CN101104870A - Method for identifying spirulina strain production characteristics - Google Patents
Method for identifying spirulina strain production characteristics Download PDFInfo
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- CN101104870A CN101104870A CNA2007100699622A CN200710069962A CN101104870A CN 101104870 A CN101104870 A CN 101104870A CN A2007100699622 A CNA2007100699622 A CN A2007100699622A CN 200710069962 A CN200710069962 A CN 200710069962A CN 101104870 A CN101104870 A CN 101104870A
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- 235000016425 Arthrospira platensis Nutrition 0.000 title claims abstract description 33
- 240000002900 Arthrospira platensis Species 0.000 title claims abstract description 33
- 229940082787 spirulina Drugs 0.000 title claims abstract description 33
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 26
- 238000000034 method Methods 0.000 title claims abstract description 9
- 238000001962 electrophoresis Methods 0.000 claims abstract description 11
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- 241000195493 Cryptophyta Species 0.000 claims description 7
- 239000000047 product Substances 0.000 claims description 7
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 6
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 6
- 239000006228 supernatant Substances 0.000 claims description 6
- 108010067770 Endopeptidase K Proteins 0.000 claims description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 4
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 claims description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 4
- 239000003153 chemical reaction reagent Substances 0.000 claims description 4
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- 229920000936 Agarose Polymers 0.000 claims description 2
- 238000007400 DNA extraction Methods 0.000 claims description 2
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- 102000006382 Ribonucleases Human genes 0.000 claims description 2
- 108010083644 Ribonucleases Proteins 0.000 claims description 2
- 229920002684 Sepharose Polymers 0.000 claims description 2
- 150000001298 alcohols Chemical class 0.000 claims description 2
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Abstract
Disclosed is an identification method for spirulina strain production characters. Through extracting total DNA of 5 strains of spirulina sp-1, sp-2, sp-3, sp-10, sp-NC and electrophoresis analysis, the 5 strains can be classified into two main categories according to the chromosome external DNA numbers(exDNA): wherein sp-1, sp-2, sp-10 and sp-NC fall into one category with only one exDNA; sp-3 falls into the other category with two exDNA . When two exDNAs are contained in total DNA of candidate strain, the production character is much better and is good for large scale production; when only one exDNA is contained in total DNA, the production character is probably not good and can not be used for plant production.
Description
Technical field
The invention belongs to the technology of spirulina Application and Development
Background technology
Spirulina (Spirulina), be a kind of photosynthetic oxygen evolution, be the thread little algae of protokaryon of regular helix, be a genus [Wuhan phytology research of Cyanophyta (Cyanophyta), Oscillatoriales (Oscillatoriales), Oscillariaceae (Oscillatoriaceae), 1997,15 (4): 369-374].Receive very big concern both domestic and external because of being rich in quality protein and multiple biologically active substance, and formed huge spirulina industry on the basis of big quantity research, it is largest to become the research and development of the present whole world, the most economic little algae of application prospect.It is worthy of note, numerous experimental studies and long-term production practice show, spirulina different varieties (being), to temperature, light quality, intensity of illumination, and there are significant difference in the requirement and the adaptability of environmental factors such as the salinity of nutrient solution, pH and nutritive ingredient, consequent economic benefit also differ greatly [hydrobiont journal, 1999,23 (1): 59-64].So, the same with other agricultural with biotechnology industry, seed selection moral character hold concurrently excellent kind (being) be effectively the current spirulina industrial economy benefit of lifting, solve the production practical problems conscientiously, the most direct and one of important approach.
The general ordinary method that screening both at home and abroad at present is fit to scale operation spirulina kind (being) is as follows: 1, the kind (being) that newly is separated to is numbered; 2, under laboratory condition, make the combined crosswise culture experiment of various environmental factors, and make preliminary screening according to indexs such as the growth curve of being measured, photosynthetic oxygen evolution, biochemical compositions; 3, the laboratory just is sieved to candidate's kind (being) of producing the breed potentiality and under Various Seasonal, weather and nutritional condition, cultures lab scale, pilot scale and industrial experimentation, rescreen and select target variety (being).Though this method is practical, effective, program is loaded down with trivial details, workload is big, the cycle is long, cost is high.Therefore, be necessary to set up simply, estimate fast and effectively and screening method, to satisfy the constantly actual needs of development of current domestic and international spirulina industry.
Summary of the invention
The object of the invention provides a kind of novel method of screening the high-quality spirulina strain that is fit to scale operation simply, fast and effectively.
We have extracted total DNA of 5 strain spirulina Sp-1, Sp-2, Sp-3, Sp-10 and Sp-NC, find by electrophoretic analysis, quantity 5 strain strains according to exchromosomal DNA (exDNA) among total DNA can be divided into two big classes: wherein Sp-1, Sp-2, Sp-10 and Sp-NC are a class, only contain an exDNA; Sp-3 is another kind of, contains 2 exDNA.Secular experimental study and production practice show, the Sp-3 product tie up to actual production culture in performance good, and Sp-1, Sp-2, Sp-10 and Sp-NC can not produce as batch production because of the adaptive faculty difference and culture.This shows, there are dependency in the exDNA and the spirulina production traits among the total DNA of spirulina, can be used as the molecule marker of high-quality spirulina strain screening, when even containing 2 exDNA among the total DNA of candidate's strain, may be production traits strain preferably, be fit to scale operation; If when only containing 1 exDNA among total DNA, may be the strain of production traits difference, be not useable for batch production production.
Remarkable advantage of the present invention:
The screening that the present invention set up is fit to the method for the high-quality spirulina strain of scale operation to be compared with ordinary method, not only simple, quick, effective, and cost is low, and is fit to extensive, high flux screening.
Description of drawings
Fig. 1 is total DNA electrophorogram of 4 strain spirulina strains;
M: standard molecular weight; 1～5: the corresponding Sp-3 of difference, Sp-NC, Sp-1, Sp-2, Sp-10
Fig. 2 is total DNA electrophorogram of being differentiated algae strain Sp-5 and Sp-9.
M: standard molecular weight; 6 and 7: corresponding Sp-5 of difference and Sp-9
Embodiment
Detailed technology scheme of the present invention can be implemented by following steps:
1, select materials is known 5 strain spirulina strains, i.e. Sp-1, Sp-2, Sp-3, Sp-10 and Sp-NC (also there is preservation in Zhejiang University's nucleus research of agricultural science institute's Biological resources and molecular engineering laboratory).Wherein, Sp-3 is strong to adaptive capacity to environment, be widely used in scale operation is cultured, and Sp-1, Sp-2, Sp-10 and Sp-NC can not be used as batch production production because of the adaptive faculty difference.
2, reagent and instrument
Agarose is that worker bio-engineering corporation product is given birth in Shanghai; Proteinase K is a Merck company product; RNaseA is Japanese TaKaRa company product.Other reagent is analytical pure.
3, artrospira spirulina total DNA extraction
(1) gets the algae mud of the fresh algae mud of about 200mg or-20 ℃ of freezing preservations, change 5ml in advance in the centrifuge tube of sterilization, earlier with liquid nitrogen to sample freeze thawing treatment 2～3 times after, add 2ml and be preheated to 65 ℃ CTAB extracting solution [1.5%CTAB, 50mmol/L Tris-HCl (pH 8.0), 10mmol/L EDTA, 0.7mol/L NaCl, 1% (V/V) beta-mercaptoethanol (adding before using)], and add an amount of Proteinase K solution to final concentration 200 μ g/ml; Again sample is put upside down and is mixed, in 62 ℃ of water-bath 1h, during every about 10min, sample is shaken up once gently.
(2) under the room temperature 10,000rpm is centrifugal, and 5min removes impurity such as cell debris, and supernatant liquor adds equal-volume chloroform/primary isoamyl alcohol (24: 1), puts upside down behind the mixing in 12 the centrifugal 10min of 000rpm gently repeatedly.
(3) get supernatant liquor in another sterilization centrifuge tube, add-20 ℃ of precooling dehydrated alcohols of 3 times of volumes and the 3mol/L NaAc (pH 4.8) of 1/10 volume, put upside down several times the back gently and place 1h down in-20 ℃, DNA is fully precipitated.
The centrifugal 10min of (4) 12,000rpm.Precipitate the washing with alcohol with 70%, vacuum is drained, and is dissolved in 500 μ l TE-Proteinase K damping fluid (10mmol/L Tris-HCl, 1mmol/L EDTA adds Proteinase K to final concentration 100 μ g/ml, pH8.0), also shake gently frequently in 50 ℃ of water-bath 1h, clarify until solution.
(5) add isopyknic phenol/chloroform/primary isoamyl alcohol (25: 24: 1), put upside down mixing gently, the centrifugal 10min of 12,000 rpm.
(6) get supernatant liquor, add 3 mol/LNaAc of 1/10 volume and the dehydrated alcohol of 3 times of volume-20 ℃ precoolings, place 1h in-20 ℃.
(7) 12,4 ℃ of centrifugal 10min of 000rpm, precipitation is with 70% washing with alcohol 2 times, and vacuum is drained, and adds 100 μ lTE-RNase damping fluid (10mmol/L Tris-HCl, 1mmol/L EDTA, 50 μ g/ml RNase, pH 8.0), in 37 ℃ of water-baths, be incubated 30min, make the DNA dissolving, put-20 ℃ then and preserve standby down.
4, electrophoretic analysis and observation
With amplified production electrophoresis on 1% sepharose, voltage 4 V/cm.The intact back of electrophoresis is with EB about 30 min that dye, again at VersaDoc
TMImaging System 3000 gel imaging systems (Bio-Rad, USA) observation and photograph.Through observing,, then may be fit to scale operation for production traits strain preferably if screened spirulina strain contains 2 exDNA.
Result and analysis
The electrophoretic analysis of exDNA band
Extracted total DNA of 5 strain spirulina Sp-1, Sp-2, Sp-3, Sp-10 and Sp-NC, find by electrophoretic analysis, number difference according to exchromosomal DNA (exDNA) among total DNA, 5 strain strains can be divided into two big classes: wherein Sp-1, Sp-2, Sp-10 and Sp-NC are a class, only contain an exDNA; Sp-3 is another kind of, contains 2 exDNA.
Secular experimental study and production practice show, the Sp-3 product tie up to actual production culture in performance good, and Sp-1, Sp-2, Sp-10 and Sp-NC can not produce as batch production because of the adaptive faculty difference and culture.This shows, there are dependency in the band number of exDNA and spirulina production traits quality among the total DNA of spirulina, can be used as the molecule marker of high-quality spirulina strain screening, when even containing 2 exDNA among the total DNA of candidate's strain, may be production traits strain preferably, be fit to scale operation; If when only containing 1 exDNA among total DNA, may be the strain of production traits difference, be not useable for batch production production.
As an example, we have chosen Sp-5 and this 2 strain strain of Sp-9 is verified practicality of the present invention, and wherein, the Sp-5 strain is the breeding that is applicable to that scale operation is cultured, and Sp-9 can not be used for scale operation because of bad adaptability.By extracting total DNA of Sp-5 and Sp-9, electrophoresis detection finds that Sp-5 contains 2 exDNA, and Sp-9 only contains 1 exDNA.The above results further specifies, and the band number of exDNA can be used as a kind of molecule marker and screens the high-quality spirulina strain that is fit to scale operation among the total DNA of spirulina.
Claims (2)
1. the discrimination method of good and bad production of spirurina strain is characterized in that utilizing the band number of exDNA among the total DNA of spirulina, screens high-quality spirulina in candidate's strain; Its proterties is that high-quality can be used when containing 2 exDNA among the total DNA of candidate's strain; Then its proterties is that poor quality can not be used when containing 1 exDNA among the DNA.
2. by the method for the described selection spirulina of claim 1 strain, it is characterized in that adopting following operation steps:
(1) select materials is known 5 strain spirulina strain, i.e. Sp-1, Sp-2, Sp-3, Sp-10 and Sp-NC;
(2) reagent and instrument
Agarose is that worker bio-engineering corporation product is given birth in Shanghai; RNaseA is Japanese TaKaRa company product, and other reagent is analytical pure;
(3) artrospira spirulina total DNA extraction
1. get the algae mud of the fresh algae mud of about 200mg or-20 ℃ of freezing preservations, change 5ml in advance in the centrifuge tube of sterilization, earlier with liquid nitrogen to sample freeze thawing treatment 2～3 times after, add 2ml and be preheated to 65 ℃ CTAB extracting solution: 1.5%CTAB, 50mmol/L Tris-HCl, pH8.0,10mmol/L EDTA, 0.7mol/L NaCl adds before 1% (V/V) beta-mercaptoethanol uses, and adds an amount of Proteinase K solution to final concentration 200 μ g/ml; Again sample is put upside down and is mixed, in 62 ℃ of water-bath 1h, during every about 10min, sample is shaken up once gently;
2. under the room temperature 10,000rpm is centrifugal, and 5min removes impurity such as cell debris, and it is 24: 1 that supernatant liquor adds equal-volume chloroform/primary isoamyl alcohol, puts upside down behind the mixing in 12 the centrifugal 10min of 000rpm gently repeatedly;
3. get supernatant liquor in another sterilization centrifuge tube, add-20 ℃ of precooling dehydrated alcohols of 3 times of volumes and the 3mol/LNaAc (pH4.8) of 1/10 volume, put upside down several times the back gently and place 1h down in-20 ℃, DNA is fully precipitated;
4. 12, the centrifugal 10min of 000rpm.Precipitate the washing with alcohol with 70%, vacuum is drained, and is dissolved in 500 μ l TE-Proteinase K damping fluids: 10mmol/L Tris-HCl, 1mmol/L EDTA adds Proteinase K to final concentration 100 μ g/ml, pH8.0, also shake gently frequently in 50 ℃ of water-bath 1h, clarify until solution;
5. adding isopyknic phenol/chloroform/primary isoamyl alcohol is 25: 24: 1, puts upside down mixing gently, 12, and the centrifugal 10min of 000rpm;
6. get supernatant liquor, add the 3mol/L NaAc of 1/10 volume and the dehydrated alcohol of 3 times of volume-20 ℃ precoolings, place 1h in-20 ℃;
7. 12,4 ℃ of centrifugal 10min of 000rpm, precipitation is with 70% washing with alcohol 2 times, and vacuum is drained, and adds 100 μ 1TE-RNase damping fluids: 10mmol/L Tris-HCl, 1mmol/L EDTA, 50 μ g/ml RNase, pH8.0 is incubated 30min in 37 ℃ of water-baths, make the DNA dissolving, put-20 ℃ then and preserve standby down;
(4) electrophoretic analysis and observation
With amplified production electrophoresis on 1% sepharose, voltage 4V/cm.The intact back of electrophoresis is with the about 30min of EB dyeing, again at VersaDoc
TMImaging System 3000 gel imaging system Bio-Rad, USA observe also and take a picture, and through observing, if screened spirulina strain is identical with SP-3, contain 2 exDNA, and then its production traits is strain preferably, suitable scale operation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007100699622A CN101104870A (en) | 2007-07-09 | 2007-07-09 | Method for identifying spirulina strain production characteristics |
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| Application Number | Priority Date | Filing Date | Title |
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| CNA2007100699622A CN101104870A (en) | 2007-07-09 | 2007-07-09 | Method for identifying spirulina strain production characteristics |
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| CN101104870A true CN101104870A (en) | 2008-01-16 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102807978A (en) * | 2012-04-17 | 2012-12-05 | 浙江省海洋开发研究院 | Alga ribonucleic acid (RNA) extractant and using method |
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- 2007-07-09 CN CNA2007100699622A patent/CN101104870A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102807978A (en) * | 2012-04-17 | 2012-12-05 | 浙江省海洋开发研究院 | Alga ribonucleic acid (RNA) extractant and using method |
| CN102807978B (en) * | 2012-04-17 | 2014-02-26 | 浙江省海洋开发研究院 | Alga ribonucleic acid (RNA) extractant and using method |
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