CN1904071B - Method of discriminating good and bad production of spirurina strain - Google Patents
Method of discriminating good and bad production of spirurina strain Download PDFInfo
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- CN1904071B CN1904071B CN2006100522331A CN200610052233A CN1904071B CN 1904071 B CN1904071 B CN 1904071B CN 2006100522331 A CN2006100522331 A CN 2006100522331A CN 200610052233 A CN200610052233 A CN 200610052233A CN 1904071 B CN1904071 B CN 1904071B
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Abstract
The present invention relates to a method for identifying spirulina line production character quality. Said method includes the following steps: selecting four plants of Sp-1, Sp-2, Sp-3 and Sp-5 from abtuse acron spirulina line; utilizing upstream primer PF:5'-CAATACATCTTCG CCGATTT-3' and downstream primer PR: 5'-CGTATTATCGGTAGTCATCGG-3'; in PCR reaction system making reaction: 94 DEG.C 5 min, 94 DEG.C 30s, 58 DEG.C 1 min, 72 DEG.C 2 min, 30 cycles; 72 DEG.C 8 min; connecting PCR product recovered and purified by DNA gel onto PMD18-T carrier to screen male clone; extracting plasmid and making enzyme incision identification, sequencing the recombinant plasmid containing target fragment; making the cpc HID operon gene sequences of above-mentioned four plants and correspondent sequence of identified spirulina plant undergo the process of multiple sequence comparison and creating phylogenetic tree so as to identify spirulina line character quality.
Description
Technical field
The invention belongs to the technology of tenaculat Habenaria Application and Development
Background technology
Tenaculat Habenaria (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], be rich in quality protein and multiple biologically active substance receives very big concern both domestic and external because of it; On the basis of big quantity research, form huge tenaculat Habenaria industry at present, and be applied to fields such as food, biological medicine health care, feed, fine chemistry industry.Spirulina plalensis in this genus (Spirulinaplatensis) 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, and they are to temperature, light quality, intensity of illumination, and the requirement and the flexibility 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 are difficult to artificial adjustment, show good production performance though some spirulina plalensis article ties up to the laboratory or produces in the lab scale; But after getting into 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.
Differentiate both at home and abroad that at present the good and bad ordinary method of tenaculat Habenaria strain is, make various environmental factors combined crosswise culture experiment in the laboratory earlier, make 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.
Summary of the invention
The objective of the invention is the weak point to prior art, set up both practical, easy again, consuming time less, the good and bad discriminating novel method of tenaculat Habenaria strain cheaply, to satisfy the constantly actual needs of development of current domestic and international tenaculat Habenaria industry.
The present invention is in the tenaculat Habenaria molecular genetics background that studies for a long period of time, and on the basis of the good and bad molecular marker gene-cpcHID operon gene of research tenaculat Habenaria strain, sets up a kind of novel method of differentiating good and bad production of spirurina strain.
The cpcHID operon gene is that minority algae such as blue-green algae institute is peculiar, and 3 kinds of bar-shaped polypeptide of its coding play ligation in the phycobilisome assembling.We are through the specific primer of design; Utilize PCR equimolecular biological method to clone and measured the cpcHID operon gene sequence of 4 strain spirulina plalensis strains (being respectively Sp-1, Sp-2, Sp-3, Sp-5); And then utilize information biology and molecular system taxonomy etc. analyses to show; According to the difference number of sites and the similarity degree of cpcHID operon gene sequence, this 4 strain strain is divided into two big types that there were significant differences.Be that Sp-3 and Sp-5 are one type; Sp-1 and Sp-2 are one type.Secular experimental study and production practice show; These 4 strain spirulina plalensis article tie up to environmental factors such as temperature, illumination and all show in can the laboratory culture test of auto-controls good; But have only these two strains article of Sp-3 and Sp-5 tie up to actual production culture in performance good, Sp-1 and Sp-2 can not be as the production breedings because of the adaptive faculty difference.This shows that the cpcHID operon gene sequence signature and the tenaculat Habenaria production traits exist dependency.Therefore, use the cpcHID operon gene and can be used as the molecule marker of differentiating that the tenaculat Habenaria production traits is good and bad, promptly differentiated strain cpcHID operon gene sequence if with gather into one type of Sp-3 and Sp-5, then the possibility production traits is good; If with gather into one type of Sp-1, Sp-2, then possibly not can be applicable to produce.
Remarkable advantage of the present invention:
Application of CPC HID operon gene sequence differentiates that the novel method of good and bad production of spirurina strain compares 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 that 4 strains are used to make up the tenaculat Habenaria strain of production traits judging standard;
Fig. 2 is for being differentiated the classification figure of algae strain Sp-10 in building judging standard.
Embodiment
Detailed technology scheme of the present invention can be implemented through following steps:
1, select materials: the 4 strain spirulina plalensis strains that are used to make up judging standard are known Sp-1, Sp-2, Sp-3 and Sp-5 (also there is preservation in Zhejiang University's nucleus research of agricultural science institute's Biological resources and molecular engineering laboratory); In of the laboratory culture test of environment such as temperature, illumination, all show good because of the ability auto-control; Wherein Sp-3 and Sp-5 are strong to adaptive capacity to environment, in scale operation is cultured, are widely used; And Sp-1 and Sp-2 can not be as producing breeding because of the adaptive faculty difference.
2, reagent of selecting for use 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 products; Dna gel reclaims test kit and purchases the Ying Jun company in Shanghai; The PCR primer is synthetic by Shanghai Ying Jun company.Other reagent is analytical pure.Sequence amplification is with the Thermal Cycler PCR appearance of U.S. Hybaid company, and order-checking is with 3730 type sequenators of American AB I company.
3, PCR design of primers and amplification
Utilize Primer 5.0 primer-design softwares to obtain the upstream and downstream primer and be respectively PF:5 '-CAATACATCTTCGCCGATTT-3 ', PR:5 '-CGTATTATCGGTAGTCATCGG-3 '.Contain each 0.5 μ mol/L of primer PF and PR in the 25 uLPCR 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; 50ng genomic dna [process for extracting is with reference to " Oceanologia et Limnologia Sinica ", 2002,33 (2): 203-208].Response procedures: 94 ℃ of 5min; 94 ℃ of 30s, 58 ℃ of 1min, 72 ℃ of 2min, 30 circulations; 72 ℃ of 8min.
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.
Record the cpcHID operon gene sequence such as the SEQNO1 of Sp-1, Sp-2, Sp-3 and Sp-5 genomic dna.
5, sequence alignment and phylogenetic tree make up
Clustal X 1.81 softwares are adopted in Sp-1, Sp-2, Sp-3 and the comparison of Sp-5 multiple sequence to SEQNO1, and the structure of its phylogenetic tree adopts Phylip 3.65 software packages.
Interpretation of result of the present invention
Analyze and the phylogenetic tree structure based on the difference number of sites of cpcHID operon gene sequence
The genomic dna of Sp-1, Sp-2, Sp-3 and Sp-5 is respectively through the specific primer pcr amplification and the clone of cpcHID operon gene, and records corresponding SEQNO1 sequence.
Utilization Clustal X 1.81 softwares are denoted as underscore to Sp-1, these two strains of Sp-2 and Sp-3 or Sp-5 base difference site between any two among the multiple sequence compare of analysis result of the cpcHID operon sequence of above-mentioned 4 strain tenaculat Habenaria strains;
The same loci of the cpcHID operon sequence of 4 strains of " * " expression is shown in SEQNO2.
Statistics obtains the difference number of sites of base between strain.
Can know that by table 1 the difference number of sites between Sp-1 and the Sp-2 is 0; Difference number of sites between Sp-3 and the Sp-5 also is merely 4; Yet Sp-1 or Sp-2 and Sp-3 and Sp-5 difference number of sites between any two is for being respectively 58 and 59.Further the multiple sequence comparison result of the cpcHID operon gene sequence of above-mentioned 4 strain strains is imported the dnamlk program construction phylogenetic tree of Phylip 3.65 software packages.The result is shown in accompanying drawing 1, and 4 strain strains can be divided into two big types: Sp-3 and Sp-5 are another kind of; Sp-1 and Sp-2 are one type, and the result of this and the analysis of above-mentioned difference number of sites matches.Secular experimental study practice shows; These 4 strain spirulina plalensis article tie up to environmental factors such as temperature, illumination and all show in can the laboratory culture test of auto-controls good; But have only these two strains article of Sp-3 and Sp-5 tie up to actual production culture in performance good, Sp-1 and Sp-2 can not be as the production breedings because of the adaptive faculty difference.According to above-mentioned; There are dependency in the cpcHID operon gene sequence signature and the tenaculat Habenaria production traits; Therefore; The cpcHID operon gene can be used as the molecule marker of differentiating that the tenaculat Habenaria production traits is good and bad, promptly differentiated strain cpcHID operon gene sequence if with gather into one type of Sp-3 and Sp-5, then the possibility production traits is good; If with gather into one type of Sp-1, Sp-2, then not can be applicable to produce.
The difference number of sites of the cpcHID operon gene sequence of table 14 strain strains
| The algae strain | Sp-1 | Sp-2 | Sp-3 | Sp-5 |
| Sp-1 Sp-2 Sp-3 Sp-5 | 0 0 58 59 | 0 58 59 | 0 4 | 0 |
As embodiment, we have chosen this strain strain of Sp-10 and have verified practicality of the present invention.Though Sp-10 performance in laboratory culture is tested is good, in actual production, can not be used as the production breeding owing to bad adaptability.CpcHID operon gene sequence such as SEQNO3 through clone and mensuration Sp-10.
Utilize analyses such as information biology and molecular system taxonomy to show, the cpcHID operon gene sequence of this strain and gather into one type of Sp-1 and Sp-2, as shown in Figure 2.This example further specifies, and it is feasible that the cpcHID operon gene is compared the quality of differentiating the tenaculat Habenaria production traits as a kind of molecule marker.Prove that discrimination method of the present invention can realize.
SEQNO1：
The cpcHID operon gene sequence of Sp-1
1 CAATACATCT TCGCCGATTT CTCCAGCCGG GAGCGGACAA GCTCTGGTGG GGGCTAATGG
61 TGGCTCGCGC GGTCAATTGT ATCGTGTGGT AGTAGTACAA AAGCCCACAC AGTTGACACC
121 GAGAATGCGT AAAGCTACAG CAGAGTATAC AGTAGCCTAC GAGCAACTTT CAGGACAGTT
181 GCAGCGGATC AACCGCATGG GCGGACGGGT CATCAGCGTG ACCCCTGCTT AGAACAGCAA
241 GTTGTTAACT GTTAACCAGT TAATAACAAT AGCATAATCA AGTTAGATTG TCTAGGACTA
301 AATCCGCAAG TAATTCGGTT GTGATCATAG GAGGATTTTA GTGGCCATTA CAACCCAAGC
361 GTCTCGGTTA GGGACAACAG CTTTCCAGGA GAGTTCTCTG GTAGAGTTGC GCCCCAATTG
421 GAGTCGTGAC AATGCCCAAG AGGTAATTCG TGCGGTATAC CGTCAATTAC TGGGTAATGA
481 TTATCTGATG AGTTCTGAGC GCCTCACGAG TGCGGAGTCA CTGTTGTGCG ATGGTAGCAT
541 CACAGTACGG GAGTTGGTGC GGTGTGTGGC GAAGTCAGAG CTTTATAAAA AGAAATTCTT
601 TTACCCGAAT TTCCAGACCC GTGTAATTGA ACTAAATTAC AAACACCTGT TGGGTCGGGC
661 TCCCTATGAT GAGTCAGAGG TGGTTTTCCA CTTAGACTTA TATCAAAACG AAGGGTACGA
721 TGCCGATATC GATTCTTATA TTGATTCTCC AGAATATCTG GAGAGCTTCG GCGAAAATAT
781 TGTTCCCTAC TACCGTGGTT TCGAGACTCA GCGGGGACAG AAAACGGTTG GTTTTACCCG
841 GATGTTCCGC CTGTATCGGG GTTATGCTAA TAGTGATAGC GCTCAAGGTG AAGGCACTAT
901 GTCTCGTCTG GCGCAGGAAC TAGGTGCTAA CCGCGCTTCT GTGGTAGTGG CTCCTTCTGG
961 GGATTCGGAT GGTTGGTCCT ATCGTCCATC TGGACAGAGT GTGGCTCCTA GCACTGGATT
1021 CAGCCAAGGT GGAGTTCTGC AAGCGGGACG GACTTACCGT GTAGAGGTTT CTGGAATCCG
1081 TGAGCGCCGT TATCCCAGAG TACGTCGTTG CAGTAAGGCT TTTATTGTTC CTTATGATCA
1141 GCTTTCGGCA CAACTGAGGG AAATTCAGCG CCAAGGTGGC ACAATTGCCA GCATTACGCC
1201 TATTTAGAAT GGCAAGAGAA AACTTGATTG TTTTGGGAGA TTAATAGGGA CAATGTTGGG
1261 TTACATCAGT GCCGGAAAAA TGGGCAGTAG TCCTTCGGGC GCTCGCTTCT TCAAATATGA
1321 AGTGGTGGGA ATGCGTCAGA ATGACGAAAC CGACAAGACA GAATATCAGA TTCGCTCGAG
1381 TGGGAGTCAA TTTGTAATTG TTCCTTACAA TAGGATGAAT CAGGAAATGC GGCGGATTAA
1441 CCGCATGGGT GGTAAAATTG TTAGCATTGA GCCGATGACT ACCGATAATA CG
The cpcHID operon gene sequence of Sp-2
1 CAATACATCT TCGCCGATTT CTCCAGCCGG GAGCGGACAA GCTCTGGTGG GGGCTAATGG
61 TGGCTCGCGC GGTCAATTGT ATCGTGTGGT AGTAGTACAA AAGCCCACAC AGTTGACACC
121 GAGAATGCGT AAAGCTACAG CAGAGTATAC AGTAGCCTAC GAGCAACTTT CAGGACAGTT
181 GCAGCGGATC AACCGCATGG GCGGACGGGT CATCAGCGTG ACCCCTGCTT AGAACAGCAA
241 GTTGTTAACT GTTAACCAGT TAATAACAAT AGCATAATCA AGTTAGATTG TCTAGGACTA
301 AATCCGCAAG TAATTCGGTT GTGATCATAG GAGGATTTTA GTGGCCATTA CAACCCAAGC
361 GTCTCGGTTA GGGACAACAG CTTTCCAGGA GAGTTCTCTG GTAGAGTTGC GCCCCAATTG
421 GAGTCGTGAC AATGCCCAAG AGGTAATTCG TGCGGTATAC CGTCAATTAC TGGGTAATGA
481 TTATCTGATG AGTTCTGAGC GCCTCACGAG TGCGGAGTCA CTGTTGTGCG ATGGTAGCAT
541 CACAGTACGG GAGTTGGTGC GGTGTGTGGC GAAGTCAGAG CTTTATAAAA AGAAATTCTT
601 TTACCCGAAT TTCCAGACCC GTGTAATTGA ACTAAATTAC AAACACCTGT TGGGTCGGGC
661 TCCCTATGAT GAGTCAGAGG TGGTTTTCCA CTTAGACTTA TATCAAAACG AAGGGTACGA
721 TGCCGATATC GATTCTTATA TTGATTCTCC AGAATATCTG GAGAGCTTCG GCGAAAATAT
781 TGTTCCCTAC TACCGTGGTT TCGAGACTCA GCGGGGACAG AAAACGGTTG GTTTTACCCG
841 GATGTTCCGC CTGTATCGGG GTTATGCTAA TAGTGATAGC GCTCAAGGTG AAGGCACTAT
901 GTCTCGTCTG GCGCAGGAAC TAGGTGCTAA CCGCGCTTCT GTGGTAGTGG CTCCTTCTGG
961 GGATTCGGAT GGTTGGTCCT ATCGTCCATC TGGACAGAGT GTGGCTCCTA GCACTGGATT
1021 CAGCCAAGGT GGAGTTCTGC AAGCGGGACG GACTTACCGT GTAGAGGTTT CTGGAATCCG
1081 TGAGCGCCGT TATCCCAGAG TACGTCGTTG CAGTAAGGCT TTTATTGTTC CTTATGATCA
1141 GCTTTCGGCA CAACTGAGGG AAATTCAGCG CCAAGGTGGC ACAATTGCCA GCATTACGCC
1201 TATTTAGAAT GGCAAGAGAA AACTTGATTG TTTTGGGAGA TTAATAGGGA CAATGTTGGG
1261 TTACATCAGT GCCGGAAAAA TGGGCAGTAG TCCTTCGGGC GCTCGCTTCT TCAAATATGA
1321 AGTGGTGGGA ATGCGTCAGA ATGACGAAAC CGACAAGACA GAATATCAGA TTCGCTCGAG
1381 TGGGAGTCAA TTTGTAATTG TTCCTTACAA TAGGATGAAT CAGGAAATGC GGCGGATTAA
1441 CCGCATGGGT GGTAAAATTG TTAGCATTGA GCCGATGACT ACCGATAATA CG
The cpcHID operon gene sequence of Sp-3
1 CAATACTTCT TCGCCGATTT GTCCAGCAGG TAGCGGACAG GCTCTGGTGG GGGCCAATGG
61 TGGCGATCGC GGTCAATTAT ATCGTGTGGT AGTAGTACAA AAACCCACGC AGTTGACACC
121 GAGAATGCGT AAAGCTACAG CAGAGTACAC AGTAGCCTAC GAGCAACTTT CAGGACAGTT
181 GCAGCGGATC AACCGCATGG GCGGACGGGT CATCAGCGTG ACCCCTGCTT AGAACAGCAA
241 GTTGTTAACT GTTAACCAGT TAATAACAAT AGCATAATCA AGTTAGATTG TCTAGGACTA
301 AAGCCGCAAG TAATTCCGTT GTGATTATAG GAGGATTTAA GTGGCCATTA CAACCCAATC
361 GTCTCGGTTA GGGACAACAG CTTTCCAGGA GAGTTCTCCG GTAGAGTTGC GTCCCAATTG
421 GAGTCGTGAA AATGCCCAAG AGGTAATTCG TGCGGTATAC CGTCAATTGC TGGGTAATGA
481 TTATCTGATG AGTTCTGAGC GCCTCATCAG TGCGGAGTCG CTGTTGTGCG ATGGTAGCAT
541 CACTGTACGC GAATTTGTGC GGTGCGTGGC GAAGTCAGAA CTTTATAAAA AGAAATTCTT
601 TTACCCGAAT TTCCAGACCC GTGTAATTGA ACTAAATTAC AAACACCTGT TGGGTCGGGC
661 TCCCTATGAT GAGTCAGAGG TGGTTTTCCA CTTAGACTTA TACCAAAACG AAGGGTACGA
721 TGCCGATATC GATTCTTATA TTGATTCTCC AGAATATCTG GAGAGCTTCG GCGAGAATAT
781 TGTTCCTTAC TACCGTGGTT TCGAGACTCA GCGGGGACAA AAAACGGTTG GTTTCACCCG
841 GATGTTCCGT CTGTATCGGG GTTATGCTAA CAGTGATAGC GCCCAAGGTG AAGGCACTAT
901 GTCTCGTCTG GCGCAGGAAC TAGGTTCTAA CCGCGCTTCT GTGGTAGTGG CTCCTTCTGG
961 GGATTCGGAT GGTTGGTCCT ATCGTCCATC TGGACAGAGT GTAACTCCTA GCACTGGGTT
1021 CAGCCAAGGT GGAGTTCTGC AAGCGGGACG GACTTACCGT CTGGAGGTTT CTGGAATTCG
1081 TGAGCGCCGT TATCCCAGAG TACGTCGTTG CAGTAAGGCT TTCATTGTTC CTTATGAGCA
1141 GCTTTCGGCA CAACTGAGGG AAATTCAGCG CCAAGGTGGC ACAATTGCCA GCATTACGCC
1201 TATTTAGAAT GGCAAGAGAA AACTTGATTG TTTTGGGAGA TTAATAGGAA CAATGTTGGG
1261 TTACATCAGT GCTGGAAAAA TGGGGAGTAG TCCTTCGGGC GCTCGCTTCT TTAAATACGA
1321 AGTGGTGGGA ATACGTCAGA ATGACGAAAC CGACAAGAGT GAATATCAGA TTCGCTCGAG
1381 TGGGAGTCAA TTTGTAATTG TTCCTTACAA TCGGATGAAT CAGGAAATGC AGCGGATTAA
1441 CCGCATGGGT GGTAAAATTG TTAGCATTGA GCCGATGACT ACCGATAATA CG
The cpcHID operon gene sequence of Sp-5
1 CAATACATCT TCGCCGATTT GTCCAGCAGG TAGCGGACAG GCTCTGGTGG GGGCCAATGG
61 TGGCGATCGC GGTCAATTAT ATCGTGTGGT AGTAGTACAA AAACCCTCGC AGTTGACACC
121 GAGAATGCGT AAAGCTACAG CAGAGTACAC AGTAGCCTAC GAGCAACTTT CAGGACAGTT
181 GCAGCGGATC AACCGCATGG GCGGACGGGT CATCAGCGTG ACCCCTGCTT AGAACAGCAA
241 GTTGTTAACT GTTAACCAGT TAATAACAAT AGCATAATCA AGTTAGATTG TCTAGGACTA
301 AAGCCGCAAG TAATTCCGTT GTGATTATAG GAGGATTTAA GTGGCCATTA CAACCCAATC
361 GTCTCGGTTA GGGACAACAG CTTTCCAGGA GAGTTCTCCG GTAGAGTTGC GTCCCAATTG
421 GAGTCGTGAA AATGCCCAAG AGGTAATTCG TGCGGTATAC CGTCAATTGC TGGGTAATGA
481 TTATCTGATG AGTTCTGAGC GCCTCATCAG TGCGGAGTCG CTGTTGTGCG ATGGTAGCAT
541 CACTGTACGC GAATTTGTGC GGTGCGTGGC AAAGTCAGAA CTTTATAAAA AGAAATTCTT
601 TTACCCGAAT TTCCAGACCC GTGTAATTGA ACTAAATTAC AAACACCTGT TGGGTCGGGC
661 TCCCTATGAT GAGTCAGAGG TGGTTTTCCA CTTAGACTTA TACCAAAACG AAGGGTACGA
721 TGCCGATATC GATTCTTATA TTGATTCTCC AGAATATCTG GAGAGCTTCG GCGAGAATAT
781 TGTTCCTTAC TACCGTGGTT TCGAGACTCA GCGGGGACAA AAAACGGTTG GTTTCACCCG
841 GATGTTCCGT CTGTATCGGG GTTATGCTAA CAGTGATAGC GCCCAAGGTG AAGGCACTAT
901 GTCTCGTCTG GCGCAGGAAC TAGGTTCTAA CCGCGCTTCT GTGGTAGTGG CTCCTTCTGG
961 GGATTCGGAT GGTTGGTCCT ATCGTCCATC TGGACAGAGT GTACCTCCTA GCACTGGGTT
1021 CAGCCAAGGT GGAGTTCTGC AAGCGGGACG GACTTACCGT CTGGAGGTTT CTGGAATTCG
1081 TGAGCGCCGT TATCCCAGAG TACGTCGTTG CAGTAAGGCT TTCATTGTTC CTTATGAGCA
1141 GCTTTCGGCA CAACTGAGGG AAATTCAGCG CCAAGGTGGC ACAATTGCCA GCATTACGCC
1201 TATTTAGAAT GGCAAGAGAA AACTTGATTG TTTTGGGAGA TTAATAGGAA CAATGTTGGG
1261 TTACATCAGT GCTGGAAAAA TGGGGAGTAG TCCTTCGGGC GCTCGCTTCT TTAAATACGA
1321 AGTGGTGGGA ATACGTCAGA ATGACGAAAC CGACAAGAGT GAATATCAGA TTCGCTCGAG
1381 TGGGAGTCAA TTTGTAATTG TTCCTTACAA TCGGATGAAT CAGGAAATGC AGCGGATTAA
1441 CCGCATGGGT GGTAAAATTG TTAGCATTGA GCCGATGACT ACCGATAATA CG
SEQNO2：
Sp-1?1 CAATACATCTTCGCCGATTTCTCCAGCCGGGAGCGGACAAGCTCTGGTGGGGGCTAATGG
Sp-2 CAATACATCTTCGCCGATTTCTCCAGCCGGGAGCGGACAAGCTCTGGTGGGGGCTAATGG
Sp-3 CAATAC
TTCTTCGCCGATTT
GTCCAGC
AGG
TAGCGGACA
GGCTCTGGTGGGGGC
CAATGG
Sp-5 CAATACATCTTCGCCGATTT
GTCCAGC
AGG
TAGCGGACA
GGCTCTGGTGGGGGC
CAATGG
******?*************?******?**?********?**************?*****
Sp-1?61 TGGCTCGCGCGGTCAATTGTATCGTGTGGTAGTAGTACAAAAGCCCACACAGTTGACACC
Sp-2 TGGCTCGCGCGGTCAATTGTATCGTGTGGTAGTAGTACAAAAGCCCACACAGTTGACACC
Sp-3 TGGC
GATCGCGGTCAATT
ATATCGTGTGGTAGTAGTACAAAA
ACCCAC
GCAGTTGACACC
Sp-5 TGGC
GATCGCGGTCAATT
ATATCGTGTGGTAGTAGTACAAAA
ACCC
TC
GCAGTTGACACC
**** ***********?***********************?***?*?***********
Sp-1?121?GAGAATGCGTAAAGCTACAGCAGAGTATACAGTAGCCTACGAGCAACTTTCAGGACAGTT
Sp-2 GAGAATGCGTAAAGCTACAGCAGAGTATACAGTAGCCTACGAGCAACTTTCAGGACAGTT
Sp-3 GAGAATGCGTAAAGCTACAGCAGAGTA
CACAGTAGCCTACGAGCAACTTTCAGGACAGTT
Sp-5 GAGAATGCGTAAAGCTACAGCAGAGTA
CACAGTAGCCTACGAGCAACTTTCAGGACAGTT
***************************?*******************************
Sp-1?181?GCAGCGGATCAACCGCATGGGCGGACGGGTCATCAGCGTGACCCCTGCTTAGAACAGCAA
Sp-2 GCAGCGGATCAACCGCATGGGCGGACGGGTCATCAGCGTGACCCCTGCTTAGAACAGCAA
Sp-3 GCAGCGGATCAACCGCATGGGCGGACGGGTCATCAGCGTGACCCCTGCTTAGAACAGCAA
Sp-5 GCAGCGGATCAACCGCATGGGCGGACGGGTCATCAGCGTGACCCCTGCTTAGAACAGCAA
************************************************************
Sp-1?241?GTTGTTAACTGTTAACCAGTTAATAACAATAGCATAATCAAGTTAGATTGTCTAGGACTA
Sp-2 GTTGTTAACTGTTAACCAGTTAATAACAATAGCATAATCAAGTTAGATTGTCTAGGACTA
Sp-3 GTTGTTAACTGTTAACCAGTTAATAACAATAGCATAATCAAGTTAGATTGTCTAGGACTA
Sp-5 GTTGTTAACTGTTAACCAGTTAATAACAATAGCATAATCAAGTTAGATTGTCTAGGACTA
************************************************************
Sp-1?301?AATCCGCAAGTAATTCGGTTGTGATCATAGGAGGATTTTAGTGGCCATTACAACCCAAGC
Sp-2 AATCCGCAAGTAATTCGGTTGTGATCATAGGAGGATTTTAGTGGCCATTACAACCCAAGC
Sp-3 AA
GCCGCAAGTAATTC
CGTTGTGAT
TATAGGAGGATTT
AAGTGGCCATTACAACCCAA
TC
Sp-5 AA
GCCGCAAGTAATTC
CGTTGTGAT
TATAGGAGGATTT
AAGTGGCCATTACAACCCAA
TC
**?*************?********?************?*******************?*
Sp-1?361?GTCTCGGTTAGGGACAACAGCTTTCCAGGAGAGTTCTCTGGTAGAGTTGCGCCCCAATTG
Sp-2 GTCTCGGTTAGGGACAACAGCTTTCCAGGAGAGTTCTCTGGTAGAGTTGCGCCCCAATTG
Sp-3 GTCTCGGTTAGGGACAACAGCTTTCCAGGAGAGTTCTC
CGGTAGAGTTGCG
TCCCAATTG
Sp-5 GTCTCGGTTAGGGACAACAGCTTTCCAGGAGAGTTCTC
CGGTAGAGTTGCG
TCCCAATTG
**************************************?************?********
Sp-1?421?GAGTCGTGACAATGCCCAAGAGGTAATTCGTGCGGTATACCGTCAATTACTGGGTAATGA
Sp-2 GAGTCGTGACAATGCCCAAGAGGTAATTCGTGCGGTATACCGTCAATTACTGGGTAATGA
Sp-3 GAGTCGTGA
AAATGCCCAAGAGGTAATTCGTGCGGTATACCGTCAATT
GCTGGGTAATGA
Sp-5 GAGTCGTGA
AAATGCCCAAGAGGTAATTCGTGCGGTATACCGTCAATT
GCTGGGTAATGA
*********?**************************************?***********
Sp-1 481 ?TTATCTGATGAGTTCTGAGCGCCTCACGAGTGCGGAGTCACTGTTGTGCGATGGTAGCAT
Sp-2 TTATCTGATGAGTTCTGAGCGCCTCACGAGTGCGGAGTCACTGTTGTGCGATGGTAGCAT
Sp-3 TTATCTGATGAGTTCTGAGCGCCTCA
TCAGTGCGGAGTC
GCTGTTGTGCGATGGTAGCAT
Sp-5 TTATCTGATGAGTTCTGAGCGCCTCA
TCAGTGCGGAGTC
GCTGTTGTGCGATGGTAGCAT
************************** ***********?********************
Sp-1 541 ?CACAGTACGGGAGTTGGTGCGGTGTGTGGCGAAGTCAGAGCTTTATAAAAAGAAATTCTT
Sp-2 CACAGTACGGGAGTTGGTGCGGTGTGTGGCGAAGTCAGAGCTTTATAAAAAGAAATTCTT
Sp-3 CAC
TGTACG
CGA
ATT
TGTGCGGTG
CGTGGCGAAGTCAGA
ACTTTATAAAAAGAAATTCTT
Sp-5 CAC
TGTACG
CGA
ATT
TGTGCGGTG
CGTGGC
AAAGTCAGA
ACTTTATAAAAAGAAATTCTT
***?*****?**?**?********?*****?********?********************
Sp-1 601 ?TTACCCGAATTTCCAGACCCGTGTAATTGAACTAAATTACAAACACCTGTTGGGTCGGGC
Sp-2 TTACCCGAATTTCCAGACCCGTGTAATTGAACTAAATTACAAACACCTGTTGGGTCGGGC
Sp-3 TTACCCGAATTTCCAGACCCGTGTAATTGAACTAAATTACAAACACCTGTTGGGTCGGGC
Sp-5 TTACCCGAATTTCCAGACCCGTGTAATTGAACTAAATTACAAACACCTGTTGGGTCGGGC
***********************************************************
Sp-1 661 ?TCCCTATGATGAGTCAGAGGTGGTTTTCCACTTAGACTTATATCAAAACGAAGGGTACGA
Sp-2 TCCCTATGATGAGTCAGAGGTGGTTTTCCACTTAGACTTATATCAAAACGAAGGGTACGA
Sp-3 TCCCTATGATGAGTCAGAGGTGGTTTTCCACTTAGACTTATA
CCAAAACGAAGGGTACGA
Sp-5 TCCCTATGATGAGTCAGAGGTGGTTTTCCACTTAGACTTATA
CCAAAACGAAGGGTACGA
******************************************?*****************
Sp-1 721 ?TGCCGATATCGATTCTTATATTGATTCTCCAGAATATCTGGAGAGCTTCGGCGAAAATAT
Sp-2 TGCCGATATCGATTCTTATATTGATTCTCCAGAATATCTGGAGAGCTTCGGCGAAAATAT
Sp-3 TGCCGATATCGATTCTTATATTGATTCTCCAGAATATCTGGAGAGCTTCGGCGA
GAATAT
Sp-5 TGCCGATATCGATTCTTATATTGATTCTCCAGAATATCTGGAGAGCTTCGGCGA
GAATAT
******************************************************?*****
Sp-1 781 ?TGTTCCCTACTACCGTGGTTTCGAGACTCAGCGGGGACAGAAAACGGTTGGTTTTACCCG
Sp-2 TGTTCCCTACTACCGTGGTTTCGAGACTCAGCGGGGACAGAAAACGGTTGGTTTTACCCG
Sp-3 TGTTCC
TTACTACCGTGGTTTCGAGACTCAGCGGGGACA
AAAAACGGTTGGTTT
CACCCG
Sp-5 TGTTCC
TTACTACCGTGGTTTCGAGACTCAGCGGGGACA
AAAAACGGTTGGTTT
CACCCG
******?********************************?**************?*****
Sp-1 841 ?GATGTTCCGCCTGTATCGGGGTTATGCTAATAGTGATAGCGCTCAAGGTGAAGGCACTAT
Sp-2 GATGTTCCGCCTGTATCGGGGTTATGCTAATAGTGATAGCGCTCAAGGTGAAGGCACTAT
Sp-3 GATGTTCCG
TCTGTATCGGGGTTATGCTAA
CAGTGATAGCGC
CCAAGGTGAAGGCACTAT
Sp-5 GATGTTCCG
TCTGTATCGGGGTTATGCTAA
CAGTGATAGCGC
CCAAGGTGAAGGCACTAT
*********?********************?***********?*****************
Sp-1 901 ?GTCTCGTCTGGCGCAGGAACTAGGTGCTAACCGCGCTTCTGTGGTAGTGGCTCCTTCTGG
Sp-2 GTCTCGTCTGGCGCAGGAACTAGGTGCTAACCGCGCTTCTGTGGTAGTGGCTCCTTCTGG
Sp-3 GTCTCGTCTGGCGCAGGAACTAGGT
TCTAACCGCGCTTCTGTGGTAGTGGCTCCTTCTGG
Sp-5 GTCTCGTCTGGCGCAGGAACTAGGT
TCTAACCGCGCTTCTGTGGTAGTGGCTCCTTCTGG
*************************?**********************************
Sp-1 961 ?GGATTCGGATGGTTGGTCCTATCGTCCATCTGGACAGAGTGTGGCTCCTAGCACTGGATT
Sp-2 GGATTCGGATGGTTGGTCCTATCGTCCATCTGGACAGAGTGTGGCTCCTAGCACTGGATT
Sp-3 GGATTCGGATGGTTGGTCCTATCGTCCATCTGGACAGAGTGT
AACTCCTAGCACTGG
GTT
Sp-5 GGATTCGGATGGTTGGTCCTATCGTCCATCTGGACAGAGTGT
ACCTCCTAGCACTGG
GTT
******************************************?*************?**
Sp-1?1021?CAGCCAAGGTGGAGTTCTGCAAGCGGGACGGACTTACCGTGTAGAGGTTTCTGGAATCCG
Sp-2 CAGCCAAGGTGGAGTTCTGCAAGCGGGACGGACTTACCGTGTAGAGGTTTCTGGAATCCG
Sp-3 CAGCCAAGGTGGAGTTCTGCAAGCGGGACGGACTTACCGT
CT
GGAGGTTTCTGGAAT
TCG
Sp-5 CAGCCAAGGTGGAGTTCTGCAAGCGGGACGGACTTACCGT
CT
GGAGGTTTCTGGAAT
TCG
****************************************?*?**************?**
Sp-1?1081?TGAGCGCCGTTATCCCAGAGTACGTCGTTGCAGTAAGGCTTTTATTGTTCCTTATGATCA
Sp-2 TGAGCGCCGTTATCCCAGAGTACGTCGTTGCAGTAAGGCTTTTATTGTTCCTTATGATCA
Sp-3 TGAGCGCCGTTATCCCAGAGTACGTCGTTGCAGTAAGGCTTT
CATTGTTCCTTATGA
GCA
Sp-5 TGAGCGCCGTTATCCCAGAGTACGTCGTTGCAGTAAGGCTTT
CATTGTTCCTTATGA
GCA
******************************************?**************?**
Sp-1?1141?GCTTTCGGCACAACTGAGGGAAATTCAGCGCCAAGGTGGCACAATTGCCAGCATTACGCC
Sp-2 GCTTTCGGCACAACTGAGGGAAATTCAGCGCCAAGGTGGCACAATTGCCAGCATTACGCC
Sp-3 GCTTTCGGCACAACTGAGGGAAATTCAGCGCCAAGGTGGCACAATTGCCAGCATTACGCC
Sp-5 GCTTTCGGCACAACTGAGGGAAATTCAGCGCCAAGGTGGCACAATTGCCAGCATTACGCC
***********************************************************
Sp-1?1201?TATTTAGAATGGCAAGAGAAAACTTGATTGTTTTGGGAGATTAATAGGGACAATGTTGGG
Sp-2 TATTTAGAATGGCAAGAGAAAACTTGATTGTTTTGGGAGATTAATAGGGACAATGTTGGG
Sp-3 TATTTAGAATGGCAAGAGAAAACTTGATTGTTTTGGGAGATTAATAGG
AACAATGTTGGG
Sp-5 TATTTAGAATGGCAAGAGAAAACTTGATTGTTTTGGGAGATTAATAGG
AACAATGTTGGG
*?***********************************************?***********
Sp-1?1261?TTACATCAGTGCCGGAAAAATGGGCAGTAGTCCTTCGGGCGCTCGCTTCTTCAAATATGA
Sp-2 TTACATCAGTGCCGGAAAAATGGGCAGTAGTCCTTCGGGCGCTCGCTTCTTCAAATATGA
Sp-3 TTACATCAGTGC
TGGAAAAATGGG
GAGTAGTCCTTCGGGCGCTCGCTTCTT
TAAATA
CGA
Sp-5 TTACATCAGTGC
TGGAAAAATGGG
GAGTAGTCCTTCGGGCGCTCGCTTCTT
TAAATA
CGA
************?***********?**************************?*****?**
Sp-1?1321?AGTGGTGGGAATGCGTCAGAATGACGAAACCGACAAGACAGAATATCAGATTCGCTCGAG
Sp-2 AGTGGTGGGAATGCGTCAGAATGACGAAACCGACAAGACAGAATATCAGATTCGCTCGAG
Sp-3 AGTGGTGGGAAT
ACGTCAGAATGACGAAACCGACAAGA
GTGAATATCAGATTCGCTCGAG
Sp-5 AGTGGTGGGAAT
ACGTCAGAATGACGAAACCGACAAGA
GTGAATATCAGATTCGCTCGAG
************?*************************?********************
Sp-1?1381?TGGGAGTCAATTTGTAATTGTTCCTTACAATAGGATGAATCAGGAAATGCGGCGGATTAA
Sp-2 TGGGAGTCAATTTGTAATTGTTCCTTACAATAGGATGAATCAGGAAATGCGGCGGATTAA
Sp-3 TGGGAGTCAATTTGTAATTGTTCCTTACAAT
CGGATGAATCAGGAAATGC
AGCGGATTAA
Sp-5 TGGGAGTCAATTTGTAATTGTTCCTTACAAT
CGGATGAATCAGGAAATGC
AGCGGATTAA
*******************************?******************?*********
Sp-1?1441?CCGCATGGGTGGTAAAATTGTTAGCATTGAGCCGATGACTACCGATAATACG
Sp-2 CCGCATGGGTGGTAAAATTGTTAGCATTGAGCCGATGACTACCGATAATACG
Sp-3 CCGCATGGGTGGTAAAATTGTTAGCATTGAGCCGATGACTACCGATAATACG
Sp-5 CCGCATGGGTGGTAAAATTGTTAGCATTGAGCCGATGACTACCGATAATACG
***************************************************
SEQNO3：
The cpcHID operon gene sequence of Sp-10
1 CAATACATCT TCGCCGATTT CTCCAGCCGG GAGCGGACAA GCTCTGGTGG GGGCTAATGG
61 TGGCTCGCGC GGTCAATTGT ATCGTGTGGT AGTAGTACAA AAGCCCACAC AGTTGACACC
121 GAGAATGCGT AAAGCTACAG CAGAGTATAC AGTAGCCTAC GAGCAACTTT CAGGACAGTT
181 GCAGCGGATC AACCGCATGG GCGGACGGGT CATCAGCGTG ACCCCTGCTT AGAACAGCAA
241 GTTGTTAACT GTTAACCAGT TAATAACAAT AGCATAATCA AGTTAGATTG TCTAGGACTA
301 AATCCGCAAG TAATTCGGTT GTGATCATAG GAGGATTTTA GTGGCCATTA CAACCCAAGC
361 GTCTCGGTTA GGGACAACAG CTTTCCAGGA GAGTTCTCTG GTAGAGTTGC GCCCCAATTG
421 GAGTCGTGAC AATGCCCAAG AGGTAATTCG TGCGGTATAC CGTCAATTAC TGGGTAATGA
481 TTATCTGATG AGTTCTGAGC GCCTCACGAG TGCGGAGTCA CTGTTGTGCG ATGGTAGCAT
541 CACAGTACGG GAGTTGGTGC GGTGTGTGGC GAAGTCAGAG CTTTATAAAA AGAAATTCTT
601 TTACCCGAAT TTCCAGACCC GTGTAATTGA ACTAAATTAC AAACACCTGT TGGGTCGGGC
661 TCCCTATGAT GAGTCAGAGG TGGTTTTCCA CTTAGACTTA TATCAAAACG AAGGGTACGA
721 TGCCGATATC GATTCTTATA TTGATTCTCC AGAATATCTG GAGAGCTTCG GCGAAAATAT
781 TGTTCCCTAC TACCGTGGTT TCGAGACTCA GCGGGGACAG AAAACGGTTG GTTTTACCCG
841 GATGTTCCGC CTGTATCGGG GGTATGCTAA TAGTGATAGC GCTCAAGGTG AAGGCACTAT
901 GTCTCGTCTG GCGCAGGAAC TAGGTGCTAA CCGCGCTTCT GTGGTAGTGG CTCCTTCTGG
961 GGATTCGGAT GGTTGGTCCT ATCGTCCATC TGGACAGAGT GTGGCTCCTA GCACTGGATT
1021 CAGCCAAGGT GGAGTTCTGC AAGCGGGACG GACTTACCGT GTAGAGGTTT CTGGAATCCG
1081 TGAGCGCCGT TATCCCAGAG TACGTCGTTG CAGTAAGGCT TTTATTGTTC CTTATGATCA
1141 GCTTTCGGCA CAACTGAGGG AAATTCAGCG CCAAGGTGGC ACAATTGCCA GCATTACGCC
1201 TATTTAGAAT GGCAAGAGAA AACTTGATTG TTTTGGGAGA TTAATAGGGA CAATGTTGGG
1261 TTACATCAGT GCCGGAAAAA TGGGCAGTAG TCCTTCGGGC GCTCGCTTCT TCAAATATGA
1321 AGTGGTGGGA ATGCGTCAGA ATGACGAAAC CGACAAGACA GAATATCAGA TTCGCTCGAG
1381 TGGGAGTCAA TTTGTAATTG TTCCTTACAA TAGGATGAAT CAGGAAATGC GGCGGATTAA
1441 CCGCATGGGT GGTAAAATTG TTAGCATTGA GCCGATGACT ACCGATAATA CG
Claims (1)
1. a method of differentiating good and bad production of spirurina strain is characterized in that 4 strain spirulina plalensis strain Sp-1, Sp-2, Sp-3 and Sp-5 have the cpcHID operon gene sequence of SEQNO1 respectively; To be differentiated the cpcHID operon gene sequence of tenaculat Habenaria strain and the quality that above-mentioned 4 sequence alignments are differentiated its production traits;
This method specifically adopts the following operations step:
(1) select materials: choose Sp-1, Sp-2, Sp-3, Sp-5 in the 4 strain spirulina plalensis strains, wherein Sp-3 and Sp-5 are strong to adaptive capacity to environment, in scale operation is cultured, are widely used; And Sp-1 and Sp-2 can not be as producing breeding because of the adaptive faculty difference;
(2) reagent of selecting for use 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, T4DNA ligase enzyme are Japanese TaKaRa Company products; Dna gel reclaims test kit and purchases the Ying Jun company in Shanghai; The PCR primer is synthetic by Shanghai Ying Jun company, and other reagent is analytical pure, and sequence amplification is with the Thermal Cycler PCR appearance of U.S. Hybaid company, and order-checking is with 3730 type sequenators of American AB I company;
(3) PCR design of primers and amplification
Utilize the Primer5.0 primer-design software to obtain upstream primer PF:5 '-CAATACATCTTCGCCGATTT-3 '; Downstream primer PR:5 '-CGTATTATCGGTAGTCATCGG-3 ' contains 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, 50ng genomic dna, response procedures: 94 ℃ of 5min; 94 ℃ of 30s, 58 ℃ of 1min, 72 ℃ of 2min, 30 circulations; 72 ℃ of 8min;
(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, will contain the segmental recombinant plasmid of purpose and check order, the cpcHID operon gene sequence that records Sp-1, Sp-2, Sp-3 and Sp-5 genomic dna is shown in SEQNO1;
(5) sequence alignment and phylogenetic tree make up
To above-mentioned Sp-1, Sp-2, Sp-3 and Sp-5 multiple sequence comparison adopt Clustal X 1.81 softwares; The structure of its phylogenetic tree adopts Phylip 3.65 software packages, compares the quality of differentiating the tenaculat Habenaria production traits as a kind of molecule marker with this cpcHID operon gene.
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