CN1904051A - Salt algae NADP glyceral dehyde-3-phosdehydrogenase gene clone and protein expression method - Google Patents
Salt algae NADP glyceral dehyde-3-phosdehydrogenase gene clone and protein expression method Download PDFInfo
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Abstract
The present invention relates to a kind of saline alga photosynthetic metabolic pathway key enzyme NADP-glyceraldehyde-3-phosphate dehydrogenase gene, coded protein and its clone and protein expression method. The invented saline alga photosynthetic metabolic pathway key enzyme NADP-glyceraldehyde-3-phosphate dehydrogenase gene has the base sequence showed by SEQ NO 5, and its coded protein has the amino acid sequence showed by SEQ NO 6. Said invention uses homologous fragment of glyceraldehydes-3-phosphate dehydrogenase gene originated from chlamydomonas as probe and firstly clones a glyceraldehydes-3 phosphate dehydrogenase specialized by saline alga. Besides, said invention makes primary analysis for its sequence and coded protein sequence, at the same time makes primary function analysis for said gene.
Description
Technical field
The present invention relates to a kind of salt algae photosynthetic metabolite pathway key enzyme NADP-glyceraldehyde-3-phosphate dehydrogenase gene clone and protein expression thereof.
Background technology
(glyceraldehyd-3-phosphate dehydrogenase GAPDH) is a kind of ancient and widely distributed enzyme to glyceraldehyde-3-phosphate dehydrogenase.It is prevalent in protobiont, prokaryotic organism, the eukaryote, with the sugar phosphate metabolism approach of organism closely related (Fothergill-Gilmore etc., 1993).
Glycerose 3-phosphate dehydrogenase (GAPDH) is main in organism to participate in Calvin cycle approach in glycolytic pathway in the tenuigenin and the plant chloroplast.GAPDH in the glycolytic pathway (EC1.2.1.12) with NADH as of the conversion of its electron donor catalysis phosphodihydroxyacetone to glyceraldehyde 3-phosphate; And the GAPDH in the chloroplast(id) (EC1.2.1.13) with NADPH or NADH as its electron donor (Cerff R. etc., 1978,1982) catalysis 1,3-diphosphoglyceric acid (BPGA) is to reversible reduction of glyceraldehyde 3-phosphate and dephosphorylation, photosynthetic carbon reduction approach-Calvin cycle in the involved in plant body.Because glycolytic pathway and Calvin cycle approach all are the main paties of organism cell energy metabolism, therefore, GAPDH receives numerous scientists' concern all the time, and its classical mode albumen as a sugar phosphate metabolism approach carried out number of research projects (Cerff R. etc. from aspects such as structure, zymetology mechanism and expression regulations, 1979,1982); In addition, because the conservative property of gene order, protein sequence and the higher structure of different plant species, make it in the research of species phylogeny, have extremely special status, the plastosome that is released to classics of a large amount of gene orders and protein sequence and chloroplast(id) endosymbiosis origin theory provide ample evidence (Brinkmann H. etc., 1989; Cerff R. etc., 1995; Kwon HB etc., 1995; Martinez P. etc., 1989; Meyer-GauenG. etc., 1994; Meyer-Gauen G. etc., 1998);
The salt algae is as a kind of unicell green alga that extreme environments such as salt, radiation and high light are had high resistance, all the time by scientists as the model animals of vegetable cell to the environment-stress Study on Molecular Mechanism.When high-salt stress, the salt algae mainly comes osmotic pressure inside and outside the statocyte by quick synthetic glycerine, 13C-NMR studies show that (Degani K etc., 1985), when high-salt stress just began, the glycerine in the salt frustule mainly produced by degraded starch, and after 45 minutes, the lasting synthetic of glycerine then mainly finished by photosynthesis, and the glyceraldehyde-3-phosphate dehydrogenase of chloroplast(id) Calvin cycle approach is one of key enzyme of this metabolic process (Goyal A. etc., 1987).For better from the molecular level to the research of salt algae photosynthesizer and its height salt tolerant mechanism relation, this area press for from the molecular level to the research of salt algae photosynthesizer and its height salt tolerant mechanism relation, this area presses for the key gene of its photosynthetic metabolite approach of clone.But also there are not salt algae NADP-glyceraldehyde-3-phosphate dehydrogenase gene and relevant report in the prior art.
Summary of the invention
One of purpose of the present invention is to provide salt algae photosynthetic metabolite pathway key enzyme NADP-glyceraldehyde-3-phosphate dehydrogenase gene.
Two of purpose of the present invention is to provide salt algae photosynthetic metabolite pathway key enzyme NADP-glyceraldehyde-3-phosphate dehydrogenase gene coded aminoacid sequence.
Three of purpose of the present invention be to be used to come from the nearer conservative section of chlamydomonas glyceraldehyde-3-phosphate dehydrogenase gene of salt algae sibship be probe, from salt algae cDNA library, be cloned into a salt algae photosynthetic metabolite pathway key enzyme NADP-glyceraldehyde-3-phosphate dehydrogenase gene first, and this gene has been carried out preliminary functional analysis.
Four of purpose of the present invention is to provide carries out protein expression to salt algae photosynthetic metabolite pathway key enzyme NADP-glyceraldehyde-3-phosphate dehydrogenase gene in prokaryotic expression system.
In order to achieve the above object, the following technical scheme of employing of the present invention:
A kind of salt algae photosynthetic metabolite pathway key enzyme NADP-glyceraldehyde-3-phosphate dehydrogenase gene is characterized in that this gene has the base sequence shown in the SEQ NO 5.
A kind of salt algae photosynthetic metabolite pathway key enzyme NADP-glyceraldehyde-3-phosphate dehydrogenase gene proteins encoded according to claim 1 is characterized in that having the aminoacid sequence shown in the SEQ NO 6.
A kind of cloning process of salt algae photosynthetic metabolite pathway key enzyme NADP-glyceraldehyde-3-phosphate dehydrogenase gene according to claim 1 is characterized in that the concrete steps of this method are:
A. the conserved sequence according to chlamydomonas W80 NADP-glyceraldehyde-3-phosphate dehydrogenase gene cDNA has synthesized a pair of primer CRGAPAF and CRGAPAR:
CRGAPAF：1?AAGGGCACCA?TGACCACC?18，
CRGAPAR：1?ATGCCGTTGA?GCTTGCC?17；
B. the method by RT-PCR obtains the cDNA fragment from total RNA amplification of chlamydomonas CC2677, and total length is 176bp, with this gene fragment called after CRGAPAf;
C. the gene fragment CRGAPAf that obtains among the step b is carried out probe mark with DIG, the cDNA library of screening salt algae, promptly 3.0M NaCl cDNA library through the two-wheeled screening, obtains 5 independently phage positive colonies, is converted into plasmid by external excision then; So promptly obtain salt algae photosynthetic metabolite pathway key enzyme NADP-glyceraldehyde-3-phosphate dehydrogenase gene and proteins encoded thereof.
A kind of protein expression of salt algae photosynthetic metabolite pathway key enzyme NADP-glyceraldehyde-3-phosphate dehydrogenase gene according to claim 1, it is characterized in that this method is: the coding region of salt algae photosynthetic metabolite pathway key enzyme NADP-glyceraldehyde-3-phosphate dehydrogenase gene is cloned among the prokaryotic expression carrier pTWIN1, and in BL21 (DE3), carries out protein expression.
Above-mentioned protein expression is specially: according to the multiple clone site of carrier and the zymogram of salt algae photosynthetic metabolite pathway key enzyme NADP-glyceraldehyde-3-phosphate dehydrogenase A subunit (DvGapA1) gene, respectively prokaryotic expression carrier pTWIN1 (7375bp) and plasmid pBlueSK (-)-DvGapA are carried out EcoR I/Xho I double digestion, utilize carrier and the target gene fragment of QIAquick Gel Extraction Kit recovery through double digestion; Utilize the T4 dna ligase that an amount of carrier is connected at 16 ℃ with target gene fragment then and spend the night, will connect product again and transform DH5 α competent cell, and be coated with LB ammonia benzyl resistant panel, 37 ℃ of incubated overnight; Select resistance clone and identify, boiling method extracts plasmid in a small amount to carry out enzyme and cuts and identify whether institute's selected clone has the segmental insertion of purpose; The purpose clone pWIN-DvGapA1 that identifies is transformed BL21 (DE3) host bacterium, and carry out induction expression of protein; Be cultured to OD
600When being 0.5, adding 1/1000 IPTG (1M) and continue shaking culture at 30 ℃ in volume of culture, and in different time (0,1,2,3,4,5,6hrs) sampling is carried out SDS-PAGE and is detected to determine the time of the high expression level of albumen, and the result is presented at and induces 4 hours expression levels the highest.
The present invention will derive from the glyceraldehyde-3-phosphate dehydrogenase gene (Miyasaka of chlamydomonas, H. etc., 1994) homologous fragment is cloned into a glyceraldehyde-3-phosphate dehydrogenase gene that the salt algae is special first as probe, and its sequence and encoded protein sequence carried out preliminary analysis, simultaneously this gene has been carried out preliminary functional analysis, inquired into this unicell green alga of salt algae on this basis in the status of plant from low higher plant evolutionary process by the time; The more important thing is, the present invention studies its transcriptional level expression under the high-salt stress situation, with the further conservative property of the biological function of zymoprotein during participating in photosynthesis Calvin cycle process of clear and definite this gene and coding thereof from molecular level, disclose this gene simultaneously and coerce the particularly specificity of the biological function in the osmotic stress process of process at salt algae response environment.
Description of drawings
Fig. 1 has shown the different plant species glyceraldehyde-3-phosphate dehydrogenase, and (GapA, GapB) protein sequence relatively.
Fig. 2 has shown the glyceraldehyde-3-phosphate dehydrogenase systematic evolution tree in different plant species source.
Fig. 3 has shown the Northern expression analysis of salt algae photosynthetic metabolite pathway key enzyme NADP-glyceraldehyde-3-phosphate dehydrogenase gene under constant NaCl culture condition and NaCl high-salt stress condition.
Fig. 4 has shown the Northern expression analysis of salt algae photosynthetic metabolite pathway key enzyme NADP-glyceraldehyde-3-phosphate dehydrogenase gene under NaCl high-salt stress condition.
Fig. 5 has shown the abduction delivering of salt algae photosynthetic metabolite pathway key enzyme NADP-glyceraldehyde-3-phosphate dehydrogenase gene in prokaryotic expression system.
Embodiment:
Embodiment one: the clone of salt algae photosynthetic metabolite pathway key enzyme NADP-glyceraldehyde-3-phosphate dehydrogenase gene
At first the conserved sequence according to chlamydomonas W80 NADP-glyceraldehyde-3-phosphate dehydrogenase gene cDNA (AB035312) has synthesized a pair of primer (CRGAPAF:(SEQ ID NO:1); CRGAPAR (SEQ ID NO:2)), the method by RT-PCR obtains and estimates the cDNA fragment (176bp) that size conforms to (SEQ ID NO:3) from total RNA amplification of chlamydomonas CC2677.The cDNA fragment that obtains is carried out sequential analysis, find with the homology of the corresponding sequence of AB035312 to be 89.2%, and the homology of corresponding aminoacid sequence (SEQ ID NO:4) is 93.1%, has only 4 amino acid whose sequences different, and we are with this fragment called after CRGAPAf.Then, the above-mentioned chlamydomonas that obtains 2677 glyceraldehyde-3-phosphate dehydrogenase gene fragment CRGAPAf (SEQ ID NO:3) are carried out probe mark with DIG, the cDNA library (3.0M NaCl cDNA library) of screening salt algae, through the two-wheeled screening, obtain 5 independently phage positive colonies, be converted into plasmid by external excision then, by PCR (primer: CRGAPAF, CRGAPAR) identify further proof, we may obtain deriving from the glyceraldehyde-3-phosphate dehydrogenase gene in the salt algae genome.And picking 1 clone wherein carried out further sequencing.Sequencing is the result show, this clone's correct coding a salt algae glyceraldehyde-3-phosphate dehydrogenase, we are with this gene and its encoded protein called after DvGapA1 (SEQ ID NO:5) and DvGapA1 (1478bp) (SEQ ID NO:6) respectively.
Embodiment two: the sequential analysis of salt algae photosynthetic metabolite pathway key enzyme NADP-glyceraldehyde-3-phosphate dehydrogenase gene: DvGapA1 cDNA sequence total length encoded protein sequence is 376 amino acid whose albumen.Infer that according to sequence its molecular weight is 40.5kDa, iso-electric point is 9.05 basic protein.The protein sequence that supposition is obtained carries out BlastP and relatively finds, itself and the homology that glyceraldehyde-3-phosphate dehydrogenase gene encoded protein on the chloroplast(id) all has height that is positioned that derives from different plant species, wherein with the homology of Glycerose 3-phosphate dehydrogenase (BAA94304) aminoacid sequence of chlamydomonas up to 72%.By the prediction of online chloroplast transit peptides (
Http:// www.cbs.dtu.dk/services/ChloroP/) find that preceding 35 amino acid of the coded by said gene albumen that we are separated to are possible transit peptides, this distribution with the chloroplast transit peptides of the glyceraldehyde-3-phosphate dehydrogenase that is positioned chloroplast(id) of other source of species is consistent.Above-mentioned data show, we have obtained being positioned at the cDNA of the glyceraldehyde-3-phosphate dehydrogenase gene of salt algae chloroplast(id) really, our temporary transient called after DvGapA1 of its encoded protein.
Embodiment four: (GapA, GapB) protein sequence relatively for the different plant species glyceraldehyde-3-phosphate dehydrogenase
The chloroplast(id) type glyceraldehyde-3-phosphate dehydrogenase that derives from spinach, Arabidopis thaliana and pea and salt algae is carried out sequence relatively, the protein sequence that the result shows the GapB type significantly has more one section than the protein sequence of GapA type at C-terminal, and this C-terminal sequence homology in different plant species source is also than higher.The glyceraldehyde-3-phosphate dehydrogenase DvGapA1 of salt algae obviously belongs to the GapA type, comprises to combine with NADP and the conservative amino acid residues of zymoprotein catalyst structure domain, and referring to Fig. 1, this shows that salt algae glyceraldehyde-3-phosphate dehydrogenase may be in the conservative property of higher structure.
Embodiment five: the glyceraldehyde-3-phosphate dehydrogenase systematic evolution tree in different plant species source
By will be from archeobacteria, Euglena, red algae, general living stonewort, blue-green algae, chlamydomonas, marchantia, sphagnum moss, Arabidopis thaliana, capsicum, the cytoplasm type in source such as pea and corn and the protein sequence (CAC80388: marchantia GapA of chloroplast(id) type glyceraldehyde-3-phosphate dehydrogenase, CAC80393: sphagnum moss GapA1, CAC80392: the continuous GapA of water, CAC80372: capsicum GapCP1, CAC80373: capsicum GapCP2, CAA33264: pea GapA, CAA30152: corn GapA, AAA32793: Arabidopis thaliana GapA1, P25856: Arabidopis thaliana GapA, T09668: pine tree GapA, CAC80391: green alga Kf GapA, CAC81011: grid algae GapA, AAA86855: chlamydomonas GapA1, BAA94304: chlamydomonas GapA2, CAC80389: marchantia GapB, CAC80390: sheath hair algae GapB, CAC80374: capsicum GapB, AAA32795: Arabidopis thaliana GapB, CAC80378: general living stonewort GapB, BAA18633: cytoalgae GapA, BAC88471: no thylakoid blue-green algae GapA, P30724: red algae GapA, AAD10216: Euglena GapA, Q48335: archeobacteria Gap, AAC37245: Euglena Gap1, BAA17609: cytoalgae Gap1, BAB74265: anabena Gap1, BAC91266: no thylakoid blue-green algae Gap1, CAC80386: marchantia GapC, CAC80387: Herba Funariae Hygrometricae GapC, CAC80384: sphagnum moss GapC2, CAC80377: capsicum GapC, CAC80385: marchantia GapC, AAB59010: Selaginella tamariscina GapC, CAC80381: sheath hair algae GapC, CAA55116: resurrection plant GapC, CAC80375: capsicum GapC, AAA32796: Arabidopis thaliana GapC, CAC80376: capsicum GapC, AAM92008: potato GapC, AAA03442: Taiwan saltbush, Q39769: pine tree GapC, Q41595: pine tree GapC, AAA87880: corn GapC, CAC80383: sphagnum moss GapC, CAC80379: general living stonewort GapC, CAC80382: green alga Kf GapC, P49644: chlamydomonas GapC) and DvGapA1 carry out homology relatively, at first by smart and Pfam, predicted the position of two domain, the sequence that two is irrelevant is removed, with archeobacteria (Q48335) is outer group, adopting ClustalX software that above-mentioned homologous sequence is carried out the multisequencing coupling arranges, by Gene Doc the multisequencing matching result is proofreaied and correct, utilize MEGA3.1 software to carry out the structure of systematic evolution tree then, it is according to the Neighbor-joining method, analyzes through 1000 bootstrap by the Poissondistance method.The result shows that (Fig. 5) DvGapA1 significantly is distributed in GapA one class, nearest with the sibship of chlamydomonas and grid algae, and this proves that further we have obtained participating in the glyceraldehyde-3-phosphate dehydrogenase of one of the key gene of salt algae photosynthesis Calvin cycle.Because the glyceraldehyde-3-phosphate dehydrogenase of archeobacteria can be nonselective is prothetic group with NAD or NADP, therefore be considered to comparatively primary Evolutionary Type, and the glyceraldehyde-3-phosphate dehydrogenase of thin matter type and chloroplast(id) type also just begins differentiation from archeobacteria, the evolution route of chloroplast(id) type glyceraldehyde-3-phosphate dehydrogenase is as can be seen from Figure 2: archeobacteria GapA → Euglena GapA → blue-green algae (no thylakoid blue-green algae, cytoalgae) GapA → red algae GapA, begin branch again by red algae GapA then, wherein branch one is further to green alga (green alga Kf), low terrestrial plant (the marchantia that waits, narrow leaf sphagnum moss) and high terrestrial plant (pine tree, Arabidopis thaliana, corn, pea) GapA evolves, and branch two is further to green alga (salt algae simultaneously, chlamydomonas, the grid algae) GapA and lower plant (general living stonewort, sheath hair algae, marchantia) and higher plant (Arabidopis thaliana, capsicum) GapB evolves.From here as can be seen, GapA and GapB should evolve to red algae GapA just to begin later on to break up, and in a part of green alga, keep in the GapA Evolutionary Type, part lower plant (general living stonewort) and higher plant have occurred by the next a kind of novel glyceraldehyde-3-phosphate dehydrogenase (GapB) of GapA evolution, also do not find the glyceraldehyde-3-phosphate dehydrogenase gene of GapB type up to now in other unicell green alga, this shows that general living stonewort may be that hydrobiontic algae is to a low transitional type that waits terrestrial plant to evolve.
Embodiment six: the Northern expression analysis of DvGapA1 under constant NaCl culture condition and NaCl high-salt stress condition
Research in the past is verified, and most glyceraldehyde-3-phosphate dehydrogenase genes that participate in the Calvin cycle approach are generally the gene of constitutive expression, are not subjected to the regulation and control of the external environment factor.In order to verify further whether the DvGapA1 that we are separated to also is constitutive expression on transcriptional level, we have extracted total RNA of the salt frustule of long term growth under different N aCl concentration culture condition, with the DvGapA1 full-length cDNA is probe, has carried out the Northern analysis, referring to Fig. 3.The result shows that the hybridization signal that different N aCl culture condition is obtained does not down have tangible difference, and the expression that shows DvGapA1 is not subjected to the influence of NaCl in the environment.
Show that through the salt frustule Northern analytical results after 1.0M NaCl → 2.0M NaCl concussion is cultivated DvGapA1 is not subjected to inducing of NaCl infiltration concussion, referring to Fig. 4, its expression also is a composing type.
The abduction delivering of embodiment 7 salt algae DvGapA1 in prokaryotic expression system
According to the multiple clone site of carrier and the zymogram of salt algae Glycerose 3-phosphate dehydrogenase A subunit (DvGapA1) gene, respectively prokaryotic expression carrier pTWIN1 (7375bp) and plasmid pBlueSK (-)-DvGapA are carried out EcoR I/XhoI double digestion, utilize carrier and the target gene fragment of QIAquick Gel Extraction Kit recovery through double digestion.Utilize the T4DNA ligase enzyme that an amount of carrier is connected at 16 ℃ with target gene fragment then and spend the night, will connect product again and transform DH5 α competent cell, and be coated with LB ammonia benzyl resistant panel, 37 ℃ of incubated overnight.Select resistance clone and identify, boiling method extracts plasmid in a small amount to carry out enzyme and cuts and identify whether institute's selected clone has the segmental insertion of purpose.The purpose clone pWIN-DvGapA1 that identifies is transformed BL21 (DE3) host bacterium, and carry out induction expression of protein.Be cultured to OD
600When being 0.5, add 1/1000 IPTG (1M) and continue shaking culture at 30 ℃ in volume of culture, and in different time (0,1,2,3,4,5,6hrs) sampling is carried out SDS-PAGE and is detected to determine the time of the high expression level of albumen, the result is presented at and induces 4 hours expression levels the highest, referring to Fig. 5.
Sequence table
＜110〉Shanghai University
＜120〉salt algae photosynthetic metabolite pathway key enzyme NADP-glyceraldehyde-3-phosphate dehydrogenase gene clone and protein expression thereof
<160>6
<170>PatentIn?version?3.3
<210>1
<211>18
<212>DNA
＜213〉artificial sequence (Artificial)
<220>
<221>misc?feature
＜223〉primer
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aagggcacca?tgaccacc 18
<210>2
<211>17
<212>DNA
＜213〉artificial sequence (Artificial)
<220>
<221>misc?feature
＜223〉primer
<400>2
atgccgttga?gcttgcc 17
<210>3
<211>176
<212>DNA
＜213〉chlamydomonas (Chlamydomonas reinhardtii)
<400>3
aagggcacga?tgaccaccac?ccactcctac?accggtgacc?agcgcctgct?ggacgcgtcc 60
caccgcgacc?tgcgccgcgc?ccgcgccgcc?gccctgaaca?ttgtgcccac?caccaccggt?120
gccgccaagg?ccgtgtcgct?ggtgctgccc?agcctgaagg?gcaagctgaa?cggcat 176
<210>4
<211>58
<212>PRT
＜213〉chlamydomonas (Chlamydomonas reinhardtii)
<400>4
Lys?Gly?Thr?Met?Thr?Thr?Thr?His?Ser?Tyr?Thr?Gly?Asp?Gln?Arg?Leu
1 5 10 15
Leu?Asp?Ala?Ser?His?Arg?Asp?Leu?Arg?Arg?Ala?Arg?Ala?Ala?Ala?Leu
20 25 30
Asn?Ile?Val?Pro?Thr?Thr?Thr?Gly?Ala?Ala?Lys?Ala?Val?Ser?Leu?Val
35 40 45
Leu?Pro?Ser?Leu?Lys?Gly?Lys?Leu?Asn?Gly
50 55
<210>5
<211>1478
<212>DNA
＜213〉salt algae (Dunaliella viridis)
<220>
<221>CDS
<222>(13)..(1143)
<400>5
cgccagcgaa?aa?atg?gcc?acc?tca?atg?gcg?aag?tcc?gcc?ttc?acc?ggc?48
Met?Ala?Thr?Ser?Met?Ala?Lys?Ser?Ala?Phe?Thr?Gly
1 5 10
aac?atg?gcc?ggc?ctg?aag?aac?ttc?cag?cgc?gtg?cag?ccc?gcc?agg?ggc 96
Asn?Met?Ala?Gly?Leu?Lys?Asn?Phe?Gln?Arg?Val?Gln?Pro?Ala?Arg?Gly
15 20 25
gcc?gtg?aag?atg?gag?gtt?gtg?gcg?cag?aag?aag?gtg?cgc?gtg?gcc?atc 144
Ala?Val?Lys?Met?Glu?Val?Val?Ala?Gln?Lys?Lys?Val?Arg?Val?Ala?Ile
30 35 40
aac?ggc?ttc?ggc?cgc?att?ggc?cgc?aac?ttc?ctg?cgc?tgc?tgg?gag?ggc 192
Asn?Gly?Phe?Gly?Arg?Ile?Gly?Arg?Asn?Phe?Leu?Arg?Cys?Trp?Glu?Gly
45 50 55 60
cgc?aag?gac?tcc?ctg?ctg?gac?gtg?gtg?tgc?gtg?aac?gac?tcc?ggc?ggt 240
Arg?Lys?Asp?Ser?Leu?Leu?Asp?Val?Val?Cys?Val?Asn?Asp?Ser?Gly?Gly
65 70 75
gtg?aag?cag?gcc?tct?cac?ctg?ctg?aag?tac?gac?acc?acg?ctg?ggc?aag 288
Val?Lys?Gln?Ala?Ser?His?Leu?Leu?Lys?Tyr?Asp?Thr?Thr?Leu?Gly?Lys
80 85 90
ttc?gac?gct?gat?gtc?aag?gcc?gtg?gac?gac?aag?acc?atc?agc?gtg?aac 336
Phe?Asp?Ala?Asp?Val?Lys?Ala?Val?Asp?Asp?Lys?Thr?Ile?Ser?Val?Asn
95 100 105
ggc?aag?aac?atc?gcc?gtg?gtg?tcc?agc?cgc?gac?ccc?acc?cag?ctg?ccc 384
Gly?Lys?Asn?Ile?Ala?Val?Val?Ser?Ser?Arg?Asp?Pro?Thr?Gln?Leu?Pro
110 115 120
tgg?aag?gcc?atg?gac?atc?gac?ctg?gtc?atc?gag?ggc?acc?ggc?gtg?ttc 432
Trp?Lys?Ala?Met?Asp?Ile?Asp?Leu?Val?Ile?Glu?Gly?Thr?Gly?Val?Phe
125 130 135 140
gtg?gac?acc?ccc?ggc?gct?ggc?aag?cac?atc?cag?gcc?ggc?gcc?aag?aag 480
Val?Asp?Thr?Pro?Gly?Ala?Gly?Lys?His?Ile?Gln?Ala?Gly?Ala?Lys?Lys
145 150 155
gtg?ctg?atc?acc?gcc?ccc?gcc?aag?ggc?aac?gac?atc?ccc?acc?ttc?gtg 528
Val?Leu?Ile?Thr?Ala?Pro?Ala?Lys?Gly?Asn?Asp?Ile?Pro?Thr?Phe?Val
160 165 170
gtc?ggc?gtg?aac?tgc?gac?ggc?tac?aac?cac?tcc?tac?ccc?atc?atc?tcc 576
Val?Gly?Val?Asn?Cys?Asp?Gly?Tyr?Asn?His?Ser?Tyr?Pro?Ile?Ile?Ser
175 180 185
aac?gcc?tcc?tgc?acc?acc?aac?tgc?ctg?gca?ccc?ttc?gtc?aag?gtg?ctg 624
Asn?Ala?Ser?Cys?Thr?Thr?Asn?Cys?Leu?Ala?Pro?Phe?Val?Lys?Val?Leu
190 195 200
cac?gag?aag?ttc?cgc?atc?gtg?aag?ggc?acc?atg?acc?acc?acc?cac?tcc 672
His?Glu?Lys?Phe?Arg?Ile?Val?Lys?Gly?Thr?Met?Thr?Thr?Thr?His?Ser
205 210 215 220
tac?act?ggt?gac?cag?cgt?ctg?ctg?gat?gct?tct?cac?cgg?gac?ctg?cgc 720
Tyr?Thr?Gly?Asp?Gln?Arg?Leu?Leu?Asp?Ala?Ser?His?Arg?Asp?Leu?Arg
225 230 235
cgc?gct?cgt?gcc?gcc?gcc?ctg?aac?att?gtg?ccc?acc?acc?act?ggt?gcg 768
Arg?Ala?Arg?Ala?Ala?Ala?Leu?Asn?Ile?Val?Pro?Thr?Thr?Thr?Gly?Ala
240 245 250
gcc?aag?gct?gtg?gcc?ctg?gtg?ctg?cct?gac?ctg?aag?ggc?aag?ctg?aac 816
Ala?Lys?Ala?Val?Ala?Leu?Val?Leu?Pro?Asp?Leu?Lys?Gly?Lys?Leu?Asn
255 260 265
ggt?gtg?gcc?ctg?cgt?gtg?ccc?acc?ccc?aac?gtg?tcc?att?gtg?gat?ctg 864
Gly?Val?Ala?Leu?Arg?Val?Pro?Thr?Pro?Asn?Val?Ser?Ile?Val?Asp?Leu
270 275 280
gtg?gtg?cag?gtc?gag?aag?aag?acc?ttc?gct?gag?gag?gtg?aac?aac?gcc 912
Val?Val?Gln?Val?Glu?Lys?Lys?Thr?Phe?Ala?Glu?Glu?Val?Asn?Asn?Ala
285 290 295 300
ttc?cgc?gag?gcc?gcc?gct?ggc?ccc?atg?agc?aac?gtg?ctg?gcc?gtg?gct 960
Phe?Arg?Glu?Ala?Ala?Ala?Gly?Pro?Met?Ser?Asn?Val?Leu?Ala?Val?Ala
305 310 315
gat?gag?cct?ctg?gtg?tcc?gct?gac?ttc?aag?ggc?atg?gac?cag?agc?aca 1008
Asp?Glu?Pro?Leu?Val?Ser?Ala?Asp?Phe?Lys?Gly?Met?Asp?Gln?Ser?Thr
320 325 330
gcc?atc?gac?tct?gcc?ctg?acc?atg?gtc?atg?ggt?gat?gac?atg?gtc?aag 1056
Ala?Ile?Asp?Ser?Ala?Leu?Thr?Met?Val?Met?Gly?Asp?Asp?Met?Val?Lys
335 340 345
gtg?gtc?gcc?tgg?tac?gac?aac?gag?tgg?ggc?tac?tcc?cag?cgt?gtg?gtg 1104
Val?Val?Ala?Trp?Tyr?Asp?Asn?Glu?Trp?Gly?Tyr?Ser?Gln?Arg?Val?Val
350 355 360
gac?ctg?gct?gag?ctg?acc?gct?cag?cga?tgg?gcc?gca?taa?gcagctaac 1152
Asp?Leu?Ala?Glu?Leu?Thr?Ala?Gln?Arg?Trp?Ala?Ala
365 370 375
ttggctgcac?gcgcgttgag?tagctagttt?gtcggacttc?gccatcacct?cttccttcct 1212
gttagctttc?cataggtctc?taatgatgta?gattagacgc?tgcacccaca?caaacacagc 1272
ctgcgccagc?catcagcggc?actcttccca?acgatttttc?ttccgtactt?gggctgggtc 1332
ataggttgat?ggctgccaca?tatgtcctgc?ggccgcgcac?tgcagcagtg?cttgcgtgct 1392
gtaagactgt?gcaaggctgg?ctgtatttca?aaaaaaaaaa?aaaaaaaaaa?aaaaaaaaaa 1452
aaaaaaaaaa?aaaaaaaaaa?aaaaaa 1478
<210>6
<211>376
<212>PRT
＜213〉salt algae (Dunaliella viridis)
<400>6
Met?Ala?Thr?Ser?Met?Ala?Lys?Ser?Ala?Phe?Thr?Gly?Asn?Met?Ala?Gly
1 5 10 15
Leu?Lys?Asn?Phe?Gln?Arg?Val?Gln?Pro?Ala?Arg?Gly?Ala?Val?Lys?Met
20 25 30
Glu?Val?Val?Ala?Gln?Lys?Lys?Val?Arg?Val?Ala?Ile?Asn?Gly?Phe?Gly
35 40 45
Arg?Ile?Gly?Arg?Asn?Phe?Leu?Arg?Cys?Trp?Glu?Gly?Arg?Lys?Asp?Ser
50 55 60
Leu?Leu?Asp?Val?Val?Cys?Val?Asn?Asp?Ser?Gly?Gly?Val?Lys?Gln?Ala
65 70 75 80
Ser?His?Leu?Leu?Lys?Tyr?Asp?Thr?Thr?Leu?Gly?Lys?Phe?Asp?Ala?Asp
85 90 95
Val?Lys?Ala?Val?Asp?Asp?Lys?Thr?Ile?Ser?Val?Asn?Gly?Lys?Asn?Ile
100 105 110
Ala?Val?Val?Ser?Ser?Arg?Asp?Pro?Thr?Gln?Leu?Pro?Trp?Lys?Ala?Met
115 120 125
Asp?Ile?Asp?Leu?Val?Ile?Glu?Gly?Thr?Gly?Val?Phe?Val?Asp?Thr?Pro
130 135 140
Gly?Ala?Gly?Lys?His?Ile?Gln?Ala?Gly?Ala?Lys?Lys?Val?Leu?Ile?Thr
145 150 155 160
Ala?Pro?Ala?Lys?Gly?Asn?Asp?Ile?Pro?Thr?Phe?Val?Val?Gly?Val?Asn
165 170 175
Cys?Asp?Gly?Tyr?Asn?His?Ser?Tyr?Pro?Ile?Ile?Ser?Asn?Ala?Ser?Cys
180 185 190
Thr?Thr?Asn?Cys?Leu?Ala?Pro?Phe?Val?Lys?Val?Leu?His?Glu?Lys?Phe
195 200 205
Arg?Ile?Val?Lys?Gly?Thr?Met?Thr?Thr?Thr?His?Ser?Tyr?Thr?Gly?Asp
210 215 220
Gln?Arg?Leu?Leu?Asp?Ala?Ser?His?Arg?Asp?Leu?Arg?Arg?Ala?Arg?Ala
225 230 235 240
Ala?Ala?Leu?Asn?Ile?Val?Pro?Thr?Thr?Thr?Gly?Ala?Ala?Lys?Ala?Val
245 250 255
Ala?Leu?Val?Leu?Pro?Asp?Leu?Lys?Gly?Lys?Leu?Asn?Gly?Val?Ala?Leu
260 265 270
Arg?Val?Pro?Thr?Pro?Asn?Val?Ser?Ile?Val?Asp?Leu?Val?Val?Gln?Val
275 280 285
Glu?Lys?Lys?Thr?Phe?Ala?Glu?Glu?Val?Asn?Asn?Ala?Phe?Arg?Glu?Ala
290 295 300
Ala?Ala?Gly?Pro?Met?Ser?Asn?Val?Leu?Ala?Val?Ala?Asp?Glu?Pro?Leu
305 310 315 320
Val?Ser?Ala?Asp?Phe?Lys?Gly?Met?Asp?Gln?Ser?Thr?Ala?Ile?Asp?Ser
325 330 335
Ala?Leu?Thr?Met?Val?Met?Gly?Asp?Asp?Met?Val?Lys?Val?Val?Ala?Trp
340 345 350
Tyr?Asp?Asn?Glu?Trp?Gly?Tyr?Ser?Gln?Arg?Val?Val?Asp?Leu?Ala?Glu
355 360 365
Leu?Thr?Ala?Gln?Arg?Trp?Ala?Ala
370 375
Claims (5)
1. a salt algae photosynthetic metabolite pathway key enzyme NADP-glyceraldehyde-3-phosphate dehydrogenase gene is characterized in that this gene has the base sequence shown in the SEQ NO 5.
2. a salt algae photosynthetic metabolite pathway key enzyme NADP-glyceraldehyde-3-phosphate dehydrogenase gene proteins encoded according to claim 1 is characterized in that having the aminoacid sequence shown in the SEQ NO 6.
3. the cloning process of a salt algae photosynthetic metabolite pathway key enzyme NADP-glyceraldehyde-3-phosphate dehydrogenase gene according to claim 1 is characterized in that the concrete steps of this method are:
A. according to synthetic a pair of primer CRGAPAF of the conserved sequence of chlamydomonas W80NADP-glyceraldehyde-3-phosphate dehydrogenase gene cDNA and CRGAPAR:
CRGAPAF：1 AAGGGCACCA?TGACCACC 18，
CRGAPAR：1 ATGCCGTTGA?GCTTGCC 17；
B. the method by RT-PCR obtains the cDNA fragment from total RNA amplification of chlamydomonas CC2677, and total length 176bp is with this cDNA fragment called after CRGAPAf;
C. the cDNA fragment CRGAPAf that obtains among the step b is carried out probe mark with DIG, the cDNA library of screening salt algae, promptly 3.0M NaCl cDNA library through the two-wheeled screening, obtains 5 independently phage positive colonies, is converted into plasmid by external excision then; So promptly obtain salt algae photosynthetic metabolite pathway key enzyme NADP-glyceraldehyde-3-phosphate dehydrogenase gene.
4. the protein expression of a salt algae photosynthetic metabolite pathway key enzyme NADP-glyceraldehyde-3-phosphate dehydrogenase gene according to claim 1, it is characterized in that this method is: the coding region of salt algae photosynthetic metabolite pathway key enzyme NADP-glyceraldehyde-3-phosphate dehydrogenase gene is cloned among the prokaryotic expression carrier pTWIN1, and in BL21 (DE3), carries out protein expression.
5. the protein expression of salt algae photosynthetic metabolite pathway key enzyme NADP-glyceraldehyde-3-phosphate dehydrogenase gene according to claim 4, it is characterized in that described protein expression is specially: according to the multiple clone site of carrier and the zymogram of salt algae photosynthetic metabolite pathway key enzyme NADP-glyceraldehyde-3-phosphate dehydrogenase A subunit (DvGapA1) gene, respectively prokaryotic expression carrier pTWIN1 (7375bp) and plasmid pBlueSK (-)-DvGapA are carried out the EcoRI/XhoI double digestion, utilize carrier and the target gene fragment of QIAquick Gel Extraction Kit recovery through double digestion; Utilize the T4DNA ligase enzyme that an amount of carrier is connected at 16 ℃ with target gene fragment then and spend the night, will connect product again and transform DH5 α competent cell, and be coated with LB ammonia benzyl resistant panel, 37 ℃ of incubated overnight; Select resistance clone and identify, boiling method extracts plasmid in a small amount to carry out enzyme and cuts and identify whether institute's selected clone has the segmental insertion of purpose; The purpose clone pWIN-DvGapA1 that identifies is transformed BL21 (DE3) host bacterium, and carry out induction expression of protein; Be cultured to OD
600When being 0.5, adding 1/1000 IPTG (1M) and continue shaking culture at 30 ℃ in volume of culture, and in different time (0,1,2,3,4,5,6hrs) sampling is carried out SDS-PAGE and is detected to determine the time of the high expression level of albumen, and the result is presented at and induces 4 hours expression levels the highest.
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| CN 200610025228 CN1904051A (en) | 2006-03-30 | 2006-03-30 | Salt algae NADP glyceral dehyde-3-phosdehydrogenase gene clone and protein expression method |
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| CN 200610025228 CN1904051A (en) | 2006-03-30 | 2006-03-30 | Salt algae NADP glyceral dehyde-3-phosdehydrogenase gene clone and protein expression method |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102181369A (en) * | 2011-02-24 | 2011-09-14 | 中国科学院植物研究所 | Genetic engineering chlamydomonas having increased hydrogen producing capacity and application thereof |
| CN103468655A (en) * | 2013-09-03 | 2013-12-25 | 中国农业科学院棉花研究所 | Method for preparing GAPDH (reduced glyceradehyde-phosphate dehydrogenase) transgenic cotton |
| CN109825441A (en) * | 2017-11-23 | 2019-05-31 | 中国科学院大连化学物理研究所 | A kind of method and transgenosis chlamydomonas and application for improving microalgae carbon sequestration efficiency |
| CN110499315A (en) * | 2019-08-21 | 2019-11-26 | 华南理工大学 | Pasteur Du algae glyceraldehyde-3-phosphate dehydrogenase promoter and its application |
| CN114317468A (en) * | 2021-12-06 | 2022-04-12 | 嘉必优生物技术（武汉）股份有限公司 | Escherichia coli underpan cell for enhancing carbon source metabolism |
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2006
- 2006-03-30 CN CN 200610025228 patent/CN1904051A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102181369A (en) * | 2011-02-24 | 2011-09-14 | 中国科学院植物研究所 | Genetic engineering chlamydomonas having increased hydrogen producing capacity and application thereof |
| CN103468655A (en) * | 2013-09-03 | 2013-12-25 | 中国农业科学院棉花研究所 | Method for preparing GAPDH (reduced glyceradehyde-phosphate dehydrogenase) transgenic cotton |
| CN103468655B (en) * | 2013-09-03 | 2016-03-30 | 中国农业科学院棉花研究所 | A kind ofly prepare the method turning GAPDH gene cotton |
| CN109825441A (en) * | 2017-11-23 | 2019-05-31 | 中国科学院大连化学物理研究所 | A kind of method and transgenosis chlamydomonas and application for improving microalgae carbon sequestration efficiency |
| CN109825441B (en) * | 2017-11-23 | 2021-11-30 | 中国科学院大连化学物理研究所 | Method for improving carbon sequestration efficiency of microalgae, transgenic chlamydomonas and application |
| CN110499315A (en) * | 2019-08-21 | 2019-11-26 | 华南理工大学 | Pasteur Du algae glyceraldehyde-3-phosphate dehydrogenase promoter and its application |
| CN114317468A (en) * | 2021-12-06 | 2022-04-12 | 嘉必优生物技术（武汉）股份有限公司 | Escherichia coli underpan cell for enhancing carbon source metabolism |
| CN114317468B (en) * | 2021-12-06 | 2023-10-20 | 嘉必优生物技术（武汉）股份有限公司 | Escherichia coli chassis cell for enhancing carbon source metabolism |
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