CN1563068A - Encoding gene of synthetase for phytoene of Dushi salt alga - Google Patents
Encoding gene of synthetase for phytoene of Dushi salt alga Download PDFInfo
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- CN1563068A CN1563068A CN 200410026481 CN200410026481A CN1563068A CN 1563068 A CN1563068 A CN 1563068A CN 200410026481 CN200410026481 CN 200410026481 CN 200410026481 A CN200410026481 A CN 200410026481A CN 1563068 A CN1563068 A CN 1563068A
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- phytoene
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- synthetase
- psy
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Abstract
The invention provides a new gene of coded phytoene synthetase (PSY) separated from dunaliella saline, and can lay the foundation for utilizing gene recombination technology to obtain high-efficiency high-yield beta-carrotene engineering bacteria or making natural saline algae under the process of biological transformation to raise the yield of beta-carrotene.
Description
Technical field
The present invention relates to a kind of new gene, relate in particular to the new gene of separated coding phytoene synthetase (PSY) from Dunaliella salina (Dunaliellasaline).
Background technology
β-Hu Luobusu is the precursor of vitamin A in human body, is widely used in additive and the medicine and the healthcare products aspect of food, makeup.In the market to the demand of β-Hu Luobusu mainly by the blakeslea trispora fermentation, from the salt algae of culturing, directly extract and chemosynthesis satisfied, but still be that supply falls short of demand.Dunaliella salina (Dunaliella salina) is called for short the salt algae, is a kind of unicell green alga, can accumulate a large amount of β-Hu Luobusus under suitable condition, and the highest 10% of the dry weight that surpasses of its content is the best natural resource of product β-Hu Luobusu of generally acknowledging.Phytoene synthetase is the key enzyme in the β-Hu Luobusu metabolic process, its function is the synthetic phytoene of the geranyl geranyl tetra-sodium (GGPP) of catalysis 2 molecules, and phytoene is the precursor substance in the β-Hu Luobusu route of synthesis, is first colourless carotenoid molecule.Report is arranged even claim that this enzyme is the rate-limiting enzyme of whole pathways metabolism.About the PSY gene, obtained in tomato early than 1987 (GBAN A21360, L23424).Now isolated the plant of PSY gene and algae also have capsicum (GBAN X68017), Arabidopis thaliana (GBANAF009954, L25812), daffodil (GBAN X78814), muskmelon (GBANZ37543), corn (GBAN U32636) etc.
In the algae, now isolated this gene kind and had only pasteur Du algae (Dunaliellabardawil) (GBAN U91900) and Haematocoocus Pluvialls (Haematococcus pluvialis) (GBAN AF305430), and these two kinds of algaes have all only been reported the mRNA sequence of this gene.
Summary of the invention
The object of the present invention is to provide the dna sequence dna that from the Yan Shi Dunaliella salina, separates the gene complete of the phytoene synthetase that obtains.
The present invention by with two kinds of nearer algae (Dunaliella bardawil of salt algae sibship, Haematococcus pluvialis) and six kind of plant (Arabidopsis thaliana, Oryzasative, Tagetes erecta, Lycopersicon esculentum, Capsicum annuum, the aminoacid sequence of phytoene synthase gene Daucus carota) (PSY) is analyzed, infer possible conserved regions, directly design a pair of special primer in the position of conserved regions: upstream primer F:5 '-GAGTACGCCAAGACCTTCTA-3 ' according to the nucleotide sequence of Dunaliellabardawil; Downstream primer R:5 '-GTTGTCATAGTCATTCTTCTC-3 ' is a template with salt algae genomic dna, amplifies the partial sequence of salt algae PSY gene.And then obtain the total length of psy gene by the chromosome walking technology, design upstream walking primer UWR:5 '-GTGCGTGGCTTGAGCGGCTTAGAAATG-3 ' respectively according to the 5 ' end and the 3 ' terminal sequence of acquired PSY partial sequence; Downstream walking primer DWF:5 '-ATGGTCATCACAGCGGCTTGCTTCACAG-3 '; With the genomic dna that has connected Adaptor is template, the primer AP1 on the Adaptor that provides with test kit respectively with UWR, DWF pairing carrying out inhibition pcr amplification.Carry out the inhibition pcr amplification, and the back of checking order obtains PSY gene order (SEQ ID NO.1).
The acquisition of phytoene synthetase sequence in the salt algae obtains efficiently to produce the β-Hu Luobusu engineering bacteria or natural salt algae is carried out biogenic reworking to lay the foundation with the output that improves β-Hu Luobusu for utilizing gene recombination technology.
Brief description of drawings
The inhibition PCR schematic diagram that Fig. 1 adopts for the present invention.
The embodiment of invention
By with two kinds of nearer algae (Dunaliella bardawil of salt algae sibship, Haematococcus pluvialis) and six kind of plant (Arabidopsis thaliana, Oryzasative, Tagetes erecta, Lycopersicon esculentum, Capsicum annuum, the aminoacid sequence of phytoene synthase gene Daucus carota) (PSY) is analyzed, infer possible conserved regions, directly design a pair of special primer in the position of conserved regions: upstream primer F:5 '-GAGTACGCCAAGACCTTCTA-3 ' according to the nucleotide sequence of Dunaliellabardawil; Downstream primer R:5 '-GTTGTCATAGTCATTCTTCTC-3 ' is a template with salt algae genomic dna, amplifies the partial sequence of salt algae PSY gene.The PCR program is: 94 ℃ of pre-sex change 5min enter working cycle; 94 ℃ of sex change 45sec, 46 ℃ of annealing 40sec, 72 ℃ are extended 2min, and next the annealing temperature with 0.5 ℃ of every cycle down circulates 22 times, and last annealing temperature with 35 ℃ circulates 5 times, extends 10min with 72 ℃ after circulation is finished.Behind 1.5% agarose gel electrophoresis, reclaim 2 main amplified bands, and be connected respectively on the pMD18-Tvector, with the heat shock method recombinant plasmid is changed over to E.coli DH5 α at last.The upward marine Ke Kairui of trust company checks order simultaneously, and with BLAST sequence is carried out homology analysis, determines that wherein the band of 2.5kb is possible salt algae phytoene synthetase fragment.
Next obtain the total length of psy gene by the chromosome walking technology: chromosome walking carries out according to CLONTECH Laboratories company test kit specification sheets, use restriction enzyme EcoRV, Dra I, Pvu II and Stu I are respectively to salt algae genomic dna complete degestion, and enzyme is cut the purified back of product and is connected with the Adaptor that test kit provides.5 ' end and 3 ' terminal sequence according to acquired PSY partial sequence design upstream walking primer UWR:5 '-GTGCGTGGCTTGAGCGGCTTAGAAATG-3 ' respectively; Downstream walking primer DWF:5 '-ATGGTCATCACAGCGGCTTGCTTCACAG-3 '; With the genomic dna that has connected Adaptor is template, the primer AP1 on the Adaptor that provides with test kit respectively with UWR, DWF pairing carrying out inhibition pcr amplification.
So-called inhibition PCR is meant because Adaptor is a bit of double chain nucleotide with 3 ' recessed end, in going on foot the pcr amplification process of moving, there is the 3 ' end of a fraction of Adaptor can extend to form the flush end joint, in the result of amplification, will some be the template strand that comes out by the direct amplification of the primer on the joint like this, but not the purpose fragment that the specially primer of known array and joint primer amplification come out.Inhibition PCR is exactly by annealing temperature being arranged on certain scope and so on, guarantee that major part has the template formation panhandle structure of flush end joint, and do not combine with the joint primer, having reduced the segmental background amplification of non-purpose (as Fig. 1) PCR program is: 94 ℃ of sex change 2sec, with 72 ℃ serves as to anneal and elongating temperature 3min, so carries out 7 circulations; Serve as that annealing and elongating temperature carry out 32 circulations with 67 ℃ again; After finishing, circulation extends 4min with 67 ℃.Upstream and downstream is gone on foot the fragment of moving acquisition respectively entrust the order-checking of Beijing three rich polygala root biotechnology limited liability companys, splice the complete sequence of 3 times sequencing result acquisition PSY.The gene order of PSY is shown in SEQ ID.1.
Sequence .ST25.txt
SEQUENCE?LISTING
＜110〉South China Science ﹠ Engineering University
＜120〉gene of coding Dunaliella salina phytoene synthetase
<130>200403
<160>1
<170>PatentIn?version?3.1
<210>1
<211>2983
<212>DNA
＜213〉Dunaliella salina (Dunaliella salina)
<400>1
atgccctcca?cttccggtgc?gtcgcccttc?ctcccagcag?cgccagcact?tgccaggcgg 60
tgcagtcgcg?gccccaacgg?cagcagcagg?cggtgcagca?gggcggtgcc?agcctccagt 120
gtcagcagga?gcccgacggt?ggccgtgcag?gcgacgcttg?ccatgcccag?ccccgattcc 180
cagcgcctgc?gcctgcagca?gcagctgcag?cagcaagccc?agcagcagca?ggcccagcag 240
cagctgtccg?gcaaggacgt?ggagcaggca?gcgatgcagg?cgtgcatacg?gacagccacc 300
tccgtgcccc?cctcgtccgg?cgtgctggac?cccagcggcc?tgcgctggag?gggcggagcg 360
ctggaggcag?cgtacgagcg?gtgtggcgcg?gtgtgcaagg?agtacgccaa?gaccttctac 420
ctgggcacgc?agctcatgac?ccccgtccag?gcacgctgca?tctgggccat?ctacgtgtgg 480
tgcaggcgca?cggatgagct?ggtggatggc?cctaatgcct?ccaagatcac?accacaggtg 540
cgcgtgcggg?tgctgcggca?gcgcaggaga?tgatggggag?cgggcatctt?gcgtgggagc 600
atgcgcgcga?gcggttggtc?cagtgttagc?attcgtgtgt?gcacgctgtg?gccaatcctg 660
ctgcacagcc?agctgcagtg?aggagtgcag?atgcaagcag?ctgtgtgttg?ctcctggtgt 720
cacgcgtccg?gtggacatgt?ggacttagaa?atgggggtta?catttctaac?ccgctcaagc 780
cacgcacacc?tgagcattgc?cacacggtcg?ctcatgcacc?ctctgcatcc?cacactacct 840
gcgcatggaa?catctgcacg?gcatgaaggg?ttcattggca?ttgtgcactc?acatggtgca 900
Sequence .ST25.txt
ttccacactg?cctcctgcca?cacacacaca?tgcgctctct?gctgcactcg?tatctgattg 960
ccacgactcc?tcactgccaa?catgcaccag?cagcttcatg?ctcattcact?gtagtactgg 1020
agggggagca?tgcactcatg?gcccaactga?aggcacctgc?tgccaggaca?agccactgtg 1080
tactcccaac?agcagcaagg?gatgcagcac?cagctcctca?gcatcctcct?gttgtcataa 1140
tgccagggtt?ccagacaaat?gacacgcatg?catacacatg?cacgcgcatg?cacgcatcca 1200
tggacttgcg?cgcacgcgcc?aacgcacacc?cctccccccc?ttcacacgca?cacacaggcc 1260
ctggaccggt?gggaggagcg?gctaaacggc?gtcttccaag?gcaggcctta?cgacgtgctg 1320
gacgcagcgc?tgacagacac?catctccaag?ttccctctgg?aggtgcagcc?attccgtgac 1380
atgattgagg?gcatgcgcat?ggacctgttc?aagtcacggt?accagacgtt?tgacgagctg 1440
tatgagtact?gctatcgggt?agccgggact?gtgggcctga?tgaccgtgcc?tgtcatgggc 1500
atcgacccca?actacaaggt?gagtgggcat?atgcggaaac?tgagcaaggc?tttgccaacc 1560
tgctcctgga?atcaagggca?tggtattttt?caagtgggat?tgccttcgcc?gattccttga 1620
tgaatgattt?gaaggtacac?tcatcaaaag?gagggttgga?actggcgctg?cattcattca 1680
tgcgtggctg?ccgcatgctg?aagcactgca?accacgtgca?cttatgtgtc?cgggcagcca 1740
cctgctcctt?cgttgcaacc?acactgcaca?cacttgctca?agcactgcaa?ccacatgcac 1800
gcactcacag?ggccccttgg?acaaggtgta?ccgggctgcg?ctcgcgctgg?gcaccgccaa 1860
ccagctgaca?aacattctga?gagacgtagg?agaggacatt?cgggaacggg?accgcatcta 1920
cctgcccttg?gatgagctac?ggggagtttg?gcatctcgga?agatgaggta?tgtgtgcggc 1980
gtcaacgtcg?tttctcatct?catttcctcc?gcaaaactgc?ttgtgccgat?atcagcagta 2040
ggcctggcct?tgagagcctg?catttcatgc?agaggaggtc?tggaagggcg?gtttcacctg 2100
gcaggcttgt?gagagtgttt?cttatggtca?tcacagcggc?ttgcttcaca?gccctcaaca 2160
agcactctga?gcctgaccta?aactctttgt?tcccaccact?gccaactcgt?aaccacaggt 2220
gcgggcaggc?atccacaagc?cctcacaagg?caaggtcgac?gagcggtggc?gcaagttcat 2280
gaagttccag?atacagcggg?cgcgggagta?cttccaggag?gctgaggatg?gtgtggacta 2340
cttggacgtg?aaggcgcggt?ggcctgtgtg?gtgagtggca?gcagtagctg?tgtgtcttgc 2400
tttgttccct?tgaagggctt?ctctttggac?tgccggtgca?gcctgtgttc?agaggcgctc 2460
gtttcgaact?tccctctcct?gtgacttaca?ggaggtatct?cgaatgcttg?actgccatga 2520
atgtcgtgaa?ccagacagct?gagggcctgc?ctgcagaaat?gcaagcctgc?ctgcttaaat 2580
gaaaaagtgt?tctagccaaa?agatgatcca?tgctaggttc?acagctgcat?gttcatagcg 2640
agacagtcgc?tcctcctgaa?tccttcttat?ctcagcagct?gtgattgtgt?gacctcgcag 2700
ggctcgctgc?ccttcttggt?ggaatcacaa?aagcaagcgg?aattctcatg?ccgagagcgc 2760
agcaagcgct?ctgtaagcaa?tattaaacgg?actgtcgagt?attgaccctc?cctatgtcga 2820
Sequence .ST25.txt
ctgtcgcagg?tctgcgctca?tcttgtaccg?ccagatcctt?gatgtcattg?agaagaacga 2880
ctacgacaac?ttctccatgc?gtgcgtatgt?gtccaagtca?aagaagctgg?cctccctgcc 2940
cctggccttg?ctgcgtgcca?tgatgcccaa?gagcccacag?tag 2983
Claims (5)
1, the Nucleotide that has SEQ ID NO.1 sequence.
2, the described Nucleotide of claim 1 is characterized in that it separates from Dunaliella salina.
3, by the aminoacid sequence of the nucleotide coding of claim 1.
4, the expression vector that contains the described Nucleotide of claim 1.
5, the host cell that contains the described Nucleotide of claim 1.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410026481 CN1563068A (en) | 2004-03-15 | 2004-03-15 | Encoding gene of synthetase for phytoene of Dushi salt alga |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410026481 CN1563068A (en) | 2004-03-15 | 2004-03-15 | Encoding gene of synthetase for phytoene of Dushi salt alga |
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| CN 200410026481 Pending CN1563068A (en) | 2004-03-15 | 2004-03-15 | Encoding gene of synthetase for phytoene of Dushi salt alga |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101985623A (en) * | 2010-07-20 | 2011-03-16 | 华中农业大学 | Cloning and application of key gene Wo for controlling tomato hair generation |
| CN102154328A (en) * | 2011-01-14 | 2011-08-17 | 华南理工大学 | Lycopene engineering bacteria based on metabolic pathway of dunaliella salina and construction method |
| CN102260692A (en) * | 2011-07-08 | 2011-11-30 | 山东大学 | Phytoene dehydrogenase gene and application thereof |
-
2004
- 2004-03-15 CN CN 200410026481 patent/CN1563068A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101985623A (en) * | 2010-07-20 | 2011-03-16 | 华中农业大学 | Cloning and application of key gene Wo for controlling tomato hair generation |
| CN101985623B (en) * | 2010-07-20 | 2012-08-29 | 华中农业大学 | Cloning and application of key gene Wo for controlling tomato hair generation |
| CN102154328A (en) * | 2011-01-14 | 2011-08-17 | 华南理工大学 | Lycopene engineering bacteria based on metabolic pathway of dunaliella salina and construction method |
| CN102260692A (en) * | 2011-07-08 | 2011-11-30 | 山东大学 | Phytoene dehydrogenase gene and application thereof |
| CN102260692B (en) * | 2011-07-08 | 2012-08-15 | 山东大学 | Phytoene dehydrogenase gene and application thereof |
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