CN1670212A - Gene for coding phytoene dehydrogenase of Dunaliella salina - Google Patents
Gene for coding phytoene dehydrogenase of Dunaliella salina Download PDFInfo
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- CN1670212A CN1670212A CN 200510033238 CN200510033238A CN1670212A CN 1670212 A CN1670212 A CN 1670212A CN 200510033238 CN200510033238 CN 200510033238 CN 200510033238 A CN200510033238 A CN 200510033238A CN 1670212 A CN1670212 A CN 1670212A
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- dunaliella salina
- phytoene dehydrogenase
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Abstract
The invention provides genes for coding phytoene dehydrogenase of Dunaliella salina which is separated from Dunaliella salena, thus laying the groundwork for obtaining highly effective beta-carotene engineering bacterium through gene recombination technology or for increasing output of beta-carotene through biological reconstruction to the natural salt algae.
Description
Technical field
The present invention relates to a kind of new gene, relate in particular to the new gene of separated coding phytoene dehydrogenase (PDS) 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 is the precursor substance in the β-Hu Luobusu route of synthesis, is first colourless carotenoid molecule.The function of phytoene dehydrogenase is a catalysis phytoene generation successive dehydrogenation reaction, Lyeopene or its intermediate that formation can Cheng Huan.
In the green alga, the species of now having isolated this gene have pasteur Du algae (Dunaliellabardawil) (GBAN Y14807) and Haematocoocus Pluvialls (Haematococcus pluvialis) (GBAN X86783), and the just mRNA sequence of this gene of report.Also reported the est sequence of Chlamydomonas reinhardtii (Chlamydomonas reinhardtii) PDS in addition
Summary of the invention
The object of the present invention is to provide the cDNA sequence of from the Yan Shi Dunaliella salina, separating the phytoene dehydrogenase gene that obtains.
The present invention by with nearer three kinds of green algas (Dunaliella bardawil, the Haematococcus pluvialis of salt algae sibship; Chlamydomonas reinhardtii) and five kind of plant (Arabidopsis thaliana, zea mays, Lycopersicon esculentum, Capsicumannuum, Oryza sative), and the aminoacid sequence of the phytoene dehydrogenase gene (PDS) of uredo erwinia phage (Erwinia uredovora) is analyzed, infer possible conserved regions, directly according to 6 special primers of nucleotide sequence design of Dunaliella bardawil, utilize the method for RT-PCR to amplify the partial sequence of salt algae PDS cDNA in the position of conserved regions.And then obtain to comprise the upstream sequence of initiator codon by the RACE-PCR technology.
Brief description of drawings
The RACE-PCR schematic diagram that Fig. 1 adopts for the present invention.
The embodiment of invention
By with two kinds of nearer green alga (Dunaliella bardawil of salt algae sibship, Haematococcus pluvialis) and five kind of plant (Arabidopsis thaliana, zea mays, Lycopersicon esculentum, Capsicum annuum, the aminoacid sequence of phytoene dehydrogenase gene (PDS) Oryza sative) is analyzed, infer possible conserved regions, directly design following 6 special primers according to the nucleotide sequence of Dunaliella bardawil in the position of conserved regions:
Upstream primer: PDS921-F1,5 '-CTTTGGTGCTTACCCCAACA-3 ',
PDS921-F2，5’-GAGCAGGATGAGCTGAC-3’，
PDS921-F3，5’-TGGCGAAGGCCCTGAACT-3’；
Downstream primer: PDS921-R1,5 '-GCAGGAAACGGTTCAGTG-3 '
PDS921-R2，5’-CTTCATGATGTCCACTGGCA-3’
PDS921-R3，5’-TCACCCGCCAGGTAGAAG-3’。
From the salt algae in logarithm later stage, extract total RNA, amplify the partial sequence of salt algae PDS cDNA with the synthesizing single-stranded cDNA of BD Clontech powerscript ThermoScript II as pcr template.Response procedures is: 94 ℃ of pre-sex change 5min enter working cycle; 94 ℃ of sex change 30sec, 47 ℃ of annealing 30sec, 72 ℃ are extended 1min, 30 circulations.Extend 10min with 72 ℃ at last.Carry out electrophoretic analysis through 1% sepharose.
The salt algae RNA that gets the different time sections cultivation as above reacts.It is better that combination of primers PDS921-F1/R2 can amplify consistence, the band of the 650bp that specificity is stronger, and gel is connected on pGEM-T easy (Promega) carrier after reclaiming, and the recombinant plasmid electricity is transformed into E.coli DH5 α competent cell.Positive colony is checked order, and sequence is carried out homology analysis with blast program.The amino acid of this fragment and Dunaliella bardawil PDS and the consistence of nucleotide sequence reach 92% and 88% respectively, tentatively determine the salt algae phytoene dehydrogenase cDNA fragment that it is.
According to known this section sequences Design gene specific downstream primer (5 '-GGGCTGGGATGTCTGGGAACTCAA-3 ') carry out 5 '-RACE.Its principle is to utilize 3 ' terminal poly (A) tail of eukaryote mRNA as a primer binding site, with Oligo (dT) before this
18VN is as locking primer under the effect of ThermoScript II PowerscriptMMLV ThermoScript II, the reverse transcription synthetic standards first chain cDNA.The terminal enzyme (DNA) activity of utilizing this ThermoScript II to have, reach 5 of first chain in reverse transcription and ' add 3-5 (dC) residue during end automatically, after annealing back (dC) residue and the universal joint primer that contains oligonucleotide sequence Oliogo (dG) (5 '-GGGTCTAGAGAATTCGGATCCAGGGGG-3 ') pairing, being converted to this primer sequence is that template continues to extend.As upstream primer, as downstream primer, is template with the first chain cDNA with above-mentioned gene specific primer with this primer sequence, carries out the touchdown PCR circulation, and the cDNA fragment amplification of end of target gene 5 ' is come out.
Through similarly doing the BLAST analysis after clone and the order-checking, find that 5 ' the terminal cDNA sequence of this segment length 394bp comprises initiator codon.The salt algae PDS cDNA sequence of a segment length 1047bp will be obtained behind above-mentioned two sections sequence assemblies.
The gene order of PDS is shown in SEQ ID.1.
03 sequence ~ 1
SEQUENCE?LISTING
＜110〉South China Science ﹠ Engineering University
＜120〉gene of coding phytoene dehydrogenase of Dunaliella salina
<130>200502
<160>1
<170>PatentIn?version?3.1
<210>1
<211>1037
<212>cDNA
＜213〉Dunaliella salina (Dunaliella salina)
<400>1
atgcaggtta?tgcaaggcag?ggcacacgcg?caggccgctt?cttttaacag?caagcctgtc 60
cagcagaggc?gcacgcagcg?gaaagtaggc?aggtcccgcc?tgcaggtgta?cgccagggat 120
ttcccagctc?ctcagttcga?tggcacggag?acgtaccagg?aggccgtggc?cctgtccaca 180
aagctgcaaa?atgccccccg?gccagtaaag?ccacagcgtg?tcgtcattgc?tggagccggc 240
ctggctggcc?tacccgctgc?caagtacctt?tctgaacgct?ggccacatcc?ccgtcgtgct 300
ggaggcccgc?gatgtgctgg?ggggcaaggt?ggccgcatgg?aaggacgaag?atggggactg 360
gtatgagact?ggcctgcaca?tctttttcgg?cgcatacccc?aacatgcagc?ggctgttcaa 420
ggagctcaac?atctctgaca?ggttacagtg?gaagagccac?tctatgattt?ttgccatgca 480
agacaagcct?ggagagttct?cgccttttga?gttcccagac?atcccagccc?catggaacgg 540
tgtcattgcc?atcctacgca?acaatgagat?gctgtcgtgg?cctgagaaga?tccagtttgc 600
tgttggcctg?ctgccggcca?tcatcttcgg?ccagaagtat?gtggaagagc?aggatgaact 660
gacagtaact?cagtggatgc?agaagcaggg?cgtgcccagc?cgagtaaacg?atgaggtctt 720
cattgccatg?gccaaggccc?tcaacttcat?caaccccgat?gagctgtcca?tgactgtcgt 780
gctgacagcg?ctgaaccgct?tcttgcaaga?gcgacatgga?agcaagatgg?cctttcttga 840
tggtgctcct?ccagagcgct?tgtgccagcc?catggtggac?tacttcactt?ccaggggtgg 900
agagctaaag?atgaacgcac?gcatcaagca?aatcgtgctc?aatgaggaca?acagcgtcaa 960
gcacttcgag?ctgctgaacg?gagagattgt?tgagggagat?gcgtacatgt?ctgcaatgcc 1020
aggtggacat?catgaag 1037
Claims (5)
1, a kind of isolating Nucleotide, the coding phytoene dehydrogenase, it has sequence shown in the SEQ IDNO.1.
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.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200510033238 CN1670212A (en) | 2005-02-22 | 2005-02-22 | Gene for coding phytoene dehydrogenase of Dunaliella salina |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200510033238 CN1670212A (en) | 2005-02-22 | 2005-02-22 | Gene for coding phytoene dehydrogenase of Dunaliella salina |
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| CN1670212A true CN1670212A (en) | 2005-09-21 |
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| CN 200510033238 Pending CN1670212A (en) | 2005-02-22 | 2005-02-22 | Gene for coding phytoene dehydrogenase of Dunaliella salina |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101979587A (en) * | 2010-10-14 | 2011-02-23 | 浙江大学 | Phytoene desaturase gene of sphingomonas sp. and application thereof |
| 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 |
| CN103146743A (en) * | 2012-11-18 | 2013-06-12 | 西北农林科技大学 | Method for improving currant tomato endogenous gene silencing efficiency by viruses through induction |
| CN110499272A (en) * | 2019-07-23 | 2019-11-26 | 华南理工大学 | Beta carotene high production bacteria and its construction method and application based on Du Shi algae metabolic pathway |
-
2005
- 2005-02-22 CN CN 200510033238 patent/CN1670212A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101979587A (en) * | 2010-10-14 | 2011-02-23 | 浙江大学 | Phytoene desaturase gene of sphingomonas sp. and application thereof |
| CN101979587B (en) * | 2010-10-14 | 2013-05-01 | 浙江大学 | Phytoene desaturase gene of sphingomonas sp. and application thereof |
| 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 |
| CN103146743A (en) * | 2012-11-18 | 2013-06-12 | 西北农林科技大学 | Method for improving currant tomato endogenous gene silencing efficiency by viruses through induction |
| CN103146743B (en) * | 2012-11-18 | 2015-01-07 | 西北农林科技大学 | Method for improving currant tomato endogenous gene silencing efficiency by viruses through induction |
| CN110499272A (en) * | 2019-07-23 | 2019-11-26 | 华南理工大学 | Beta carotene high production bacteria and its construction method and application based on Du Shi algae metabolic pathway |
| CN110499272B (en) * | 2019-07-23 | 2021-03-30 | 华南理工大学 | Beta-carotene high-yield engineering bacterium based on Dunaliella metabolic pathway and construction method and application thereof |
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