CN103149211B - Distinguish the method for good and bad production of spirurina strain degree - Google Patents
Distinguish the method for good and bad production of spirurina strain degree Download PDFInfo
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- CN103149211B CN103149211B CN201310066963.7A CN201310066963A CN103149211B CN 103149211 B CN103149211 B CN 103149211B CN 201310066963 A CN201310066963 A CN 201310066963A CN 103149211 B CN103149211 B CN 103149211B
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Abstract
The present invention relates to spirulina Application and Development technology, aim to provide a kind of method distinguishing good and bad production of spirurina strain degree.The method extracts with Tris-HCl extract the water-solubility protein obtaining spirulina cells, after sodium dodecyl sulfate-polyacrylamide gel electrophoresis is separated protein sample, detect the optical density value of electrophoretic protein pattern 102kD place protein band, and build the dendrogram between algae strain according to its value; If the optical density value that candidate ties up to 102kD place protein band is greater than 140, and together with the strain good with the known production traits gather, then illustrates that this strain production traits is good, be applicable to cultivate production on a large scale; If be less than 40 in the optical density value of 102kD place protein band, and together with the strain bad with the known production traits gather, then illustrate that this strain production traits is poor, be not suitable for and cultivate production on a large scale.The present invention is simple, efficient, reliable, cost is low, need not carry out nucleic acid sequencing with bioinformatics than reciprocity sophisticated testing and analysis, thus be applicable to extensive, high flux screening.<!--1-->
Description
Technical field
The invention belongs to the technology of spirulina Application and Development, particularly a kind of method distinguishing good and bad production of spirurina strain degree.
Background technology
Spirulina (Spirulina), a kind of photosynthetic oxygen evolution, the thread micro-algae of protokaryon in regular helix, be a genus [Wuhan botany research of Cyanophyta (Cyanophyta), Oscillatoriales (Oscillatoriales), Oscillariaceae (Oscillatoriaceae), 1997,15 (4): 369-374].Receive very big concern both domestic and external because being rich in high-quality protein and various bioactivators, and on the basis of large quantity research, defined huge spirulina industry, become largest, the application prospect the most economic micro-algae of whole world research and development at present.It is worthy of note, numerous experimental studies and long-term production practices show: spirulina different cultivars (being), significant difference is existed to the salinity of temperature, light quality, intensity of illumination and nutrient solution, the requirement of the envirment factor such as pH and nutritional labeling and adaptability, consequent economic benefit also differs greatly [hydrobiont journal, 1999,23 (1): 59-64].So the same with biotechnology industry with other agricultural, the kind (being) that seed selection moral character is held concurrently excellent effectively promotes one of current spirulina industrial economy benefit, the most direct and important approach conscientiously solving production practical problems.
The conventional method that screening both at home and abroad is at present applicable to large-scale production spirulina kind (being) is as follows: 1, be numbered the kind be newly separated to (being); 2, do the combined crosswise culture experiment of various environmental factors in laboratory conditions, and carry out preliminary screening according to indexs such as measured growth curve, photosynthetic oxygen evolution and biochemical compositions; 3, under Various Seasonal, weather and nutritional condition, carry out cultivation lab scale, pilot scale and industrial experimentation to being sieved to the candidate variety (being) producing cultivation potentiality at the beginning of laboratory, then filter out target variety (being).Although this method is practical, effectively, program is loaded down with trivial details, workload is large, the cycle is long, and cost is high.Therefore, in the urgent need to setting up a kind of evaluation and screening technique of simple, efficient, low cost, to meet the actual needs of Present Domestic external spiral algae industry development.
Summary of the invention
The technical problem to be solved in the present invention is, overcomes deficiency of the prior art, provides a kind of method of differentiation good and bad production of spirurina strain degree of simple, efficient, low cost.
For technical solution problem, solution of the present invention is:
A kind of method distinguishing good and bad production of spirurina strain degree is provided, comprises the following steps:
The water-solubility protein obtaining spirulina cells is extracted with Tris-HCl extract, after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is separated protein sample, utilize QuantityOne analysis software to detect the optical density value of electrophoretic protein pattern 102kD place protein band, and build the dendrogram between algae strain according to its value; If the electrophoretic protein pattern of candidate's strain is greater than 140 in the optical density value of 102kD place protein band, and together with the strain good with the known production traits gather, then illustrates that this strain production traits is good, be applicable to cultivate production on a large scale; If be less than 40 in the optical density value of 102kD place protein band, and together with the strain bad with the known production traits gather, then illustrate that this strain production traits is poor, be not suitable for and cultivate production on a large scale.
The present invention specifically comprises the steps:
1, select 9 strain spirulina strains as sample, comprise the strain that the 4 strain production traits are good: Sp-4, Sp-5, Sp-15 and Sp-17; The strain that the 5 strain production traits are bad: Sp-1, Sp-2, Sp-6, Sp-9 and Sp-10;
2, reagent and instrument
Protein molecular weight standard is purchased from the GEHealthcareBiosciences(U.S.); Acrylamide, methene acrylamide, Tris, glycocoll, sodium dodecylsulphonate (SDS), ammonium persulfate (APS) and tetramethylethylenediamine (TEMED) etc. are purchased from the Amresco(U.S.).Vertical electrophoresis system used and ultraviolet-visible scanning spectrophotometer etc. are the AmershamPharmciaBiotech(U.S.) product; Freeze drier is the VirTis(U.S.) product; Scanner GS-800 and QuantityOne analysis software etc. are the Bio-Rad(U.S.) product;
3, the preparation of water-solubility protein
Get 0.75g blunt top spirulina algae mud in 5ml centrifuge tube, add 3mlTris-HCl extract (125mMTris-HCI, 0.9%NaCl, pH6.8), after-20 DEG C of freezing 1.5h, melt 2h again at 4 DEG C, until there is deep blue purple color fluorescence in multigelation like this 3 times.At 4 DEG C, the centrifugal 20min of 12000r/min obtains supernatant and is water-soluble spirulina albumen; In above-mentioned carried supernatant, add the acetone soln of 3 times of volume precoolings ,-20 DEG C of standing 2h after mixing, at 4 DEG C, the centrifugal 20min of 12000r/min, abandons supernatant; Precipitation, after vacuum freeze drying, adds appropriate SDS sample-loading buffer and makes it dissolve, namely obtain the protein sample for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
4, SDS-PAGE and software analysis
Determination of protein concentration carries out with reference to the Coomassie brilliant G-250 method of Bradford [AnalBiochem, 1976,72:248-254]; The method that protein s DS-PAGE analyzes with reference to Laemmli [Nature, 1970,227:680-685] is carried out, and resolving gel concentration is 12.5%, and concentrated gum concentration is 5%.First with after 80V electrophoresis 45min, then make bottom bromophenol blue to glue with 120V electrophoresis.Dye with Coomassie brilliant G-250, gel imaging after decolouring, utilize QuantityOne analysis software to carry out detection to electrophoretic protein pattern in the optical density value of 102kD place protein band to analyze, and utilize the dendrogram between the strain of SPSS13.0 statistical analysis software structure algae according to its value, build the dendrogram between algae strain, differentiated the quality of this strain production traits by cluster situation.
The invention has the beneficial effects as follows:
The method of the differentiation spirulina production traits excellent strain that the present invention sets up is compared with conventional method, not only simply, efficiently, reliably, cost is low, and nucleic acid sequencing need not be carried out with bioinformatics than reciprocity sophisticated testing and analysis, be thus applicable to extensive, high flux screening.
Accompanying drawing explanation
Fig. 1 is the SDS-PAGE gel figure of 9 strain spirulina strain water-solubility proteins; Wherein M: standard molecular weight; 1 ~ 9: be corresponding in turn to Sp-1, Sp-2, Sp-6, Sp-10, Sp-9, Sp-4, Sp-5, Sp-17 and Sp-15.
Fig. 2 is that 9 strains are for building the dendrogram of the spirulina strain of production traits judging standard.
Fig. 3 is differentiated the SDS-PAGE gel figure of algae strain Sp-3, Sp-16, Sp-18 and Sp-37; Wherein M: standard molecular weight; 1 ~ 4: be corresponding in turn to 1:Sp-18; 2:Sp-3; 3:Sp-16; 4:Sp-37.
Fig. 4 for differentiated algae strain Sp-3, Sp-16, Sp-18 and Sp-37 the classification of building in judging standard scheme.
Embodiment
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) technology of protein has been widely used in the fields such as the biological classification such as tobacco, paddy rice, xylophyta and marine alga and qualification, but about SDS-PAGE finger-print and the application of correlation analysis method in classification and qualification etc. of hydrolysis of protein spirulina, but rarely have report.The present invention establishes a kind of to detect the size of protein fingerprint spectrum in 102kD place band optical density value, and differentiate the excellent of the spirulina production traits by cluster and can be applied to large-scale production cultivation new method.
We with the known spirulina Sp-1 of 9 strain, Sp-2, Sp-4, Sp-5, Sp-6, Sp-9, Sp-10, Sp-15 and Sp-17 be for control material, through protein SDS-PAGE and utilize its electrophoretic protein pattern of QuantityOne software detection in the size of 102kD place protein band optical density value, and carry out cluster analysis according to its size, found that, 9 strain strains can be divided into two large classes: Sp-4, Sp-5, Sp-15 and Sp-17 are I class; Sp-1, Sp-2, Sp-6, Sp-9 and Sp-10 are II class.Long-term scientific research and production practices show, 4 strain product in above-mentioned I class tie up to and show excellent in actual production cultivation, and 5 strain strains in II class should not be used as factorial praluction cultivation because adaptive faculty difference.As can be seen here, electrophoretic protein pattern can be used for differentiating the excellent of the spirulina production traits in the size of 102kD place protein band optical density value.Even the electrophoretic protein pattern of candidate's strain is greater than 140 in the optical density value of 102kD place protein band, and together with the strain good with the known production traits gather, then illustrates that this strain production traits is good, be applicable to cultivate production on a large scale; If be less than 40 in the optical density value of 102kD place protein band, and together with the strain bad with the known production traits gather, then illustrate that this strain production traits is poor, be not suitable for and cultivate production on a large scale.Detailed technology scheme of the present invention can be implemented by following steps:
1, the selection of material is known 9 strain spirulina strain Sp-1, Sp-2, Sp-4, Sp-5, Sp-6, Sp-9, Sp-10, Sp-15 and Sp-17, also has preservation in Zhejiang University's atomic nucleus Institute of agricultural sciences living resources and molecular engineering laboratory.Wherein, Sp-4, Sp-5, Sp-15 and Sp-17 are good because of the production traits, are widely used in large-scale farming and produce; And Sp-1, Sp-2, Sp-6, Sp-9 and Sp-10 are not suitable for factorial praluction because of production traits difference.
2, reagent and instrument
Protein molecular weight standard is purchased from the GEHealthcareBiosciences(U.S.); Acrylamide, methene acrylamide, Tris, glycocoll, sodium dodecylsulphonate (SDS), ammonium persulfate (APS) and tetramethylethylenediamine (TEMED) etc. are purchased from the Amresco(U.S.).Vertical electrophoresis system used and ultraviolet-visible scanning spectrophotometer etc. are the AmershamPharmciaBiotech(U.S.) product; Freeze drier is the VirTis(U.S.) product; Scanner GS-800 and QuantityOne analysis software etc. are the Bio-Rad(U.S.) product;
3, the preparation of water-solubility protein
Get 0.75g blunt top spirulina algae mud in 5ml centrifuge tube, and add Tris-HCl extract (125mMTris-HCI, 0.9%NaCl, pH6.8) 3ml, 2h is melted at 4 DEG C again after-20 DEG C of freezing 1.5h, multigelation like this 5 times to occurring deep blue purple color fluorescence, and under the microscope microscopy without intact cell.At 4 DEG C, the centrifugal 20min of 12000r/min obtains supernatant and is water-soluble spirulina albumen;
4, SDS-PAGE and software analysis
Determination of protein concentration carries out with reference to the Coomassie brilliant G-250 method of Bradford [AnalBiochem, 1976,72:248-254]; The method that protein s DS-PAGE analyzes with reference to Laemmli [Nature, 1970,227:680-685] is carried out, and resolving gel concentration is 12.5%, and concentrated gum concentration is 5%.First with after 80V electrophoresis 45min, then make bottom bromophenol blue to glue with 120V electrophoresis.Dye with Coomassie brilliant G-250, gel imaging after decolouring, utilize QuantityOne analysis software to carry out detection to electrophoretic protein pattern in the optical density value of 102kD place protein band to analyze, and utilize the dendrogram between the strain of SPSS13.0 statistical analysis software structure algae according to its value, the quality of this strain production traits is differentiated by cluster situation.
5, results and analysis
Fig. 1 is the SDS-PAGE gel figure of 9 strain spirulina strain water-solubility proteins; QuantityOne software is utilized to detect in 102kD place protein band optical density value electrophoretic protein pattern, and carry out cluster analysis according to its value, as shown in Figure 2,9 strain spirulina strains can be divided into two large classes: Sp-4, Sp-5, Sp-15 and Sp-17 are I class; Sp-1, Sp-2, Sp-6, Sp-9 and Sp-10 are II class.
Long-term scientific research and production practices show, 4 strain product in above-mentioned I class tie up to and show excellent in actual production cultivation, and 5 strain strains in II class should not be used as factorial praluction cultivation because adaptive faculty difference.As can be seen here, protein electrophoresis collection of illustrative plates can be used in the size of 102kD place protein band optical density value the quality differentiating the spirulina production traits.Even the protein electrophoresis collection of illustrative plates of candidate's strain is greater than 140 in the optical density value of 102kD place protein band, and together with the strain good with the known production traits gather, then illustrates that this strain production traits is good, be applicable to cultivate production on a large scale; If be less than 40 in the optical density value of 102kD place protein band, and together with the strain bad with the known production traits gather, then illustrate that this strain production traits is poor, be not suitable for and cultivate production on a large scale.
NanoLC-MS/MS is utilized to carry out Mass Spectrometric Identification and bioinformatic analysis to the protein band at 102kD place on the good and bad two groups of arthrospira strain SDS-PAGE of the production traits and compare discovery further, the strain that the production traits is good has species specific albumen a---malt oligosaccharide based mycose synthetase, and does not have in the strain of production traits difference.Malt oligosaccharide based mycose synthetase can catalyze and synthesize trehalose.And trehalose can form unique diaphragm under the extraneous severe environmental conditions such as high temperature, high and cold, hyperosmosis and dry dehydration on biological cell surface; protected protein matter molecule unchangeability inactivation effectively; the normal life process of the body that sustains life and biological characteristic; thus make biosome to external world environment have better adaptive faculty (Shao Yingang etc. the application of trehalose and genetic engineering research. Agriculture of Anhui science .2008,36 (3): 857-859).Therefore, the trehalose synthesized by distinctive malt oligosaccharide based mycose synthetase in the strain that the production traits is good, can resist the various poor environment such as high temperature and osmotic pressure cataclysm in arthrospira cultivation effectively, the right to use its show the better production traits; And the strain of production traits difference, for want of this synzyme and can not trehalose synthesis, cause it more responsive and show the worse production traits to changes such as environment.Visible, on arthrospira SDS-PAGE the protein band at 102kD place and the production traits of strain closely related.
As an example, we choose this 4 strain strain of known Sp-3, Sp-16, Sp-18 and Sp-37 to verify practicality of the present invention, wherein, the production traits of Sp-3 and Sp-16 strain is good, be widely used in large-scale farming to produce, and Sp-18 and Sp-37 is not suitable for factorial praluction because of production traits difference.The water-solubility protein of this 4 strain strain of Sp-3, Sp-16, Sp-18 and Sp-37 obtains electrophoretic protein pattern (Fig. 3) through SDS-PAGE, utilize QuantityOne software detection protein to be swim collection of illustrative plates in the optical density value of 102kD place protein band, then carry out cluster analysis according to its value.As shown in Figure 4, the protein fingerprint spectrum of Sp-3 and Sp-16 is all greater than 140 in the optical density value of 102kD place protein band to result, and together with the strain good with the known production traits gather, can illustrate that this strain production traits is good, be applicable to cultivate production on a large scale; And the electrophoretic protein pattern of Sp-18 and Sp-37 is all less than 40 in the optical density value of 102kD place protein band, and together with can gathering with the strain of known production traits difference, illustrates that this strain production traits is poor, be not suitable for and cultivate production on a large scale.This example further illustrates, and differentiates the excellent of this strain production traits by detecting electrophoretic protein pattern in the optical density value of 102kD place protein band, can be used as a kind of simple, efficiently, the method for low cost to be to differentiate the quality of the spirulina production traits.
Claims (1)
1. distinguish a method for good and bad production of spirurina strain degree, it is characterized in that, comprise the following steps:
The water-solubility protein obtaining spirulina cells is extracted with Tris-HCl extract, after sodium dodecyl sulfate-polyacrylamide gel electrophoresis is separated protein sample, utilize QuantityOne analysis software to detect the optical density value of electrophoretic protein pattern 102kD place protein band, and build the dendrogram between algae strain according to its value; If the electrophoretic protein pattern of candidate's strain is greater than 140 in the optical density value of 102kD place protein band, and together with the strain good with the known production traits gather, then illustrates that this strain production traits is good, be applicable to cultivate production on a large scale; If be less than 40 in the optical density value of 102kD place protein band, and together with the strain bad with the known production traits gather, then illustrate that this strain production traits is poor, be not suitable for and cultivate production on a large scale;
The strain that the described known production traits is good refers to spirulina Sp-4, Sp-5, Sp-15 and Sp-17; The bad strain of the described known production traits refers to spirulina Sp-1, Sp-2, Sp-6, Sp-9 and Sp-10;
The method specifically comprises the steps:
Get 0.75g blunt top spirulina algae mud in 5ml centrifuge tube, add 3mlTris-HCl extract and utilize the method for freeze thawing broken wall to raise water-solubility protein;
Carry out protein s DS-PAGE analysis, resolving gel concentration is 12.5%, and concentrated gum concentration is 5%; First with after 80V electrophoresis 45min, then make bottom bromophenol blue to glue with 120V electrophoresis; With Coomassie brilliant G-250 dyeing, gel imaging after decolouring;
Utilize QuantityOne analysis software to detect the optical density value of electrophoretic protein pattern at 102kD place protein band, utilize the dendrogram between the strain of SPSS13.0 statistical analysis software structure algae according to its value, and differentiated the quality of this strain production traits by cluster situation.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102329867A (en) * | 2011-09-26 | 2012-01-25 | 浙江大学 | Method for screening good and bad production traits of spirulina strains |
| CN102634576A (en) * | 2012-03-27 | 2012-08-15 | 浙江大学 | Method for identifying spirulina strains with favorable production traits |
| CN103018311A (en) * | 2012-12-25 | 2013-04-03 | 浙江大学 | Method for detecting production trait goodness and badness of spirulina strain |
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| CN102329867A (en) * | 2011-09-26 | 2012-01-25 | 浙江大学 | Method for screening good and bad production traits of spirulina strains |
| CN102634576A (en) * | 2012-03-27 | 2012-08-15 | 浙江大学 | Method for identifying spirulina strains with favorable production traits |
| CN103018311A (en) * | 2012-12-25 | 2013-04-03 | 浙江大学 | Method for detecting production trait goodness and badness of spirulina strain |
Non-Patent Citations (1)
| Title |
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| 钝顶节旋藻（Arthrospira platensis）2-DE分析蛋白制备方法改进及试用于螺旋手性差异蛋白研究;王景梅等;《海洋与湖沼》;20130131;第44卷(第1期);141-147 * |
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