CN102127563A - Method for expressing exogenous gene by using synechocystis pevalekii PCC6803 - Google Patents
Method for expressing exogenous gene by using synechocystis pevalekii PCC6803 Download PDFInfo
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Abstract
The invention discloses a method for expressing an exogenous gene by using synechocystis pevalekii PCC6803. The method comprises the following steps of: (1) constructing an rbcL promotor (Prbc,1.3kb) and a termination sequence rbc terminator (Trbc,0.2kb) of synechocystis pevalekii PCC6803, 2kb of spectinomycin resistant maker gene omega and a reporter gene lacZ on a synechocystis pevalekii PCC6803 genome integration plasmid platform to obtain a platform plasmid pFQ20, which contains the reporter gene and is used for expressing the exogenous gene by using the synechocystis pevalekii PCC6803 and supporting genomic integration; and (2) transforming a synechocystis pevalekii PCC6803 strain by using the pFQ20, performing spectinomycin resistant screening to obtain a transformer, identifying a gene type by using a specific primer PCR (Polymerase Chain Reaction) and measuring the beta-galactosidase enzyme activity of the gene engineering synechocystis pevalekii PCC6803 strain which transforms an expression platform and the exogenous gene to verify the expression efficiency of an exogenous gene expression platform of the synechocystis pevalekii PCC6803.
Description
Technical field
The invention belongs to bioengineering field, be specifically related to a kind of method of cyanobacteria cytoalgae PCC6803 efficiently expressing exogenous gene.
Background technology
Cyanobacteria claims blue-green algae again, is a kind ofly can carry out photosynthetic prokaryotic micro-organisms, can directly utilize sun power, carbonic acid gas and water to grow, and has fast growth, photosynthetic efficiency advantages of higher.Therefore, cyanobacteria is with a wide range of applications at bioenergy and field of biological pharmacy as a kind of engineered pattern host microorganism.
Cyanobacteria has special genetic manipulation background, and domestic and international many scientific research team is studied the exogenous gene expression of cyanobacteria, but compares with traditional genetically engineered host e. coli, and cyanobacteria is still very low to expression of exogenous gene efficient.How to make goal gene in blue-green algae, obtain efficiently expressing and remain a difficult problem.Therefore, exploitation platform technology of efficiently expressing exogenous gene in cyanobacteria is extremely important.
Genetic expression platform in the cyanobacteria is meant a kind of expression vector, in the intravital existence form of cyanobacteria, is divided into two kinds in shuttle vector and genome conformity plasmid carrier according to carrier again.Two types expression vector can mainly import goal gene in the cyanobacteria body by means of the natural conversion (mainly being unicellular blue green algae) or the method for conjugal transfer.Shuttle vector can import cyanobacteria by conjugal transfer, self-replicating in tenuigenin.The genome conformity carrier can be incorporated into the mode that goal gene and even whole operon are integrated by homology in the blue-green algae genome, and its advantage is to express stablely, can overcome the unsettled shortcoming of autonomously replicating plasmid.
The selection of promotor is most important for genetic expression.Rbc (ribulose-1,5-bisphosphatecarboxylase/oxygenase gene) promotor is the intravital strong promoter of cyanobacteria, its product 1, contracting of 5-ribulose diphosphate enzyme/oxydase (RuBisCO) is a main soluble proteins in the cyanobacteria body, plays an important role in photosynthesis.The rbc promotor possesses the ability that efficiently expresses gene in the cyanobacteria body, by successful Application expressing gene in multiple cyanobacteria bacterial strain, comprising synechococcus 6301 (Synechococcus PCC 6301), anabena 7120 (Anabaena sp.strain PCC7120), synechococcus 7002 (Synechococcus PCC 7002), synechococcus 7942 (Synechococcussp.strain PCC 7942) etc.
Cytoalgae 6803 (Synechocystis PCC6803) is the type strain in the cyanobacteria, and its genome sequence is determined in 1996, and genetic manipulation method is ripe relatively, is suitable as a strain pattern algae strain and carries out transgenic research.Cytoalgae 6803 is unicellular blue green algaes, can utilize nature to transform and accept foreign DNA, also can carry out genetic manipulation by the method for conjugal transfer.In cytoalgae 6803 (Synechocystis PCC6803), the specifying information of mentioning the rbc promotor and having inferred rbc promotor in the cytoalgae 6803 in the research of Doron Amichay and Ruth Levitz etc., comprise promoter element and rbcL in the rbc operon, rbcX and rbcS gene, whole operon begins at rbcL upstream region of gene 250bp place, and infer that in rbcS terminator codon downstream one section 40bp reverse complementary sequence being arranged is terminator sequence, Amichay etc. are by order-checking and think in rbcL upstream-15～and there is similar SD sequence in-10 districts, and the sequence of finding similar-10 districts, two places and-35 districts at 60bp place, translation initiation codon upstream is [referring to (Construction of a Synechocystis PCC6803mutant suitable for thestudy of variant hexadecameric ribulose bisphosphate carboxylase/oxygenaseenzymes, Doron Amichay, Ruth Levitz and Michael Gurevitz, Plant MolecularBiology 23:465-476,1993.)].
The human superoxide dismutase gene hSOD of the rbc promoter expression of people's utilizations in synechococcus 6301 (also being called the nido cyanobacteria of falling capsule Anacystis nidulans 6301) such as Yasunobu Takeshima of Japan own, finally the content of human superoxide-dismutase reaches 3% of total soluble protein in the strain of sudden change algae.In this experiment, promotor length is the sequence of rbc upstream region of gene 343bp, and has added the rbc terminator sequence of 294bp in hSOD gene downstream.People such as Yasunobu Takeshima have studied in the synechococcus 6301rbc promoter element distance between the SD sequence and ATG simultaneously to the influence of expression of gene efficient, prove by change the SD sequence with and and ATG between distance all can make the enzymic activity change of expression product hSOD.Its achievement is published in (High-levelexpression of human superoxide dismutase in the cyanobacterium Anacystisnidulans 6301, YASUNOBU TAKESHIMA on the PNAS
*, NOBORUTAKATSUGU
*, Proc.Nadl.Acad.Sci.USA, Vol.91, pp.9685-9689,1994).
The Ming-de Deng of University of Toronto and John R.Coleman utilize the rbc promoter expression pdc of self and two genes of adh to produce ethanol in synechococcus 7942 (Synechococcus sp.strain PCC 7942).The promotor length that it adopts has been added the terminator sequence of pdc self 27bp equally at 361bp in the downstream of goal gene pdc.And in their research, used a kind of translation convergence strategy, this strategy can make pdc expression of gene amount improve nearly 2 times [(Ethanol Synthesis by Genetic so that the distance between ATG and the SD sequence remains unchanged
Engineering?in?Cyanobacteria，MING-DE?DENG?AND?JOHN?R.COLEMAN，APPLIED?AND?ENVIRONMENTAL?MICROBIOLOGY，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 cytoalgae PCC6803 expression alien gene.
For achieving the above object, the method for cytoalgae PCC6803 expression alien gene provided by the invention, main process comprises:
1) respectively with the rbcL promotor (Prbc of cytoalgae 6803,1.3kb) and terminator sequence rbcterminator (Trbc, 0.2kb), the spectinomycin resistant maker gene Ω of 2kb, and reporter gene lacZ is implemented in a cytoalgae 6803 genome conformity plasmid platforms, and what comprised reporter gene is used for cytoalgae 6803 expression alien genes and platform plasmid pFQ20 that can genome conformity;
2) pFQ20 transforms cytoalgae 6803 algae strains, obtain transformant through the spectinomycin resistance screening, behind special primer PCR identified gene type, mensuration has transformed the beta-galactosidase enzymes enzyme of the genetically engineered cytoalgae 6803 algae strains of expressing platform and foreign gene and has lived, with the expression efficiency of checking cytoalgae 6803 exogenous gene expression platforms.
In the described method, the rbcL promotor of cytoalgae 6803 (Prbc, 1.3kb) and terminator sequence rbc terminator (Trbc, 0.2kb), the spectinomycin resistant maker gene Ω of 2kb, and reporter gene lacZ obtains by the clone.
In the described method, cytoalgae 6803 genome conformity plasmid platforms are to utilize the EcoRI enzyme to cut pKW1188, from connecting, obtain pKW1188SL after the removal C.K2 fragment.
The present invention has set up a kind of exogenous gene high-efficient expressed method of using cytoalgae 6803 self rbc strong promoter, SD sequence, rbc terminator.
By cloning experimentation with reference to the promotor in different algae strains source, compare rbcL strong promoter element sequences in other algae kinds, final this upstream region of gene of clone 1300bp is as promotor, clone rbcS terminator codon TAA downstream 200bp as terminator sequence, and guaranteed that the distance between SD sequence and SD sequence and the ATG is identical with wild-type.Provide simultaneously among the present invention a kind of in unicellular cyanobacteria cytoalgae 6803 the genome conformity carrier of efficiently expressing exogenous gene platform.
Construction strategy of the present invention is not only applicable to the rbc promotor, is equally applicable to the promotor of other types, finishes goal gene efficiently expressing in cyanobacteria.
Construction strategy of the present invention not only can be used for expressing the lacZ gene, can express other foreign genes equally.
The construction strategy that proposes among 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 that makes up among the present invention can be applied to the blue-green algae expression vector of other any kinds, comprises other shuttle expression carriers and other genome conformity carriers.
The present invention has detected expression of exogenous gene efficient by the lacZ gene as reporter gene.
Description of drawings
Fig. 1 is the basic structure of plasmid pFQ1, and Prbc (0.3kb) clones the carrier in pMD18T, and the gene fragment two ends have SalI and XbaI site;
Fig. 2 is the basic structure of plasmid pFQ2, and Trbc clones the carrier in pMD18T, and the gene fragment two ends have SmaI and SacI site;
Fig. 3 is the basic structure of plasmid pQL4, and grand resistant gene Ω clones the carrier in pMD18T, and SphI and SalI restriction enzyme site are contained in the gene two ends;
Fig. 4 is the basic structure of plasmid pFQ6, and the carrier in pMD18T is cloned in Prbc and Trbc series connection, and the gene fragment two ends have SalI and SacI site;
Fig. 5 is the basic structure of plasmid pKW1188SL, includes slr0168Nterminal and slr0168C terminal, is the N end and the C end of slr0168 gene, and restriction enzyme site is EcoRI between the two;
Fig. 6 is the basic structure of plasmid pFQ15, comprises to insert site EcoRI, and remove in the SmaI site in this plasmid;
Fig. 7 is the basic structure of plasmid pFQ9Forward, and the carrier in pFQ15 is cloned in Ω, Prbc and Trbc series connection, and XbaI and SmaI site are that foreign gene inserts the site;
Fig. 8 is the basic structure of plasmid pQL12, and the lacZ gene clone is in the pMD18T carrier, and the gene two ends are XbaI and SmaI site;
Fig. 9 is the basic structure of plasmid pFQ19, and the lacZ gene clone is in carrier pFQ9Forward, and the 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 the carrier pFQ19, and two ends have SalI and XbaI enzyme cutting site;
Figure 11 is the basic structure of plasmid pLY2, and grand resistant gene Ω fragment cloning is in the pKW1188SL plasmid;
Figure 12 is the basic structure of plasmid pHB 1567, and Ω fragment, PpetE and lacZ gene oppositely are inserted into clones the plasmid in pKW1188SL.
Embodiment
The objective of the invention is to reach by following measure:
1) will clone the rbcL promotor (Prbc of the cytoalgae 6803 that is obtained respectively, 1.3kb) and terminator sequence rbc terminator (Trbc, 0.2kb), the spectinomycin resistant maker gene Ω of 2kb, and reporter gene lacZ, being implemented in a cytoalgae 6803 genome conformity plasmid platforms (utilizes the EcoRI enzyme to cut pKW1188, certainly connect after removing the C.K2 fragment, obtain pKW1188SL), what comprised reporter gene is used for cytoalgae 6803 expression alien genes and platform plasmid pFQ20 that can genome conformity.
2) pFQ20 transforms cytoalgae 6803 algae strains, obtain transformant through the spectinomycin resistance screening, with special primer PCR identified gene type errorless after, mensuration has transformed the beta-galactosidase enzymes enzyme of the genetically engineered cytoalgae 6803 algae strains that efficiently express platform and foreign gene and has lived, and then has verified the expression efficiency of the constructed cytoalgae of the present invention 6803 exogenous gene expression platforms.
Further specify the present invention with specific examples below.
Embodiment 1: respectively lacZ gene, Prbc, Trbc are cloned on the pMD18T carrier
1) PCR clone Prbc and Trbc fragment from cytoalgae 6803; Plasmid pFQ1 is that Prbc is connected to the pMD18T carrier and obtains; Plasmid pFQ2 is that Trbc is connected to the pMD18T carrier and obtains.Clone lacZ gene (3.1kb) from intestinal bacteria E.coli (BL21DE3) is inserted in the pUC19 plasmid.Obtain plasmid pQL12.
2) all plasmids all identify that through order-checking the segmental nucleotide sequence of purpose is errorless.
Embodiment 2: grand resistant gene Ω and Prbc, Trbc are connected respectively clone on same pMD18T carrier
1) pQL4 is that what to contain grand resistant gene Ω is the carrier that the source makes up with pMD18T.With XbaI and SalI digested plasmid pQL4, reclaim linear fragment pQL4EH; With XbaI and SalI digested plasmid pFQ1, reclaim linear DNA fragment Prbc equally; The pQL4EH fragment is connected with the T4 ligase enzyme with fragment Prbc, transformed into escherichia coli, with the two anti-screening transformants of spectinomycin and ammonia benzyl mycin, again by the upgrading granzyme cut Screening and Identification errorless after, promptly obtain plasmid pFQ5;
2) same quadrat method is cut pFQ2 with the SmaI-SacI enzyme, and glue reclaims the Trbc fragment of 200bp; Simultaneously cut pFQ5, with obtaining double digestion fragment pFQ5EH behind the PCR product purification test kit purifying with the SmaI-SacI enzyme; Utilize the T4 ligase enzyme that Trbc is inserted into the downstream of pFQ5, transformed into escherichia coli, with spectinomycin and the two anti-screening transformants of ammonia benzyl mycin, again by the upgrading granzyme cut Screening and Identification errorless after, promptly obtain plasmid pFQ6; Ω and Prbc, Trbc fragment have been comprised among the pFQ6;
Embodiment 3: grand resistant gene Ω and Prbc, Trbc and lacZ gene are connected in series on the blue-green algae expression vector
1) structure of expression vector and treating processes: pKW1188 is a plasmid platform that is used for cytoalgae 6803 genome conformities.Utilize the EcoRI enzyme to cut pKW1188, from connecting, obtain pKW1188SL after the removal CK2 fragment.PKW1188SL obtains plasmid pFQ15 after removing the SmaI site.
2) processing of goal gene: cut pFQ6 glue with the HindIII-SacI enzyme and reclaim to obtain Ω and Prbc, Trbc fragment, utilize the T4 polysaccharase to mend flat 30min for 37 ℃, obtain mending fragment after flat with PCR product purification test kit purifying; Use with quadrat method after expression vector pFQ15 cuts with EcoRI and mend flat and purifying; The pFQ15 fragment of mending Ω and Prbc, the Trbc fragment after flat and mending after flat is connected with the T4 ligase enzyme, transformed into escherichia coli, the transformant that obtains utilizes special primer PCR to identify the segmental direction of insertion of series connection, screen the clone that purpose fragment forward inserts, extract plasmid enzyme restriction at last and identify errorless back called after pFQ9Forward.
3) will cut pQL12 with XbaI and SmaI enzyme, glue reclaims the lacZ gene fragment, is inserted among the pFQ9Forward, and transformed into escherichia coli gets transformant and identifies the lacZ fragment with special primer PCR, extracts plasmid enzyme restriction at last and identifies errorless back called after pFQ19.The Prbc of pcr amplification 1.3kb utilizes same loci SalI and XbaI, and the Prbc of 0.3kb is replaced, and obtains pFQ20.
Embodiment 4: expression vector is to conversion, the genotype identification of cytoalgae 6803 and the activity that detects the gene expression product beta-galactosidase enzymes.
1) method of pFQ20 being utilized nature transform transforms cytoalgae 6803 wild-type cells.Transformant grows after about 1 week, and transformant enlarged culturing in having the BG11 liquid of spectinomycin is gathered in the crops algae liquid after 1 week, extracts genome.Utilize special primer PCR to identify in the transformant whether contain goal gene lacZ.Identify the sudden change algae strain called after SynechocystisPCC6803 (FQ20) after errorless.
2) treat that Synechocystis PCC6803 (FQ20) is cultured to vegetative period in liquid after, results algae liquid is also measured the beta galactosidase enzyme activity.With Synechocystis PCC6803 wild-type as negative control 1, with Synechocystis PCC6803 (LY2) as negative control 2, with SynechocystisPCC6803 (QL15) as negative control 2.With a strain algae strain Synechocystis PCC6803 (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 that the present invention is constructed under the same conditions, surpasses and to have reported positive control, and all negative controls all fail to detect activity.
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 (Escherichia coli DH5 α) FQ20 and 1 strain cytoalgae (Synechocystis PCC6803) FQ20; Above bacterial strain and algae strain all are deposited in Institute of Microorganism, Academia Sinica China common micro-organisms culture presevation administrative center (CGMCC).
Deposit number (following form is " a CGMCC numbering bacterial strain host bacterium Latin (plasmid number) "):
3458?FQ20?Escherichia?coli?DH5α(pFQ20)
3462?FQ20?Synechocystis?PCC6803(pFQ20)
Claims (3)
1. the method for a cytoalgae PCC6803 expression alien gene, main process comprises:
1) respectively with the rbcL promotor (Prbc of cytoalgae 6803,1.3kb) and terminator sequence rbcterminator (Trbc, 0.2kb), the spectinomycin resistant maker gene Ω of 2kb, and reporter gene lacZ is implemented in a cytoalgae 6803 genome conformity plasmid platforms, and what comprised reporter gene is used for cytoalgae 6803 expression alien genes and platform plasmid pFQ20 that can genome conformity;
2) pFQ20 transforms cytoalgae 6803 algae strains, obtain transformant through the spectinomycin resistance screening, behind special primer PCR identified gene type, mensuration has transformed the beta-galactosidase enzymes enzyme of the genetically engineered cytoalgae 6803 algae strains of expressing platform and foreign gene and has lived, with the expression efficiency of checking cytoalgae 6803 exogenous gene expression platforms.
2. the method for claim 1, wherein rbcL promotor (Prbc of cytoalgae 6803,1.3kb) and terminator sequence rbc terminator (Trbc, 0.2kb), the spectinomycin resistant maker gene Ω of 2kb, and reporter gene lacZ obtains by the clone.
3. the method for claim 1, wherein cytoalgae 6803 genome conformity plasmid platforms are to utilize the EcoRI enzyme to cut pKW1188, from connecting, obtain pKW1188SL after the removal C.K2 fragment.
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| CN201010034404.4A CN102127563B (en) | 2010-01-15 | 2010-01-15 | A kind of method of expressing exogenous gene by using synechocystis pevalekii PCC 6803 |
| 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" |
| 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 |
| 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 |
| 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 |
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Cited By (7)
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| CN102952818A (en) * | 2011-08-26 | 2013-03-06 | 中国科学院青岛生物能源与过程研究所 | Construction body and method for improving fatty alcohol yield in cyanobacteria |
| CN103014053A (en) * | 2012-12-10 | 2013-04-03 | 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 |
| CN103352048A (en) * | 2013-06-27 | 2013-10-16 | 暨南大学 | Microalgae chloroplast carrier for efficient cloning and expression and application thereof |
| CN103361375A (en) * | 2012-04-05 | 2013-10-23 | 中国科学院青岛生物能源与过程研究所 | Construction body for biosynthesizing ethanol in cyanobacteria, bacterial strain and method |
| 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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2010
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102952818A (en) * | 2011-08-26 | 2013-03-06 | 中国科学院青岛生物能源与过程研究所 | Construction body and method for improving fatty alcohol yield in cyanobacteria |
| 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 |
| CN103361375A (en) * | 2012-04-05 | 2013-10-23 | 中国科学院青岛生物能源与过程研究所 | Construction body for biosynthesizing ethanol in cyanobacteria, bacterial strain and method |
| 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 |
| CN103014053A (en) * | 2012-12-10 | 2013-04-03 | 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 |
| 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 |
| CN103352048A (en) * | 2013-06-27 | 2013-10-16 | 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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