CN106893725A - A kind of Plant Light regulation type promoter and application - Google Patents
A kind of Plant Light regulation type promoter and application Download PDFInfo
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- CN106893725A CN106893725A CN201510964291.0A CN201510964291A CN106893725A CN 106893725 A CN106893725 A CN 106893725A CN 201510964291 A CN201510964291 A CN 201510964291A CN 106893725 A CN106893725 A CN 106893725A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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- 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
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
- C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
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Abstract
The present invention relates to a kind of Plant Light regulation type promoter and application.The Plant Light regulation type promoter, containing selected from it is following 1) or 2) or 3) in any one and there is the nucleotide sequence of promoter function：1) its nucleotide sequence is sequence 1 in sequence table；2) hybridize and the DNA molecular related with vegetable protein expression is started to the DNA fragmentation that sequence 1 is limited under strict conditions；3) there is more than 90% homology, and the DNA molecular related to vegetable protein expression is started with gene 1) or 2).The present invention replaces constitutive promoter using inducible promoter, can obtain the promoter of the specifically expressing of light regulation and control；It is conducted into Plant Genome using genetic transfoumation, it is possible to achieve to the specific expressed of genes of interest.
Description
Technical field
The present invention relates to plant genetic engineering field, in particular, it is related to what a kind of light from corn regulated and controled
The clone of specific promoter, expression and its application in plant.
Background technology
Promoter is the section of DNA sequence for being located at gene 5 ' end upstream, and it includes enhancing, starts or repressor gene table
Hormone or the cis-acting elements of stress response effect in the sequence that reaches and response external environment, transcription factor by with these yuan
Part interacts to realize the transcriptional control to certain gene.
Plant promoter can be divided into constitutive promoter, tissue specific promoter and induction type and open by its Transcript patterns
Mover.Constitutive promoter is widely used in plant transgene, conventional such as CaMV35S promoters, ubiquitin promoters and
Actin promoters etc., the startup exogenous gene expression that they can be efficient, lasting and non-specific, but foreign protein are planted in transgenosis
Constitutive expression also increases the metabolism burden of plant in thing, directly affects growing for plant；Meanwhile, constitutive promoter
Possible modificator gene silence, influences transgenic technology application；Compared with constitutive promoter, inducible promoter is only in some things
Under the stimulation of reason or chemical signal or development of plants is to the specific stage, the just transcription of startup foreign gene.It can not only make
The expression product of genes of interest is accumulated in certain space-time, increase Zonal expression amount, improve plant resistance, while can avoid by
Constitutive promoter starts the negative effect to plant development that foreign gene overexpression causes, it is also possible to a certain extent
The gene silencing in transgenosis application is solved the problems, such as, is the important startup that appliable plant transgenic technology carries out genetic engineering breeding
Son.
One of most important factor of growth and development of plants is just influenceed, because it serves not only as the signal that plant perceives environment
Source, also originates as photosynthetic energy.Therefore, the research to light-inducible promoter contributes to understanding plant gene to exist
Transcriptional control expression pattern and its regulatory mechanism under light, can be applied to make foreign gene with illumination and plant in genetic engineering
Physiological status is expressed, and maintains the healthy growth of transfer-gen plant.
The content of the invention
The present invention clones a kind of promoter of light regulation and control from corn, its nucleotide sequence total length 2058bp, entitled
PZm169o, from corn B73, nucleotide sequence total length is as follows：
GAGAGGGAGAGGGAAGTTGCTGCGCGGGAAAAGAAAATGAGATAGGGGAGGGGGCGCATGGGGGAAGGGGCGCCAGG
GGCCGGGTTGGGCCACGGGCCGGGCCGGACCACGAGCTGGGCCGAAAACCCACTGCACGCACGACCACTAATCGAAA
ACAAATCATGATTCGAAGACCAAAACGAGACGGACGCGCGATTAGACACAACATTAGACAAAAGAAATATGCTTCAG
CATGAAGCAAAACCTATGTCAACTTAGGTTTTTGTTTACACGCGATACGGACACCAGTCACTATACTGCTTTGAAAA
TTAGAAGAAGGAGCAAAACGGGAAGAGAAAAGAGAGTAACGCCTGAATTTGCTGAGTATCGGAGAAGAAAAATTATA
CTCCCAAATTCAGGGCGTTACAACATCTCTTGGTCAAGGTGTGACATGCTGGCTATAGAGGATGGTACTCTGCTTCC
CAATTATGGAACATAGAGTGAGGGAGATACTAGATGCGACGAGGGTGAGGAAGACCGGTGGTGGCGATGGCAACCTC
GGGGAGAGGCAGGAGAAACTGCAGTAGTGGGCCAAG CGTGTAGATGCGAAGGGGTATAGGGGTATT
CTCACGGGAACCGCTCGTTTCGTCCCAAAGAGGCCTGTAAACCGATTTGGCATTCACCTTGAAAACCAAGCTACCGG
AAAAATCAGTTCAGGGAAAAACTGACCCAAAATCAGAGCGTTTTGCACTCAGATCAGCTTTTGGCGGTCACAATCTA
AAAAAGGGGTTCCAAACGGGGCATGCACTACAACTCTCTATAGCTCTTGGAAGAGGTTCAAATAATATTCTAGCAAT
CAGGGCTATGCCTAAGATTTTAGGGGCCCTGGGCGAAAGCTTATATGGATGCCCTTTTGGCCGATGTATATACAATA
ACTATATACATAGACACATTTTGATGGGCCAAATAGTAAATGCAATTAGCTAGCTCCTACATATGGTATACATG
TATGTGCGAAGGTTGATCGTCGATGGCATGTCTTGGCGTTTGCAACCGTGTCTAGACTGTCCAGTTCTTTGGCAACA
TGGTGGTGGCACCACGACATGAATTAGGTTTTCTAGATGTATTCTATTATGAGTCCTGACTTGGGATCTGGCTTACA
TGAACATGCTAGCATTTGCTTCCGATTTACGTTACATGAGCAAGATAGTAGAGGCTGAGCTACCCGCTTATGTGAGC
ATGATAGCAGTTCATAGTAAGGATGACAATAAGTACCCGAAACCCGAACACCCAACAGATTTTACCTAATTAGAAGG
TAGGTATGAGATAATTTCTTTAACCGTGAGTATGTTAATAGGTAATACCCATACTCGTCTAACCGCGGGTAAAATAT
ACCGACATCATATCACTATTACCATCTAATAACTCATCTTAGCTAAAAGAAAATCCGTATTCGACTATTGTTTGTTT
AAATACCATATTATGTAATCATATGAGATGTTATATTTGAAGTTAAACTTATTTTTATATGTTTGTTTGATATTTTG
AGTGATTGATATATTACAATTTAAGATTTTCTTAATGGGTG CCTTAATGGGTAAAATTACACCCGC
GGATATGGGTACGGATGGGATTCTATACCCGCGAGTGTGCATGGTAACCTAATGGATATAATTTTTTTACGAATAAC
GGCATAAAATGGTACTAGATATATTCCATTTCGTAGCAGTGGACGGTTGAACCAACCAAGTGAACACTATTTAAACT
GATGAAATGATTAAATATTATACTTAGATATATACTTAAGGGGACTTCAACATTTTGAGACGCTTTAGCCTGCTAGT
TCAATCCTTGAAACTGAAGAACAAGATATGGCAGAAGCTGCTCACCCAAAATACCTGACCCCTCTCCTTGTCCACAC
CATAAATACCAGGCGTATGCAACCAGTGCTGCCACACACACACACACAGACACCAAACGCCACAGAACTTGCAGGCA
TGGCTGAAGGTTAAGAAGAAAAAGAGGAGGGCGGCCTCACATGGAGATGGAGAGCCCCTTTTG
Blackened in above-mentioned sequence part "”、“”、“" it is photoresponse unit
Part.
Present invention also offers the construction method of above-mentioned smooth regulation type gene promoter, concretely comprise the following steps:With corn B73 systems
Genomic DNA is template, and specific primer is designed according to Zm-miR169o gene orders, enters performing PCR amplification, obtains above-mentioned photo-induction
Conductivity type gene promoter.
Present invention also offers the plant expression vector containing above-mentioned smooth regulation type promoter, pPZm169o is named as::GUS.
The present invention also provides the transgenosis that above-mentioned promoter obtains light regulating and expressing genes of interest in plant genetic engineering
Application in milpa.
The present invention replaces constitutive promoter using inducible promoter, can obtain the startup of the specifically expressing of light regulation and control
Son；It is conducted into Plant Genome using genetic transfoumation, it is possible to achieve to the specific expressed of genes of interest.
Brief description of the drawings
Technical scheme in order to illustrate more clearly the embodiments of the present invention, below will be to that will make needed for embodiment description
Accompanying drawing is briefly described.
Fig. 1 is the carrier pPZm169o that GUS expression is driven using PZm169o::GUS schematic diagrames；
Fig. 2 is GUS coloration result figures in different corn strain blades in embodiment 4；
Fig. 3 is PZm169o in embodiment 5::GUS transgenic corn plants processed through dim light and far-red light after GUS table
Up to figure.
Specific embodiment
Below in conjunction with embodiment, the technical scheme in the embodiment of the present invention is clearly and completely described.
The clone of the promoter of embodiment 1
According to the corn B73 whole genome sequences provided in maize genetics and genomic database (maizeGDB),
Upstream 2058bp sequences Design amplimers according to Zm-miR169o genes, primer sequence is as follows：
PZm169oF：5’-GAGAGGGAGAGGGAAGTTGC-3’；
PZm169oR：5’-CAAAAGGGGCTCTCCATCTC-3’.
Wherein, PCR amplification programs are as follows：
72 DEG C of extension 10min.4 DEG C of preservations.Amplify DNA fragmentation to be reclaimed from agarose gel, be cloned into pEASY-T1 loads
Body.Digestion and sequencing identification, are named as pTP169o, and sequencing result shows：Promoter sequence is obtained shown in Fig. 1, this to be started
Son is named as PZm169o.
The structure of the plant expression vector of embodiment 2
1) primer of amplification PZm169o total lengths is redesigned, 20nt carrier homologous sequences is respectively added at primer two ends,
PZm169oF2：
5'-TATGACCATGATTACGAATT GAGAGGGAGAGGGAAGTTGC-3'；
PZm169oR2：
5'-TACCCTCAGATCTACCATGG CAAAAGGGGCTCTCCATCTC-3'。
With pTP169o plasmids as template, enter performing PCR amplification；
2) amplified production is reclaimed, and product carries out weight with through the pCAMBIA3301 of EcoRI/NcoI double digestions after recovery
Group reaction, agents useful for same is GBclonart Seamless Cloning Kit, and reaction system is as follows：
3) digestion identification positive colony, positive colony is pPZm169o::GUS carriers.Positive colony is carried out into sequencing to test
Card, obtains positive plasmid as shown in figure 1, being used for subsequent experimental.
The pPZm169o of embodiment 3::The acquisition of GUS transgenic corns
1) Agrobacterium-mediated Transformation and corn transformation
The plant expression vector pPZm169o that will be obtained in embodiment 2::GUS is transformed into Agrobacterium EHA105.Corn turns
Change method is carried out with reference to Frame et al..
Transgenic corns PCR is detected
Forward primer：PZm169oFW:5’-GGACGGTTGAACCAACCAAGTG-3’
Reverse primer：GUS3R:5’-CGGCGAACTGATCGTTAAAACT-3’
Reaction system：
Reaction condition is as follows：
72 DEG C of extension 5min.Filter out PCR positive plants.
2) transgenic corns Basta foliage spray detection and the acquisition of pure lines
When the PCR positive plants piece leaf to 5-6 long, sprayed with the Basta of 1000 times of dilutions, once in a week, continuously spray 3
Plant survival condition is observed after week.The positive seedling selfing of survival obtains T2 generation pure lines, and planting different tissues organ to be taken carries out GUS
Analysis.
The GUS histochemical stains of embodiment 4
With reference to Jefferson (Jefferson RA et al., 1987) method, tissue to be dyed is immersed into tissue fluid right
After vacuumize, 37 DEG C of stained over night, secondary daily 95% ethanol decolorization, to negative control material into white.
By GUS tissue stainings, detection promoter PZm169o is in corn gene plant to the startup activity of GUS.Figure
2 is GUS coloration results in different corn strain blades.Result shows that positive control pCAMBIA3301 (comes from pCAMBIA companies)
(A) during transgenic corn plant is respectively organized, GUS activity is detected, there is substantially dyeing.And PZm169o::GUS (B) and feminine gender
GUS activity is not detected by control (C).
The PZm169o of embodiment 5::GUS transgenic corn plants are through GUS expression analysis after dim light and far-red light treatment
To the PZm169o to three leaves wholeheartedly long::GUS transgenic corns seedling, carries out 16h/8h dim lights/dark training respectively
Support and 50 μm of ol/m2/s far-red lights are processed 15 minutes, the blade of seedling leaves and before processing after processing is taken respectively carries out GUS activity
Analysis, Fig. 3 is the expression of results of GUS after transgenic corn plant is processed through dim light and far-red light, as a result shows dim light/dark
The GUS activity for the treatment of (A) and far-red light treatment 15min (B) afterwards in transgenic line is remarkably reinforced, the wild type pair of non-transgenosis
According to (C) without GUS activity, PZm169o promoters are illustrated by far-red light and dark induced expression, be an induction type for light regulation and control
Promoter.
Obviously, embodiment described above is only a part of embodiment of the invention, rather than whole embodiments.It is based on
Embodiment in the present invention, those of ordinary skill in the art obtained on the premise of creative work is not made it is all its
His embodiment, belongs to the scope of protection of the invention.
Sequence table
<110>Nong Ke Group Co., Ltd of Shenzhen, Biological Technology institute, Chinese Academy of Agricultural Sciences
<120>A kind of Plant Light regulation type promoter and application
<130>
<160> 1
<210> 1
<211> 2058
<212> DNA
<213>Corn variety " B73 "
<400> 1
GAGAGGGAGA GGGAAGTTGC TGCGCGGGAA AAGAAAATGA GATAGGGGAG GGGGCGCATG 60
GGGGAAGGGG CGCCAGGGGC CGGGTTGGGC CACGGGCCGG GCCGGACCAC GAGCTGGGCC 120
GAAAACCCAC TGCACGCACG ACCACTAATC GAAAACAAAT CATGATTCGA AGACCAAAAC 180
GAGACGGACG CGCGATTAGA CACAACATTA GACAAAAGAA ATATGCTTCA GCATGAAGCA 240
AAACCTATGT CAACTTAGGT TTTTGTTTAC ACGCGATACG GACACCAGTC ACTATACTGC 300
TTTGAAAATT AGAAGAAGGA GCAAAACGGG AAGAGAAAAG AGAGTAACGC CTGAATTTGC 360
TGAGTATCGG AGAAGAAAAA TTATACTCCC AAATTCAGGG CGTTACAACA TCTCTTGGTC 420
AAGGTGTGAC ATGCTGGCTA TAGAGGATGG TACTCTGCTT CCCAATTATG GAACATAGAG 480
TGAGGGAGAT ACTAGATGCG ACGAGGGTGA GGAAGACCGG TGGTGGCGAT GGCAACCTCG 540
GGGAGAGGCA GGAGAAACTG CAGTAGTGGG CCAAGCACGT GCGTGTAGAT GCGAAGGGGT 600
ATAGGGGTAT TCTCACGGGA ACCGCTCGTT TCGTCCCAAA GAGGCCTGTA AACCGATTTG 660
GCATTCACCT TGAAAACCAA GCTACCGGAA AAATCAGTTC AGGGAAAAAC TGACCCAAAA 720
TCAGAGCGTT TTGCACTCAG ATCAGCTTTT GGCGGTCACA ATCTAAAAAA GGGGTTCCAA 780
ACGGGGCATG CACTACAACT CTCTATAGCT CTTGGAAGAG GTTCAAATAA TATTCTAGCA 840
ATCAGGGCTA TGCCTAAGAT TTTAGGGGCC CTGGGCGAAA GCTTATATGG ATGCCCTTTT 900
GGCCGATGTA TATACAATAA CTATATACAT AGACACATTT TGATGGGCCA AATAGTAAAT 960
GCAATTAGCT AGCTCCTACA TATGGTATAC ATGTACGTGT ATGTGCGAAG GTTGATCGTC 1020
GATGGCATGT CTTGGCGTTT GCAACCGTGT CTAGACTGTC CAGTTCTTTG GCAACATGGT 1080
GGTGGCACCA CGACATGAAT TAGGTTTTCT AGATGTATTC TATTATGAGT CCTGACTTGG 1140
GATCTGGCTT ACATGAACAT GCTAGCATTT GCTTCCGATT TACGTTACAT GAGCAAGATA 1200
GTAGAGGCTG AGCTACCCGC TTATGTGAGC ATGATAGCAG TTCATAGTAA GGATGACAAT 1260
AAGTACCCGA AACCCGAACA CCCAACAGAT TTTACCTAAT TAGAAGGTAG GTATGAGATA 1320
ATTTCTTTAA CCGTGAGTAT GTTAATAGGT AATACCCATA CTCGTCTAAC CGCGGGTAAA 1380
ATATACCGAC ATCATATCAC TATTACCATC TAATAACTCA TCTTAGCTAA AAGAAAATCC 1440
GTATTCGACT ATTGTTTGTT TAAATACCAT ATTATGTAAT CATATGAGAT GTTATATTTG 1500
AAGTTAAACT TATTTTTATA TGTTTGTTTG ATATTTTGAG TGATTGATAT ATTACAATTT 1560
AAGATTTTCT TAATGGGTGC ACGTTCCTTA ATGGGTAAAA TTACACCCGC GGATATGGGT 1620
ACGGATGGGA TTCTATACCC GCGAGTGTGC ATGGTAACCT AATGGATATA ATTTTTTTAC 1680
GAATAACGGC ATAAAATGGT ACTAGATATA TTCCATTTCG TAGCAGTGGA CGGTTGAACC 1740
AACCAAGTGA ACACTATTTA AACTGATGAA ATGATTAAAT ATTATACTTA GATATATACT 1800
TAAGGGGACT TCAACATTTT GAGACGCTTT AGCCTGCTAG TTCAATCCTT GAAACTGAAG 1860
AACAAGATAT GGCAGAAGCT GCTCACCCAA AATACCTGAC CCCTCTCCTT GTCCACACCA 1920
TAAATACCAG GCGTATGCAA CCAGTGCTGC CACACACACA CACACAGACA CCAAACGCCA 1980
CAGAACTTGC AGGCATGGCT GAAGGTTAAG AAGAAAAAGA GGAGGGCGGC CTCACATGGA 2040
GATGGAGAGC CCCTTTTG 2058
Claims (3)
1. a kind of Plant Light regulation type promoter, it is characterised in that：The promoter contain selected from it is following 1) or 2) or 3) in appoint
Meaning one and the nucleotide sequence with promoter function：
1) its nucleotide sequence is sequence 1 in sequence table；
2) hybridize and the DNA molecular related with vegetable protein expression is started to the DNA fragmentation that sequence 1 is limited under strict conditions；
3) there is more than 90% homology, and the DNA molecular related to vegetable protein expression is started with gene 1) or 2).
2. the recombinant expression carrier of Plant Light regulation type promoter described in claim 1 is contained.
3. Plant Light regulation type promoter, recombinant expression carrier described in claim 2 described in claim 1 are in genetically modified plants
Application.
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Cited By (1)
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