CN1528892A - Artrospira spirulina protein gene promotor and preparing method thereof - Google Patents
Artrospira spirulina protein gene promotor and preparing method thereof Download PDFInfo
- Publication number
- CN1528892A CN1528892A CNA2003101054161A CN200310105416A CN1528892A CN 1528892 A CN1528892 A CN 1528892A CN A2003101054161 A CNA2003101054161 A CN A2003101054161A CN 200310105416 A CN200310105416 A CN 200310105416A CN 1528892 A CN1528892 A CN 1528892A
- Authority
- CN
- China
- Prior art keywords
- promotor
- gene
- sequence
- primer
- beta
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a promotor for beta and alpha subunit gene of algae blue protein Jiexuan algae, the segment length of the DNA is: 427bp, the series description is: algae blue protein beta and alpha subunit gene use the same promotor. The series includes following functional factors- 10 district and 35 district main function districts and DOFCOREZM, EBOXBNNAPA, GT1CONSENSUS, GTGANTG10, IBOXCORE and so on. The distance length between the two function districts verifies that it is a high efficient promotor, thus it can realizes to rerecord the algae gene project with high efficient promotor, the outer gene expression upgrades the efficiency, thus obtains more improved gene product. The promotor is expressed with high efficiency when being used in promotor detecting carrier green fluorescence protein, it verifies that it is developable.
Description
Technical field:
The present invention relates to the improvement of algae genetic engineering technique, specifically is a kind of promotor and preparation method thereof of phycocyanin gene of artrospira spirulina.Be the promoter sequence and the preparation method thereof of artrospira spirulina Arthrospira platensis FACHB341 phycocyanin beta and α subunit gene.It belongs to molecular biology and biological technical field.
Background technology:
In the prior art, a kind of artrospira spirulina Arthrospira platensis is arranged, it belongs to blue algae, and it contains rich in protein.Phycocyanins, C-wherein is the chief component of a kind of important photosynthetic pigments of blue-green algae.Phycocyanins, C-is to be made of β and α subunit and chromophoric group.The gene order of two these subunits obtains (De Rossi E by De Rossi in clone in 1985, RiccardiG, Sanangelantoni AM and Ciferri O.Construction of cosmid library of Spirulina platensis as anapproach to DNA physical mapping.FEMS Microbiol.Letters, 1985,30:239.), but up to now, the upstream sequence of the gene order of two these subunits does not but have revealed, and it has influenced the engineered development of artrospira spirulina.Therefore, fully exploitation is the problem that modern algae genetically engineered faces with rationally utilizing artrospira spirulina, and how obtaining efficiently, the algae gene promoter is to hinder the key factor that the algae transgenosis is succeedd always.Simultaneously, the expression efficiency of foreign gene in transgenic plant is low is a ubiquity and problem demanding prompt solution.The promotor of bacterium and eukaryotic gene engineering efficient in the algae genetically engineered is lower, does not even work sometimes.This has just incured loss through delay the engineered development of algae.
Current, phycocyanin beta and α subunit blue-green algae development of molecular biology are laid a good foundation for the research of blue-green algae genetically engineered, have the foreign gene of introducing to clone in blue-green algae or express, to improve the research of blue-green algae hereditary quality; Also has the research that the blue-green algae gene is expressed in other expression system.There is research report to prove: edible artrospira spirulina safety non-toxic.This exploitation for the large-scale farming of artrospira spirulina and protective foods and medicine is laid a good foundation.It is found that artrospira spirulina not only has comprehensive nutritive value, also has multiple medical functions.As everyone knows, artrospira spirulina contains rich in protein, VITAMIN, unsaturated fatty acids, polysaccharide and is of value to the trace element of HUMAN HEALTH, and this algae is balanced in nutrition, safety non-toxic.
The present invention is on the basis of the gene order of above-mentioned artrospira spirulina phycocyanin beta of having reported and α subunit, designed degenerated primer Y1 (ttgatgcctt cactaaggtg g, 21bp) and Y2 (gtagtagcc (t/g) at (g/a) tcacgagc, 21bp), and carry out the polymerase chain reaction, through in 48 ℃ of-58 ℃ of scopes the test of renaturation temperature, amplification obtains the dna fragmentation of long 900bp under best renaturation temperature.Show through the sequencing result analysis: the structure of this gene fragment is as follows: 1-494bp is the cpcB gene; 495-605bp is a subunit transcribed spacer sequence (IGSB-A); 606-857bp is the cpcA gene, and this gene order has been submitted GenBank to, and the sequence registration number is: AY244668.The nucleotide sequencing result is as follows:
tttctcaagc?tgatactcgc?ggcgaaatgc?tgagtacagc?tcaaatcgat?gctctgagcc60
aaatggttgc?tgaaagcaac?aaacgtttgg?gttctgttaa?ccgcattacc?agcaacgctt120
ccaccattgt?ttccaacgct?gctcgttctt?tgttcgcaga?gcaaccccaa?ctgattgctc180
ccggtggaaa?cgcctacacc?agccgtcgta?tggctgcttg?cttgcgtgac?atggaaatca240
tcctgcgcta?tgtaacctac?gctgtgtttg?ctggcgatgc?aagtgttctc?gaagatcgtt300
gcttgaacgg?tttgcgtgaa?acttacctgg?ctttgggaac?tcccggttct?tccgttgctg360
tcggtgttgg?caaaatgaaa?gaagctgctc?tggcgatcgt?taacgatccc?gcaggtatca420
ctcctggtga?ttgtagcgct?ttggcttcag?aaatcgctgg?ttactttgac?cgtgctgctg480
cagcagtttc?ctaatcaagc?agatccatag?catataacaa?ttgaaacagt?ttagctgaag540
tctaagtgac?tggacttctg?tttgttacct?aattttttgt?aaaccaatcg?ggagataact600
cgagaatgaa?aaccccccta?accgaagcag?tttctatcgc?tgattcccaa?ggtcgtttcc660
taagcagcac?cgaaatccaa?gtagcttttg?gccgttttcg?tcaagccaaa?gctggtctgg720
aagctgctaa?agctctgacc?tctaaagctg?atagtctgat?cagtggtgct?gcccaagcag780
tgtacaacaa?gttcccctac?accacccaaa?tgcagggacc?taactacgcg?gcagaccaac840
gcggtaagga?caaatgt857
The inventor on the basis of the above work, the algae gene promoter has designed following technical scheme in order to develop efficiently.
Summary of the invention:
The present invention is from the algae genetically engineered, on the basis of above-mentioned work, expect the engineered expression product of algae, and this gene can be expressed, and the prerequisite of expressing at first will be transcribed, and this is transcribed and depends on promotor efficiently.Therefore, the objective of the invention is basic function structure according to gene, select and design a promotor of algae genetically engineered urgent need efficiently, finish the engineered transcription stage task of algae, expression of exogenous gene can be raised the efficiency, and then obtain the required gene product of more algae genetically engineered.The present invention will select and design artrospira spirulina (Arthrospira platensisFACHB341) phycocyanin beta that obtains and promotor of α subunit gene and preparation method thereof, and utilize artrospira spirulina to establish a good basis for improvement.
The objective of the invention is to be realized by following technical scheme, developed the promotor of the phycocyanin beta and the α subunit gene of artrospira spirulina, it is that length is the dna fragmentation in the operon sequence of phycocyanin gene of 1545bp.This dna fragmentation length is: 427bp, and its sequence description is: the promotor that phycocyanin beta and α subunit gene are shared, the sequence characterization of this promotor is:
tcaatatttt?aatgcttgta?ttgacccacg?gattcgctat?taattccctg?tcagaaaata?60
tacttagtta?ttaatactta?gttatttttc?ttaataaatt?ttaacaaacc?aaagggcgtt?120
ggctgtgtat?aaaggatatt?cgaagaggtg?agaaggaagg?cgaatgttga?tttatgaagt?180
ttgattaaca?tttgtatcaa?aatataaaat?tcttctcata?aaccctgtag?aatcttttaa?240
gatttcggaa?agtgttctag?gatactgaag?aaatgaacca?cggggcaatt?gttaaaagcc?300
tttgtcgatg?gttcgccccg?gaaggggtct?taggaggtga?caccgatgga?ttgattgtcg?360
tgatcattca?tggtgtgtcc?aatcccaact?caactctaag?caagtcaaca?agtaggagat?420
aaatcc?426
This sequence includes following functional factor: DOFCOREZM, EBOXBNNAPA, GT1CONSENSUS, GTGANTG10, IBOXCORE, MYBGAHV, MYBST1, POLASIG1, POLLEN1LELAT52, RAV1AAT, REALPHALGLHCB21, ROOTMOTIFTAPOX1, TAAAGSTKST1, WBOXATNPR1 ,-10 districts and-35 districts.
The factor that has the transcriptional control function in described-10 districts has: TATAAA, GATACTG, GATCATT and TCCAAT; Wherein the CAAT factor is positioned at the 2nd, 287,381 sites; Tata factor is positioned at the 39th, 70 and 83 site; The GATA factor is nuclear zinc fingers DNA, and it lays respectively at the 136th, 262,419 and 464 sites.
The factor that has the transcriptional control function in described-35 districts has: TTGATT, TTAAGA, GTGACA and TTGTCG.
Described Phycocyanins, C-operon sequence, it comprises three open reading frame:
1), be connected on the promotor back, length is the beta subunit gene of 519bp---cpcB, this gene is a structure gene, the polypeptide (β subunit) of 173 amino-acid residues of its coding;
2)---cpcB back, length be the transcribed spacer sequence of the 110bp---IGSB-A that, is connected on beta subunit gene;
3), being connected on the transcribed spacer sequence,---IGSB-A back, length be the α subunit gene of 489bp---cpcA, this gene is a structure gene, the polypeptide (α subunit) of 163 amino-acid residues of its coding.
The preparation method of the promotor of artrospira spirulina phycocyanin beta of the present invention and α subunit gene, this method is to be template with artrospira spirulina (Arthrospira platensis FACHB341) genome, adopt the method for chromosome walking, that is: with suitable restriction enzyme with genomic dna complete digestion (this experiment adopt Sau3AI restriction endonuclease), carry out ligation with the joint with corresponding restriction enzyme site (providing), carry out the PCR reaction first time with the joint primer with according to the primer that known region designs by test kit; Use a part of PCR product and the inboard primer (the inboard primer of joint is provided by test kit) of last amplification to carry out for the second time special DNA amplification again.The test kit that adopts in the present invention's experiment is that the precious biotech firm in Dalian produces: TaKaRa LAPCR
TMIn vitro Cloning Kit test kit.
Described PCR reaction, its experimental system is as follows:
DNA????????????????????????????????????1-5μl；
10 * PCR damping fluid (PCR buffer), 5 μ l;
Mg
2+(25mmol/L)????????????????????????5-8μl；
Deoxynucleoside triphosphate (dNTP10mmol/L) 2-5 μ l;
Primer C1 (50pmol/L) 0.5-3 μ l;
Or primer C2 (50pmol/L) 0.5-3 μ l;
Primer S1 (50pmol/L) 0.5-3 μ l;
Or primer S2 (50pmol/L) 0.5-3 μ l;
Archaeal dna polymerase (TaqE) 0.5-1 μ l;
Supply reaction system to 50 μ l with distilled water; Be equipped with corresponding experiment condition again and carry out pcr amplification.
Described primer C1, its sequence is: gtacatattg tcgttagaac gcgtaatacg actca;
Described primer C2, its sequence is: cgttagaacg cgtaatacga ctcactatag ggaga;
Described primer S1, its sequence is: tgcatcgcca gcaaacacag c;
Described primer S2, its sequence is: gagctgtact cagcatttcg cc.
Positively effect of the present invention is: owing to designed a kind of promotor of phycocyanin beta and α subunit gene of artrospira spirulina, it is that length is: 427bp, its sequence description is: the promotor that phycocyanin beta and α subunit gene are shared, the sequence table of this promotor is obtained so clear, complete, make when phycocyanin beta and α subunit gene transcription initiation,-10 districts that contact with the RNA polymerase specificity and-35 district's functional factors can promptly form mixture, and transcribe rapidly at the transcription initiation site place.This efficient of transcribing depends on the based composition that characterizes in the promotor of the present invention: as the factor with transcriptional control function of the present invention: TATAAA, and GATACTG, GATCATT and TCCAAT, wherein the CAAT factor is positioned at the 2nd, 287,381 sites; Tata factor is positioned at the 39th, 70 and 83 site; The GATA factor is nuclear zinc fingers DNA, and it lays respectively at the 136th, 262,419 and 464 sites; Functional factor with-35 districts: TTGATT, TTAAGA, GTGACA and TTGTCG have determined that to a great extent promotor of the present invention is a strong promoter efficiently.Prove that through the inventor applied chemistry method synthetic oligonucleotide primer induces first of TATA of 39-10 districts of promotor of the present invention and second bit base to undergo mutation, obtain 2 mutant (AATA, TGTA), by detecting the protein-bonded ability of mutant, the near and activity of indirect detection promotor.The gel retardation assasy result shows that the activity of this promotor changes to some extent.The ability of single base mutation binding protein precursor obviously descends, and instruction book base mutation body promoter activity descends.Mutation research shows, the base mutation in promotor-10 district and-35 districts, or the distance change between the different elements all can make the gene transcription activity be affected.
This promotor has complete-35 districts and-10 districts, the gap length of two functional zone holds itself out to be efficient promoter, this just can realize purpose of the present invention---finish the engineered transcription stage task of algae with strong promoter efficiently, make expression of exogenous gene improve efficient, and then more improved and utilize the required gene product of artrospira spirulina.
Accompanying drawing and embodiment:
Protection scope of the present invention not only is confined in the embodiments of the invention.
Fig. 1. the promotor body outer clone PCR principle schematic of the phycocyanin gene of artrospira spirulina
Fig. 2. the promotor of the phycocyanin gene of artrospira spirulina and the structural representation of operon
Referring to Fig. 1-2, Target DNA is artrospira spirulina (Arthrospira platensis FACHB341) genomic dna among Fig. 1, after Restriction Enzyme Digestion is Restriction Enzyme digestion (that select in our experiment is Sau3AI), the Cassette of the Sau3AI restriction enzyme site that has with test kit is that joint carries out after Ligation just is connected, as template, with PrimerC1 and S1, be primer C1 and S1, wherein C1 is that test kit has, S1 is that we design according to genomic dna sequence, carrying out 1st PCR is the polymerase chain reaction first time, a part of product of reaction carries out 2nd PCR as template, polymerase chain reaction for the second time just, the PrimerC2 of use and S2, i.e. primer C2 and S2, wherein C2 is that test kit has, S2 is that we design according to genomic dna sequence, and it is positioned at the inboard of S1, has guaranteed specific amplification.
1 is USSB phycocyanin beta subunit upstream sequence---promoter element among Fig. 2; 2 is cpcB phycocyanin beta subunit gene order; 3 is IGSB-A albumen β subunit and α subunit gene transcribed spacer sequence; 4 is cpcA Phycocyanins, C-α subunit gene sequence.
The preparation process of promotor of the present invention is:
The partial sequence design primer of the Phycocyanins, C-operon coding beta subunit gene that obtains according to amplification, at first the upstream sequence to the β subunit moves, and it is made up of the twice PCR reaction.The primer that first set reaction adopted is S1, C1; The primer that second reaction is adopted is S2 and C2.The cpcB gene that S1 and S2 clone according to previous step designed (seeing Table 1).
Table 1. experiment the primer of the present invention and Cassette sequence
| Primer | Sequence |
| ????Y1 | ????ttgatgcctt?cactaaggtg?g |
| ????Y2 | ????gtagtagcc(t/g)at(g/a)tcacgagc |
| ????C1 | ????gtacatattg?tcgttagaac?gcgtaatacg?actca |
| ????C2 | ????cgttagaacg?cgtaatacga?ctcactatag?ggaga |
| ????S1 | ????tgcatcgcca?gcaaacacag?c |
| ????S2 | ????gagctgtact?cagcatttcg?cc |
| ????PS1 | ????tccatcggtg?gcacctccta?ag |
| ????PS2 | ????tgccccgtgg?ttcatttctt?cag |
| ????Sau3AI ????Cassette | ????5′ho?gtacatattg?tcgttagaac?gcgtaatacg?actcactata?ggga ????3′catgtataac?agcaatcttg?cgcattatgc?tgagtgatat?ccctctag?oh?5′ |
The experimental system of table 2. promotor PCR reaction of the present invention design
| Or primer C2 (μ l) | ??0.5 | ??1 | ??1.5 | |||
| Primer S1 (μ l) | ??0.5 | ??1 | ????1.5 | |||
| Or primer S2 (μ l) | ??0.5 | ??1 | ??1.5 | |||
| TaqE(μl) | ??0.2 | ??0.2 | ??0.3 | ??0.3 | ????0.5 | ??0.5 |
The reaction conditions of twice PCR reaction is: 94 ℃ of pre-sex change 5min, and 94 ℃ of sex change 60s, 50 ℃ of renaturation 60s, 72 ℃ are extended 80s, 30 circulations, 72 ℃ are extended 10min after the loop ends.Finally obtain the promotor of efficient specific phycocyanin beta and α subunit gene.
The promoter element sequence that infer the upstream of table 3. Phycocyanins, C-operon cpcB gene
| Initiating terminal | Terminal | Promoter sequence |
| ??175 | ??220 | ?atgaagt ttg?attaacattt?gtatcaaaa t?ataaaattct?tctcataaa |
| ??233 | ??278 | ?atctt ttaa?gatttc ggaa?agtgttctag? gatactgaag?aaatgaacca?c |
| ??334 | ??379 | ?ggag gtgaca?ccgatggatt?gattgtcgt g?atcattcatg?gtgtgtccaa |
| ??350 | ??395 | ?gattga ttgt?cgtgatcatt?catggtgtg tccaatcccaa?ctcaactcta |
Annotate: transcription initiation site is the color burn italics in table.-10 districts that infer and-35 region sequence underscorings.
The upstream sequence that shows the cpcB gene through the promoter element analysis software contains promoter sequence, TATAAA wherein, and GATACTG, GATCATT and TCCAAT are-10 sequences, GATACTG and P
LSequence (GT
GATACTGAGCACA) identical; TTGATT wherein, TTAAGA, GTGACA and TTGTCG are-35 sequences (seeing Table 3 sequence underscoring).
In the reaction of α subunit downstream sequence amplification, the primer that is adopted is PS1, PS2, C1 and C2 (seeing Table 1).The sequence of PS1 and PS2 obtains according to the sequencing result of the cloned sequence of last time reaction, and the step is moved the complete sequence that the result has obtained the cpcA gene.
The above-mentioned sequence that obtains all is stitched together, and obtaining length is the promotor and the operon sequence (see figure 2) of the phycocyanin gene of 1545bp, comprising four open reading frame:
The cpcB gene of 519bp, the polypeptide (β subunit) of 173 amino-acid residues of its coding;
The cpcA gene of 489bp, the polypeptide (α subunit) of 163 amino-acid residues of coding;
The cpcB gene of 110bp and the transcribed spacer sequence of cpcA gene;
Upstream sequence---the promoter element of the cpcB gene of 427bp.
As shown in Figure 1: 1 is USSB phycocyanin beta subunit upstream sequence---promoter element; 2 is cpcB phycocyanin beta subunit gene order; 3 is IGSB-A albumen β subunit and α subunit gene transcribed spacer sequence; 4 is cpcA Phycocyanins, C-α subunit gene sequence.
The artrospira spirulina Phycocyanins, C-promoter element among the present invention and the gene order of operon are as follows:
The ribosome bind site (RBS) that the present invention infers is marked in this sequence
tcaatatttt?aatgcttgta?ttgacccacg?gattcgctat?taattccetg?tcagaaaata?60
tacttagtta?ttaatactta?gttatttttc?ttaataaatt?ttaacaaacc?aaagggcgtt?120
ggctgtgtat?aaaggatatt?cgaagaggtg?agaaggaagg?cgaatgttga?tttatgaagt?180
ttgattaaca?tttgtatcaa?aatataaaat?tcttctcata?aaccctgtag?aatcttttaa?240
gatttcggaa?agtgttctag?gatactgaag?aaatgaacca?cggggcaatt?gttaaaagcc?300
tttgtcgatg?gttcgccccg?gaaggggtct?taggaggtga?caccgatgga?ttgattgtcg?360
tgatcattca?tggtgtgtcc?aatcccaact?caactctaag?caagtcaaca?agta
ggagat?420
aaatcc?426??????????????????????????????????????????????RBS
→CPCB
atgtttgatg?ccttcaccaa?ggtggtttct?caagctgata?ctcgcggcga?aatgctgagt?486
acagctcaaa?tcgatgctct?gagccaaatg?gttgctgaaa?gcaacaaac?gtttggattc?546
tgttaaccgc?attaccagca?acgcttccac?cattgtttcc?aacgctgctc?gttctttgtt?606
cgcagagcaa?ccccaactga?ttgctcccgg?tggaaacgcc?tacaccagcc?gtcgtatggc?666
tgcttgcttg?cgtgacatgg?aaatcatcct?gcgctatgta?acctacgctg?tgtttgctgg?726
cgatgcaagt?gttctcgaag?atcgttgctt?gaacggtttg?cgtgaaactt?acctggcttt?786
gggaactccc?ggttcttccg?ttgctgtcgg?tgttggcaaa?atgaaagaag?ctgctctggc?846
gatcgttaac?gatcccgcag?gtatcactcc?tggtgattgt?agcgctttgg?cttcagaaat?906
cgctggttac?tttgaccgtg?ctgctgcagc?agtttcctaa?tcaagcagat?ccatagcata?966
taacaattga?aacagtttag?ctgaagtcta?agtgactgga?cttctgtttg?ttacctaatt?1026
ttttgtaaac?caatc
gggag?ataactcgag?a?1057
RBS
→CPCA
atgaaaaccc?ccctaaccga?agcagtttct?atcgctgatt?cccaaggtcg?tttcctaagc?1117
agcaccgaaa?tccaagtagc?ttttggccgt?tttcgtcaag?ccaaagctgg?tctggaagct?1177
gctaaagctt?tgacctctaa?agctgatagt?ctgatcagtg?gtgctgccca?agcagtgtac?1237
aacaagttcc?cctacaccac?ccaaatgcag?ggacctaact?acgcggcaga?ccaacgcggt?1297
aaggacaaat?gtgctcgtga?cataggctac?tacctgcgga?tggtaactta?ttgcctgatt?1357
gctggtggaa?ctggccccat?ggatgagtac?ctgattgccg?gtattgatga?aatcaaccgg?1417
actttcgagc?tttctccaag?ctggtacatt?gaagccctga?aatacatcaa?agctaaccac?1477
ggtttgtctg?gtgacgctgc?tgttgaagct?aactcctacc?tcgactacgc?gatcaacgcc?1537
ctgagctag?1546
In this sequence, the shared promoter element of beta subunit gene and α subunit gene.Some sequence in this promoter element is as-10 region sequences (TATAAA, GATACTG, GATCATT and TCCAAT) and-35 region sequences (TTGATT, TTAAGA, GTGACA and TTGTCG) have the function of transcriptional regulator, be that gene expression regulation is necessary, if disappearance causes the promotor inactivation.The 2nd, 287,381 of its middle and upper reaches is the CAAT site, and tata factor is positioned at the 39th, 70 and 83.The GATA factor is considered to examine the conjugated protein family of zinc fingers DNA, can regulate the specific target expression of gene, and they lay respectively at the 136th, 262,419 and 464.
Artrospira spirulina Arthrospira platensis FACHB341 phycocyanin beta and α subunit gene promotor that the present invention obtains are impelled the artrospira spirulina gene to efficiently express in other acceptors and are become possibility.
Promotor Application Example 4 of the present invention:
Promotor of the present invention is connected to the promotor carrier detection---the egfp grain, forward in the artrospira spirulina with conventional ultrasonic conversion method, observe the fluorescence situation after 24 hours.Concrete experimental design is as follows:
Upstream sequence according to the cpcB gene, design primer PS2 and PS3 again, the upstream from start codon of conventional PCR method amplification cpcB gene, this has removed the coding region of Phycocyanins, C-operon during to primer amplification, thereby the reading frame that has guaranteed the gfp gene in multiple clone site downstream on the promotor carrier detection is not destroyed.After promotor carrier detection green fluorescent protein reporter gene cut the flat end of generation with Restriction enzyme Sma I, self-control T carrier connected and adopts the connection test kit to carry out.Reaction system is at 16 ℃ of incubation 3hrs.Connect product transformed into escherichia coli (E.coli) DH5 α competent cell.With PCR reaction screening positive recombinant (PT).After the cultivation of positive colony bacterium colony, determine to be inserted as forward by sequencing.Extracting plasmid forwards in the artrospira spirulina with conventional ultrasonic conversion method, carry out fluoroscopic examination with moral home-made fluorescent microscope (AxiophotPhotomicroscope From Carl Zeiss HB050) after 24 hours, selected excitation wavelength is 488nm, and emission wavelength is 507nm.Through comparing with control group, laboratory sample group cell sends very strong green fluorescence.Measure its fluorescence intensity through flow cytometer, compare fluorescent with control group and greatly strengthen, the control group average fluorescent strength is 14.8, and laboratory sample group average fluorescent strength is 30.5.
The acquisition of artrospira spirulina Arthrospira platensis FACHB341 phycocyanin beta of the present invention and α subunit promotor, promoted the engineered development of algae, for algae germplasm improvement provides prerequisite, make foreign gene in artrospira spirulina or the artrospira spirulina gene at other acceptors---realized efficiently expressing in the bacillus coli DH 5 alpha.
Claims (8)
1, the promotor of a kind of phycocyanin beta of artrospira spirulina and α subunit gene, it is that length is the dna fragmentation in the operon sequence of phycocyanin gene of 1545bp, it is characterized in that: this dna fragmentation length is: 427bp, its sequence description is: the promotor that phycocyanin beta and α subunit gene are shared, and the sequence characterization of this promotor is:
tcaatatttt?aatgcttgta?ttgacccacg?gattcgctat?taattccctg?tcagaaaata?60
tacttagtta?ttaatactta?gttatttttc?ttaataaatt?ttaacaaacc?aaagggcgtt?120
ggctgtgtat?aaaggatatt?cgaagaggtg?agaaggaagg?cgaatgttga?tttatgaagt?180
ttgattaaca?tttgtatcaa?aatataaaat?tcttctcata?aaccctgtag?aatcttttaa?240
gatttcggaa?agtgttctag?gatactgaag?aaatgaacca?cggggcaatt?gttaaaagcc?300
tttgtcgatg?gttcgccccg?gaaggggtct?taggaggtga?caccgatgga?ttgattgtcg?360
tgatcattca?tggtgtgtcc?aatcccaact?caactctaag?caagtcaaca?agtaggagat?420
aaatcc?426。
2, according to the promotor of the phycocyanin beta and the α subunit gene of the described artrospira spirulina of claim 1, it is characterized in that: include following functional factor: DOFCOREZM, EBOXBNNAPA, GT1CONSENSUS, GTGANTG10, IBOXCORE, MYBGAHV, MYBST1, POLASIG1, POLLEN1LELAT52, RAV1AAT, REALPHALGLHCB21, ROOTMOTIFTAPOX1, TAAAGSTKST1, WBOXATNPR1 ,-10 districts and-35 districts in the sequence of described this promotor.
3, according to the promotor of the phycocyanin beta and the α subunit gene of the described artrospira spirulina of claim 2, it is characterized in that: the factor that has the transcriptional control function in described-10 districts has: TATAAA, GATACTG, GATCATT and TCCAAT; Wherein the CAAT factor is positioned at the 2nd, 287,381 sites; Tata factor is positioned at the 39th, 70 and 83 site; The GATA factor is nuclear zinc fingers DNA, and it lays respectively at the 136th, 262,419 and 464 sites.
4, according to the promotor of the phycocyanin beta and the α subunit gene of the described artrospira spirulina of claim 2, it is characterized in that: the factor that has the transcriptional control function in described-35 districts has: TTGATT, TTAAGA, GTGACA and TTGTCG.
5, according to the promotor of the phycocyanin beta and the α subunit gene of the described artrospira spirulina of claim 1, it is characterized in that: described Phycocyanins, C-operon sequence, it comprises three open reading frame:
1), be connected on the promotor back, length is the beta subunit gene of 519bp---cpcB, this gene is a structure gene, the polypeptide (β subunit) of 173 amino-acid residues of its coding;
2)---cpcB back, length be the transcribed spacer sequence of the 110bp---IGSB-A that, is connected on beta subunit gene;
3), being connected on the transcribed spacer sequence,---IGSB-A back, length be the α subunit gene of 489bp---cpcA, this gene is a structure gene, the polypeptide (α subunit) of 163 amino-acid residues of its coding.
6, according to the preparation method of the promotor of the phycocyanin beta of the described artrospira spirulina of claim 1 and α subunit gene, described this method is to be template with artrospira spirulina (Arthrospira platensis FACHB341) genome, adopt the method for chromosome walking, that is: with suitable restriction enzyme with the genomic dna complete digestion, with joint with corresponding restriction enzyme site, carry out ligation, it is characterized in that: this reaction is to have carried out PCR reaction for the first time with joint primer C1 with according to the primer S1 that known region designs; A part of PCR product and inboard primer C2, S2 with last amplification carries out for the second time special DNA amplification again; Described PCR reaction, its experimental system is as follows:
DNA?????????????????????????????1-5μl；
10 * PCR damping fluid (PCR buffer), 5 μ l;
Mg
2+(25mmol/L)?????????????????5-8μl；
Deoxynucleoside triphosphate (dNTP10mmol/L) 2-5 μ l;
Primer C1 (50pmol/L) 0.5-3 μ l;
Or primer C2 (50pmol/L) 0.5-3 μ l;
Primer S1 (50pmol/L) 0.5-3 μ l;
Or primer S2 (50pmol/L) 0.5-3 μ l;
Archaeal dna polymerase (TaqE) 0.5-1 μ l;
Supply reaction system to 50 μ l with distilled water; Be equipped with corresponding experiment condition again and carry out pcr amplification.
7, according to the preparation method of the promotor of the phycocyanin beta of the described artrospira spirulina of claim 6 and α subunit gene, it is characterized in that: described primer S1, its sequence is: tgcatcgcca gcaaacacag c;
8, according to the preparation method of the promotor of the phycocyanin beta of the described artrospira spirulina of claim 6 and α subunit gene, it is characterized in that: described primer S2, its sequence is: gagctgtact cagcatttcg cc.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200310105416 CN1283794C (en) | 2003-10-16 | 2003-10-16 | Artrospira spirulina protein gene promotor and preparing method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200310105416 CN1283794C (en) | 2003-10-16 | 2003-10-16 | Artrospira spirulina protein gene promotor and preparing method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1528892A true CN1528892A (en) | 2004-09-15 |
| CN1283794C CN1283794C (en) | 2006-11-08 |
Family
ID=34304333
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200310105416 Expired - Fee Related CN1283794C (en) | 2003-10-16 | 2003-10-16 | Artrospira spirulina protein gene promotor and preparing method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1283794C (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110734927A (en) * | 2019-11-07 | 2020-01-31 | 江南大学 | recombinant escherichia coli and application thereof in screening erythritol production strains |
-
2003
- 2003-10-16 CN CN 200310105416 patent/CN1283794C/en not_active Expired - Fee Related
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110734927A (en) * | 2019-11-07 | 2020-01-31 | 江南大学 | recombinant escherichia coli and application thereof in screening erythritol production strains |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1283794C (en) | 2006-11-08 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN1276082C (en) | Method for synthesizing C DNA | |
| CN101063104A (en) | Engineering bacterium producing 5-glycyl ethylformic acid and construction method thereof | |
| CN1974768A (en) | Transcriptional control element of human lung carcinoma cell NGAL gene promoter region | |
| CN1766109A (en) | Chinese prawn C-type agglutinin gene and its coded C-type agglutinin peptide and uses | |
| CN1283794C (en) | Artrospira spirulina protein gene promotor and preparing method thereof | |
| CN1880480A (en) | PCR method for detection of allele specific primer and use in detection of KLOTHO gene polymorphism | |
| CN1699573A (en) | V. alginolyticus outer-membrane protein W gene, process for preparing the same and uses thereof | |
| CN1300313C (en) | Clone of gene MAC30 related to pig backfat thickness and its use in mark-assisted selection | |
| CN1238378C (en) | An antibiotic peptide and its coded sequence and use | |
| CN1740322A (en) | Para-gene sequence for exogenous insertion vector of corn strain MON863 | |
| CN1171998C (en) | Antibacterial peptide gene of Chinese prawn and its colon technique | |
| CN1974593A (en) | Genome specificity amplifying primer and the method of labeling wheat alpha-prolamine therewith | |
| CN1257987C (en) | Molecule labelling method for specific PCR detection of genes related to chicken growth and egg-laying deseription by employing allelomorph and use thereof | |
| CN1818068A (en) | Construction of recombinant human leucocyte medium-2(125ser) expression carrier | |
| CN100342011C (en) | Clone of pig immunity character relative gene PSMC5 and its use | |
| CN101063132A (en) | Lateolabrax interleukins 8 gene order | |
| CN1869229A (en) | Perch interlenkin 8 gene sequence | |
| CN1715406A (en) | Cloning for pig immunity character relative gene PSMB6 and its use | |
| CN101063160A (en) | Sub-clone method for polygene fragment | |
| CN1273595C (en) | Meiosis gene of paddy, coded protein and application | |
| CN1710075A (en) | Wheat somatic cell hybrid analogous 13 glutelin sub-gene nucleic acid sequence and use | |
| CN1262657C (en) | Method for producing human leukemia inhibitory factor using transgenic plant | |
| CN1472219A (en) | Recombined human parathyroid hormone gene, its expression carrier and preparing method of recombined human parathyroid protein | |
| CN1737138A (en) | Pig ouclar muscles area character-related protein and its coding gene and uses | |
| CN1220775C (en) | ST13 gene 5' terminal promotion function zone sequence and use thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20061108 Termination date: 20151016 |
|
| EXPY | Termination of patent right or utility model |