CN113046382A - Method for one-step transformation and marking of blue algae chromosome and application - Google Patents
Method for one-step transformation and marking of blue algae chromosome and application Download PDFInfo
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Abstract
The invention discloses a method for one-step transformation and marking of blue algae chromosomes and application. According to the invention, the specificity of a suppressor protein TetR in a tetracycline operon is combined with a manipulation DNA sequence TetO, a TetR-GFP fusion protein and a TetO x n sequence array are transferred to a blue-green algae genome chromosome through one-step genetic transformation, then a promoter is used for inducing the expression of the TetR-GFP fusion protein, the expressed TetR-GFP fusion protein is combined with the TetO x n sequence on the chromosome, finally, a blue-green algae positive strain with a green fluorescence emitting chromosome is obtained, the number and dynamic information of the chromosome can be observed under a fluorescence microscope, and thus, the chromosome fluorescence labeling algae strain can be used as an ideal material for researching the copying and separating mechanism of the blue-green algae chromosome.
Description
Technical Field
The invention belongs to the field of microbial genetic engineering, and particularly relates to a method for finally realizing fluorescence labeling of blue algae chromosomes by only one-step genetic transformation and application.
Background
Replication and isolation of bacterial chromosomes has been extensively and extensively studied in species such as Escherichia coli, Lactobacillus crescentus, Vibrio cholerae and Bacillus subtilis. Bacteria have no nucleus, only one membrane-enclosed pseudo-nucleus, and to distinguish them from plasmid DNA, the present scholars generally refer to the macrocyclic DNA molecule in the pseudo-nucleus as a chromosome. Bacteria typically have 1 or 2 different chromosomes, the second chromosome typically serving as an aid.
However, cyanobacteria, as a prokaryotic bacterium, has a chromosome that is very different from other bacteria: 1. a cyanobacterial cell contains multiple copies of the same chromosome, typically 2-8. 2. The chromosome number per cyanobacterial cell is not fixed, and the current literature tends to suggest that the chromosome number within cyanobacterial cells is related to cell volume. 3. The chromosome replication and segregation of cyanobacteria may not have a particularly strict mechanism, and it has been found through staining and real-time quantitative PCR that the daughter cells of cyanobacteria that just divide contain a difference in the amount of chromosomes, and that the replication of each chromosome of cyanobacteria is independent from cell to cell and even from chromosome within cells, and that multiple chromosomes will replicate simultaneously in no obvious period of time.
Unlike other bacteria, current research on the replication and isolation of cyanobacteria chromosomes is limited, mainly due to the lack of methods for efficiently labeling cyanobacteria chromosomes.
Disclosure of Invention
The invention aims to provide a method for carrying out one-step genetic operation on blue-green algae to finally carry out fluorescence labeling on blue-green algae chromosomes, and provides an effective means for researching the replication and separation mechanism of the blue-green algae chromosomes in the future.
In order to achieve the purpose, the specificity of a suppressor protein TetR (Tet decompressor) in a tetracycline operator is combined with an operation DNA sequence TetO (Tet operator), a TetR-GFP fusion protein and a TetO xn sequence array are transferred to a blue-green algae genome chromosome through one-step genetic transformation, then a promoter is used for inducing the expression of the TetR-GFP fusion protein, and the expressed TetR-GFP fusion protein is combined with the TetO xn sequence on the chromosome, so that the blue-green algae positive strain with the chromosome capable of emitting green fluorescence is finally obtained. Specifically, the technical scheme of the invention is as follows:
a method for marking blue algae chromosomes comprises the following steps:
(1) constructing a TetO sequence array TetO multiplied by n times of repetition of TetO sequences, wherein 10-30 nt random base sequences R1, R2, … … and Rn are inserted between every two TetO sequences because a section of continuous repeated sequences are very unstable in the genetic replication of bacteria, wherein n is a positive integer and is not less than 60;
(2) designing and constructing a chromosome marker vector which has a core element Upstream-P1-TetR-GFP-TetO × n-S-Downstem, wherein: upstream and Downstream represent Upstream and Downstream homologous fragments which connect the core element into a certain locus of a blue algae chromosome gene through homologous double exchange; p1Represents a promoter that can function in cyanobacteria; TetR-GFP is a fusion protein of the suppressor protein TetR with Green Fluorescent Protein (GFP) in the tetracycline operon; TetO × n is a TetO sequence array formed by repeating the TetO sequence n times, and S represents a selectable marker gene;
(3) transferring the chromosome marker vector into blue-green algae, continuously streaking and passaging positive clones on a plate culture medium with selective pressure, and screening homozygous positive strains;
(4) the positive homozygous strain is taken to detect the green fluorescence, and then the fluorescent bright spots of the blue algae chromosome can be observed.
In the step (1), the TetO sequence is: 5'-tctctatcactgataggga-3' (SEQ ID No:1), in one embodiment of the invention, a TetO sequence array TetO x 120 formed by repeating a TetO sequence 120 times is designed, and the sequence is shown as SEQ ID No:2 in the sequence table.
In the step (2), the promoter P1An inducible promoter or a constitutive promoter which is suitable for selection can be selected according to different blue algae species, such as: cu can be selected from Anabaena sp2+An induced promoter PpetE; fe can be selected from Synechococcus sp.PCC 7002 and Synechocystis sp.PCC 68033+The inducible promoter PisiAB; zn can be selected from Synechococcus sp.PCC 79422+The inducible promoter Psmt.
In the step (2), the Upstream homologous fragment of the Downstream homologous fragment of the Upstream homologous fragment of the Downstream homologous fragment of the Upstream homologous.
In the step (2), the selectable marker gene is usually a resistance marker gene, such as erythromycin resistance gene Em, kanamycin resistance gene Kan, neomycin resistance gene Neo, streptomycin resistance gene Sm, and the like.
In the step (3), the chromosome marker vector may be transferred into cyanobacteria by a natural transformation method, or may be transferred by a triparental mating genetic transformation method. The filamentous blue-green algae of anabaena or nostoc is generally genetically transformed by triparental mating, while common unicellular blue-green algae such as Synechococcus sp.PCC 7002, Synechococcus sp.PCC 7942 and Synechocystis sp.PCC 6803 are naturally competent and can be selected by natural transformation methods.
In one embodiment of the present invention, a chromosome marker vector 277ARY is constructed, which has a core element Upstream-PpetE-TetR-GFP-TetO × 120-Em-downlnstream thereon, wherein a cyanobacterial promoter PpetE induced by copper ions is capable of regulating the expression of TetR-GFP fusion protein according to the concentration of copper ions in a culture environment; carrying out genetic transformation on a chromosome marker vector 277ARY through triparental mating to enter Anabaena sp.PCC 7120, then continuously streaking and passaging positive clones on an erythromycin resistance plate, and screening a homozygous positive strain ARY; the positive homozygous strain ARY is taken to detect the green fluorescence, and then the fluorescent bright point of the blue algae chromosome can be observed.
The marking method of blue-green algae chromosomes provided by the invention creatively combines a TetO DNA sequence array formed by repeating a TetO sequence for multiple times with a TetR-GFP fusion protein, and the number and dynamic information of chromosomes can be observed under a fluorescence microscope by only carrying out one-step genetic transformation and transferring the obtained positive algae into blue-green algae genomes. The chromosome fluorescence labeling strain obtained by the invention can be used as an ideal material for researching the replication and separation mechanism of blue algae chromosomes in the future.
Drawings
FIG. 1 is a schematic diagram of construction of a chromosome marker vector 277ARY and a schematic diagram of obtaining a chromosome marker strain ARY by homologous recombination.
FIG. 2 electrophoretogram of PCR test of chromosome marker positive strain ARY.
FIG. 3 is a fluorescent picture of chromosome marker positive strain ARY, each dot of green fluorescence representing each marked cyanobacteria chromosome.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. In the following examples,% is by mass unless otherwise specified.
The pRL277 vector is owned by the laboratory and was given in 1998 by professor Donald A.Bryant, university of State university of Pennsylvania, original literature sources T.A.Black, Y.P.Cai and C.P.Wolk, Spatial Expression and Autorestriction of Hetr, a Gene analyzed in the Control of heterologous Development in Anabaena (Vol 9, Pg 77,1993), Mol Microbiol 10(5), 1153-. Escherichia coli HB 101: from takara, catalog No.: D9051. the cyanobacterium Anabaena sp.pcc 7120 (derived from freshwater algae seed bank (FACHB) of chinese academy of sciences) and mutants derived therefrom are grown in BG11 liquid or solid medium. BG11 solid medium: agar was added to BG11 liquid medium to a final concentration of 1.2% (mass percent, g/mL). During liquid culture, a fluorescent lamp light source is illuminated, and the light intensity is 25uE/m2S, temperature 30 ℃ and introduction of 1% CO2(v/v) air. Erythromycin Em for selection of growth+The antibiotic concentrations were: 10. mu.g/mL.
The experimental E.coli strain DH5 a was grown in conventional LB medium, with the antibiotic concentrations selected for growth: 75 mu g/mL Sm+，150μg/mL Em+。
BG11 medium: per liter of culture medium
Wherein the Trace metal mix comprises the following components:
example 1 construction of chromosome marker vector 277ARY
(1) Construction of TetO repeat sequence TetO × 120
A schematic diagram of a TetO sequence array TetO × 120 in which TetO sequences are repeated 120 times is shown in FIG. 1A, in which the base sequence of TetO is tctctatcactgataggga. Because a continuous multiple repeat sequence is quite unstable in the genetic replication of bacteria, 10-30 nt random base sequences R are inserted between every two TetO sequences. In one example of our implementation, the entire base sequence of TetO.times.120 was designed as shown in SEQ ID No. 2 of the sequence Listing. Then TetO X120 is handed in Beijing Ongke Biotechnology Co., Ltd. to obtain the gene synthesis technology.
(2) Construction of chromosome marker vector 277ARY
A schematic diagram of the construction of the chromosomal marker vector 277ARY having the core element Upstream-P is shown in FIG. 1B1-TetR-GFP-TetO × 120-Em-downlnstream, wherein: upstream and Downstream represent Upstream and Downstream homologous fragments which are connected into a certain site of a blue algae gene through homologous double exchange; promoter P1Selecting a cyanobacteria promoter PpetE; TetR-GFP is a fusion protein of the suppressor protein TetR with Green Fluorescent Protein (GFP) in the tetracycline operon; TetO.times.120 is a TetO sequence array formed by repeating the TetO sequence 120 times, and Em represents an erythromycin resistance gene. Using wild Anabaena sp.PCC 7120 genome total DNA as a template, amplifying Upstream and Downstream fragments by using primer pairs P1/P2 and P3/P4, and using primer pair PP amplification from 5/P6, P7/P81And a TetR-GFP fragment, and amplifying a resistance gene erythromycin Em gene fragment by using a primer pair P9/P10; then using the whole gold one-step seamless cloning kit (
Uni Senamless Cloning and Assembly Kit) six fragments, Upstream, P1, TetR-GFP, TetO × 120, Em, Downstram, were ligated simultaneously into pRL277 plasmid to obtain chromosome marker vector 277 ARY. Wherein the base sequence of the TetR-GFP is shown as SEQ ID No. 3 in the sequence table.
Example 2 screening of chromosome marker-positive Strain ARY and chromosome fluorescence detection
(1) Screening and homozygous verification of chromosome marker positive strain ARY
Transferring the constructed chromosome marker vector 277ARY into a wild Anabaena sp.PCC 7120 genome through triparental mating, wherein the schematic diagram of homologous double exchange between the chromosome marker vector 277ARY and the genome is shown as C in figure 1, and P in the chromosome marker vector 277ARY is subjected to homologous double exchange1The method for triparental mating by swapping the TetR-GFP-TetO X120-Em fragment between all1730 and all1729 genes of the chromosome of the Anabaena sp.PCC 7120 genome is as follows: 1. the combined plasmid pRL443 and the helper plasmid pRL528 are transformed into Escherichia coli HB101, and recombinant bacteria A containing the resistance genes Amp (ampicillin), Sm (streptomycin), Cm (chloramphenicol), and Tc (tetracycline) are obtained. 2. Transferring the chromosome marker vector 277ARY into the recombinant bacterium A to obtain a recombinant bacterium B, and washing the recombinant bacterium B for 3 times by using an LB culture medium. 3. Wild-type Anabaena sp.pcc 7120 was washed 3 times with BG11 liquid medium. 4. And (3) centrifuging the recombinant strain B in the step (2) and the wild Anabaena sp.PCC 7120 in the step (3), placing the centrifuged recombinant strain B in a suspension culture medium (a culture medium obtained by mixing an LB culture medium and a BG11 culture medium in equal volumes), fully mixing the centrifuged recombinant strain B and the suspension culture medium, and standing the mixture for 4 hours at room temperature under a low-light culture condition. 5. And covering a sterile nitrocellulose membrane on BG11 solid culture medium, then coating the thallus mixture on the sterile nitrocellulose membrane, and culturing by illumination until new cyanobacterial colonies grow out.
Positive strains were screened on plates containing BG11 solids of sucrose erythromycin Em and serial streaking (about 3 generations) allowed homozygous mutants. The positive strains were identified by PCR using the primer pairs P11 and P12 (see FIG. 2 for the identification results, the size of the PCR band of WT is 0.79Kb, and the size of the PCR band of the positive strains is 11.8Kb), and the identification results show that the positive strains completely do not see the specific bands of the wild strains, i.e., the positive chromosome marker strain ARY is homozygous.
(2) Chromosome detection of ARY using fluorescence microscopy
After culturing the positive homozygous strain ARY in liquid BG11, a sample was prepared and observed under a fluorescent microscope. The green fluorescence of ARY was observed using 488nm excitation fluorescence. As shown in the fluorescence picture of fig. 3, each dot of green GFP fluorescence represents a labeled blue algae chromosome.
TABLE 1 primer sequences used in the present invention
SEQUENCE LISTING
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gatcctcgag tcgagtcgag cattacagtt tacgaaccga acaggcttat gtcaactggg 60
tgctagatag tctctcatct ctatcacgat agggaagtgc ccctgtctct atcactgata 120
gggaacttct tgtctctcta tcactgatag ggaccgcatt ttttgctaga ggcccccatc 180
ttctctatca ctgataggga atgcgccctc tctctatcac tggtagggac cccccctttt 240
ctctatcact gatagggatc tttgcgcgtg ctagagattc ccccgctctc tatcactgat 300
agggattcct ccccttctct atcactgata gggacctgct gcgttctcta tcactgatag 360
ggagcacccg cgtgctagag tgccagtacc tctctatcac tgatagggaa ttgtcccttt 420
ctctatcact gatagggaag tttttccatc tctatcactg atagggatgt catgccttgc 480
tagagtctat atttatctct atcactgata gggagtcccc ctcatctcta tcactgatag 540
ggatacgtcc cgatctctat cactgatagg gtctcgctct cttgctagag tgcccggttt 600
tctctatcac tgatagggac ggaacgccgt ctctatcact gatagggacg ctatctctct 660
ctatcactga tagggacccc catgcctgct agagtggctt cttttctcta tcactgatag 720
ggactgcacg tcgtctctat cactgatagg gattccgccg cttctctatc actgataggg 780
acacgcggcc gtgctagagt gatgcgtttt ctctatcact gatagggacc tcccctgctc 840
tctatcactg atagggatac cactccttct ctatcactga tagggatttg tttcgttgct 900
agagcactag ggggtctcta tcactgatag ggaccctgct aggtctctat cactgatagg 960
gaagagcgtt tatctctatc actgataggg attcccccgt ctgctagagg gcccttccct 1020
ctctatcact gataggggtg gtctgctctc tctaccacag atagggatca acaactttct 1080
ctatcactga tagggaactg ctactttgct agagtgtggc cctttctcta tcactgatag 1140
ggaaccatct ctttctctat cactgatagg gatattctgg tttctctatc acttagggac 1200
agctaccact gctagagtcc caggccctct ctatcactga tagggaacga ccttactctc 1260
tatcactgat agggagttcc ccctttctct atcctgatag ggatcgtcca tactgctaga 1320
ggctgccctc ctctctatca ctatagggat ccccctcatt ctctatcact gatagggatg 1380
tggtctcctc tctatcactg atagggatct ccgccgttgc tagagtgccc gcgcctctct 1440
atcactgata gggatatgcg tgcttctcta tcactgatag ggagccattg ccttctctat 1500
cactgatagg gattattcct tctgctagag ctactattct tctctatcac tgatagggac 1560
ccccggcgct ctctatcact gatagggagg catcgccctc tctatcactg atagggaccc 1620
cctggcttgc tagagatgcg ttacctctct atcactgata gggaccggcc cttttctcta 1680
tcactgatag ggagccgcct ctctctctat cactgatagg gatcatttan gctgctagag 1740
tgctgcttcc tctctaccac tgatagggac gcttttatat ctctatcact gatagggatt 1800
ttggcccttc tctatcactg agagggacgt ttggccctgc tagagctccc cgccctctct 1860
atcactgata gggattcctg atagggacct gccgcgctct ctatcactga tagggacgtt 1920
ggcacctgct agagcgctct accctctcta tcactgatag ggaccctgtg ctttctctat 1980
cactgatagg actcaggcct ctctctatca ctgataggga catccccccg tgctagagac 2040
tctttttcgt ctctatcact gatagggact ctccggaatc tctatcactg gtagggacgt 2100
ttgtctctct ctatcactga tagggactag ttgcaatgct agagtctccc cgcctctcta 2160
tcactgatag ggactttttt ccctctctat cactgatagg gattttcgtt gttctctatc 2220
actgataggg atacctcacc tgctagagtt tttaacggtc tctatcactg atagggagtc 2280
ggctcgctct ctatcactga tagggattta tatgactctc tatcactgat agggattagc 2340
tcccctgcta gagattataa ggctctctat cactgatagg gatcccccct cgtctctatc 2400
actaataggg atcactccgc gtctctatca ctgataggga gtcgctctca tgctagagtc 2460
cgtctctttc tctatcactg atagggactt agcactctct ctatcactga tagggagctt 2520
acttcctctc tatcactgat agggacgcct ctctctgcta gagtaccttc cactctctat 2580
cactgatagg gatacatctt cctccctatc actgataggg acgtgnataa ttctctatca 2640
ctatagggat agcggcctnt gntagagcct accattctct ctatcactga tagggacctt 2700
ctccacgtct ctatcactga tagggacctc ctctcttctc tatcactgat agggacacgg 2760
ctccgtgcta gagtccccgc ggttctctat cactgatagg gatttaaacc gctctctatc 2820
actgataggg atgtctcccg gtctcnatcn ctnataggga tcagactcca tgctagagcc 2880
tccttacntn tcnatcactg atagggatcc gccngngtct ctatcantga tagggaaccg 2940
ccnccatctc tatcactgat agggatggnc cccatngcta gagttatccc ttgtctctat 3000
cactgatngg gatggctctt cgtctctatc actgataggg agntctgcgc tctntctatc 3060
nactgatagg gactanagac cctgctanag agtagccccc tcnctatcnc tgatagggac 3120
acccgagatt ctctatcnct gagggattcn tnagtgtctc tntcnctgat agggannaca 3180
cttcatgnta gagtgntgat ctgtctctat cnctgatagg gacatctccc cctctctatc 3240
nctgagggac ctctnacttt ctctgtcact gatagggacn actctttctg ctagagtctc 3300
tctccatctc tatcactgat agggactcgg ctttatctct atcactgata gggacggtcc 3360
cgtctctcta tcactgatag ggantgngcc cnctgctaga gccagctcgc ttctctatca 3420
ctgataggga acatttcggc tctctatcac tgatagggag gccccgcatt ctctatcact 3480
gatagggatt ctcggccctg ctagagatgc accccgtctc tatcactgat agggatagtc 3540
cgcagtctct atcactgata gggatgcgtt ctgttctcta tcactgtagg gaccattgct 3600
agagttgtcc cttctctcta tcactgatag ggactccggc gtctctctat cactgatagg 3660
gacaagcgcg cctctctatc actgataggg accgtctccc ctgctagagc ttcgtccctt 3720
ctctatcact gatagggatc catcctgttc tctatcactg ataggatcta taggtctctc 3780
tatcactgat agggaccttc ccttctgcta gagctggccc cattctctat cactgatagg 3840
gaccgtctcc tttctctatc actgataggg accacctgtc tctctatcac tgatagggac 3900
cccctcgctt gctagagcga ttacctttct ctatcactga tagggacctt ccgtcgtctc 3960
tatcactgat agggatctcc tgttctctct atcactgata gggaccctgc ccgctgctag 4020
agccacacac tgtctttatc actgataggg acctccccac ttctctatca ctgataggga 4080
cgcgcaagat agggacccgc ctccgtgcta gagtcccact tcgtctctat cactgatagg 4140
gatcctttcc cctctctatc actgataggg acggttcgtc ttctctatca ctaaagcttg 4200
gatagggtct cgctctcttg ctagagtgcc cggttttctc tatcactgat agggacggaa 4260
cgccgtctct atcactgata gggacgctat ctctctctat cactgatagg gacccccatg 4320
cctgctagag tggcttcttt tctctatcac tgatagggac tgcacgtcgt ctctatcact 4380
gatagggatt ccgccgcttc tctatcactg atagggacac gcggccgtgc tagagtgatg 4440
gatccactag ttctagagcg gccgc 4465
<210> 3
<211> 1389
<212> DNA
<213> Artificial sequence
<400> 3
atgtctagat tagataaaag taaagtgatt aacagcgcat tagagctgct taatgaggtc 60
ggaatcgaag gtttaacaac ccgtaaactc gcccagaagc taggtgtaga gcagcctaca 120
ttgtattggc atgtaaaaaa taagcgggct ttgctcgacg ccttagccat tgagatgtta 180
gataggcacc atactcactt ttgcccttta gaaggggaaa gctggcaaga ttttttacgt 240
aataacgcta aaagttttag atgtgcttta ctaagtcatc gcgatggagc aaaagtacat 300
ttaggtacac ggcctacaga aaaacagtat gaaactctcg aaaatcaatt agccttttta 360
tgccaacaag gtttttcact agagaatgca ttatatgcac tcagcgctgt ggggcatttt 420
actttaggtt gcgtattgga agatcaagag catcaagtcg ctaaagaaga aagggaaaca 480
cctactactg atagtatgcc gccattatta cgacaagcta tcgaattatt tgatcaccaa 540
ggtgcagagc cagccttctt attcggcctt gaattgatca tatgcggatt agaaaaacaa 600
cttaaatgtg aaagtgggtc cgcgtacagc gctagcggtg atgacgatga caagctggga 660
attgatccct tcaccagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc 720
gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat 780
gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc 840
tggcccaccc tcgtgaccac cttcggctac ggcctgcagt gcttcgcccg ctaccccgac 900
cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc 960
accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc 1020
gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc 1080
ctggggcaca agctggagta caactacaac agccacaacg tctatatcat ggccgacaag 1140
cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg 1200
cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc 1260
gacaaccact acctgagcta ccagtccgcc ctgagcaaag accccaacga gaagcgcgat 1320
cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg 1380
tacaagtaa 1389
<210> 4
<211> 42
<212> DNA
<213> Artificial sequence
<400> 4
gaatggcaga aattcgatat ctggcgtagc ctgtgcttgc gt 42
<210> 5
<211> 46
<212> DNA
<213> Artificial sequence
<400> 5
tgtttaaggc taagatggtc gagatctggc taggggaatt accttg 46
<210> 6
<211> 43
<212> DNA
<213> Artificial sequence
<400> 6
ctataaatta tttaataagt aactgttgaa tgttgactgt atg 43
<210> 7
<211> 45
<212> DNA
<213> Artificial sequence
<400> 7
aacgttgttg ccattgctgc aggttaagtg attttgaccg tgtag 45
<210> 8
<211> 49
<212> DNA
<213> Artificial sequence
<400> 8
caaggtaatt cccctagcca gatctcgacc atcttagcct taaacaagt 49
<210> 9
<211> 40
<212> DNA
<213> Artificial sequence
<400> 9
cttttatcta atctagacat cctgtagttt tatttttctt 40
<210> 10
<211> 40
<212> DNA
<213> Artificial sequence
<400> 10
aagaaaaata aaactacagg atgtctagat tagataaaag 40
<210> 11
<211> 50
<212> DNA
<213> Artificial sequence
<400> 11
ctcgactcga ctcgaggatc attacttgta cagctcgtcc atgccgagag 50
<210> 12
<211> 48
<212> DNA
<213> Artificial sequence
<400> 12
gatccactag ttctagagcg gccgcgcgtg ctataattat actaattt 48
<210> 13
<211> 43
<212> DNA
<213> Artificial sequence
<400> 13
catacagtca acattcaaca gttacttatt aaataattta tag 43
<210> 14
<211> 20
<212> DNA
<213> Artificial sequence
<400> 14
atctccctta cctgatgaat 20
<210> 15
<211> 20
<212> DNA
<213> Artificial sequence
<400> 15
aataaaaggc ttccaagtct 20
Claims (10)
1. A method for marking blue algae chromosomes comprises the following steps:
1) constructing a TetO sequence array TetO multiplied by n times of repetition of TetO sequences, and inserting 10-30 nt random base sequences R1, R2, … … and Rn between every two TetO sequences, wherein n is a positive integer and is more than or equal to 60;
2) designing and constructing the Upestream-P with the core element1-a chromosomal marking vector of TetR-GFP-TetO xn-S-downlnstream, wherein: the Upstream and Downstream homologous fragments of the core element connected to a certain locus of the blue algae chromosome gene through homologous double exchange are represented by Upstream and Downstream; p1Represents a promoter; the TetR-GFP is a fusion protein of a suppressor protein TetR and green fluorescent protein in a tetracycline operator; TetO × n is a TetO sequence array formed by repeating the TetO sequence n times, and S represents a selectable marker gene;
3) transferring the chromosome marker vector into blue-green algae, continuously streaking and passaging positive clones on a plate culture medium with selective pressure, and screening homozygous positive strains;
4) the positive homozygous strain is taken to detect the green fluorescence, and then the fluorescent bright spots of the blue algae chromosome can be observed.
2. The labeling method of claim 1, wherein the TetO sequence is 5'-tctctatcactgataggga-3', and a TetO sequence array TetO x 120 is constructed in step 1), and the sequence is shown as SEQ ID No. 2 in the sequence Listing.
3. The labeling method of claim 1, wherein the promoter P in step 2)1Is inducible or constitutive.
4. The labeling method of claim 3, wherein the promoter P1Selected from Cu2+Inducible promoters PpetE, Fe3+Inducible promoter pisiAB, Zn2+The inducible promoter Psmt.
5. The labeling method according to claim 1, wherein in step 2), the length of the filamentous cyanobacteria Upstream and Downflow is 3Kb, and the length of the single-cell cyanobacteria Upstream and Downflow is 1 Kb.
6. The method according to claim 1, wherein the selectable marker gene in step 2) is a resistance marker gene.
7. The labeling method according to claim 1, wherein the resistance marker gene is an erythromycin resistance gene Em, a kanamycin resistance gene Kan, a neomycin resistance gene Neo, or a streptomycin resistance gene Sm.
8. The method as claimed in claim 1, wherein in step 3), the chromosome marker vector is transferred into unicellular cyanobacteria by natural transformation or transferred into filamentous cyanobacteria by triparental mating genetic transformation.
9. The labeling method according to claim 1, wherein the cyanobacteria is Anabaena sp.pcc 7120, the chromosome marker vector 277ARY with the core element Upstream-PpetE-TetR-GFP-TetO x 120-Em-downlnsteam is constructed, then the chromosome marker vector 277ARY is genetically transformed into Anabaena sp.pcc 7120 through triparental mating, then positive clones are continuously streaked on an erythromycin resistant plate for passage, and the homozygous positive strains ARY are screened, and green fluorescence of the cyanobacteria chromosome is detected, so that the fluorescent bright spot of the cyanobacteria chromosome can be observed.
10. The application of the blue algae chromosome marking method of any one of claims 1-9 in researching blue algae chromosome replication and separation mechanisms.
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| CN110438053A (en) * | 2019-07-23 | 2019-11-12 | 天津大学 | Biological sequestration system suitable for synechococcus, construction method and application |
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