CN100366755C - Method for screening high quality spirulina princeps strain for large scale production - Google Patents
Method for screening high quality spirulina princeps strain for large scale production Download PDFInfo
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- CN100366755C CN100366755C CNB2006100522346A CN200610052234A CN100366755C CN 100366755 C CN100366755 C CN 100366755C CN B2006100522346 A CNB2006100522346 A CN B2006100522346A CN 200610052234 A CN200610052234 A CN 200610052234A CN 100366755 C CN100366755 C CN 100366755C
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Abstract
The method of screening high quality spirulina strain for large scale production includes designing primer of 16S-23S rRNA transcription spacer, cloning and measuring 16S-23S rRNAITS sequences shown in SEQ No. 1 of 7 spirulina strains Sp-1, Sp-2, Sp-3, Sp-5, Sp-9, Sp-10 and Sp-15 by means of PCR technology; separating the 7 spirulina strains into two groups, including the first group comprising Sp-3, Sp-5 and Sp-15, and the second group comprising Sp-1, Sp-2, Sp-9 and Sp-10 according to the similarity of the sequences; and measuring the 16S-23S rRNAITS sequence of the candidate strain and comparing with those of the said strain. When the candidate strain may be classified into the first group, it has excellent character and is suitable for large scale production; when the candidate strain may be classified into the first group, it is not suitable for large scale 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 shape, be a genus [Wuhan phytology research of Cyanophyta (Cyanophyta), Oscillatoriales (Oscillatoriales), Oscillariaceae (Oscillatoriaceae), 1997,15 (4): 369-374], because of it is rich in quality protein and multiple biologically active substance receives very big concern both domestic and external, on the basis of big quantity research, form huge spirulina industry at present, and be applied to fields such as food, biological medicine health care, feed, fine chemistry industry.Spirulina plalensis in this genus (Spirulina platensis) be both at home and abroad the commercialization breeding production with the exploitation in most widely used kind.It is worthy of note, numerous experimental studies and long-term production practice show, many strains are arranged under the spirulina plalensis kind, they are to temperature, light quality, intensity of illumination, and the requirement and the adaptability of environmental factors such as the salinity of nutrient solution, pH and nutritive ingredient have significant difference [hydrobiont journal, 1999,23 (1): 59-64].Nearly all adopt open or semiclosed, this more extensive breeding production pattern of the circulating cultivation pool of racetrack at present both at home and abroad, many environmental factors, particularly temperature and intensity of illumination, be difficult to artificial adjustment, though tying up to the laboratory or produce in the lab scale, some spirulina plalensis product shows good production performance, but after entering the production pond, because of the bigger variation of the factor that is difficult to conform can't be applied to scale operation.Therefore, seed selection moral character hold concurrently excellent strain be spirulina breeding production always with exploitation in one of most important link and technical essential.
The method that the high-quality spirulina strain of scale operation is closed in suitable both at home and abroad at present screening is that elder generation makes various environmental factors combined crosswise culture experiment in the laboratory, makes preliminary screening according to indexs such as the growth curve of being measured, photosynthetic oxygen evolution, biochemical compositions; Be transferred to outdoor about 5m more successively
2, 50m
2And 500m
2Cultivation pool under Various Seasonal and weather and nutritional condition, culture lab scale, pilot scale and industrial experimentation, and then filter out good algae strain.Though this method is practical, program is loaded down with trivial details, workload is big, the cycle is long, cost is high.Therefore, it was both practical to press for foundation, easy again, consuming time less, the screening novel method of high-quality spirulina strain cheaply, 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 high-quality spirulina strain of suitable scale operation.
(Internally Transcribed Spacer ITS) is the ancient sequence of a class that is present in all biological gene groups to the 16S-23SrRNA transcribed spacer.We are by its specific primer of design, utilize PCR equimolecular biological method to clone and measured the 16S-23SrRNA ITS sequence of 7 strain spirulina plalensis strain Sp-1, Sp-2, Sp-3, Sp-5, Sp-9, Sp-10 and Sp-15, and then utilize information biology and molecular systematics etc. analyses to show, the 16S-23SrRNA ITS sequence of this 7 strain strain is not quite similar, according to the similarity degree of 16S-23SrRNA ITS sequence, 7 strain strains can be divided into two big classes: wherein Sp-3, Sp-5 and Sp-15 are a class; Sp-1, Sp-2, Sp-9 and Sp-10 are another kind of.Secular experimental study and practice show, above-mentioned 7 strain spirulina plalensis product tie up to environmental factors such as temperature, illumination and all show in can the laboratory culture test of auto-controls good, also show in culturing goodly but have only these 3 strain product of Sp-3, Sp-5 and Sp-15 to tie up to actual production, and Sp-1, Sp-2, Sp-9 and Sp-10 can not be as producing breeding because of the adaptive faculty difference.This shows, there are dependency in the 16S-23SrRNA ITS sequence signature and the spirulina production traits, therefore, 16S-23SrRNA ITS sequence is expected to the molecule marker as screening high-quality spirulina strain, be candidate's strain 16S-23SrRNA ITS sequence if with the class of gathering into of Sp-3, Sp-5, Sp-15, then the possibility production traits is good, is fit to scale operation; If with the class of gathering into of Sp-1, Sp-2, Sp-9, Sp-10, then may not can be applicable to produce.
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 fast, standard is clear and definite, and cost is low, and is fit to extensive, high flux screening.
Description of drawings
Fig. 1 is the phylogenetic tree synoptic diagram of 7 strain strains;
Fig. 2 is the phylogenetic tree synoptic diagram of 10 strain strains.
Embodiment
Technical scheme of the present invention can realize by following steps
1, select materials is known 7 strain obtusatus arthrospira strains, i.e. Sp-1, Sp-2, Sp-3, Sp-5, Sp-9, Sp-10 and Sp-15 (also there is preservation in Zhejiang University's nucleus research of agricultural science institute's Biological resources and molecular engineering laboratory).Wherein, Sp-3, Sp-5 and Sp-15 are strong to adaptive capacity to environment, be widely used in scale operation is cultured, and Sp-1, Sp-2, Sp-9 and Sp-10 can not be as producing breeding because of the adaptive faculty difference;
2, reagent and instrument: Taq archaeal dna polymerase and agarose are that worker bio-engineering corporation product is given birth in Shanghai; Cloning vector pMD18-T, and dNTP, DNA restriction enzyme, T4 dna ligase are Japanese TaKaRa company product; Dna gel reclaims test kit and purchases the company in Shanghai Ying Jun; The PCR primer is synthetic by Shanghai Ying Jun company.Other reagent is analytical pure.Sequence amplification checks order with 3730 type sequenators of American AB I company with the Thermal Cycler PCR instrument of U.S. Hybaid company;
3, PCR design of primers and amplification
Utilize Primer 5.0 primer-design softwares to obtain upstream primer SF:5 '-TTAGGGAGACCTACTTCAGGACA-3 ', downstream primer SR:5 '-TACATTGGAATTGTCTTTACGA-3 '.Contain each 0.5 μ mol/L of primer PF and PR in the 25 μ L PCR reaction systems, 4 kinds of each 1 μ mol/L of dNTP, the Taq archaeal dna polymerase of 2.5U, 10 times PCR damping fluid 2.5 μ L, 100ng genomic dna [extracting method reference---Oceanologia et Limnologia Sinica, 2002,33 (2): 203-208].Response procedures: 94 ℃ of 5min; 94 ℃ of 30s, 55 ℃ of 1min, 72 ℃ of 1min, 30 circulations; 72 ℃ of 5min;
4, the clone of PCR product and order-checking
To reclaim the PCR product that test kit reclaims purifying through dna gel and be connected on the pMD18-T carrier, transformed competence colibacillus E.coli TG1, blue hickie method screening positive clone extracts plasmid and cuts evaluation as enzyme, will contain the segmental recombinant plasmid of purpose and check order.
5, sequence alignment and phylogenetic tree make up
Clustal X 1.81 softwares are adopted in the multiple sequence comparison, the structure of phylogenetic tree adopts Phylip 3.65 software packages, through comparison, if screened spirulina plalensis strain can three strains gather to become with Sp-15 with Sp-3, Sp-5, then this strain excellent property is fit to scale operation and cultures.
Result and analysis
Analyze and the phylogenetic tree structure based on the difference number of sites of 16S-23S rRNA ITS sequence
The genomic dna of Sp-1, Sp-2, Sp-3, Sp-5, Sp-9, Sp-10 and Sp-15 is respectively through the specific primer pcr amplification and the clone of 16S-23SrRNA ITS sequence, and records corresponding sequence.Shown in the 16S-23SrRNA ITS sequence of SEQNO1 and SEQNO2;
Utilization Clustal X 1.81 softwares are made the multiple sequence compare of analysis to the 16S-23SrRNA ITS sequence of 7 strain strains, and statistics obtains the difference number of sites of base between strain.As shown in Table 1, Sp-1, Sp-2, Sp-9 and Sp-10 difference number of sites between any two is 0; Sp-3, Sp-5 and Sp-15 difference number of sites between any two also is 0; Yet Sp-1, Sp-2, these 4 strains of Sp-9, Sp-10 and Sp-3, Sp-5, Sp-15 difference number of sites between any two are 46.Further, with the 16S-23SrRNA ITS of above-mentioned 7 strain strains
The difference number of sites of the 16S-23SrRNAITS sequence of table 17 strain strains
| The algae strain | Sp-1 | Sp-2 | Sp-9 | Sp-10 | Sp-3 | Sp-5 | Sp-15 |
| Sp-1 | 0 | ||||||
| Sp-2 | 0 | 0 | |||||
| Sp-9 | 0 | 0 | 0 | ||||
| Sp-10 | 0 | 0 | 0 | 0 | |||
| Sp-3 | 46 | 46 | 46 | 46 | 0 | ||
| Sp-5 | 46 | 46 | 46 | 46 | 0 | 0 | |
| Sp-15 | 46 | 46 | 46 | 46 | 0 | 0 | 0 |
The multiple sequence comparison result of sequence imports the dnamlk program of Phylip 3.65 software packages and carries out the phylogenetic tree structure.The result as shown in Figure 1,7 strain strains can be divided into two big class: Sp-3, Sp-5 and Sp-15 is that a class: Sp-1, Sp-2, Sp-9 and Sp-10 are a class, the result of this and the analysis of above-mentioned difference number of sites matches.
Secular experimental study shows, above-mentioned 7 strain spirulina plalensis product tie up to environmental factors such as temperature, illumination and all show in can the laboratory culture test of auto-controls good, use relatively extensively but have only these 3 strain product of Sp-3, Sp-5 and Sp-15 to tie up in the actual production breed, and Sp-1, Sp-2, Sp-9 and Sp-10 can not be as producing breeding because of the adaptive faculty difference.This shows, there are dependency in the 16S-23S rRNA ITS sequence signature and the spirulina production traits, therefore, 16S-23S rRNA ITS sequence can be used as the molecule marker of screening high-quality spirulina strain, be candidate's strain 16S-23S rRNA ITS sequence if with the class of gathering into of Sp-3, Sp-5, Sp-15, then may the production traits good; If with the class of gathering into of Sp-1, Sp-2, Sp-9, Sp-10, then may not can be applicable to produce.
As an example, we have chosen Sp-6, Sp-12 and this 3 strain strain of Sp-16 is verified practicality of the present invention, wherein, these two strains of Sp-12 and Sp-16 are the breedings that are applicable to that scale operation is cultured, and Sp-6 can not be used for scale operation because of bad adaptability.By cloning and measure the 16S-23S rRNA ITS sequence of this 3 strain strain: utilize analyses such as information biology and molecular systematics to show, shown in SEQNO2 and accompanying drawing 2, the 16S-23S rRNA ITS sequence of Sp-12 and Sp-16 and the class of gathering into of Sp-3, Sp-5 and Sp-15, and sequence is in full accord, and the class of gathering into of the 16S-23S rRNA ITS sequence of Sp-6 and Sp-1, Sp-2, Sp-9 and Sp-10, and sequence is in full accord.The above results further specifies, and 16S-23S rRNA ITS sequence can be used as a kind of molecule marker and screens the high-quality spirulina strain that is fit to scale operation.
The multiple sequence compare of analysis result of the 16S-23S rRNA ITS sequence of SEQNO1 7 strain spirulina strains
Wherein Sp-1, Sp-2, these 4 strains of Sp-9, Sp-10 and Sp-3, Sp-5, Sp-15 difference between any two
Underscore is marked in the site; The same loci of the 16S-23S rRNAITS sequence of 7 strains of " * " expression.
Sp-1 1 TTAGGGAGACCTACTTCAGGACATCGTGCGATGATAATAATAGCCGAGTCTTGAGGTCAT
Sp-2 TTAGGGAGACCTACTTCAGGACATCGTGCGATGATAATAATAGCCGAGTCTTGAGGTCAT
Sp-9 TTAGGGAGACCTACTTCAGGACATCGTGCGATGATAATAATAGCCGAGTCTTGAGGTCAT
Sp-10 TTAGGGAGACCTACTTCAGGACATCGTGCGATGATAATAATAGCCGAGTCTTGAGGTCAT
Sp-3 TTAGGGAGACCTACTTC
GAGA
TATCG
CGC
CTT
AA
CAA
CTATAGCCG
TGTCTTGAGGTCAT
Sp-5 TTAGGGAGACCTACTTC
GAGA
TATCG
CGC
CTT
AA
CAA
CTATAGCCG
TGTCTTGAGGTCAT
Sp-15 TTAGGGAGACCTACTTC
GAGA
TATCG
CGC
CTT
AA
CAA
CTATAGCCG
TGTCTTGAGGTCAT
***************** ** **** ** * * ** ******* *************
Sp-1 61 CCTTAGGTCGGATGGGGCGGTCAGAGAGCTTTCAAACTTTAGGGTTCGTGTTATGGGCTA
Sp-2 CCTTAGGTCGGATGGGGCGGTCAGAGAGCTTTCAAACTTTAGGGTTCGTGTTATGGGCTA
Sp-9 CCTTAGGTCGGATGGGGCGGTCAGAGAGCTTTCAAACTTTAGGGTTCGTGTTATGGGCTA
Sp-10 CCTTAGGTCGGATGGGGCGGTCAGAGAGCTTTCAAACTTTAGGGTTCGTGTTATGGGCTA
Sp-3 CCTTAGGTCGGATGGGGCGGTCAGAGAGCTTTCAAACTTTAGGGTTCGTGTTATGGGCTA
Sp-5 CCTTAGGTCGGATGGGGCGGTCAGAGAGCTTTCAAACTTTAGGGTTCGTGTTATGGGCTA
Sp-15 CCTTAGGTCGGATGGGGCGGTCAGAGACCTTTCAAACTTTAGGGTTCGTGTTATGGGCTA
************************************************************
Sp-1 121 TTAGCTCAGGTGGTTAGAGCGCACCCCTGATAAGGGTGAGGTCCCTGGTTCAAGTCCAGG
Sp-2 TTAGCTCAGGTGGTTAGAGCGCACCCCTGATAAGGGTGAGGTCCCTGGTTCAAGTCCAGG
Sp-9 TTAGCTCAGGTGGTTAGAGCGCACCCCTGATAAGGGTGAGGTCCCTGGTTCAAGTCCAGG
Sp-10 TTAGCTCAGGTGGTTAGAGCGCACCCCTGATAAGGGTGAGGTCCCTGGTTCAAGTCCAGG
Sp-3 TTAGCTCAGGTGGTTAGAGCGCACCCCTGATAAGGGTGAGGTCCCTGGTTCAAGTCCAGG
Sp-5 TTAGCTCAGGTGGTTAGAGCGCACCCCTGATAAGGGTGAGGTCCCTGGTTCAAGTCCAGG
Sp-15 TTAGCTCAGGTGGTTAGAGCGCACCCCTGATAAGGGTGAGGTCCCTGGTTCAAGTCCAGG
************************************************************
Sp-1 181 ATGGCCCACATCCACCCCAAACTGGGGGTATAGCTCAGTTGGTAGAGCGCTGCCTTTGCA
Sp-2 ATGGCCCACATCCACCCCAAACTGGGGGTATAGCTCAGTTGGTAGAGCGCTGCCTTTGCA
Sp-9 ATGGCCCACATCCACCCCAAACTGGGGGTATAGCTCAGTTGGTAGAGCGCTGCCTTTGCA
Sp-10 ATGGCCCACATCCACCCCAAACTGGGGGTATAGCTCAGTTGGTAGAGCGCTGCCTTTGCA
Sp-3 ATGGCCCACATCCACCCCAAACTGGGGGTATAGCTCAGTTGGTAGAGCGCTGCCTTTGCA
Sp-5 ATGGCCCACATCCACCCCAAACTGGGGGTATAGCTCAGTTGGTAGAGCGCTGCCTTTGCA
Sp-15 ATGGCCCACATCCACCCCAAACTGGGGGTATAGCTCAGTTGGTAGAGCGCTGCCTTTGCA
*************************************************************
Sp-1 241 CGGCAGAAGTCAGCGGTTCGAGTCCGCTTACCTCCACTCTCCTAGAATTAGGTGCTAGTT
Sp-2 CGGCAGAAGTCAGCGGTTCGAGTCCGCTTACCTCCACTCTCCTAGAATTAGGTGCTAGTT
Sp-9 CGGCAGAAGTCAGCGGTTCGAGTCCGCTTACCTCCACTCTCCTAGAATTAGGTGCTAGTT
Sp-10 CGGCAGAAGTCAGCGGTTCGAGTCCGCTTACCTCCACTCTCCTAGAATTAGGTGCTAGTT
Sp-3 CGGCAGAAGTCAGCGGTTCGAGTCCGCTTACCTCCACTCTCCT
TTGTGATGGTGCTAGTT
Sp-5 CGGCAGAAGTCAGCGGTTCGAGTCCGCTTACCTCCACTCTCCT
TTGTGATGGTGCTAGTT
Sp-15 CGGCAGAAGTCAGCGGTTCGAGTCCGCTTACCTCCACTCTCCT
TTGTGATGGTGCTAGTT
******************************************* **********
Sp-1 301 GGGGTGAGGTAGTCTTGAATTGAGA--AATTGAGAGTTGGTGACTGTACAGCTCCTAAGT
Sp-2 GGGGTGAGGTAGTCTTGAATTGAGA--AATTGAGAGTTGGTGACTGTACAGCTCCTAAGT
Sp-9 GGGGTGAGGTAGTCTTGAATTGAGA--AATTGAGAGTTGGTGACTGTACAGCTCCTAAGT
Sp-10 GGGGTGAGGTAGTCTTGAATTGAGA--AATTGAGAGTTGGTGACTGTACAGCTCCTAAGT
Sp-3 GGGGTGAG
AT
GAGAT
GAGAT
GACCTCTGAT
AGA
TA
ATT
TAT
CACTGTACAGCTCCTAA
AT
Sp-5 GGGGTGAG
AT
GAGAT
GAGAT
GACCTCTGAT
AGA
TA
ATT
TAT
CACTGTACAGCTCCTAA
AT
Sp-15 GGGGTGAG
AT
GAGAT
GAGAT
GACCTCTGAT
AGA
TA
ATT
TAT
CACTGTACAGCTCCTAA
AT
******** * * ** ** ** * ** * **************** *
Sp-1 361 CTGTAGATGTTAATCTAGGACTAGATAGCTGGACATAAGTTCCAGTCAGAACCTTGAAAA
Sp-2 CTGTAGATGTTAATCTAGGACTAGATAGCTGGACATAAGTTCCAGTCAGAACCTTGAAAA
Sp-9 CTGTAGATGTTAATCTAGGACTAGATAGCTGGACATAAGTTCCAGTCAGAACCTTGAAAA
Sp-10 CTGTAGATGTTAATCTAGGACTAGATAGCTGGACATAAGTTCCAGTCAGAACCTTGAAAA
Sp-3 CT
TTAGATGTTA
GTCT
GAGA
TT
GGATAGCTGGACAT
CTGTTCCAGTCAGAACCTTGAAAA
Sp-5 CT
TTAGATGTTA
GTCT
GAGA
TT
GGATAGCTGGACAT
CTGTTCCAGTCAGAACCTTGAAAA
Sp-15 CT
TTAGATGTTA
GTCT
GAGA
TT
GGATAGCTGGACAT
CTGTTCCAGTCAGAACCTTGAAAA
** ********* *** ** * ************* **********************
Sp-1 421 CTGCATAGAGAAAAGCATAATGGTGTAGGAAAACGTCGTAAAGACAATTCCAATGTA
Sp-2 CTGCATAGAGAAAAGCATAATGGTGTAGGAAAACGTCGTAAAGACAATTCCAATGTA
Sp-9 CTGCATAGAGAAAAGCATAATGGTGTAGGAAAACGTCGTAAAGACAATTCCAATGTA
Sp-10 CTGCATAGAGAAAAGCATAATGGTGTAGGAAAACGTCGTAAAGACAATTCCAATGTA
Sp-3 CTGCATAGAGAAAAGCATAATGGTGTAGGAAAACGTCGTAAAGACAATTCCAATGTA
Sp-5 CTGCATAGAGAAAAGCATAATGGTGTAGGAAAACGTCGTAAAGACAATTCCAATGTA
Sp-15 CTGCATAGAGAAAAGCATAATGGTGTAGGAAAACGTCGTAAAGACAATTCCAATGTA
*********************************************************
The multiple sequence compare of analysis result of the 16S-23S rRNA ITS sequence of this 10 strain spirulina strain of SEQNO2
Sp-6 1 TTAGGGAGACCTACTTCAGGACATCGTGCGATGATAATAATAGCCGAGTCTTGAGGTCAT
Sp-1 TTAGGGAGACCTACTTCAGGACATCGTGCGATGATAATAATAGCCGAGTCTTGAGGTCAT
Sp-2 TTAGGGAGACCTACTTCAGGACATCGTGCGATGATAATAaTAGCCGAGTCTTGAGGTCAT
Sp-9 TTAGGGAGACCTACTTCAGGACATCGTGCGATGATAATAATAGCCGAGTCTTGAGGTCAT
Sp-10 TTAGGGAGACCTACTTCAGGACATCGTGCGATGATAATAATAGCCGAGTCTTGAGGTCAT
Sp-3 TTAGGGAGACCTACTTC
GAGA
TATCG
CGC
CTT
AA
CAA
CTATAGCCG
TGTCTTGAGGTCAT
Sp-5 TTAGGGAGACCTACTTC
GAGA
TATCG
CGC
CTT
AA
CAA
CTATAGCCG
TGTCTTGAGGTCAT
Sp-15 TTAGGGAGACCTACTTC
GAGA
TATCG
CGC
CTT
AA
CAA
CTATAGCCG
TGTCTTGAGGTCAT
Sp-12 TTAGGGAGACCTACTTC
GAGA
TATCG
CGC
CTT
AA
CAA
CTATAGCCG
TGTCTTGAGGTCAT
Sp-16 TTAGGGAGACCTACTTC
GAGA
TATCG
CGC
CTT
AA
CAA
CTATAGCCG
TGTCTTGAGGTCAT
***************** ** **** ** * * ** ******* *************
Sp-6 61 CCTTAGGTCGGATGGGGCGGTCAGAGAGCTTTCAAACTTTAGGGTTCGTGTTATGGGCTA
Sp-1 CCTTAGGTCGGATGGGGCGGTCAGAGAGCTTTCAAACTTTAGGGTTCGTGTTATGGGCTA
Sp-2 CCTTAGGTCGGATGGGGCGGTCAGAGAGCTTTCAAACTTTAGGGTTCGTGTTATGGGCTA
Sp-9 CCTTAGGTCGGATGGGGCGGTCAGAGAGCTTTCAAACTTTAGGGTTCGTGTTATGGGCTA
Sp-10 CCTTAGGTCGGATGGGGCGGTCAGAGAGCTTTCAAACTTTAGGGTTCGTGTTATGGGCTA
Sp-3 CCTTAGGTCGGATGGGGCGGTCAGAGAGCTTTCAAACTTTAGGGTTCGTGTTATGGGCTA
Sp-5 CCTTAGGTCGGATGGGGCGGTCAGAGAGCTTTCAAACTTTAGGGTTCGTGTTATGGGCTA
Sp-15 CCTTAGGTCGGATGGGGCGGTCAGAGAGCTTTCAAACTTTAGGGTTCGTGTTATGGGCTA
Sp-12 CCTTAGGTCGGATGGGGCGGTCAGAGAGCTTTCAAACTTTAGGGTTCGTGTTATGGGCTA
Sp-16 CCTTAGGTCGGATGGGGCGGTCAGAGAGCTTTCAAACTTTAGGGTTCGTGTTATGGGCTA
************************************************************
Sp-6 121 TTAGCTCAGGTGGTTAGAGCGCACCCCTGATAAGGGTGAGGTCCCTGGTTCAAGTCCAGG
Sp-1 TTAGCTCAGGTGGTTAGAGCGCACCCCTGATAAGGGTGAGGTCCCTGGTTCAAGTCCAGG
Sp-2 TTAGCTCAGGTGGTTAGAGCGCACCCCTGATAAGGGTGAGGTCCCTGGTTCAAGTCCAGG
Sp-9 TTAGCTCAGGTGGTTAGAGCGCACCCCTGATAAGGGTGAGGTCCCTGGTTCAAGTCCAGG
Sp-10 TTAGCTCAGGTGGTTAGAGCGCACCCCTGATAAGGGTGAGGTCCCTGGTTCAAGTCCAGG
Sp-3 TTAGCTCAGGTGGTTAGAGCGCACCCCTGATAAGGGTGAGGTCCCTGGTTCAAGTCCAGG
Sp-5 TTAGCTCAGGTGGTTAGAGCGCACCCCTGATAAGGGTGAGGTCCCTGGTTCAAGTCCAGG
Sp-15 TTAGCTCAGGTGGTTAGAGCGCACCCCTGATAAGGGTGAGGTCCCTGGTTCAAGTCCAGG
Sp-12 TTAGCTCAGGTGGTTAGAGCGCACCCCTGATAAGGGTGAGGTCCCTGGTTCAAGTCCAGG
Sp-16 TTAGCTCAGGTGGTTAGAGCGCACCCCTGATAAGGGTGAGGTCCCTGGTTCAAGTCCAGG
************************************************************
Sp-6 181 ATGGCCCACATCCACCCCAAACTGGGGGTATAGCTCAGTTGGTAGAGCGCTGCCTTTGCA
Sp-1 ATGGCCCACATCCACCCCAAACTGGGGGTATAGCTCAGTTGGTAGAGCGCTGCCTTTGCA
Sp-2 ATGGCCCACATCCACCCCAAACTGGGGGTATAGCTCAGTTGGTAGAGCGCTGCCTTTGCA
Sp-9 ATGGCCCACATCCACCCCAAACTGGGGGTATAGCTCAGTTGGTAGAGCGCTGCCTTTGCA
Sp-10 ATGGCCCACATCCACCCCAAACTGGGGGTATAGCTCAGTTGGTAGAGCGCTGCCTTTGCA
Sp-3 ATGGCCCACATCCACCCCAAACTGGGGGTATAGCTCAGTTGGTAGAGCGCTGCCTTTGCA
Sp-5 ATGGCCCACATCCACCCCAAACTGGGGGTATAGCTCAGTTGGTAGAGCGCTGCCTTTGCA
Sp-15 ATGGCCCACATCCACCCCAAACTGGGGGTATAGCTCAGTTGGTAGAGCGCTGCCTTTGCA
Sp-12 ATGGCCCACATCCACCCCAAACTGGGGGTATAGCTCAGTTGGTAGAGCGCTGCCTTTGCA
Sp-16 ATGGCCCACATCCACCCCAAACTGGGGGTATAGCTCAGTTGGTAGAGCGCTGCCTTTGCA
************************************************************
Sp-6 241 CGGCAGTCGTCAGCGGTTCGAGTCCGCTTACCTCCACTCTCCTAGAATTAGGTGCTAGTT
Sp-1 CGGCAGAAGTCAGCGGTTCGAGTCCGCTTACCTCCACTCTCCTAGAATTAGGTGCTAGTT
Sp-2 CGGCAGAAGTCAGCGGTTCGAGTCCGCTTACCTCCACTCTCCTAGAATTAGGTGCTAGTT
Sp-9 CGGCAGAAGTCAGCGGTTCGAGTCCGCTTACCTCCACTCTCCTAGAATTAGGTGCTAGTT
Sp-10 CGGCAGTCGTCAGCGGTTCGAGTCCGCTTACCTCCACTCTCCTAGAATTAGGTGCTAGTT
Sp-3 CGGCAGAAGTCAGCGGTTCGAGTCCCCTTACCTCCACTCTCCT
TTGTGATGGTGCTAGTT
Sp-5 CGGCAGAAGTCAGCGGTTCGAGTCCGCTTACCTCCACTCTCCT
TTGTGATGGTGCTAGTT
Sp-15 CGGCAGAAGTCAGCGGTTCGAGTCCGCTTACCTCCACTCTCCT
TTGTGATGGTGCTAGTT
Sp-12 CGGCAGAAGTCAGCGGTTCGAGTCCGCTTACCTCCACTCTCCT
TTGTGATGGTGCTAGTT
Sp-16 CGGCAGAAGTCAGCGGTTCGAGTCCGCTTACCTCCACTCTCCT
TTGTGATGGTGCTAGTT
******************************************* **********
Sp-6 301 GGGGTGAGGTAGTCTTGAATTGAGA--AATTGAGAGTTGGTGACTGTACAGCTCCTAAGT
Sp-1 GGGGTGAGGTAGTCTTGAATTGAGA--AATTGAGAGTTGGTGACTGTACAGCTCCTAAGT
Sp-2 GGGGTGAGGTAGTCTTGAATTGAGA--AATTGAGAGTTGGTGACTGTACAGCTCCTAAGT
Sp-9 GGGGTGAGGTAGTCTTGAATTGAGA--AATTGAGAGTTGGTGACTGTACAGCTCCTAAGT
Sp-10 GGGGTGAGGTAGTCTTGAATTGAGA--AATTGAGAGTTGGTGACTGTACAGCTCCTAAGT
Sp-3 GGGGTGAG
AT
GAGAT
GAGAT
GACCTCTGAT
AGA
TA
ATT
TAT
CACTGTACAGCTCCTAA
AT
Sp-5 GGGGTGAG
AT
GAGAT
GAGAT
GACCTCTGAT
AGA
TA
ATT
TAT
CACTGTACAGCTCCTAA
AT
Sp-15 GGGGTGAG
AT
GAGAT
GAGAT
GACCTCTGAT
AGA
TA
ATT
TAT
CACTGTACAGCTCCTAA
AT
Sp-12 GGGGTGAG
AT
GAGAT
GAGAT
GACCTCTGAT
AGA
TA
ATT
TAT
CACTGTACAGCTCCTAA
AT
Sp-16 GGGGTGAG
AT
GAGAT
GAGAT
GACCTCTGAT
AGA
TA
ATT
TAT
CACTGTACAGCTCCTAA
AT
******** * * ** ** ** * ** * **************** *
Sp-6 361 CTGTAGATGTTAATCTAGGACTAGATAGCTGGACATAAGTTCCAGTCAGAACCTTGAAAA
Sp-1 CTGTAGATGTTAATCTAGGACTAGATAGCTGGACATAAGTTCCAGTCAGAACCTTGAAAA
Sp-2 CTGTAGATGTTAATCTAGGACTAGATAGCTGGACATAAGTTCCAGTCAGAACCTTGAAAA
Sp-9 CTGTAGATGTTAATCTAGGACTAGATAGCTGGACATAAGTTCCAGTCAGAACCTTGAAAA
Sp-10 CTGTAGATGTTAATCTAGGACTAGATAGCTGGACATAAGTTCCAGTCAGAACCTTGAAAA
Sp-3 CT
TTAGATGTTA
GTCT
GAGA
TT
GGATAGCTGGACAT
CTGTTCCAGTCAGAACCTTGAAAA
Sp-5 CT
TTAGATGTTA
GTCT
GAGA
TT
GGATAGCTGGACAT
CTGTTCCAGTCAGAACCTTGAAAA
Sp-15 CT
TTAGATGTTA
GTCT
GAGA
TT
GGATAGCTGGACAT
CTGTTCCAGTCAGAACCTTGAAAA
Sp-12 CT
TTAGATGTTA
GTCT
GAGA
TT
GGATAGCTGGACAT
CTGTTCCAGTCAGAACCTTGAAAA
Sp-16 CT
TTAGATGTTA
GTCT
GAGA
TT
GGATAGCTGGACAT
CTGTTCCAGTCAGAACCTTGAAAA
** ********* *** ** * * ************ **********************
Sp-6 421 CTGCATAGAGAAAAGCATAATGGTGTAGGAAAACGTCGTAAAGACAATTCCAATGTA
Sp-1 CTGCATAGAGAAAAGCATAATGGTGTAGGAAAACGTCGTAAAGACAATTCCAATGTA
Sp-2 CTGCATAGAGAAAAGCATAATGGTGTAGGAAAACGTCGTAAAGACAATTCCAATGTA
Sp-9 CTGCATAGAGAAAAGCATAATGGTGTAGGAAAACGTCGTAAAGACAATTCCAATGTA
Sp-10 CTGCATAGAGAAAAGCATAATGGTGTAGGAAAACGTCGTAAAGACAATTCCAATGTA
Sp-3 CTGCATAGAGAAAAGCATAATGGTGTAGGAAAACGTCGTAAAGACAATTCCAATGTA
Sp-5 CTGCATAGAGAAAAGCATAATGGTGTAGGAAAACGTCGTAAAGACAATTCCAATGTA
Sp-15 CTGCATAGAGAAAAGCATAATGGTGTAGGAAAACGTCGTAAAGACAATTCCAATGTA
Sp-12 CTGCATAGAGAAAAGCATAATGGTGTAGGAAAACGTCGTAAAGACAATTCCAATGTA
Sp-16 CTGCATAGAGAAAAGCATAATGGTGTAGGAAAACGTCGTAAAGACAATTCCAATGTA
*********************************************************
Claims (2)
1. a method of screening high-quality spirulina strain is characterized in that 7 strain spirulina plalensis strain Sp-1, Sp-2, Sp-3, Sp-5, Sp-9, Sp-1O, Sp-15 have the 16S-23S rRNA ITS sequence of SEQNO1; The 16S-23SrRNA ITS sequence and the above-mentioned strain sequence multiple ratio of candidate's strain is right, and when the sequence of candidate's strain is gathered into a class with above-mentioned Sp-3, Sp-5 with the Sp-15 sequence, and sequence is when in full accord, and then this spirulina candidate strain proterties is good; And gather into a class with Sp-1, Sp-2, Sp-9, Sp-10 when candidate's strain sequence, and sequence then can not be used for production when in full accord.
2. by the method for the described screening high-quality of claim 1 spirulina strain, it is characterized in that adopting following operation steps:
(1) select materials is 7 strain obtusatus arthrospira strains, be Sp-1, Sp-2, Sp-3, Sp-5, Sp-9, Sp-10 and Sp-15 wherein, Sp-3, Sp-5 and Sp-15 are strong to adaptive capacity to environment, in scale operation is cultured, be widely used, and Sp-1, Sp-2, Sp-9 and Sp-10 can not be as producing breeding because of the adaptive faculty difference;
(2) reagent and instrument: Taq archaeal dna polymerase and agarose are that worker bio-engineering corporation product is given birth in Shanghai; Cloning vector pMD18-T, and dNTP, DNA restriction enzyme, T4 dna ligase are Japanese TaKaRa company product; Dna gel reclaims test kit and purchases the company in Shanghai Ying Jun; The PCR primer is synthetic by Shanghai Ying Jun company, and other reagent is analytical pure, and sequence amplification checks order with 3730 type sequenators of American AB I company with the Thermal Cycler PCR instrument of U.S. Hybaid company;
(3) PCR design of primers and amplification
Utilize Primer 5.0 primer-design softwares to obtain upstream primer SF:5 '-TTAGGGAGACCTACTTCAGGACA-3 ', downstream primer SR:5 '-TACATTGGAATTGTCTTTACGA-3 ', contain each 0.5 μ mol/L of primer PF and PR in the 25 μ L PCR reaction systems, 4 kinds of each 1 μ mol/L of dNTP, 2.5U the Taq archaeal dna polymerase, 10 times PCR damping fluid 2.5 μ L, 100ng genomic dna, response procedures: 94 ℃ of 5min; 94 ℃ of 30s, 55 ℃ of 1min, 72 ℃ of 1min, 30 circulations; 72 ℃ of 5min;
(4) clone of PCR product and order-checking
To reclaim the PCR product that test kit reclaims purifying through dna gel is connected on the pMD18-T carrier, transformed competence colibacillus E.coli TG1, blue hickie method screening positive clone, extract plasmid and cut evaluation as enzyme, to contain the segmental recombinant plasmid of purpose and check order, shown in the 16S-23S rRNA ITS sequence of table SEQNO1 and table SEQNO2;
(5) sequence alignment and phylogenetic tree make up
Clustal X 1.81 softwares are adopted in the multiple sequence comparison, the structure of phylogenetic tree adopts Phylip 3.65 software packages, through comparison, if screened spirulina plalensis strain can three strains gather into a class with Sp-15 with Sp-3, Sp-5, then this strain excellent property is fit to scale operation and cultures.
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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 |
| CN102605077A (en) * | 2012-03-27 | 2012-07-25 | 浙江大学 | Selection method for massively produced and used spirulina strains |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1144037A (en) * | 1995-09-01 | 1997-03-05 | 中国科学院武汉植物研究所 | Selection and breeding method for new strain of blunt top spirulina |
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| CN1144037A (en) * | 1995-09-01 | 1997-03-05 | 中国科学院武汉植物研究所 | Selection and breeding method for new strain of blunt top spirulina |
Non-Patent Citations (4)
| Title |
|---|
| RAPD分子标记技术用于螺旋藻分类的研究. 李晋楠等.海洋与湖沼，第33卷第2期. 2002 * |
| 螺旋藻遗传选育研究进展. 汪志平等.微生物学通报，第27卷第4期. 2000 * |
| 钝顶螺旋藻藻种的紫外诱变初步筛选. 陈新美等.生物技术，第16卷第2期. 2006 * |
| 高产多糖钝顶螺旋藻新品系的选育及蛋白质SDS-PAGE鉴定. 汪志平等.核农学报，第18卷第5期. 2004 * |
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