CN109517048A - A kind of light genetic tool that blue light down regulation chromatin interacts over long distances - Google Patents

A kind of light genetic tool that blue light down regulation chromatin interacts over long distances Download PDF

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CN109517048A
CN109517048A CN201811474709.XA CN201811474709A CN109517048A CN 109517048 A CN109517048 A CN 109517048A CN 201811474709 A CN201811474709 A CN 201811474709A CN 109517048 A CN109517048 A CN 109517048A
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blue light
cry2
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CN109517048B (en
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欧阳鑫昊
黄烯
邱蕾蕾
王笑颖
庄桂
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Xiamen University
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Abstract

A kind of light genetic tool that blue light down regulation chromatin interacts over long distances, is related to light genetic arts.Protein complexes are formed for Induced by Blue Light blue light receptor CRY2 and CIB1, the light genetic tool of regulation chromatin interaction over long distances interacts over long distances, it can be achieved that regulating and controlling chromatin in eucaryote body.Protein complexes are formed in Induced by Blue Light blue light receptor CRY2 and CIB1, regulate and control in the chromatin light genetic tool of interaction over long distances, blue light the light receptor CRY2 and CIB1 can form complex in yeast, can also realize that the interaction of light receptor CRY2 and CIB1 form complex by transgenic animal model in animal experiments.Complex is formed by the interaction of the light receptor CRY2 and CIB1 of Induced by Blue Light to realize the long range interaction of regulation chromosome, to prove that this tool has the application potential of regulation Chromosome.

Description

A kind of light genetic tool that blue light down regulation chromatin interacts over long distances
Technical field
The present invention relates to light genetic arts, and it is multiple to form albumen more particularly, to Induced by Blue Light blue light receptor CRY2 and CIB1 A kind of light genetic tool of fit blue light down regulation chromatin interaction over long distances.
Background technique
Chromosome interaction over long distances can occur between the region on same chromosome or in different chromosomes Region between, on same chromosome, the interaction between promoter and enhancer generally requires mutual between bridging albumen Effect.For example, the interaction between TRAJECTORY CONTROL region (LCR) and beta-globin gene need GATA1 and EKLF1 transcribe because The combination of son and enhancer and target gene.Meanwhile the interaction of GATA1 and EKLF1 transcription factor promotes enhancer and target The combination of gene.In plant, the interaction of CCAAT nuclear factor Y (NF-Y) compound and transcription factor CO albumen promotes FT Chromatin cyclization on gene promoter, and adjust arabidopsis flowering time ([1] Shuanghe Caoet al., The Plant Cell,Vol.26:1009-1017,March 2014)。
The interior interaction of another type of chromosome is the interaction that insulator mediates, and this interaction is by dividing Every the region that difference the is adjusted region that genomic organization is different at function.CTCT binding factor (CTCF) is considered as lactation The sub- albumen of the primary insulation of animal, CTCF boundary element isolation gene group region, to genome is divided into active and non-live Property region.The study found that the forfeiture in the specific site CTCF will lead to serious disorders such as cancers and heart failure.
Traditionally, fluorescence in situ hybridization (FISH) technology is for detecting genome interaction.Recently, a kind of chromosome structure As capture Chromosome Conformation Capture (3C) technology can accurately detect genome interaction.This Item technology and its mutation 4C, 5C and Hi-C facilitate us and deeply understand chromosome interaction ([2] Satish over long distances Sati,Giacomo Cavalli.Chromosoma,Vol 126:33-44,February 2017).However, all 3C skills Art and its mutation only rest on the detection of already existing chromatin interaction.If chromatin interaction can be controlled arbitrarily System, it is believed that the door of a new 3D genome research will be opened.Currently, the Method and kit for of light-operated interaction between chromosomes is still In blank.
The development of light genetics technology makes it possible that arbitrarily regulating and controlling chromatin interacts, and CRY2 is a kind of exists With the light receptor of maximum absorption band under 450nm wavelength blue light, CRY2 is to interact under blue light with downstream elements CIB1 ([3]Hongtao Liu et al.,Science,Vol 322,1535-1539,December 2008)。
Summary of the invention
The purpose of the present invention is to provide a kind of indigo plants that Induced by Blue Light blue light receptor CRY2 and CIB1 form protein complexes The light genetic tool of light down regulation chromatin interaction over long distances.
The light genetic tool of blue light down regulation chromatin of the present invention interaction over long distances is Induced by Blue Light blue light Receptor CRY2 and CIB1 form protein complexes, the light genetic tool of regulation chromatin interaction over long distances, comprising:
1) the blue light receptor CRY2 and downstream elements CIB1 of model plant arabidopsis are cloned into Yeast expression carrier；
2) confirm that blue light can induce that blue light receptor CRY2's and downstream elements CIB1 is mutual by yeast two-hybrid assay Effect forms protein complexes；
3) the two artificial constructed chromosome segments that will separately include the binding sequence of LexA and GAL4BD pass through linearly The mode for changing integration and homologous recombination is building up on No. 5 chromosome of saccharomyces cerevisiae EGY48 bacterial strain, at a distance of 12.1kb；
4) pLexA-CRY2 and pBridge-CIB1, the pLexA-CRY2 expressed fusion protein are converted in yeast cells BD-CRY2, the pBridge-CIB1 expressed fusion protein GAL4BD-CIB1；Fusion protein BD-CRY2 is identified The binding sequence of LexA, fusion protein GAL4BD-CIB1 can identify the binding sequence of GAL4BD；
5) after using blue light illumination yeast cells, phase occurs for fusion protein BD-CRY2 and fusion protein GAL4BD-CIB1 Interaction forms protein complexes；To the binding sequence and GAL4BD of the LexA to interact with them that spatially furthers Binding sequence；Finally, so that long range occurs at a distance of the binding sequence of the LexA of 12.1kb and the binding sequence of GAL4BD Chromatin interaction.
The clone can pass through the correctness of the method validation sequence of sequencing；The blue light receptor of the model plant arabidopsis CRY2 and downstream elements CIB1 gene are cloned in Arabidopsisecotype Col-0.
By the aobvious indigo plant of yeast for turning to have p8op-lacZ reporter plasmid, to determine whether CRY2 and CIB1 has phase under blue light Interaction.
It is whether true using combination of the ChIP-qPCR experimental identification BD-CRY2 and GAL4BD-CIB1 to corresponding binding sequence In the presence of；
Using between chromosomal conformation capture Chromosome Conformation Capture technical appraisement target fragment Over long distances interaction whether necessary being.
The light genetic tool of blue light down regulation chromatin interaction over long distances be a kind of Induced by Blue Light blue light by Body CRY2 and CIB1 form protein complexes, the light genetic tool of regulation chromatin interaction over long distances, this tool is realized Regulate and control chromatin in eucaryote body to interact over long distances.
Protein complexes are formed in Induced by Blue Light blue light receptor CRY2 and CIB1, the interaction over long distances of regulation chromatin In light genetic tool, blue light the light receptor CRY2 and CIB1 can form complex in yeast, can also be in animal reality Realize that the interaction of light receptor CRY2 and CIB1 form complex by transgenic animal model in testing.
The present invention provides:
1, the artificial sequence LexAops- of blue light regulation chromatin interaction over long distances is carried out in a kind of yeast GAL1minimal promoter-YFP, base sequence is as shown in SEQIDNO.1:
2, the artificial sequence Homologous of blue light regulation chromatin interaction over long distances is carried out in a kind of yeast Arm1-UEE (PGK1)-GAL4ops-proTEF1-KanMX-TerTEF1-Homologous arm2, base sequence is such as Shown in SEQIDNO.2:
3, a kind of for controlling the PROTEIN C RY2 of blue light regulation chromatin interaction over long distances, amino acid sequence is such as Shown in SEQIDNO.3:
In above-mentioned smooth genetic tool, the blue light receptor CRY2 is applied to chromatin long range phase interaction in yeast With, can also in mammal and plant for chromatin over long distances interact.
In above-mentioned smooth genetic tool, the blue light receptor CRY2 is cloned by arabidopsis, can also be by having indigo plant It is cloned in the other plant of light receptor CRY2.
4, a kind of for controlling the PROTEIN C IB1 of blue light regulation chromatin interaction over long distances, amino acid sequence is such as Shown in SEQIDNO.4:
In above-mentioned smooth genetic tool, the CIB1 can be applied to chromatin in yeast and interact over long distances, It can interact over long distances in mammal and plant for chromatin.
In above-mentioned smooth genetic tool, the CIB1 is cloned by arabidopsis, can also be by other with CIB1 It is cloned in plant.
Therefore, the present invention forms complex by the interaction of the light receptor CRY2 and CIB1 of Induced by Blue Light to realize and adjust The long range interaction of chromosome is controlled, to prove that this tool has the application potential of regulation Chromosome.
The present invention provides a kind of smooth genetic tool using plant Arabidopsis thaliana the light receptor CRY2 and CIB1 of Induced by Blue Light Interaction forms the long range interaction of complex regulation chromosome.
The beneficial effects of the present invention are: realizing raw in eukaryon based on blue wave band light receptor CRY2 and downstream elements CIB1 The interaction between chromosomes of regulation long range in object, have the characteristics that it is non-toxic, contactless, light science of heredity it is applicable This tool can be used in field.
Detailed description of the invention
Fig. 1 is the overall schematic of the embodiment of the present invention.
Fig. 2 is the phase interaction that yeast two-hybrid verifying blue light can induce the CRY2 and CIB1 of heterogenous expression in yeast With.
Fig. 3 is the binding sequence that ChIP-qPCR experimental verification BD-CRY2 can identify LexA, and GAL4BD-CIB1 can know The binding sequence of other GAL4BD.
Fig. 4 is that chromosomal conformation captures Chromosome Conformation Capture (3C) experimental verification blue light Jie BD-CRY2 and GAL4BD-CIB1 is led to interact so that the phase interaction of long range occurs for two artificial constructed chromosome segments With
Fig. 5 is that Time course chromosomal conformation captures Chromosome Conformation Capture (Timecourse3C) sampling flowsheet figure is tested.
Fig. 6 is Timecourse3C experiments have shown that long range interaction between chromosomes occur rapidly simultaneously under UV-B prolonged exposure It keeps stablizing.
Specific embodiment
Embodiments of the present invention are described in detail with reference to the accompanying drawing.
According to the strategy of Fig. 1, it is necessary first to which building has the yeast of artificial constructed chromosome segment, removes p8op-YFP 2 μ ori of carrier, then with ApaI restriction endonuclease linearized vector, main element LexAops-GAL1minimal promoter-YFP Nucleic acid sequence such as SEQIDNO.1.
Using the principle of homologous recombination, the carrier sequence after linearisation is integrated into No. 5 of saccharomyces cerevisiae EGY48 bacterial strain On chromosome.Construct Homologous arm1-UEE (PGK1)-GAL4ops-proTEF1-KanMX-TerTEF1- Homologous arm2 nucleic acid sequence, the control element that the DNA sequence dna of building includes have Homologous arm1-UEE (PGK1) this DNA sequence dna is constructed by homologous recombination and is being located at No. 5 dyeing by-GAL4ops-KanMX-Homologous arm2 The position in body LexAops-GAL1mini promoter-YFP insetion sequence downstream, LexAops and GAL4ops are at a distance of 12.1kb. Finally obtain yeast QY1 as shown in Figure 1.
Homologous arm1-UEE (the PGK1)-GAL4ops-proTEF1-KanMX-TerTEF1- Homologous arm2 nucleic acid sequence such as SEQIDNO.2.
The coded sequence for encoding the protein sequence of CRY2, CIB1 is expanded to obtain from the cDNA of arabidopsis Col-0.CRY2 grams At the grand XhoI restriction enzyme site to pLexA carrier；CIB1 is cloned among EcorI the and XhoI restriction enzyme site of pB42AD carrier. By obtained recombinant vector corotation into the bacterial strain EGY48 (Clontech) containing p8op-lacZ.Transformant is in SD/-His/- Trp/-Ura is darkling cultivated, and is then transferred to containing 5-bromo-4-chloro-3-indolyl- β-D- In the SD/Gal/Raff/-His/-Trp/-Ura induced medium of galactopyranoside, it is respectively placed in Dark and 450nm Blue light (5.5uW/cm2) growth 36h colour developing.Blue light as shown in Figure 2 can induce the CRY2 and CIB1 expressed in yeast really Combination show blue, hence it is demonstrated that CRY2 and CIB1 interact in yeast.Finally, by turning have p8op- The aobvious indigo plant of the yeast of lacZ reporter plasmid, determines that CRY2 and CIB1 have interaction under blue light.CRY2 amino acid sequence is such as SEQIDNO.3.CIB1 amino acid sequence such as SEQIDNO.4.
In yeast QY1, the DNA fragmentation of the CRY2 and CIB1 coded sequence in yeast two-hybrid vector is respectively used to structure Carrier pLexA-CRY2 and pBridge-CIB1 are built, BD-CRY2 fusion protein and GAL4BD-CIB1 fusion protein are expressed.Finally Obtain yeast QY6 as shown in Figure 1.
After the formaldehyde that the 200ml QY6 yeast for growing to OD600=0.8 or so is added final concentration of 1% is normal temperature crosslinked, Cell sample is mentioned into core with Tissue lyserII (QIAGEN) is broken.Nucleus uses Mnase (New English Biolabs, M0247S) after digestion, the precipitating after centrifugation is placed in Bioruptor (Diagenode) ultrasound, merges Mnase enzyme Supernatant after cutting uses anti-LexA DNA Binding Region antibody (abcam, ab14553) and GAL4 (DBD) (RK5C1) (Santa CruZ Biotechonlogy, Inc:sc-510) carries out immunoprecipitation.Finally, heavy by ethyl alcohol Shallow lake ChIP-DNA carries out subsequent qPCR experiment.And obtain as shown in Figure 3 as a result, BD-CRY2 can be enriched in as the result is shown Near LexA recognition component, and GAL4BD-CIB1C340It can be enriched near the recognition component of GAL4BD.Finally, it uses Combination necessary being of the ChIP-qPCR experimental identification BD-CRY2 and GAL4BD-CIB1 to corresponding binding sequence.
Need the GAL4BD- of the BD-CRY2 for proving to be incorporated in LexA recognition component and the recognition component for being enriched in GAL4BD CIB1C340Interaction can occur under blue light to mediate LexA recognition component and GAL4BD the identification member at a distance of 12.1kb The interaction of part.Then, 3C experiment has been carried out.Firstly, picking QY6 yeast overnight incubation in 20ml culture medium, connects again Kind makes 0.15,30 DEG C of OD600 ≈, 200rpm cultivate 6h, yeast OD600=0.6~0.85 or so into 50ml culture medium.It is added The normal temperature crosslinked 15min of final concentration of 1% formaldehyde, glycine room temperature terminate crosslinking 5min, and the deionized water of pre-cooling is washed 3 times. Yeast is resuspended with FA lysis buffer (140mM NaCl, 50mM HEPES, 1mM EDTA, 1X PMSF, 1X Cocktail) Cell is drawn 60 μ l yeast cells in the FA lysis buffer that 740 μ l are pre-chilled, is dispensed after mixing to 4 to OD600=100 In a 1.5ml EP pipe, the glass beads (Biospec products, 11079105) of 200 μ l pre-cooling is added in every pipe.By sample Product place smudge cells in the Tissue lyserII (QIAGEN) of pre-cooling.EP pipe is punctured with the syringe needle of 22gauge, it will It is placed in 15ml centrifuge tube, 4 DEG C, 2000rpm be centrifuged 2min, abandon containing glass beads EP pipe, 12000rpm from Yeast, 65 DEG C of metal bath 10min are resuspended in heart 10min, 10mM pH=7.9Tris-HCl.The yeast being centrifuged is resuspended to use NlaIII (New English Biolabs) is in 37 DEG C of digestion 8h.Digestion products T4DNA ligase (New English Biolabs, M0202L) in 16 DEG C of connections 13h, 22 DEG C of connection 1h.3C DNA after connection is precipitated with ethyl alcohol.After purification 3C DNA detects the interaction frequency that LexA identifies sequence and GAL4 identifies sequence by TaqMan qpcr.With saccharomyces cerevisiae The promoter of middle HEM3 gene and the interaction frequency of terminator are as internal reference.As a result as shown in figure 4,450nm blue light (5.5uW/cm2) can induce LexA identification sequence and GAL4 identification sequence that the interaction of long range occurs.Finally, using dye Colour solid conformation captures the long range interaction between Chromosome Conformation Capture technical appraisement target fragment Necessary being.
According to sample handling characteristics shown in fig. 5, the culture medium that single QY6 yeast clone is transferred to 20ml is trained overnight It supports.Then, yeast cells, which is averaged, is assigned to 5 plastic bottles and keeps OD600 ≈ 0.15.Following 5 plastic bottles are by tinfoil paper Paper bag, which is wrapped up in, is put in 450nm blue light (5.5uW/cm2) cultivate in environment, and masking foil is removed respectively at 0,3,5,5.5 and 6h.Most Afterwards, sample is collected in 6h, carries out Time course3C experiment.Experimental result as shown in fig. 6, long range chromosome phase interaction Occur rapidly used in indigo plant irradiation early stage, and stablizes under blue light illumination and keep.
In eucaryote, chromatin interaction over long distances is adjusting gene expression, control cell differentiation, is influencing biology It plays an important role in body developmental process.Currently, being still in blank by the exploitation that light regulates and controls the tool of chromatin cyclization. The present invention relates to a kind of methods about blue light regulation chromatin conformation.Using saccharomyces cerevisiae as experimental subjects, it was demonstrated that Induced by Blue Light The interaction of blue light receptor CRY2 and CIB1 of model plant arabidopsis can regulate and control same dye in eukaryotic gene group DNA fragmentation on colour solid at a distance of 10kb or more interacts.The invention of the genetic tool for passing through optical signal in the future The change that noninvasive targeting changes chromatin higher order structure in active somatic cell has important application value.
Sequence table
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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
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Arg Gly Ile Gly Leu Arg Glu Tyr Ser Arg Tyr Ile Cys Phe Asn Phe
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Pro Phe Thr His Glu Gln Ser Leu Leu Ser His Leu Arg Phe Phe Pro
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Trp Asp Ala Asp Val Asp Lys Phe Lys Ala Trp Arg Gln Gly Arg Thr
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Gly Tyr Pro Leu Val Asp Ala Gly Met Arg Glu Leu Trp Ala Thr Gly
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Trp Met His Asn Arg Ile Arg Val Ile Val Ser Ser Phe Ala Val Lys
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Phe Leu Leu Leu Pro Trp Lys Trp Gly Met Lys Tyr Phe Trp Asp Thr
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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
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Glu Gly Ile Gln Asp Ser Ser Asp Gln Ile Thr Thr Ser Leu Gly Lys
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Asn Gly Cys Lys
610
<210> 4
<211> 335
<212> PRT
<213>arabidopsis (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
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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 (10)

1. a kind of light genetic tool of blue light down regulation chromatin interaction over long distances, it is characterised in that blue for Induced by Blue Light Light receptor CRY2 and CIB1 form protein complexes, the light genetic tool of regulation chromatin interaction over long distances.
2. the light genetic tool of a kind of blue light down regulation chromatin interaction over long distances as described in claim 1, feature Be include:
1) the blue light receptor CRY2 and downstream elements CIB1 of model plant arabidopsis are cloned into Yeast expression carrier；
2) confirm that blue light can induce the interaction of blue light receptor CRY2 and downstream elements CIB1 by yeast two-hybrid assay Form protein complexes；
3) the two artificial constructed chromosome segments that will separately include the binding sequence of LexA and GAL4BD are whole by linearizing It closes and the mode of homologous recombination is building up on No. 5 chromosome of saccharomyces cerevisiae EGY48 bacterial strain, at a distance of 12.1kb；
4) pLexA-CRY2 and pBridge-CIB1, the pLexA-CRY2 expressed fusion protein BD- are converted in yeast cells CRY2, the pBridge-CIB1 expressed fusion protein GAL4BD-CIB1；Fusion protein BD-CRY2 is allowed to identify LexA Binding sequence, fusion protein GAL4BD-CIB1 can identify the binding sequence of GAL4BD；
5) after using blue light illumination yeast cells, phase interaction occurs for fusion protein BD-CRY2 and fusion protein GAL4BD-CIB1 With formation protein complexes；To which spatially further the binding sequence of the LexA to interact with them and the knot of GAL4BD Close sequence；Finally, so that the dyeing of long range occurs at a distance of the binding sequence of the LexA of 12.1kb and the binding sequence of GAL4BD Matter interaction.
3. the light genetic tool of a kind of blue light down regulation chromatin interaction over long distances as claimed in claim 2, feature It is correctness of the clone by the method validation sequence of sequencing；The blue light receptor CRY2 of the model plant arabidopsis and Downstream elements CIB1 gene is cloned in Arabidopsisecotype Col-0；
By the aobvious indigo plant of yeast for turning to have p8op-lacZ reporter plasmid, determine whether CRY2 and CIB1 has interaction under blue light；
Whether the combination of corresponding binding sequence is really deposited using ChIP-qPCR experimental identification BD-CRY2 and GAL4BD-CIB1 ?；
Using chromosomal conformation capture Chromosome Conformation Capture technical appraisement target fragment between length away from From interaction whether necessary being.
4. the light genetic tool of a kind of blue light down regulation chromatin interaction over long distances as claimed in claim 2, feature It is to form protein complexes in Induced by Blue Light blue light receptor CRY2 and CIB1, the light of regulation chromatin interaction over long distances is lost It passes in tool, blue light the light receptor CRY2 and CIB1 form complex in yeast or in animal experiments by turning base Because animal model realizes that the interaction of light receptor CRY2 and CIB1 form complex.
5. the light genetic tool of a kind of blue light down regulation chromatin interaction over long distances as described in claim 1, feature It is a kind of interior artificial sequence LexAops-GAL1minimal for carrying out the interaction over long distances of blue light regulation chromatin of yeast Promoter-YFP, base sequence is as shown in SEQIDNO.1:
6. the light genetic tool of a kind of blue light down regulation chromatin interaction over long distances as described in claim 1, feature It is a kind of interior artificial sequence Homologous arm1-UEE for carrying out the interaction over long distances of blue light regulation chromatin of yeast (PGK1)-GAL4ops-proTEF1-KanMX-TerTEF1-Homologous arm2, base sequence such as SEQIDNO.2 institute Show:
7. the light genetic tool of a kind of blue light down regulation chromatin interaction over long distances as described in claim 1, feature It is a kind of PROTEIN C RY2 to interact over long distances for controlling blue light regulation chromatin, amino acid sequence is such as Shown in SEQIDNO.3:
8. the light genetic tool of a kind of blue light down regulation chromatin interaction over long distances as described in claim 1, feature It is in light genetic tool, the blue light receptor CRY2 is applied to chromatin in yeast and interacts or feeding over long distances It interacts over long distances in newborn animal and plant for chromatin；
The blue light receptor CRY2 is cloned by arabidopsis or by being cloned in the other plant with blue light receptor CRY2.
9. the light genetic tool of a kind of blue light down regulation chromatin interaction over long distances as described in claim 1, feature It is a kind of PROTEIN C IB1 to interact over long distances for controlling blue light regulation chromatin, amino acid sequence is such as Shown in SEQIDNO.4:
10. the light genetic tool of a kind of blue light down regulation chromatin interaction over long distances as described in claim 1, feature Be in light genetic tool, the CIB1 be applied to chromatin interaction over long distances in yeast or in mammal and It interacts over long distances in plant for chromatin；
The CIB1 is cloned by arabidopsis or by being cloned in the other plant with CIB1.
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