps include:
1. pseudomonas is seeded in the substratum that contains NaCl 0.5mol/L, 25 ℃ of constant temperature are cultured to the exponential growth middle and later periods with the concussion of the speed of 120rpm, are about 22 hours;
2. conventional alkaline denaturation extracts its total DNA.Detect the OD of total dna solution respectively
260And OD
280Value is to confirm its purity, and both ratio is 1.82.The molecular weight that detects the DNA that extracts with 1% agarose gel electrophoresis is to be 21.5kb;
3. with restriction endonuclease E.coRI (or other produces the restriction endonuclease of sticky end) above-mentioned highly purified total DNA is cut at 37 ℃ of incomplete enzymes that carried out 80 minutes.Total DNA after enzyme is cut is through 1% agarose gel electrophoresis of brominated second pyridine, and finding molecular weight under ultraviolet lamp is the endonuclease bamhi zone of 2kb, cuts out with sharp blade to contain above-mentioned segmental gel, reclaims test kit recovery dna fragmentation wherein with gel;
With E.coRI at expression vector, as pUC18, multiple clone site its complete degestion is linear, dephosphorylation is handled the probability that himself connects to reduce.The dna fragmentation and the linear carrier that reclaim are educated altogether, add ligase enzyme and connect 20 hours, form recombinant plasmid, transform and use CaCl at 16 ℃
2Handle the competence recipient bacterium (intestinal bacteria) that obtains;
5. under aseptic condition, respectively getting 100 μ l transformants spreads upon high salt (containing NaCl 1.1mol/L) respectively and contains on the LB nutrient agar of Amp-Xgal-IPTG, cultivate after 48 hours, selecting to grow on high salt nutrient agar is containing the transformant that bacterium colony is white in color on the Amp-Xgal-IPTG substratum again;
6. in containing the LB substratum of NaCl 0.5mol/L, 25 ℃ of constant temperature are cultivated each salt tolerant transformant with the speed concussion of 120rpm.Extract 10 recombinant plasmids in the salt tolerant transformant respectively, cut out wherein insertion fragment, order-checking with former restriction endonuclease (E.coRI);
7. each that will predict inserts fragment sequence and the information in the biometric database (GenBank or other databases) and carries out gene and proteinic homology relatively, the preliminary character of judging the insertion gene;
8. according to above-mentioned prompting, further detect the product of exogenous gene expression in the transformant and the new function that transformant obtains, judge the character of this gene.
9. each transformant and contrast (without the intestinal bacteria of genetics processing) are seeded in respectively in the liquid LB substratum of high salt (containing NaCl 0.9mol/L), cultured continuously is 48 hours in the shaking table of 37 ℃ of constant temperature, 120rpm.This moment, contrast only can slowly be carried out cell fission, and each salt tolerant transformant is faster growing then.Every sampling in 6 hours detect wherein contain the bacterium number, draw the growth curve of transformant and contrast respectively, the difference of both growth conditions of comparison verifies once more that whereby this gene is a salt-resistant related gene.
Embodiment 2: similar to Example 1, its difference is pseudomonas is seeded in the substratum that contains NaCl 0.7mol/L, and 20 ℃ of constant temperature are cultured to the exponential growth middle and later periods with the concussion of the speed of 150rpm, are about 20 hours.With restriction endonuclease E.coRI (or other produces the restriction endonuclease of sticky end) above-mentioned highly purified total DNA is cut at 37 ℃ of incomplete enzymes that carried out 70 minutes.Total DNA after enzyme is cut is through 1% agarose gel electrophoresis of brominated second pyridine, finds molecular weight to be about the endonuclease bamhi zone of 3kb under ultraviolet lamp, cuts out with sharp blade to contain above-mentioned segmental gel, reclaims test kit with gel and reclaims wherein dna fragmentation.The dna fragmentation and the linear carrier that reclaim are educated altogether, add ligase enzyme and connect 18 hours, form recombinant plasmid, transform and use CaCl at 16 ℃
2Handle the competence recipient bacterium (intestinal bacteria) that obtains.In containing the LB substratum of NaCl0.7mol/L, 20 ℃ of constant temperature are cultivated each salt tolerant transformant with the speed concussion of 150rpm.Each transformant and contrast (without the intestinal bacteria of genetics processing) are seeded in respectively in the liquid LB substratum of high salt (containing NaCl 0.95mol/L), and cultured continuously is 48 hours in the shaking table of 37 ℃ of constant temperature, 130rpm.
Embodiment 3: similar to Example 1, its difference is pseudomonas is seeded in the substratum that contains NaCl 0.3mol/L, and 28 ℃ of constant temperature are cultured to the exponential growth middle and later periods with the concussion of the speed of 110rpm, are about 18 hours.With restriction endonuclease E.coRI (or other produces the restriction endonuclease of sticky end) above-mentioned highly purified total DNA is cut at 37 ℃ of incomplete enzymes that carried out 90 minutes.Total DNA after enzyme is cut is through 1% agarose gel electrophoresis of brominated second pyridine, finds molecular weight to be about the endonuclease bamhi zone of 1kb under ultraviolet lamp, cuts out with sharp blade to contain above-mentioned segmental gel, with coagulating
Glue reclaims test kit recovery dna fragmentation wherein.The dna fragmentation and the linear carrier that reclaim are educated altogether, add ligase enzyme and connect 15 hours, form recombinant plasmid, transform and use CaCl at 16 ℃
2Handle the competence recipient bacterium (intestinal bacteria) that obtains.In containing the LB substratum of NaCl 0.3mol/L, 28 ℃ of constant temperature are cultivated each salt tolerant transformant with the speed concussion of 110rpm.Each transformant and contrast (without the intestinal bacteria of genetics processing) are seeded in respectively in the liquid LB substratum of high salt (containing NaCl1.0mol/L), and cultured continuously is 48 hours in the shaking table of 37 ℃ of constant temperature, 110rpm.
Embodiment 4: similar to Example 1, its difference is pseudomonas is seeded in the substratum that contains NaCl 0.1mol/L, and 32 ℃ of constant temperature are cultured to the exponential growth middle and later periods with the concussion of the speed of 100rpm, are about 16 hours.With restriction endonuclease E.coRI (or other produces the restriction endonuclease of sticky end) above-mentioned highly purified total DNA is cut at 37 ℃ of incomplete enzymes that carried out 60 minutes.Total DNA after enzyme is cut is through 1% agarose gel electrophoresis of brominated second pyridine, finds molecular weight to be about the endonuclease bamhi zone of 2kb under ultraviolet lamp, cuts out with sharp blade to contain above-mentioned segmental gel, reclaims test kit with gel and reclaims wherein dna fragmentation.The dna fragmentation and the linear carrier that reclaim are educated altogether, add ligase enzyme and connect 10 hours, form recombinant plasmid, transform and use CaCl at 16 ℃
2Handle the competence recipient bacterium (intestinal bacteria) that obtains.In containing the LB substratum of NaCl 0.1mol/L, 32 ℃ of constant temperature are cultivated each salt tolerant transformant with the speed concussion of 100rpm.Each transformant and contrast (without the intestinal bacteria of genetics processing) are seeded in respectively in the liquid LB substratum of high salt (containing NaCl 0.95mol/L), and cultured continuously is 48 hours in the shaking table of 37 ℃ of constant temperature, 100rpm.
Embodiment 5: similar to Example 1, its difference is pseudomonas is seeded in the substratum that contains NaCl 0.9mol/L, and 15 ℃ of constant temperature are cultured to the exponential growth middle and later periods with the concussion of the speed of 150rpm, are about 24 hours.With restriction endonuclease E.coRI (or other produces the restriction endonuclease of sticky end) above-mentioned highly purified total DNA is cut at 37 ℃ of incomplete enzymes that carried out 100 minutes.Total DNA after enzyme is cut is through 1% agarose gel electrophoresis of brominated second pyridine, finds molecular weight to be about the endonuclease bamhi zone of 3kb under ultraviolet lamp, cuts out with sharp blade to contain above-mentioned segmental gel, reclaims test kit with gel and reclaims wherein dna fragmentation.The dna fragmentation and the linear carrier that reclaim are educated altogether, add ligase enzyme and connect 24 hours, form recombinant plasmid, transform and use CaCl at 16 ℃
2Handle the competence recipient bacterium (intestinal bacteria) that obtains.In containing the LB substratum of NaCl 0.9mol/L, 15 ℃ of constant temperature are cultivated each salt tolerant transformant with the speed concussion of 150rpm.Each transformant and contrast (without the intestinal bacteria of genetics processing) are seeded in respectively in the liquid LB substratum of high salt (containing NaCl 0.9mol/L), and cultured continuously is 48 hours in the shaking table of 37 ℃ of constant temperature, 150rpm.
Embodiment 6: adopt on the salt tolerant bacterium that contains the ambient growth more than the NaCl 0.1mol/L, its step is similar to Example 1, and the time that is cultured to the exponential growth middle and later periods is 12～24 hours.
Embodiment 7: adopt the salt tolerant blue-green algae to be seeded in the substratum that contains NaCl 0.1～0.9mol/L, temperature is 15～32 ℃, speed concussion with 100～150rpm under the illumination of 2000～4000lux is cultured to the exponential growth middle and later periods, and all the other steps are similar to Example 1.
Claims (6)
1, clone procaryotic biological salt-resistant related gene method, described prokaryotic organism are bacterium and blue-green algae, it is characterized in that the steps include:
1) extremely salt-tolerant bacterium and blue-green algae are seeded in the substratum that contains NaCl 0.1～0.9mol/L, temperature is 15～32 ℃, shakes with the speed of 100～150rpm to be cultured to the exponential growth middle and later periods; If the salt tolerant blue-green algae should be cultivated under illumination;
2) conventional alkaline denaturation extracts total DNA of extremely salt-tolerant bacterium or blue-green algae, detects purity, its OD through UV spectrum
260/ OD
280Ratio answers 〉=1.8.The molecular weight that detects the DNA that extracts with 1% agarose gel electrophoresis whether 〉=21kb, as if OD
260/ OD
280Ratio 〉=1.8 illustrate that gained DNA is purer, can enter next step; If OD
260/ OD
280Ratio＜1.8 illustrate that gained DNA purity is not enough, should be further purified, until OD
260/ OD
280Ratio 〉=1.8;
3) under aseptic condition, above-mentioned total DNA is cut at 37 ℃ of incomplete enzymes that carried out 60～100 minutes with restriction endonuclease (E.co RI) or other restriction endonuclease that produces sticky end, total DNA after enzyme is cut is through 1% agarose gel electrophoresis of brominated second pyridine, finding molecular weight under ultraviolet lamp is the endonuclease bamhi zone of 1～3kb, cut out the gel that contains above-mentioned this endonuclease bamhi, reclaim dna fragmentation wherein;
4) under aseptic condition with E.co RI at artificial constructed expression vector, as the multiple clone site of pUC18 that its complete degestion is linear, dephosphorylation is handled, the dna fragmentation and the linear carrier that reclaim are educated altogether, add ligase enzyme and connect 10～24 hours, form recombinant plasmid, transform and use CaCl at 16 ℃
2Handle the competence recipient bacterium that obtains;
5) transformant solution is spread upon high salt respectively and contain on the LB nutrient agar of Amp-Xgal-IPTG, cultivate after 48～60 hours, selecting to grow on high salt nutrient agar is containing the transformant that bacterium colony is white in color on the Amp-Xgal-IPTG substratum again;
6) select recombinant plasmid in the salt tolerant transformant at least in extracting above-mentioned 10, cut out wherein insertion fragment, order-checking with E.coRI;
Each that 7) will predict inserts fragment sequence and the information in the biometric database and carries out gene and proteinic homology relatively, the preliminary character of judging the insertion gene;
8) detect the product of exogenous gene expression in the transformant and the new function that transformant obtains, judge the character of this gene;
9) each transformant and contrast are seeded in respectively contain in the liquid LB substratum of high salt that NaCl is 0.9～1.0mol/L, cultured continuously in the shaking table of 37 ℃ of constant temperature, 100～150rpm, this moment, contrast only can slowly be carried out cell fission, each salt tolerant transformant is faster growing then, sampling detect wherein contain the bacterium number, draw the growth curve of transformant and contrast respectively, relatively the difference of both growth conditions verifies once more that whereby this gene is a salt-resistant related gene.
2, clone procaryotic biological salt-resistant related gene method as claimed in claim 1, the dna fragmentation in the gel that it is characterized in that in step 3), adopting the recovery of gel recovery test kit to contain endonuclease bamhi; Total DNA after enzyme is cut is through 1% agarose gel electrophoresis of brominated second pyridine, and the content of bromine second pyridine is 1 ‰.
3, clone procaryotic biological salt-resistant related gene method as claimed in claim 1 is characterized in that in step 4), and the dna fragmentation and the linear carrier that reclaim are educated altogether, and at least 1 times of the amount of carrier is to dna fragmentation; Conversion CaCl
2Handle the competence recipient bacterium that obtains and all use intestinal bacteria usually.
4, clone procaryotic biological salt-resistant related gene method as claimed in claim 1, it is characterized in that in step 5), respectively getting 100 μ L transformant solution under aseptic condition evenly spreads upon high salt and contains on the LB nutrient agar of Amp-Xgal-IPTG with the aseptic glass rod of trilateral respectively, cultivate after 48～60 hours, selecting routinely to grow on high salt nutrient agar is containing the transformant that bacterium colony is white in color on the Amp-Xgal-IPTG substratum again.
5, clone procaryotic biological salt-resistant related gene method as claimed in claim 1 is characterized in that in step 7), and said biometric database is GenBank or other databases.
6, clone procaryotic biological salt-resistant related gene method as claimed in claim 1, it is characterized in that in step 9), said contrast is the intestinal bacteria of handling without genetics, be seeded in respectively and contain in the liquid LB substratum of high salt that NaCl is 0.9～1.0mol/L, cultured continuously is 48～72 hours in the shaking table of 37 ℃ of constant temperature, 100～150rpm, every sampling in 4～8 hours detect wherein contain the bacterium number.
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| KR101326019B1 (en) * | 2010-10-27 | 2013-11-07 | 한국생명공학연구원 | Salt-resistant SyDBSP gene from Synechocystis and uses thereof |
| CN103205457B (en) * | 2013-03-18 | 2014-12-10 | 中国林业科学研究院林业研究所 | Method for using microbial stress tolerance genes to increase plant salt tolerance |
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