CN104726452A - Specific promoter PC2 of seed scutellum and leaf small vein, and applications thereof - Google Patents
Specific promoter PC2 of seed scutellum and leaf small vein, and applications thereof Download PDFInfo
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- CN104726452A CN104726452A CN201310712090.2A CN201310712090A CN104726452A CN 104726452 A CN104726452 A CN 104726452A CN 201310712090 A CN201310712090 A CN 201310712090A CN 104726452 A CN104726452 A CN 104726452A
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Abstract
The invention discloses specific promoter PC2 of seed scutellum and leaf small vein, and applications thereof. The invention provides a DNA fragment defined by 1), or 2), or 3): 1) a DNA molecule represented by sequence 1 in a sequence table; 2) a DNA molecule which is hybridized with the DNA molecule defined by 1) under strict conditions, and possesses promoter functions; and 3) a DNA molecule which possesses promoter functions, wherein homology degree of the DNA molecule with the DNA molecule defined by 1) is higher than 90%. It is confirmed by experiments that: promoter PC2 obtained via cloning of the DNA fragment is capable of driving specific expression of target genes, such as GUS, in rice scutellum or leaf small vein, so that effective regulation and controlling on seed dormancy and germination, and nutrient transportation efficiency are realized. PC2 is a first obtained leaf small vein specific promoter of grass family, so that great theoretical research significance is provided and application value is high.
Description
Technical field
The present invention relates to biological technical field, particularly relate to a kind of seed scultellum and leaf thready pulse specific expression promoter PC2 and application thereof.
Background technology
In crop seed germination process, the nutritive substance in endosperm is delivered to embryo by scultellum, forms new plant for embryo germination, and scultellum is the key position of seed germination involved enzyme and hormone secretion.In the genetic transformation system of monocotyledon rice, the scultellum of mature embryo is the transformation explant of widespread use, and in scultellum, the expression level of genes involved will affect the transformation efficiency of Different Rice Varieties.Therefore be separated and identify scultellum specific expression gene and corresponding promotor thereof, being conducive to the Effective Regulation realized seed germination and dormancy, in the crop production of hybrid seeds and food storage, all there is significant application value; Also be beneficial to the genetic transformation efficiency improving paddy rice improved seeds, thus accelerate its breeding process.
In addition, monocotyledons, if Rice Leaf thready pulse (leaf small vein) is phloem loading (phloemloading, namely photoassimilates transports to screen casing from mesophyll cell) key position, with the recycle of the synthesis transport of photosynthate, amino acid and nutritive element especially nitrogen and transport in close relations.Therefore leaf thready pulse specific expression promoter is research phloem loading and the Advantageous regulatory element of nutritive element transport mechanism, improves biological yield by the efficient output of regulating plant photosynthate.
Summary of the invention
An object of the present invention is to provide a kind of DNA fragmentation.
DNA fragmentation provided by the invention is following 1) or 2) or 3) DNA molecular:
1) DNA molecular shown in sequence 1 in sequence table;
2) under strict conditions with 1) DNA sequence dna that limits hybridizes and has the DNA molecular of promoter function;
3) with 1) DNA sequence dna that limits has more than 90% homology, and has the DNA molecular of promoter function.
Above-mentioned stringent condition can be at 0.1 × SSPE(or 0.1 × SSC), in the solution of 0.1%SDS, hybridize under 65 DEG C of conditions and wash film.
Recombinant vectors containing above-mentioned DNA fragmentation, expression cassette, transgenic cell line or recombinant bacterium are also the scope of protection of the invention.
Above-mentioned recombinant vectors is the CaMV35S promotor replaced by above-mentioned DNA fragmentation in expression vector pCAMBIA-1300-SGN, the recombinant vectors obtained.
Described recombinant vectors is specially HindIII and the BamHI enzyme above-mentioned DNA fragmentation being replaced pCAMBIA-1300-SGN and cuts the recombinant plasmid that the small segment (CaMV35S promotor) between recognition site obtains.
The total length of above-mentioned DNA fragmentation that increases or the primer pair of its any fragment are also the scope of protection of the invention.
Above-mentioned primer pair is made up of the single strand dna shown in sequence 3 in the single strand dna shown in sequence in sequence table 2 and sequence table.
The application started in plant in destination gene expression of above-mentioned DNA fragmentation is also the scope of protection of the invention.
In above-mentioned application, described destination gene expression is organizing specific expression.
In above-mentioned application, described in be organized as scultellum and/or leaf thready pulse.
In above-mentioned application, described plant is monocotyledons or dicotyledons; Described monocotyledons is specially paddy rice.
In above-mentioned application, described goal gene is gus gene.
Leaf thready pulse (leaf small vein): the vein of monocotyledon rice is for directly to go out parallel venation, and the middle arteries and veins (midrib) on blade and lateral vein (large vein) all send from the base portion of blade, and roughly parallel to each other, the top to blade is converged.Thready pulse (small vein) is had again between master pulse and lateral vein.
Experiment of the present invention proves, the present invention clone obtains promotor PC2, and it can drive goal gene as GUS specifically expressing in the scultellum and leaf thready pulse of paddy rice, thus realizes the Effective Regulation to Seed dormancy and germination and nutritive substance conveying efficiency.The PC2 that the present invention relates to is the leaf thready pulse specific expression promoter that grass obtains first, has most important theories Research Significance and using value.
Accompanying drawing explanation
Fig. 1 is the structure schematic diagram of p130-PC2 plant expression vector and the GUS staining analysis of transgenic paddy rice
Fig. 2 is the GUS staining analysis turned in PC2:GUS LIPIDS OF DRY RICE EMBRYO seed and Seed Germination
Embodiment
The experimental technique used in following embodiment if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
The acquisition of embodiment 1, paddy rice PC2 promotor
By CTAB method from paddy rice ' Japan is fine ' (Oryza sativa ssp.japonica cultivarNipponbare, be documented in Yuman Zhang, Yongsheng Yan, Lina Wang, Kun Yang, Na Xiao, Yunfeng Liu, Yaping Fu, Zongxiu Sun, Rongxiang Fang, Xiaoying Chen.A novel ricegene, NRR responds to macronutrient deficiency and regulates root growth.Mol.Plant, 2012,5 (1): the 63-72. public can obtain from Institute of Microorganism, Academia Sinica.) extract genomic dna in blade, devise forward and reverse special primer C2-Pro5-H3:5 '
-
aAGCTTaCAGCATTTCCCACGGTTATCA-3 ' (sequence 2); C2-pro3-Bgl:5 '
-
aGATCtAGGAGTTTAGGACACTAATCAC-3 ' (sequence 3) (base of band underscore is HindIII and the BglII restriction enzyme site introduced respectively), adopts TaKaRa LA-Taq archaeal dna polymerase to carry out pcr amplification.
Obtain the PCR primer of 2,086bp, this PCR primer is sent to order-checking, result is by the DNA molecular called after PC2 shown in this PCR primer, and the nucleotides sequence of this DNA molecular is classified as sequence 1 in sequence table.
The functional verification of embodiment 2, paddy rice PC2 promotor
1, the acquisition of recombinant vectors
PCR primer embodiment 1 obtained is cut through HindIII and BglII enzyme, obtain 2, the digestion products of 086bp, by this digestion products be isocaudarner through HindIII and BamHI(and BglII) the plant expression vector pCAMBIA-1300-SGN(that cuts of enzyme is documented in Yuman Zhang, Yongsheng Yan, Lina Wang, Kun Yang, NaXiao, Yunfeng Liu, Yaping Fu, Zongxiu Sun, Rongxiang Fang, Xiaoying Chen.Anovel rice gene, NRR responds to macronutrient deficiency and regulates rootgrowth.Mol.Plant, 2012, 5 (1): the 63-72. public can obtain from Institute of Microorganism, Academia Sinica.The promotor driving gus gene to express in this carrier is 35S) connect, obtain recombinant vectors.
This recombinant vectors primer C2-Pro5-H3 and C2-pro3-Bgl carries out PCR detection, and what obtain 2,086bp amplified production is positive recombinant vector.Positive recombinant vector sends to order-checking, the carrier of this recombinant vectors of result for (replacing 35S promoter) between HindIII and the BglII restriction enzyme site of the DNA molecular PC2 insertion plant expression vector pCAMBIA-1300-SGN shown in sequence in sequence table 1 is obtained, called after p130-PC2.The part-structure schematic diagram of this recombinant vectors as shown in Figure 1A, can be found out, PC2 inserts the upstream of gus gene.
2, the acquisition of recombinant bacterium
The recombinant vectors p130-PC2 obtained above-mentioned 1 proceeds to agrobacterium tumefaciens EHA105(and is documented in Yuman Zhang, Yongsheng Yan, Lina Wang, Kun Yang, Na Xiao, Yunfeng Liu, Yaping Fu, ZongxiuSun, Rongxiang Fang, Xiaoying Chen.A novel rice gene, NRR responds tomacronutrient deficiency and regulates root growth.Mol.Plant, 2012,5 (1): the 63-72. public can obtain from Institute of Microorganism, Academia Sinica), obtain recombinant bacterium.
Extract the plasmid of recombinant bacterium, send to order-checking, this plasmid is p130-PC2, the recombinant bacterium called after EHA105/p130-PC2 containing this plasmid.
3, the acquisition of PC2:GUS paddy rice is turned
1) acquisition of PC2:GUS paddy rice is turned
Recombinant bacterium EHA105/p130-PC2 is proceeded in paddy rice ' Japan is fine ' (Oryza sativa ssp.japonicacultivar Nipponbare, also referred to as wild rice), obtain 30 strain T
0in generation, turns PC2:GUS paddy rice.
2) Molecular Identification of PC2:GUS paddy rice is turned
Extract T
0for the genomic dna turning PC2:GUS rice leaf, with primer GUS-S1705:5 '-TGGCCAATGGTGATGTCAGCGTT-3 '; GUS-R2329:5 '-TCCGGTTCGTTGGCAATACTCC-3 ' carries out pcr amplification, and what obtain 625bp product is the positive, obtains the positive T of 30 strains
0in generation, turns PC2:GUS paddy rice.
From positive T
0generation turns on PC2:GUS paddy rice gathers in the crops T
1for seed, sowing, cultivation, and obtain T by Totomycin (50ug/ml) resistance screening
2for transgenosis pure lines seed.Subsequent experimental is all with T
2it is that experiment material carries out PC2 promoter Analysis that generation turns PC2:GUS paddy rice pure lines seed.
Adopt and use the same method, empty carrier pCAMBIA1300vector (Fisher Scientific catalogNO.50-513-40, this carrier does not have gus reporter gene) is proceeded in wild rice, obtains T
0in generation, turns empty carrier paddy rice, carries out PCR qualification according to the method described above, does not obtain the product of 625bp, sowing (T to it
1generation), sowing, cultivate, obtain T
2in generation, turns empty carrier paddy rice pure lines seed.
Adopt and use the same method, carrier pCAMBIA-1300-SGN is proceeded in wild rice, obtains T
0in generation, turns pCAMBIA-1300-SGN paddy rice, PCR qualification (increase with primer GUS-S1705 and GUS-R2329 equally, what obtain 625bp is the positive), sowing (T
1generation), sowing, cultivate, obtain T
2in generation, turns pCAMBIA-1300-SGN paddy rice (positive control).
4, the GUS expression characterization analysis of PC2:GUS paddy rice is turned
GUS histological staining method is adopted to detect the expression characterization of PC2 promoters driven gus reporter gene in paddy rice:
The paddy rice that following experiment adopts is 5 independently T
2in generation, turns PC2:GUS paddy rice pure lines (PC2), T
2in generation, turns empty carrier paddy rice pure lines (negative control, Vec), wild rice (WT) and T
2in generation, turns pCAMBIA-1300-SGN paddy rice pure lines (positive control, 35S), the strain of each strain 5, and experiment in triplicate.
GUS prescription of its dyeing liquor: 0.1M Na
3pO
4, pH7.0,10mM EDTA, 0.5mM K
3[Fe (CN)
6], 0.5mMK4 [Fe (CN)
6], 1.0mM X-Glucuronide, 0.1%Triton X-100 and 10%MeOH.
GUS dyeing process: by rice paddy seed through carrying out surface sterilization 20 minutes with 10% clorox, distilled water flushing for several times, sow after vernalization in 1/2MS liquid nutrient medium, 14 days are cultured at 26 DEG C of plant incubators (16h illumination/8h is dark), whole strain seedling is immersed in GUS staining fluid, stained over night, with 75% alcohol flushing several.
T in flowering period will be in
2generation turn PC2:GUS paddy rice pure lines (PC2), the leaf of wild rice (WT) plant, fringe, stem, stipes, leaf sheath and Post flowering 25 days (25DAF) seed sample respectively after, immerse in GUS staining fluid, 3min is kept under 0.08Mpa vacuum, 37 DEG C of stained over night, then use 75% ethanol decolorization three times.
1) PC2 drives GUS special high expression level in leaf thready pulse (leaf small vein)
Result as shown in Figure 1B,
At T
2in generation, turns in 14 days seedling of PC2:GUS paddy rice pure lines, and PC2 drives GUS only specifically expressing in leaf thready pulse (leaf smallvein), forms obvious blue parallel lines.Drive GUS in leaf thready pulse specifically expressing characteristic to follow the tracks of PC2 further, have detected the GUS staining analysis of paddy rice different growing stage (after planting 14-86 days) plant leaf respectively, result shows that PC2 drives GUS all to have strong expression in the blade thready pulse of 14-64 days.
Wild rice does not have blue signal, and positive control (35S) all has signal.
2) PC2 drives the specifically expressing characteristic of GUS in seed scultellum
Result as shown in Figure 1B,
In the seed of growing, only in scultellum (intersection of Fetal liver cells), there is strong GUS expression signal, and in Fetal liver cells, have no GUS signal;
Wild rice does not have blue signal, and positive control (35S) all has signal.
In order to detect the expression of GUS in seed scultellum further, have detected T respectively
2in generation, turns PC2:GUS paddy rice pure lines (PC2), T
2in generation, turns empty carrier paddy rice (negative control, Vec), wild rice (WT) and T
2in generation, turns pCAMBIA-1300-SGN paddy rice (positive control, 35S) and dyes at the GUS of Post flowering 17 days (17DAF) and 35 days (35DAF) different developmental phases seeds.
Result as shown in Figure 1 C, can be found out, in seed growth process, and T
2in generation, turns PC2 promoters driven GUS only special high expression level in scultellum in PC2:GUS paddy rice pure lines, forms the boundary line of obvious Fetal liver cells; And all without GUS expression signal in embryo (rataria or mature embryo) and endosperm.
Positive control all has signal, and WT and negative control all do not have signal.
3) when seed germination, PC2 drives the expression of GUS
By T
2in generation, turns PC2:GUS paddy rice pure lines (PC2), T
2in generation, turns empty carrier paddy rice (negative control, Vec) and T
2the dry seeds (seasoning) that generation turns pCAMBIA-1300-SGN paddy rice (positive control, 35S) is peelled off clever shell and carries out GUS dyeing after crosscut, result as shown in Figure 2, T
2generation turns PC2:GUS paddy rice pure lines (PC2) only has obvious blue signal in dry seeds scultellum, and starchy endosperm is white, does not have GUS signal.Positive control all has signal, and negative control does not all have signal.
When the dry seeds of above-mentioned each paddy rice is soaked 3 days in water, peel off GUS dyeing after clever shell, result as shown in Figure 2, T
2generation turns PC2:GUS paddy rice pure lines (PC2) only has strong GUS signal, without GUS signal in rudiment in scultellum.Positive control all has signal, and negative control does not all have signal.
When the dry seeds of above-mentioned each paddy rice is soaked 7 days in water, rice seedling is obtained to sprouting and carries out GUS dyeing, result as shown in Figure 2, T
2generation turns PC2:GUS paddy rice pure lines also only has more weak blue signal on the vein of leaf base.Positive control all has signal, and negative control does not all have signal.
In sum, DNA molecular PC2 is promotor, drives gus reporter gene specifically expressing in seed scultellum and leaf thready pulse.
Claims (10)
1. a DNA fragmentation is following 1) or 2) or 3) DNA molecular:
1) DNA molecular shown in sequence 1 in sequence table;
2) under strict conditions with 1) DNA sequence dna that limits hybridizes and has the DNA molecular of promoter function;
3) with 1) DNA sequence dna that limits has more than 90% homology, and has the DNA molecular of promoter function.
2. the recombinant vectors containing DNA fragmentation described in claim 1, expression cassette, transgenic cell line or recombinant bacterium.
3. recombinant vectors as claimed in claim 2, is characterized in that: described recombinant vectors, for DNA fragmentation described in claim 1 being replaced the CaMV35S promotor in expression vector pCAMBIA-1300-SGN, obtains recombinant vectors.
4. increase the total length of DNA fragmentation or the primer pair of its any fragment described in claim 1.
5. primer pair according to claim 4, is characterized in that: described primer pair is made up of the single strand dna shown in sequence 3 in the single strand dna shown in sequence in sequence table 2 and sequence table.
6. DNA fragmentation described in claim 1 starts the application in destination gene expression in plant.
7. apply as claimed in claim 6, it is characterized in that: described destination gene expression is organizing specific expression.
8. apply as claimed in claim 7, it is characterized in that: described in be organized as scultellum and/or leaf thready pulse.
9. apply as claimed in claim 8, it is characterized in that: described plant is monocotyledons or dicotyledons; Described monocotyledons is specially paddy rice.
10., as the application as described in arbitrary in claim 6-9, it is characterized in that: described goal gene is gus gene.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003000898A1 (en) * | 2001-06-22 | 2003-01-03 | Syngenta Participations Ag | Plant genes involved in defense against pathogens |
| US20070056055A1 (en) * | 2001-09-26 | 2007-03-08 | Syngenta Participations Ag | Rice promoters for regulation of plant expression |
| CN101063139A (en) * | 2007-05-15 | 2007-10-31 | 中国农业大学 | Seed specificity highly effective promoter and its application |
| EP1873251A1 (en) * | 2006-06-29 | 2008-01-02 | Chemotherapeutisches Forschungsinstitut Georg-Speyer-Haus | Expression vector(s) for enhanced expression of a protein of interest in eukaryotic or prokaryotic host cells |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003000898A1 (en) * | 2001-06-22 | 2003-01-03 | Syngenta Participations Ag | Plant genes involved in defense against pathogens |
| US20070056055A1 (en) * | 2001-09-26 | 2007-03-08 | Syngenta Participations Ag | Rice promoters for regulation of plant expression |
| EP1873251A1 (en) * | 2006-06-29 | 2008-01-02 | Chemotherapeutisches Forschungsinstitut Georg-Speyer-Haus | Expression vector(s) for enhanced expression of a protein of interest in eukaryotic or prokaryotic host cells |
| CN101063139A (en) * | 2007-05-15 | 2007-10-31 | 中国农业大学 | Seed specificity highly effective promoter and its application |
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