CN109517048B - Optogenetics tool for regulating long-distance interaction of chromatin under blue light - Google Patents
Optogenetics tool for regulating long-distance interaction of chromatin under blue light Download PDFInfo
- Publication number
- CN109517048B CN109517048B CN201811474709.XA CN201811474709A CN109517048B CN 109517048 B CN109517048 B CN 109517048B CN 201811474709 A CN201811474709 A CN 201811474709A CN 109517048 B CN109517048 B CN 109517048B
- Authority
- CN
- China
- Prior art keywords
- blue light
- cry2
- cib1
- long
- ser
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Mycology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Botany (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Peptides Or Proteins (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
An optogenetics tool for regulating chromatin long-distance interaction under blue light, which relates to the field of optogenetics. The optogenetic tool for blue light induction of blue light receptors CRY2 and CIB1 to form protein complexes and regulation of long-distance interaction of chromatin can realize the regulation of long-distance interaction of chromatin in eukaryotes. In a optogenetics tool for inducing blue light receptors CRY2 and CIB1 to form protein complexes and regulating long-distance interaction of chromatin, the blue light receptors CRY2 and CIB1 can form complexes in yeast, and can also realize the interaction of the light receptors CRY2 and CIB1 to form complexes through a transgenic animal model in animal experiments. The long-distance interaction of the chromosome is realized by forming a complex through interaction of a blue light-induced photoreceptor CRY2 and CIB1, so that the application potential of the tool for regulating the high-order structure of the chromosome is proved.
Description
Technical Field
The invention relates to the field of optogenetics, in particular to an optogenetics tool for controlling long-distance interaction of chromatin under blue light, wherein the optogenetics tool is used for inducing blue light receptors CRY2 and CIB1 to form a protein complex.
Background
Chromosomal long range interactions may occur between regions located on the same chromosome or between regions on different chromosomes, where interactions between promoters and enhancers often require interactions between bridging proteins. For example, the interaction between the trajectory control region (LCR) and the β -globin gene requires the binding of GATA1 and EKLF1 transcription factors to enhancer and target genes. At the same time, the interaction of GATA1 and EKLF1 transcription factors promotes the binding of enhancers and target genes. In plants, The interaction of The CCAAT nuclear factor Y (NF-Y) complex with The transcription factor CO protein promotes chromatin looping on The FT gene promoter and regulates Arabidopsis flowering time ([1] Shuanghe Caoet et al, The Plant Cell, Vol.26:1009-1017, March 2014).
Another type of intrachromosomal interaction is insulator-mediated interaction that organizes the genome into functionally distinct regions by separating differentially regulated regions. CTCT binding factor (CTCF) is considered a major insulator protein in mammals, and the CTCF border elements segregate genomic regions, thereby dividing the genome into active and inactive regions. It has been found that loss of specific CTCF sites can lead to serious diseases such as cancer and heart failure.
Traditionally, Fluorescence In Situ Hybridization (FISH) technology is used to detect genomic interactions. Recently, a Chromosome Conformation Capture (3C) technology can accurately detect genome interactions. This technique and its variants 4C, 5C and Hi-C help us to understand in depth the long distance interactions of chromosomes ([2] Satish Sati, Giacomo Cavalli. Chromosoma, Vol 126:33-44, February 2017). However, all 3C techniques and their variants only remain in the detection of chromatin interactions that already exist. If chromatin interactions can be controlled at will, it is believed that a new door to 3D genomic studies will be opened. Currently, methods and tools for light-controlled chromosome interactions remain open.
Developments in optogenetic technology have made it possible to modulate chromatin interactions at will, CRY2 being a photoreceptor with a peak of maximum absorption at a wavelength of 450nm in blue light, and CRY2 being an interaction with the downstream factor CIB1 in blue light ([3] Hongtao Liu et al, Science, Vol 322,1535-1539, December 2008).
Disclosure of Invention
The invention aims to provide an optogenetic tool for blue light to down-regulate chromatin long-distance interaction, wherein blue light induces blue light receptors CRY2 and CIB1 to form a protein complex.
The optogenetic tool for regulating chromatin long-distance interaction under blue light is an optogenetic tool for inducing blue light receptors CRY2 and CIB1 to form a protein complex and regulating chromatin long-distance interaction, and comprises the following components:
1) the blue light receptor CRY2 and downstream factor CIB1 of the model plant Arabidopsis are cloned to a yeast expression vector;
2) yeast two-hybrid experiments prove that blue light can induce the interaction of a blue light receptor CRY2 and a downstream factor CIB1 to form a protein complex;
3) two artificially constructed chromosome fragments respectively containing the binding sequences of LexA and GAL4BD are constructed on the No. 5 chromosome of the saccharomyces cerevisiae EGY48 strain by means of linearization integration and homologous recombination, and the distance between the two artificially constructed chromosome fragments is 12.1 kb;
4) transforming pLexA-CRY2 and pBridge-CIB1 in yeast cells, the pLexA-CRY2 expressing the fusion protein BD-CRY2, the pBridge-CIB1 expressing the fusion protein GAL4BD-CIB 1; so that the fusion protein BD-CRY2 can recognize the binding sequence of LexA, and the fusion protein GAL4BD-CIB1 can recognize the binding sequence of GAL4 BD;
5) when the yeast cells were irradiated with blue light, the fusion protein BD-CRY2 and the fusion protein GAL4BD-CIB1 interacted to form a protein complex; thus, the binding sequence of LexA and the binding sequence of GAL4BD that interact with them are spatially drawn closer; finally, the binding sequence of LexA and the binding sequence of GAL4BD, which are 12.1kb apart, are allowed to interact chromatin over long distances.
The clone can verify the correctness of the sequence by a sequencing method; the blue light receptor CRY2 and the downstream factor CIB1 genes of the model plant Arabidopsis are cloned in the Arabidopsis thaliana ecotype Col-0.
Whether CRY2 and CIB1 interacted under blue light was determined by blue coloration of yeast transfected with the p8op-lacZ reporter plasmid.
The binding of BD-CRY2 and GAL4BD-CIB1 to the corresponding binding sequences was indeed present using the ChIP-qPCR assay;
chromosome Conformation Capture technique was used to identify whether long-range interactions between target fragments actually exist.
The optogenetic tool for regulating the long-distance interaction of chromatin under blue light is an optogenetic tool for regulating the long-distance interaction of chromatin by using blue light to induce blue light receptors CRY2 and CIB1 to form a protein complex, and realizes the regulation of the long-distance interaction of chromatin in a eukaryote.
In a optogenetics tool for inducing blue light receptors CRY2 and CIB1 to form protein complexes and regulating long-distance interaction of chromatin, the blue light receptors CRY2 and CIB1 can form complexes in yeast, and can also realize the interaction of the light receptors CRY2 and CIB1 to form complexes through a transgenic animal model in animal experiments.
The present invention provides:
1. an artificial sequence LexAops-GAL1 minor promoter-YFP for blue light regulation of long-distance chromatin interaction in yeast, the base sequence of which is shown as SEQ ID NO. 1:
2. an artificial sequence homologus arm1-UEE (PGK1) -GAL4ops-proTEF1-KanMX-TerTEF 1-homologus arm2 for performing blue light regulation and long-distance chromatin interaction in yeast, wherein the base sequence of the artificial sequence is shown as SEQ ID NO. 2:
3. a protein CRY2 for controlling long-distance interaction of blue light regulation chromatin has an amino acid sequence shown in SEQ ID NO. 3:
in the optogenetic tools described above, the blue light receptor CRY2 is used for chromatin long distance interactions in yeast, and can also be used for chromatin long distance interactions in mammals and plants.
In the optogenetic tool described above, the blue light receptor CRY2 was cloned by arabidopsis thaliana, but also by other plants with blue light receptor CRY 2.
4. A protein CIB1 for controlling blue light to regulate long-distance interaction of chromatin has an amino acid sequence shown as SEQ ID NO. 4:
in the optogenetic tools described above, the CIB1 can be used for chromatin long-distance interactions in yeast, and can also be used for chromatin long-distance interactions in mammals and plants.
In the optogenetic tool described above, the CIB1 was cloned from Arabidopsis thaliana, but also from other plants with CIB 1.
Therefore, the invention realizes the long-distance interaction of the regulation chromosome through the interaction of the blue light induced photoreceptor CRY2 and CIB1 to form a complex, thereby proving that the tool has the application potential of regulating the higher structure of the chromosome.
The invention provides a optogenetics tool which utilizes blue light-induced interaction of plant Arabidopsis light receptors CRY2 and CIB1 to form a complex to regulate the long-distance interaction of chromosomes.
The invention has the beneficial effects that: the instrument realizes the regulation and control of long-distance chromosome interaction in eukaryote based on blue light waveband photoreceptor CRY2 and downstream factor CIB1, has the characteristics of no toxicity and no contact, and can be used in the field of application of optogenetics.
Drawings
Fig. 1 is an overall schematic diagram of an embodiment of the present invention.
FIG. 2 shows interaction of CRY2 and CIB1 in yeast two-hybrid to verify that blue light can induce heterologous expression in yeast.
FIG. 3 shows that the binding sequence of LexA can be identified by BD-CRY2 and the binding sequence of GAL4BD can be identified by GAL4BD-CIB1 as verified by ChIP-qPCR experiments.
FIG. 4 shows Chromosome Conformation Capture Chromosome Conformation transformation Capture (3C) experiment to verify that blue light mediated BD-CRY2 and GAL4BD-CIB1 interaction enables two artificially constructed Chromosome fragments to generate long-distance interaction
FIG. 5 is a flow chart of the sampling of the Time course Chromosome Conformation Capture (Time course3C) experiment.
FIG. 6 is a Timecourse3C experiment demonstrating that long range chromosomal interactions occur rapidly and remain stable under UV-B continuous irradiation.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to the accompanying drawings.
According to the strategy of FIG. 1, it is first necessary to construct yeast with artificially constructed chromosome fragment, remove 2. mu. ori of p8op-YFP vector, and then linearize the vector with ApaI endonuclease, the main element LexAops-GAL1 minor promoter-YFP nucleic acid sequence is shown as SEQ ID NO. 1.
And integrating the linearized vector sequence to chromosome 5 of the saccharomyces cerevisiae EGY48 strain by utilizing the principle of homologous recombination. The construction of the nucleic acid sequence homoloyus arm1-UEE (PGK1) -GAL4ops-protE F1-KanMX-TerTEF 1-homoloyus arm2, the constructed DNA sequence comprising the control elements homoloyus arm1-UEE (PGK1) -GAL4 ops-KanMX-homoloyus arm2, was constructed by Homologous recombination at a position downstream of the LexAops-GAL1mini promoter-YFP insertion sequence on chromosome 5, LexAops and GAL4ops being 12.1kb apart. The yeast QY1 shown in FIG. 1 was obtained.
The homologus arm1-UEE (PGK1) -GAL4ops-proTEF1-KanMX-TerTEF 1-homologus arm2 nucleic acid sequence is shown as SEQ ID NO. 2.
The coding sequence of the protein sequence for coding CRY2 and CIB1 is obtained by amplifying cDNA of arabidopsis Col-0. CRY2 is cloned to the XhoI enzyme cutting site of the pLexA vector; CIB1 was cloned in the middle of the EcoRI and XhoI cleavage sites of the pB42AD vector. The resulting recombinant vector was co-transferred to strain EGY48(Clontech) containing p8 op-lacZ. The transformants were cultured in the Dark at SD/-His/-Trp/-Ura, then transferred to SD/Gal/Raff/-His/-Trp/-Ura induction medium containing 5-bromo-4-chloro-3-indoyl- β -D-galactopyranoside, placed in Dark and 450nm blue light (5.5 uW/cm), respectively2) The color is developed after 36h of growth. Blue light did induce the combination of CRY2 and CIB1 expressed in yeast to appear blue as shown in fig. 2, thus demonstrating that CRY2 and CIB1 interact in yeast. Finally, CRY2 and CIB1 were determined to interact under blue light by blue coloration of yeast transfected with the p8op-lacZ reporter plasmid. CRY2 has amino acid sequence shown as SEQ ID NO. 3. The amino acid sequence of CIB1 is shown as SEQ ID NO. 4.
In the yeast QY1, the DNA fragments of the CRY2 and CIB1 coding sequences in the yeast two-hybrid vector are respectively used for constructing vectors pLexA-CRY2 and pBridge-CIB1, and expressing BD-CRY2 fusion protein and GAL4BD-CIB1 fusion protein. The yeast QY6 shown in FIG. 1 was obtained.
After 200ml of QY6 yeast grown to an OD600 of about 0.8 was added to a final concentration of 1% formaldehyde and crosslinked at room temperature, the cell sample was disrupted by Tissue lyser ii (QIAGEN) to extract nuclei. After the nuclei were digested with Mnase (New English Biolabs, M0247S), the centrifuged pellet was sonicated in Bioraptor (Diagenode), the supernatants after Mnase digestion were pooled, and immunoprecipitation was performed using anti-LexA DNA Binding Region antibody (abcam, ab14553) and GAL4(DBD) (RK5C1) (Santa CruZ Biotechonlogy, Inc: sc-510). Finally, the subsequent qPCR experiments were performed by ethanol precipitation of ChIP-DNA. And a junction as shown in fig. 3 is obtainedAs a result, the results showed that BD-CRY2 could be enriched near the LexA recognition element, while GAL4BD-CIB1C340May be enriched in the vicinity of the recognition element of GAL4 BD. Finally, the binding of BD-CRY2 and GAL4BD-CIB1 to the corresponding binding sequences was indeed identified using the ChIP-qPCR experiment.
It was necessary to demonstrate that BD-CRY2 bound to the LexA recognition element and GAL4BD-CIB1 enriched in the recognition element of GAL4BDC340Interaction may occur in blue light to mediate the interaction of the LexA recognition element and the GAL4BD recognition element that are 12.1kb apart. Thus, 3C experiments were performed. First, QY6 yeast was picked up and cultured overnight in 20ml of medium, and inoculated into 50ml of medium again so that OD600 ≈ 0.15, and cultured at 30 ℃ and 200rpm for 6 hours, and the yeast OD600 ≈ 0.6-0.85. Adding formaldehyde with final concentration of 1% for crosslinking at room temperature for 15min, terminating crosslinking at room temperature for 5min with glycine, and washing with precooled deionized water for 3 times. The yeast cells were resuspended in FA lysine buffer (140mM NaCl,50mM HEPES,1mM EDTA,1X PMSF,1X Cocktail) to OD600 ═ 100, 60. mu.l of yeast cells were pipetted into 740. mu.l of precooled FA lysine buffer, mixed well and dispensed into 4 1.5ml EP tubes, 200. mu.l of precooled glass beads (Biospec products,11079105) were added to each tube. The samples were placed in precooled Tissue lyserII (QIAGEN) to disrupt the cells. The EP tube was punctured with a 22gauge syringe needle, placed in a 15ml centrifuge tube, centrifuged at 2000rpm for 2min at 4 ℃, the EP tube containing glass beads was discarded, centrifuged at 12000rpm for 10min, 10mM Tris-HCl pH 7.9 was used to resuspend the yeast, and metal bath at 65 ℃ for 10 min. The resuspended centrifuged yeast was digested with NlaIII (New English Biolabs) at 37 ℃ for 8 h. The cleavage products were ligated with T4DNA ligase (New English Biolabs, M0202L) for 13h at 16 ℃ and 1h at 22 ℃. The ligated 3C DNA was precipitated with ethanol. Purified 3C DNA was tested for the frequency of interaction of the LexA recognition sequence with the GAL4 recognition sequence by TaqMan qpcr. The interaction frequency of the promoter and the terminator of the HEM3 gene in the saccharomyces cerevisiae is used as an internal reference. The results are shown in FIG. 4, 450nm blue light (5.5 uW/cm)2) The LexA recognition sequence can be induced to interact with the GAL4 recognition sequence for long distances. Finally, the Chromosome Conformation Capture technology is used for identifying the real existence of long-distance interaction between target fragments.
According to the sample handling protocol shown in FIG. 5, a single QY6 yeast clone was transferred to 20ml of medium for overnight culture. Then, the yeast cells were evenly distributed into 5 plastic bottles and kept at OD600 ≈ 0.15. Next 5 plastic bottles were wrapped in tinfoil paper at 450nm blue light (5.5 uW/cm)2) Incubate in the environment and remove the tinfoil paper at 0, 3, 5, 5.5 and 6h, respectively. Finally, samples were collected at 6h and subjected to the Time course3C experiment. As shown in fig. 6, the long-distance chromosome interaction rapidly occurs in the early stage of blue light irradiation and is stably maintained under blue light irradiation.
In eukaryotes, long-distance chromatin interactions play an important role in regulating gene expression, controlling cell differentiation, and influencing the developmental process of organisms. At present, the development of tools for the photoregulative control of chromatin looping remains open. The invention relates to a method for regulating chromatin conformation by blue light. Saccharomyces cerevisiae is used as an experimental object, and the interaction between blue light receptors CRY2 and CIB1 of blue light-induced model plant Arabidopsis thaliana can regulate the interaction of DNA fragments with a distance of more than 10kb on the same chromosome in a eukaryotic genome. The invention of the genetic tool has important application value for noninvasive targeted change of the high-order structure of chromatin in living cells through optical signals in the future.
Sequence listing
<110> university of mansion
<120> optogenetics tool for controlling long-distance interaction of chromatin under blue light
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1113
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
tcgactgctg tatataaaac cagtggttat atgtccagta ctgctgtata taaaaccagt 60
ggttatatgt acagtacgtc gactgctgta tataaaacca gtggttatat gtacagtact 120
gctgtatata aaaccagtgg ttatatgtac agtacgtcga ggggatgata atgcgattag 180
ttttttagcc ttatttctgg ggtaattaat cagcgaagcg atgatttttg atctattaac 240
agatatataa atgcaaaaac tgcataacca ctttaactaa tactttcaac attttcggtt 300
tgtattactt cttattcaaa tgtaataaaa gtatcaacaa aaaattgtta atatacctct 360
atactttaac gtcaaggaga aaaaactata atggtgagca agggcgagga gctgttcacc 420
ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg 480
tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc 540
accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccttcggcta cggcctgcag 600
tgcttcgccc gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc 660
gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc 720
gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac 780
ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac 840
gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac 900
aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc 960
gacggccccg tgctgctgcc cgacaaccac tacctgagct accagtccgc cctgagcaaa 1020
gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc 1080
actctcggca tggacgagct gtacaagtcc taa 1113
<210> 2
<211> 2873
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
aaagagcata acggacggtg taatgcgaaa ggtcattggt acggacgact ggaaattggc 60
tagacaagtg cagtttgaat tggatgatac ggtgcagttc atgagaagag cactagagta 120
aggagatagt aggcgacact tcgaaaatct aaagtcttgt aacattacta ttatatacta 180
ttttattaaa aaaatacaca cacaattctt taacagaaca atatataaca aatgaagcga 240
catgtttctc gaagtacctt caaagaatgg ggtcttatct tgttttgcaa gtaccactga 300
gcaggataat aatagaaatg ataatatact atagtagaga taacgtcgat gacttcccat 360
actgtaattg cttttagttg tgtattttta gtgtgcaagt ttctgtaaat cgattaattt 420
ttttttcttt cctcttttta ttaaccttaa tttttatttt agattcctga cttcaactca 480
agacgcacag atattataac atctgcataa taggcatttg caagaattac tcgtgagtaa 540
ggaaagagtg aggaactatc gcatacctgc atttaaagat gccgatttgg gcgcgaatcc 600
tttattttgg cttcaccctc atactattat cagggccaga aaaaggaagt gtttccctcc 660
ttcttgaatt gatgttaccc tcataaagca cgtggcctct tatcgagaaa gaaattaccg 720
tcgctcgtga tttgtttgca aaaagaacaa aactgaaaaa acccagacac gctcgacttc 780
ctgtcttcct attgattgca gcttccaatt tcgtcacaca acaaggtcct agcgacggct 840
cacaggtttt gtaacaagca atcgaaggtt ctggaatggc gggaaagggt ttagtaccac 900
atgctatgat gcccactgtg atctccagag caaagttcgt tcgatcgtac tgttactctc 960
tctctttcaa acagaattgt ccgaatcgtg tgacaacaac agcctgttct cacacactct 1020
tttcttctaa ccaagggggt ggtttagttt agtagaacct cgtgaaactt acatttacat 1080
atgaagtacg gattagaagc cgccgagcgg gtgacagccc tccgaaggaa gactctcctc 1140
cgtgcgtcct cgtcttcacc ggtcgcgttc ctgaaacgca gatgtgcctc gcgccgcact 1200
gctccgaaga tctgtttagc ttgcctcgtc cccgccgggt cacccggcca gcgacatgga 1260
ggcccagaat accctccttg acagtcttga cgtgcgcagc tcaggggcat gatgtgactg 1320
tcgcccgtac atttagccca tacatcccca tgtataatca tttgcatcca tacattttga 1380
tggccgcacg gcgcgaagca aaaattacgg ctcctcgctg cagacctgcg agcagggaaa 1440
cgctcccctc acagacgcgt tgaattgtcc ccacgccgcg cccctgtaga gaaatataaa 1500
aggttaggat ttgccactga ggttcttctt tcatatactt ccttttaaaa tcttgctagg 1560
atacagttct cacatcacat ccgaacataa acaaccatgg gtaaggaaaa gactcacgtt 1620
tcgaggccgc gattaaattc caacatggat gctgatttat atgggtataa atgggctcgc 1680
gataatgtcg ggcaatcagg tgcgacaatc tatcgattgt atgggaagcc cgatgcgcca 1740
gagttgtttc tgaaacatgg caaaggtagc gttgccaatg atgttacaga tgagatggtc 1800
agactaaact ggctgacgga atttatgcct cttccgacca tcaagcattt tatccgtact 1860
cctgatgatg catggttact caccactgcg atccccggca aaacagcatt ccaggtatta 1920
gaagaatatc ctgattcagg tgaaaatatt gttgatgcgc tggcagtgtt cctgcgccgg 1980
ttgcattcga ttcctgtttg taattgtcct tttaacagcg atcgcgtatt tcgtctcgct 2040
caggcgcaat cacgaatgaa taacggtttg gttgatgcga gtgattttga tgacgagcgt 2100
aatggctggc ctgttgaaca agtctggaaa gaaatgcata agcttttgcc attctcaccg 2160
gattcagtcg tcactcatgg tgatttctca cttgataacc ttatttttga cgaggggaaa 2220
ttaataggtt gtattgatgt tggacgagtc ggaatcgcag accgatacca ggatcttgcc 2280
atcctatgga actgcctcgg tgagttttct ccttcattac agaaacggct ttttcaaaaa 2340
tatggtattg ataatcctga tatgaataaa ttgcagtttc atttgatgct cgatgagttt 2400
ttctaatcag tactgacaat aaaaagattc ttgttttcaa gaacttgtca tttgtatagt 2460
ttttttatat tgtagttgtt ctattttaat caaatgttag cgtgatttat attttttttc 2520
gcctcgacat catctgccca gatgcgaagt taagtgcgca gaaagtaata tcatgcgtca 2580
atcgtatgtg aatgctggtc gctatactgc tgtcgattcg ataaacggga caaacaaaac 2640
gaagagcagg gtgtatttgc ccacacacgg caaacaacat ccattcacgc ccgcatgccc 2700
aaaacttttt tgcgataaaa cgcccacgca aatcacatcg ttccaacaga ggaatcgaac 2760
agcacagata caaagtagga atatagcaca cacacaggaa aaattaatta attattagaa 2820
attaaaaggg ttggggagaa aagaggtcta atttattcat taatattcag taa 2873
<210> 3
<211> 612
<212> PRT
<213> Arabidopsis thaliana (Arabidopsis thaliana)
<400> 3
Met Lys Met Asp Lys Lys Thr Ile Val Trp Phe Arg Arg Asp Leu Arg
1 5 10 15
Ile Glu Asp Asn Pro Ala Leu Ala Ala Ala Ala His Glu Gly Ser Val
20 25 30
Phe Pro Val Phe Ile Trp Cys Pro Glu Glu Glu Gly Gln Phe Tyr Pro
35 40 45
Gly Arg Ala Ser Arg Trp Trp Met Lys Gln Ser Leu Ala His Leu Ser
50 55 60
Gln Ser Leu Lys Ala Leu Gly Ser Asp Leu Thr Leu Ile Lys Thr His
65 70 75 80
Asn Thr Ile Ser Ala Ile Leu Asp Cys Ile Arg Val Thr Gly Ala Thr
85 90 95
Lys Val Val Phe Asn His Leu Tyr Asp Pro Val Ser Leu Val Arg Asp
100 105 110
His Thr Val Lys Glu Lys Leu Val Glu Arg Gly Ile Ser Val Gln Ser
115 120 125
Tyr Asn Gly Asp Leu Leu Tyr Glu Pro Trp Glu Ile Tyr Cys Glu Lys
130 135 140
Gly Lys Pro Phe Thr Ser Phe Asn Ser Tyr Trp Lys Lys Cys Leu Asp
145 150 155 160
Met Ser Ile Glu Ser Val Met Leu Pro Pro Pro Trp Arg Leu Met Pro
165 170 175
Ile Thr Ala Ala Ala Glu Ala Ile Trp Ala Cys Ser Ile Glu Glu Leu
180 185 190
Gly Leu Glu Asn Glu Ala Glu Lys Pro Ser Asn Ala Leu Leu Thr Arg
195 200 205
Ala Trp Ser Pro Gly Trp Ser Asn Ala Asp Lys Leu Leu Asn Glu Phe
210 215 220
Ile Glu Lys Gln Leu Ile Asp Tyr Ala Lys Asn Ser Lys Lys Val Val
225 230 235 240
Gly Asn Ser Thr Ser Leu Leu Ser Pro Tyr Leu His Phe Gly Glu Ile
245 250 255
Ser Val Arg His Val Phe Gln Cys Ala Arg Met Lys Gln Ile Ile Trp
260 265 270
Ala Arg Asp Lys Asn Ser Glu Gly Glu Glu Ser Ala Asp Leu Phe Leu
275 280 285
Arg Gly Ile Gly Leu Arg Glu Tyr Ser Arg Tyr Ile Cys Phe Asn Phe
290 295 300
Pro Phe Thr His Glu Gln Ser Leu Leu Ser His Leu Arg Phe Phe Pro
305 310 315 320
Trp Asp Ala Asp Val Asp Lys Phe Lys Ala Trp Arg Gln Gly Arg Thr
325 330 335
Gly Tyr Pro Leu Val Asp Ala Gly Met Arg Glu Leu Trp Ala Thr Gly
340 345 350
Trp Met His Asn Arg Ile Arg Val Ile Val Ser Ser Phe Ala Val Lys
355 360 365
Phe Leu Leu Leu Pro Trp Lys Trp Gly Met Lys Tyr Phe Trp Asp Thr
370 375 380
Leu Leu Asp Ala Asp Leu Glu Cys Asp Ile Leu Gly Trp Gln Tyr Ile
385 390 395 400
Ser Gly Ser Ile Pro Asp Gly His Glu Leu Asp Arg Leu Asp Asn Pro
405 410 415
Ala Leu Gln Gly Ala Lys Tyr Asp Pro Glu Gly Glu Tyr Ile Arg Gln
420 425 430
Trp Leu Pro Glu Leu Ala Arg Leu Pro Thr Glu Trp Ile His His Pro
435 440 445
Trp Asp Ala Pro Leu Thr Val Leu Lys Ala Ser Gly Val Glu Leu Gly
450 455 460
Thr Asn Tyr Ala Lys Pro Ile Val Asp Ile Asp Thr Ala Arg Glu Leu
465 470 475 480
Leu Ala Lys Ala Ile Ser Arg Thr Arg Glu Ala Gln Ile Met Ile Gly
485 490 495
Ala Ala Pro Asp Glu Ile Val Ala Asp Ser Phe Glu Ala Leu Gly Ala
500 505 510
Asn Thr Ile Lys Glu Pro Gly Leu Cys Pro Ser Val Ser Ser Asn Asp
515 520 525
Gln Gln Val Pro Ser Ala Val Arg Tyr Asn Gly Ser Lys Arg Val Lys
530 535 540
Pro Glu Glu Glu Glu Glu Arg Asp Met Lys Lys Ser Arg Gly Phe Asp
545 550 555 560
Glu Arg Glu Leu Phe Ser Thr Ala Glu Ser Ser Ser Ser Ser Ser Val
565 570 575
Phe Phe Val Ser Gln Ser Cys Ser Leu Ala Ser Glu Gly Lys Asn Leu
580 585 590
Glu Gly Ile Gln Asp Ser Ser Asp Gln Ile Thr Thr Ser Leu Gly Lys
595 600 605
Asn Gly Cys Lys
610
<210> 4
<211> 335
<212> PRT
<213> Arabidopsis thaliana (Arabidopsis thaliana)
<400> 4
Met Asn Gly Ala Ile Gly Gly Asp Leu Leu Leu Asn Phe Pro Asp Met
1 5 10 15
Ser Val Leu Glu Arg Gln Arg Ala His Leu Lys Tyr Leu Asn Pro Thr
20 25 30
Phe Asp Ser Pro Leu Ala Gly Phe Phe Ala Asp Ser Ser Met Ile Thr
35 40 45
Gly Gly Glu Met Asp Ser Tyr Leu Ser Thr Ala Gly Leu Asn Leu Pro
50 55 60
Met Met Tyr Gly Glu Thr Thr Val Glu Gly Asp Ser Arg Leu Ser Ile
65 70 75 80
Ser Pro Glu Thr Thr Leu Gly Thr Gly Asn Phe Lys Lys Arg Lys Phe
85 90 95
Asp Thr Glu Thr Lys Asp Cys Asn Glu Lys Lys Lys Lys Met Thr Met
100 105 110
Asn Arg Asp Asp Leu Val Glu Glu Gly Glu Glu Glu Lys Ser Lys Ile
115 120 125
Thr Glu Gln Asn Asn Gly Ser Thr Lys Ser Ile Lys Lys Met Lys His
130 135 140
Lys Ala Lys Lys Glu Glu Asn Asn Phe Ser Asn Asp Ser Ser Lys Val
145 150 155 160
Thr Lys Glu Leu Glu Lys Thr Asp Tyr Ile His Val Arg Ala Arg Arg
165 170 175
Gly Gln Ala Thr Asp Ser His Ser Ile Ala Glu Arg Val Arg Arg Glu
180 185 190
Lys Ile Ser Glu Arg Met Lys Phe Leu Gln Asp Leu Val Pro Gly Cys
195 200 205
Asp Lys Ile Thr Gly Lys Ala Gly Met Leu Asp Glu Ile Ile Asn Tyr
210 215 220
Val Gln Ser Leu Gln Arg Gln Ile Glu Phe Leu Ser Met Lys Leu Ala
225 230 235 240
Ile Val Asn Pro Arg Pro Asp Phe Asp Met Asp Asp Ile Phe Ala Lys
245 250 255
Glu Val Ala Ser Thr Pro Met Thr Val Val Pro Ser Pro Glu Met Val
260 265 270
Leu Ser Gly Tyr Ser His Glu Met Val His Ser Gly Tyr Ser Ser Glu
275 280 285
Met Val Asn Ser Gly Tyr Leu His Val Asn Pro Met Gln Gln Val Asn
290 295 300
Thr Ser Ser Asp Pro Leu Ser Cys Phe Asn Asn Gly Glu Ala Pro Ser
305 310 315 320
Met Trp Asp Ser His Val Gln Asn Leu Tyr Gly Asn Leu Gly Val
325 330 335
Claims (2)
1. A method for regulating chromatin long-distance interaction under blue light is characterized in that a blue light receptor CRY2 and CIB1 form a protein complex under blue light induction to regulate chromatin long-distance interaction, and specifically comprises the following steps:
1) the blue light receptor CRY2 gene and downstream factor CIB1 gene of the model plant Arabidopsis are cloned to a yeast expression vector;
2) the yeast two-hybrid experiment proves that blue light induces the interaction of a blue light receptor CRY2 and a downstream factor CIB1 to form a protein complex;
3) two artificially constructed chromosome fragments respectively containing the binding sequences of LexA and GAL4BD are constructed on the No. 5 chromosome of the saccharomyces cerevisiae EGY48 strain by means of linearization integration and homologous recombination, and the distance between the two artificially constructed chromosome fragments is 12.1 kb;
the main element structure of the LexA-containing vector is linearly integrated is LexAops-GAL1 minor promoter-YFP, and the base sequence of the LexA-containing vector is shown as SEQ ID NO. 1;
wherein, the main control element of the Homologous recombination vector containing the GAL4BD binding sequence is homologus arm1-UEE (PGK1) -GAL4ops-proTEF1-KanMX-TerTEF 1-homologus arm2, and the base sequence is shown as SEQ ID NO. 2;
4) transforming vectors pLexA-CRY2 and pBridge-CIB1 in yeast cells, wherein pLexA-CRY2 expresses fusion protein BD-CRY2, and pBridge-CIB1 expresses fusion protein GAL4BD-CIB 1; so that the fusion protein BD-CRY2 recognizes the binding sequence of LexA and the fusion protein GAL4BD-CIB1 recognizes the binding sequence of GAL4 BD;
the amino acid sequence of the protein CRY2 is shown as SEQ ID NO. 3;
the amino acid sequence of the protein CIB1 is shown as SEQ ID NO. 4;
5) when the yeast cells were irradiated with blue light, the fusion protein BD-CRY2 and the fusion protein GAL4BD-CIB1 interacted to form a protein complex; thus, the binding sequence of LexA and the binding sequence of GAL4BD that interact with them are spatially drawn closer; finally, the binding sequence of LexA and the binding sequence of GAL4BD, which are 12.1kb apart, are allowed to interact chromatin over long distances.
2. The method for regulating chromatin long-distance interaction under blue light as claimed in claim 1, wherein the clone is verified for sequence correctness by sequencing method; the blue light receptor CRY2 and the downstream factor CIB1 genes of the model plant Arabidopsis are cloned in an Arabidopsis thaliana ecotype Col-0;
determining whether CRY2 and CIB1 interact under blue light by blue light development of yeast transfected with p8op-lacZ reporter plasmid;
the binding of BD-CRY2 and GAL4BD-CIB1 to the corresponding binding sequences was indeed present using the ChIP-qPCR assay;
chromosome Conformation Capture technique was used to identify whether long-range interactions between target fragments actually exist.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811474709.XA CN109517048B (en) | 2018-12-04 | 2018-12-04 | Optogenetics tool for regulating long-distance interaction of chromatin under blue light |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811474709.XA CN109517048B (en) | 2018-12-04 | 2018-12-04 | Optogenetics tool for regulating long-distance interaction of chromatin under blue light |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109517048A CN109517048A (en) | 2019-03-26 |
| CN109517048B true CN109517048B (en) | 2021-05-14 |
Family
ID=65794141
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811474709.XA Active CN109517048B (en) | 2018-12-04 | 2018-12-04 | Optogenetics tool for regulating long-distance interaction of chromatin under blue light |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109517048B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110747227B (en) * | 2019-08-13 | 2023-03-31 | 华东师范大学 | Blue light induced and activated Cre recombination optimization system and application thereof |
| CN115261403B (en) * | 2021-04-29 | 2023-09-19 | 中国科学院分子植物科学卓越创新中心 | Light control system based on blue light and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106591349A (en) * | 2016-11-22 | 2017-04-26 | 深圳大学 | Chlamydomonas exogenous gene expression system based on blue light induction and application thereof |
| CN106755050A (en) * | 2016-11-22 | 2017-05-31 | 深圳大学 | A kind of light-operated gene switching system for inducing green alga type discharge hydrogen and its application |
| CN108251434A (en) * | 2018-01-24 | 2018-07-06 | 吉林大学 | A kind of method of the Induced by Blue Light Apoptosis of arabidopsis blue light receptor CRY2 mediations |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108250304A (en) * | 2017-12-28 | 2018-07-06 | 广州天宝颂原生物科技开发有限公司 | A kind of preparation method of the cyanobacteria phytochrome fluorescent marker of fluorescent orange |
| WO2019184044A1 (en) * | 2018-03-27 | 2019-10-03 | 上海欣百诺生物科技有限公司 | Fusion protein of transposase-antibody binding protein and preparation and application thereof |
-
2018
- 2018-12-04 CN CN201811474709.XA patent/CN109517048B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106591349A (en) * | 2016-11-22 | 2017-04-26 | 深圳大学 | Chlamydomonas exogenous gene expression system based on blue light induction and application thereof |
| CN106755050A (en) * | 2016-11-22 | 2017-05-31 | 深圳大学 | A kind of light-operated gene switching system for inducing green alga type discharge hydrogen and its application |
| CN108251434A (en) * | 2018-01-24 | 2018-07-06 | 吉林大学 | A kind of method of the Induced by Blue Light Apoptosis of arabidopsis blue light receptor CRY2 mediations |
Non-Patent Citations (3)
| Title |
|---|
| Light-mediated control of DNA transcription in yeast;Robert M. Hughes等;《Methods.》;20121231;第58卷(第(4)期);385–391 * |
| Optimized second generation CRY2/CIB dimerizers and photoactivatable Cre recombinase;Amir Taslimi等;《Nat Chem Biol.》;20160630;第12卷(第6期);425–430 * |
| Rapid blue light induction of protein interactions in living cells;Matthew J. Kennedy等;《Nat Methods.》;20101231;第7卷(第12期);973–975 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109517048A (en) | 2019-03-26 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| He et al. | A versatile vector toolkit for functional analysis of rice genes | |
| Lokdarshi et al. | Arabidopsis CML38, a calcium sensor that localizes to ribonucleoprotein complexes under hypoxia stress | |
| CN107400672B (en) | Application of OsCOL15 gene in regulation and control of rice heading stage | |
| EP3332005A1 (en) | Promoter variants | |
| WO2014078819A2 (en) | Site-specific enzymes and methods of use | |
| CN109517048B (en) | Optogenetics tool for regulating long-distance interaction of chromatin under blue light | |
| CN114106130B (en) | Purple sweet potato anthocyanin synthesis regulation factor IbJOX4 and application thereof | |
| CN109486850B (en) | Optogenetic tool for UV-B light-down regulation of chromatin long-distance interaction | |
| CN116949064A (en) | Cassava low-temperature response jasmonic acid synthetic gene and application thereof | |
| CN112725355B (en) | A dragon fruit HuNIP6 for promoting plant blooming in advance; 1 gene and application thereof | |
| US10526611B2 (en) | Gene targeting using mutant Agrobacterium strains | |
| CN112813097A (en) | Method for regulating and controlling salt tolerance of rice | |
| Xiao-Yi et al. | Production of marker-free transgenic tobacco plants by Flp/frt recombination system | |
| CN114085277B (en) | Method for localizing target protein in chloroplast and application thereof | |
| CN114085278B (en) | Application of Lsa25711 transit peptide in chloroplast genetic transformation | |
| CN114437191B (en) | Voltage-dependent anion channel protein OsVDAC4 and application thereof in regulation and control of rice male sterility | |
| CN113880927B (en) | Method for enhancing low-temperature tolerance of rice by over-expressing zinc finger protein OsCIP3 | |
| JP4787960B2 (en) | Conditional knockout cell of centromere localization protein gene | |
| Shao | Involvement of host and bacterial factors in Agrobacterium-mediated transformation | |
| Wang | Mammalian Artificial Chromosomes as a Synthetic Biology Tool for Transgene Expression | |
| Smithson | Investigating Replication Protein A's Role in Telomere Maintenance in Arabidopsis thaliana | |
| Shao et al. | Use of the Auxin-induced degron system to study the role of virulence protein VirD2 in the integration of T-DNA into the plant and yeast genome | |
| CN116083479A (en) | Universal multifunctional plant expression vector, construction method and application thereof | |
| Edwards | Characterization of Novel Aperture Proteins in Arabidopsis thaliana | |
| Pappas | Analysis of factors that regulate asymmetric cell division in the green alga Volvox carteri |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |