CN102127563B - A kind of method of expressing exogenous gene by using synechocystis pevalekii PCC 6803 - Google Patents
A kind of method of expressing exogenous gene by using synechocystis pevalekii PCC 6803 Download PDFInfo
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Abstract
A kind of method of expressing exogenous gene by using synechocystis pevalekii PCC 6803, comprise: 1) respectively by the rbcL promotor (Prbc of cytoalgae 6803,1.3kb) with terminator sequence rbc? terminator (Trbc, 0.2kb), the Spectinomycin resistance marker gene Ω of 2kb, and reporter gene lacZ is implemented in a cytoalgae 6803 genome conformity plasmid platform, comprised reporter gene for cytoalgae 6803 expression alien gene and can the platform plasmid pFQ20 of genome conformity; 2) pFQ20 transforms the strain of cytoalgae 6803 algae, transformant is obtained through Spectinomycin resistance screening, after specific primer PCR identified gene type, measure the beta-galactosidase enzymes enzyme having transformed the genetically engineered cytoalgae 6803 algae strain expressing platform and foreign gene to live, to verify the expression efficiency of cytoalgae 6803 exogenous gene expression platform.
Description
Technical field
The invention belongs to bioengineering field, be specifically related to a kind of method of cyanobacteria DNC wireless efficiently expressing exogenous gene.
Background technology
Cyanobacteria, also known as blue-green algae, is that one can carry out photosynthetic prokaryotic micro-organisms, sun power, carbonic acid gas and water directly can be utilized to grow, have fast growth, photosynthetic efficiency advantages of higher.Therefore, cyanobacteria, as the engineered type bost microorganism of one, is with a wide range of applications at bioenergy and field of biological pharmacy.
Cyanobacteria has special genetic manipulation background, and the exogenous gene expression of domestic and international many Research Teams to cyanobacteria is studied, but compares with traditional genetically engineered host e. coli, and cyanobacteria is still very low to the expression efficiency of foreign gene.How to make goal gene in blue-green algae, obtain high expression and remain a difficult problem.Therefore, the platform technology of efficiently expressing exogenous gene in cyanobacteria is developed extremely important.
Gene expression platform in cyanobacteria refers to a kind of expression vector, according to the existence form of carrier in cyanobacteria body, is divided into again shuttle vector and two kinds, genome conformity plasmid carrier.Goal gene can mainly import in cyanobacteria body by means of the method for Natural Transformation (mainly unicellular blue green algae) or conjugal transfer by the expression vector of two types.Shuttle vector can import cyanobacteria by conjugal transfer, self-replicating in tenuigenin.Goal gene and even whole operon can be incorporated in blue-green algae genome by the mode of Homologous integration by genomic integrated vector, and its advantage expresses to stablize, and can overcome the shortcoming of autonomously replicating plasmid instability.
The selection of promotor is most important for genetic expression.Rbc (ribulose-1,5-bisphosphatecarboxylase/oxygenasegene) promotor is the strong promoter in cyanobacteria body, its product 1,5-ribulose diphosphate contractingization enzyme/oxydase (RuBisCO) is soluble proteins main in cyanobacteria body, plays an important role in photosynthesis.Rbc promotor possesses the ability of high expression gene in cyanobacteria body, be successfully applied expressing gene in multiple cyanobacteria strains, comprising synechococcus 6301 (SynechococcusPCC6301), carbon and nitrogen ratio (Anabaenasp.strainPCC7120), Synechococcus sp.PCC7002 (SynechococcusPCC7002), synechococcus 7942 (Synechococcussp.strainPCC7942) etc.
Cytoalgae 6803 (SynechocystisPCC6803) is the type strain in cyanobacteria, and its genome sequence is determined in 1996, and genetic manipulation method relative maturity, be suitable as a strain pattern algae strain and carry out transgenic research.Cytoalgae 6803 is unicellular blue green algaes, and Natural Transformation can be utilized to accept foreign DNA, and the method also by conjugal transfer carries out genetic manipulation.In cytoalgae 6803 (SynechocystisPCC6803), mention rbc promotor in the research of DoronAmichay and RuthLevitz etc. and inferred the specifying information of rbc promotor in cytoalgae 6803, rbc operon comprises promoter element and rbcL, rbcX and rbcS gene, whole operon starts at rbcL upstream region of gene 250bp place, and inferring has one section of 40bp reverse complementary sequence to be terminator sequence in rbcS terminator codon downstream, Amichay etc. have similar SD sequence by checking order and thinking in rbcL upstream-15 ~-10 district, and the sequence in similar-10th district of 60bp place discovery two place, translation initiation codon upstream and-35th district is [see (ConstructionofaSynechocystisPCC6803mutantsuitableforthes tudyofvarianthexadecamericribulosebisphosphatecarboxylas e/oxygenaseenzymes, DoronAmichay, RuthLevitzandMichaelGurevitz, PlantMolecularBiology23:465-476, 1993.)].
The people such as the YasunobuTakeshima of Japan are utilization rbc promoter expression rhSOD gene hSOD own in synechococcus 6301 (being also called the nido cyanobacteria of falling capsule Anacystisnidulans6301), and finally in the strain of sudden change algae, the content of rhSOD reaches 3% of total soluble protein.In this experiment, promotor length is the sequence of rbc upstream region of gene 343bp, and with the addition of the rbc terminator sequence of 294bp at hSOD downstream of gene.The people such as YasunobuTakeshima have studied distance in synechococcus 6301rbc promoter element between SD sequence and ATG to the impact of the expression efficiency of gene simultaneously, prove the enzymic activity change that all can make expression product hSOD by changing SD sequence and the distance between itself and ATG.Its achievement is published in (High-levelexpressionofhumansuperoxidedismutaseinthecyano bacteriumAnacystisnidulans6301, YASUNOBUTAKESHIMA on PNAS
*, NOBORUTAKATSUGU
*, Proc.Nadl.Acad.Sci.USA, Vol.91, pp.9685-9689,1994).
Ming-deDeng and JohnR.Coleman of University of Toronto utilizes rbc promoter expression pdc and adh two genes of self to produce ethanol in synechococcus 7942 (Synechococcussp.strainPCC7942).The promotor length that it adopts, at 361bp, with the addition of the terminator sequence of pdc self 27bp equally in the downstream of goal gene pdc.And in their research, apply a kind of translation convergence strategy, this strategy can make the distance between ATG and SD sequence remain unchanged, and makes the expression amount of pdc gene improve nearly 2 times of [(EthanolSynthesisbyGenetic
EngineeringinCyanobacteria，MING-DEDENGANDJOHNR.COLEMAN，APPLIEDANDENVIRONMENTALMICROBIOLOGY，p.523-528，Vol.65，No.2，1999)]。
Summary of the invention
The object of the present invention is to provide a kind of method of expressing exogenous gene by using synechocystis pevalekii PCC 6803.
For achieving the above object, the method for expressing exogenous gene by using synechocystis pevalekii PCC 6803 provided by the invention, main process comprises:
1) respectively by the rbcL promotor (Prbc of cytoalgae 6803,1.3kb) with terminator sequence rbcterminator (Trbc, 0.2kb), the Spectinomycin resistance marker gene Ω of 2kb, and reporter gene lacZ is implemented in a cytoalgae 6803 genome conformity plasmid platform, comprised reporter gene for cytoalgae 6803 expression alien gene and can the platform plasmid pFQ20 of genome conformity;
2) pFQ20 transforms the strain of cytoalgae 6803 algae, transformant is obtained through Spectinomycin resistance screening, after specific primer PCR identified gene type, measure the beta-galactosidase enzymes enzyme having transformed the genetically engineered cytoalgae 6803 algae strain expressing platform and foreign gene to live, to verify the expression efficiency of cytoalgae 6803 exogenous gene expression platform.
In described method, the rbcL promotor (Prbc, 1.3kb) of cytoalgae 6803 and terminator sequence rbcterminator (Trbc, 0.2kb), the Spectinomycin resistance marker gene Ω of 2kb, and reporter gene lacZ is obtained by clone.
In described method, cytoalgae 6803 genome conformity plasmid platform utilizes EcoRI enzyme to cut pKW1188, from connecting after removing C.K2 fragment, obtains pKW1188SL.
The present invention establishes a kind of exogenous gene high-efficient expressed method using cytoalgae 6803 self rbc strong promoter, SD sequence, rbc terminator.
By reference to the cloning experimentation of the promotor in different algae strain source, rbcL strong promoter element sequences in other algae kinds of comparison, this upstream region of gene of final clone 1300bp is as promotor, clone rbcS terminator codon TAA downstream 200bp as terminator sequence, and ensure that SD sequence and the distance between SD sequence and ATG identical with wild-type.Provide a kind of genomic integrated vector of efficiently expressing exogenous gene platform in unicellular cyanobacteria cytoalgae 6803 in the present invention simultaneously.
Construction strategy of the present invention, is not only applicable to rbc promotor, is equally applicable to the promotor of other types, completes the high expression of goal gene in cyanobacteria.
Construction strategy of the present invention, not only can be used for expressing lacZ gene, can express other foreign genes equally.
The construction strategy proposed in the present invention, not only can be applied to cytoalgae, is equally applicable to the blue-green algae of other types.
The gene expression element built in the present invention, can be applied to the blue-green algae expression vector of other any types, comprises other shuttle expression carriers and other genomic integrated vector.
The present invention have detected the expression efficiency of foreign gene as reporter gene by lacZ gene.
Accompanying drawing explanation
Fig. 1 is the basic structure of plasmid pFQ1, and Prbc (0.3kb) is cloned in pMD18T carrier, and gene fragment two ends are with SalI and XbaI site;
Fig. 2 is the basic structure of plasmid pFQ2, and Trbc is cloned in pMD18T carrier, and gene fragment two ends are with SmaI and SacI site;
Fig. 3 is the basic structure of plasmid pQL4, and grand resistant gene Ω is cloned in pMD18T carrier, SphI and SalI restriction enzyme site is contained at gene two ends;
Fig. 4 is the basic structure of plasmid pFQ6, Prbc and Trbc series connection is cloned in pMD18T carrier, and gene fragment two ends are with SalI and SacI site;
Fig. 5 is the basic structure of plasmid pKW1188SL, includes slr0168Nterminal and slr0168Cterminal, and be N end and the C end of slr0168 gene, restriction enzyme site is EcoRI between the two;
Fig. 6 is the basic structure of plasmid pFQ15, comprises insertion point EcoRI, and the SmaI site in this plasmid is removed;
Fig. 7 is the basic structure of plasmid pFQ9Forward, and Ω, Prbc and Trbc series connection is cloned in pFQ15 carrier, XbaI and SmaI site is foreign gene insertion point;
Fig. 8 is the basic structure of plasmid pQL12, and lacZ gene clone is in pMD18T carrier, and gene two ends are XbaI and SmaI site;
Fig. 9 is the basic structure of plasmid pFQ19, and lacZ gene clone is in carrier pFQ9Forward, and gene two ends are XbaI and SmaI site;
Figure 10 is the basic structure of plasmid pFQ20, and Prbc (1.3kb) replaces the Prbc (0.3kb) in carrier pFQ19, and two ends are with SalI and XbaI enzyme cutting site;
Figure 11 is the basic structure of plasmid pLY2, and grand resistant gene Ω fragment is cloned in pKW1188SL plasmid;
Figure 12 is the basic structure of plasmid pHB1567, and Ω fragment, PpetE and lacZ gene are oppositely inserted into and are cloned in pKW1188SL plasmid.
Embodiment
The object of the invention is to be reached by following measure:
1) the rbcL promotor (Prbc of the cytoalgae 6803 obtained will be cloned respectively, 1.3kb) with terminator sequence rbcterminator (Trbc, 0.2kb), the Spectinomycin resistance marker gene Ω of 2kb, and reporter gene lacZ, being implemented in a cytoalgae 6803 genome conformity plasmid platform (utilizes EcoRI enzyme to cut pKW1188, from connecting after removing C.K2 fragment, obtain pKW1188SL), comprised reporter gene for cytoalgae 6803 expression alien gene and can the platform plasmid pFQ20 of genome conformity.
2) pFQ20 transforms the strain of cytoalgae 6803 algae, transformant is obtained through Spectinomycin resistance screening, after errorless by specific primer PCR identified gene type, measure the beta-galactosidase enzymes enzyme having transformed the genetically engineered cytoalgae 6803 algae strain of high expression platform and foreign gene to live, and then demonstrate the expression efficiency of the cytoalgae 6803 exogenous gene expression platform constructed by the present invention.
The present invention is further illustrated below with specific examples.
Embodiment 1: respectively lacZ gene, Prbc, Trbc are cloned on pMD18T carrier
1) from cytoalgae 6803, PCR clones Prbc and Trbc fragment; Plasmid pFQ1 is that Prbc is connected to pMD18T carrier and obtains; Plasmid pFQ2 is that Trbc is connected to pMD18T carrier and obtains.From the clone lacZ gene (3.1kb) of E. coli (BL21DE3), be inserted in pUC19 plasmid.Obtain plasmid pQL12.
2) nucleotide sequence of all plasmids all through order-checking qualification object fragment is errorless.
Embodiment 2: grand resistant gene Ω and Prbc, Trbc are connected respectively and is cloned on same pMD18T carrier
1) pQL4 be containing grand resistant gene Ω with pMD18T be source build carrier.With XbaI and SalI digested plasmid pQL4, reclaim linear fragment pQL4EH; Same with XbaI and SalI digested plasmid pFQ1, reclaim linear DNA fragment Prbc; PQL4EH fragment is connected with fragment Prbc T4 ligase enzyme, transformation of E. coli, with spectinomycin and the dual anti-screening transformant of ammonia benzyl mycin, then by carry plasmid enzyme restriction Screening and Identification errorless after, namely obtain plasmid pFQ5;
2) same method, cuts pFQ2 with SmaI-SacI enzyme, and glue reclaims the Trbc fragment of 200bp; Cut pFQ5 with SmaI-SacI enzyme simultaneously, obtain double digestion fragment pFQ5EH with after PCR primer Purification Kit; Utilize T4 ligase enzyme Trbc to be inserted into the downstream of pFQ5, transformation of E. coli, with spectinomycin and the dual anti-screening transformant of ammonia benzyl mycin, then by carry plasmid enzyme restriction Screening and Identification errorless after, namely obtain plasmid pFQ6; Ω and Prbc, Trbc fragment is contained in pFQ6;
Embodiment 3: by grand resistant gene Ω and Prbc, Trbc and lacZ gene tandem on blue-green algae expression vector
1) structure of expression vector and treating processes: pKW1188 are plasmid platform for cytoalgae 6803 genome conformity.Utilizing EcoRI enzyme to cut pKW1188, from connecting after removing CK2 fragment, obtaining pKW1188SL.PKW1188SL obtains plasmid pFQ15 after removing SmaI site.
2) process of goal gene: cut pFQ6 glue with HindIII-SacI enzyme and reclaim acquisition Ω and Prbc, Trbc fragment, utilize T4 polysaccharase 37 DEG C to fill 30min, obtain the fragment PCR primer Purification Kit after filling; Expression vector pFQ15 EcoRI fills and purifying with same method after cutting; Ω with Prbc after filling, Trbc fragment with fill after pFQ15 fragment be connected with T4 ligase enzyme, transformation of E. coli, the transformant obtained utilizes specific primer PCR to identify the direction of insertion of series connection fragment, screen the clone that object fragment forward inserts, finally extract the errorless rear called after pFQ9Forward of plasmid enzyme restriction qualification.
3) will cut pQL12 with XbaI and SmaI enzyme, glue reclaims lacZ gene fragment, is inserted in pFQ9Forward, transformation of E. coli, obtains transformant specific primer PCR qualification lacZ fragment, finally extracts the errorless rear called after pFQ19 of plasmid enzyme restriction qualification.The Prbc of pcr amplification 1.3kb, utilizes same loci SalI and XbaI, is replaced by the Prbc of 0.3kb, obtains pFQ20.
Embodiment 4: expression vector is to the activity of the conversion of cytoalgae 6803, genotype identification and gene expression detection product, O-galactosidase.
1) method of Natural Transformation is utilized by pFQ20 to transform cytoalgae 6803 wild-type cell.After about 1 week, transformant grows, and transformant enlarged culturing in the BG11 liquid with spectinomycin was gathered in the crops algae liquid after 1 week, extracts genome.Specific primer PCR is utilized whether to identify in transformant containing goal gene lacZ.Identify errorless after sudden change algae strain called after SynechocystisPCC6803 (FQ20).
2) after SynechocystisPCC6803 (FQ20) is cultured to vegetative period in a liquid, results algae liquid also measures beta galactosidase enzyme activity.Using SynechocystisPCC6803 wild-type as negative control 1, using SynechocystisPCC6803 (LY2) as negative control 2, using SynechocystisPCC6803 (QL15) as negative control 2.Using strain algae strain SynechocystisPCC6803 (HB1567) of the tilactase expression activity that is in the news as positive control.Wherein SynechocystisPCC6803 (LY2) is the algae strain that genome conformity has resistant gene Ω.
3) result shows, the galactosidase activity of the genetically engineered cytoalgae 6803FQ20 constructed by the present invention, under the same conditions, exceed and report positive control, and all negative controls all fails activity to be detected.
Biological material specimens preservation information
The biological material specimens that the present invention relates to is 1 constructed plasmid (pFQ20; Figure 10) with corresponding 1 strain coli strain (EscherichiacoliDH5 α) FQ20 and 1 strain cytoalgae (SynechocystisPCC6803) FQ20; Above bacterial strain and algae strain are all deposited in China General Microbiological culture presevation administrative center of Institute of Microorganism, Academia Sinica (CGMCC).
Deposit number (following form is " CGMCC numbering bacterial strain Host Strains Latin (plasmid number) "):
3458FQ20EscherichiacoliDH5α(pFQ20)
3462FQ20SynechocystisPCC6803(pFQ20)
Claims (3)
1. a coli strain FQ20EscherichiacoliDH5 α, deposit number is CGMCC3458.
2. a cytoalgae FQ20SynechocystisPCC6803, deposit number is CGMCC3462.
3. a plasmid pFQ20, is included in coli strain FQ20EscherichiacoliDH5 α as claimed in claim 1 and cytoalgae FQ20SynechocystisPCC6803 as claimed in claim 2.
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| EP11700428A EP2524035A2 (en) | 2010-01-15 | 2011-01-17 | Constructs, vectors and cyanobacteria for the synthesis of fatty alcohols, and methods for producing fatty alcohols in cyanobacteria |
| PCT/EP2011/050555 WO2011086189A2 (en) | 2010-01-15 | 2011-01-17 | Constructs, vectors and cyanobacteria for the synthesis of fatty alcohols, and methods for producing fatty alcohols in cyanobacteria |
| BR112012017456A BR112012017456A2 (en) | 2010-01-15 | 2011-01-17 | "construct, vector, cyanobacteria, method for producing fatty alcohols in a cyanobacterium, biofuel, and exogenous gene expression method" |
| CA2786244A CA2786244A1 (en) | 2010-01-15 | 2011-01-17 | Constructs, vectors and cyanobacteria for the synthesis of fatty alcohols, and methods for producing fatty alcohols in cyanobacteria |
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| CN102952818B (en) * | 2011-08-26 | 2016-03-02 | 中国科学院青岛生物能源与过程研究所 | For improving construct and the method for fatty alcohol yield in cyanobacteria |
| CN103361375B (en) * | 2012-04-05 | 2018-02-09 | 中国科学院青岛生物能源与过程研究所 | Construct, bacterial strain and the method for cyanobacteria biosynthesis ethanol |
| CN103014053B (en) * | 2012-12-10 | 2014-01-22 | 山东省农业科学院高新技术研究中心 | Synechocystis efficient double homologous recombinant vector as well as construction method and application thereof |
| CN103352048B (en) * | 2013-06-27 | 2015-02-18 | 暨南大学 | Microalgae chloroplast carrier for efficient cloning and expression and application thereof |
| CN104293803A (en) * | 2014-09-29 | 2015-01-21 | 天津大学 | Ethanol-tolerant related gene slr0982 of synechocystis 6803 and applications of gene |
| CN110157725A (en) * | 2019-05-21 | 2019-08-23 | 武汉藻优生物科技有限公司 | Make not produce the production poison microalgae that malicious microalgae generates the method for Microcystin and obtains |
| CN112646831A (en) * | 2019-10-10 | 2021-04-13 | 天津科技大学 | Shuttle plasmid, construction method and application thereof in synechocystis transformation exogenous gene |
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Non-Patent Citations (2)
| Title |
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| Construction of a synechocyst is PCC6803 mutant suitable for the study of variant hexadecameric ribulose bisphosphate carboxylase/oxygenase enzymes;Doron Amichay et al;《Plant Molecular Biology》;19931231;第23卷;465-476 * |
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Inventor after: Lv Xuefeng Inventor after: Qi Fengxia Inventor after: Tan Xiaoming Inventor after: Luo Quan Inventor before: Lv Xuefeng Inventor before: Qi Fengxia Inventor before: Tan Xiaoming Inventor before: Luo Quan |