CN102409052B - Promoter of oryza sativa rice root tip specific 3 (OsRTS3) expression gene and application thereof - Google Patents
Promoter of oryza sativa rice root tip specific 3 (OsRTS3) expression gene and application thereof Download PDFInfo
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Abstract
本发明公开了一种水稻根尖特异表达基因OsRTS3的启动子，该启动子为SEQ ID NO：1所示的核苷酸序列。本发明还同时公开了上述水稻根尖特异表达基因OsRTS3的启动子的用途：用于构建转基因水稻，其目的为建立水稻根尖特异表达品系。本发明启动子的功能在于能启动下游编码基因在水稻根尖静止中心的特异表达。
The invention discloses a promoter of rice root tip specific expression gene OsRTS3 , the promoter is the nucleotide sequence shown in SEQ ID NO:1. The present invention also discloses the use of the above-mentioned promoter of the rice root-tip-specific expression gene OsRTS3 : it is used for constructing transgenic rice, and the purpose is to establish a rice root-tip-specific expression strain. The function of the promoter of the present invention is that it can initiate the specific expression of downstream coding genes in the quiescent center of rice roots.
Description
Technical field
The invention belongs to plant genetic engineering field.Specifically, the present invention relates to the upstream promoter nucleotide sequence of cloning rice OsRTS3 (rice root tip specific 3) gene coding region, the function of this sequence is to start downstream coding gene at the specifically expressing of paddy rice root tip quiescent center.
Background technology
Root system is the vitals of plant absorbing moisture, nutrient.The structure of root is broadly divided into four parts: root cap, meristematic zone, elongation zone and root-hair zone.Cell is continuous division growth in meristematic zone, newborn cytodifferentiation and growth, the various tissues of formation root.Developmental cell volume increases, and forms the elongation zone.Full cell volume no longer increases sharply, and hair shape projection occurs the cell in the epidermis position, forms the root-hair zone.The elongation zone cell volume increases mineralization pressure to be promoted meristematic zone and moves forward, and part meristematic zone cytodifferentiation also covers the tip of root, plays the effect of protection and guiding, becomes root cap.The tip of a root is comprised of meristematic zone and root cap, and its core is quiescent center.The quiescent center cell has the lateral reactivity that is divided into other cell, produces based on this epidermis, cortex, pericycle, center pillar, the root cap cell that forms the tip of a root.Differentiation and the growth of research root-tip cells, significant to the proterties of understanding, control and improvement root system.
The growth of root system is the process of a complexity.Present research is based on mainly that the root system related mutants of dicotyledonous model plant Arabidopis thaliana (Arabidopsis thaliana) launches, and less take paddy rice (Oryza sativa) as the root system development Mechanism Study of the gramineous crop of model plant.
Genetic transfoumation is an important channel that utilizes excellent genes (especially foreign gene) improvement species.Except the needs fine genes, also need to be applicable to the promotor that target gene is brought into play function in the best condition by genetic transfoumation improvement species.The quantity of international and domestic available functional gene for the farm crop character improvement is increasing gradually.But by comparison, alternative promotor value volume and range of product is but very limited, especially lacks the promotor that is applicable to improve the farm crop proterties.Promotor has become one of bottleneck that adopts genetic transfoumation improvement species.International and domestic promotor for the farm crop genetic transformation mainly is the composition type expression promoters such as corn ubiquitin (ubiquitin) gene promoter, rice actin (actin) gene promoter, viral 35S at present.Their goal of regulation and control genes are all expressed in a organized way in institute, and do not have space-time to limit.When expressing with these promoter regulation target genes, because a large amount of target protein occurs, often cause easily species physiology and morphology dysfunction in unwanted cells.Although the report of a small amount of specific promoter separating clone is also arranged in the world, the kind of the farm crop that relate to seldom, quantity is very limited, and we do not have intellecture property, will be subject to production application.
Summary of the invention
The technical problem to be solved in the present invention provides the promotor of a kind of paddy rice root tip specific expression gene OsRTS3, utilizes this promotor (OsRTS3 gene promoter) can start foreign gene at paddy rice root tip quiescent center specifically expressing.
In order to solve the problems of the technologies described above, the invention provides the promotor of a kind of paddy rice root tip specific expression gene OsRTS3, this promotor is the nucleotide sequence shown in the SEQ ID NO:1.
Improvement as the promotor of paddy rice root tip specific expression gene OsRTS3 of the present invention: promotor also is included in the nucleotide sequence shown in the SEQ IDNO:1 to be added, replace, inserts and lack mutant, allelotrope and the derivative that one or more Nucleotide generate.
Further improvement as the promotor of paddy rice root tip specific expression gene OsRTS3 of the present invention: promotor also comprises it being can and have the nucleotide sequence of identical function with the nucleotide sequence hybridization shown in the SEQ ID NO:1 under the rigorous condition of height.
The present invention also discloses the purposes of the promotor of above-mentioned paddy rice root tip specific expression gene OsRTS3 simultaneously: be used for the organizing specific expression cell marking.
Promotor of the present invention is the upstream sequence of the gene coding region of paddy rice root tip specific expression gene OsRTS3.The plant expression vector that contains this promotor by structure, it is transformed in the transgenic paddy rice, the multiple different tissues such as common leaf, sword-like leave, flower and root to transgenic line have carried out GUS histochemical stain detection, the result has confirmed this promotor only at the Meristernatic zone specifically expressing, and further clearly the expression position of this promotor concentrate near the quiescent center.
Utilize promotor of the present invention to make up transgenic paddy rice, have following advantage: can start downstream gene and be expressed near the quiescent center specifically, and not start this gene in the expression at other position of root.The mode that this specific character helps to connect the reporter genes such as fluorescin comes the cell of above-mentioned position and on every side other cell differentiation.In the present invention, we have connected a reporter gene (GUS) in the downstream of OsRTS3 promotor. because the position that GUS expresses can be dyed to blueness, so can intuitively demonstrate the tissue specificity of promotor.The application of this patent is intended in the future that the promotor downstream connects specific fluorescent mark gene, makes it specifically expressing near quiescent center, is convenient under condition of living body the research gene and regulates and control and interaction in the root system of plant early development.This only have very large tachnical storage for the targeting genetic marker of particular organization or cell to correlative study and be worth.
Description of drawings
Below in conjunction with accompanying drawing the specific embodiment of the present invention is described in further detail.
Fig. 1 is pBI101.1-GUS plus carrier synoptic diagram.
Fig. 2 is pCAMBIA-GUSplus carrier synoptic diagram
Fig. 3 is the coloration result that the OsRTS3 promotor merges the gus gene transgenic line.
Among the figure: A, stereoscope photo show that the OsRTS3 promotor is at tip of a root quiescent center specifically expressing; B, resin slicer demonstration OsRTS3 promotor is expressed near quiescent center; C, the organization of root tips structure is divided.bars＝500μm(A)，bar＝100μm(B)。
Embodiment
Following examples of the present invention use molecular biological method to be known technology.The Current Protocols in Molecular Biology that publishes in the John Wiley and Sons company that Ausubel writes, write the Molecular Cloning:A Labortory Manual that Cold Spring Harbor Laboratory Press (2001) publishes with J.Sambrook etc., the documents such as 3rd ED. all have detailed explanation.
According to correlative study, know that OsRTS3 may be the organization of root tips specific expression gene.Upstream sequence design special primer according to the OsRTS3 gene coding region, take the genomic dna of wild-type Nipponbare as template, amplify the OsRTS3 gene promoter of total length 2607bp, its nucleotides sequence is classified shown in the SEQ ID NO:l (namely as sequence table as described in) as.
Primer sequence is as follows:
Upstream primer: AGGCCGAAAGACTAAGAGTTGAG
Downstream primer: ATACCATGGAACGGCTGCATAACTAA
The extracting method of the genomic dna of wild-type Nipponbare is:
Wild-type rice varieties Nipponbare blade is ground with cooled with liquid nitrogen, extract genomic dna with the CTAB method, detailed process is as follows:
1) adds the 2-ME/CTAB of 65 ℃ of preheatings in the tissue of pulverizing, mix making it fully moistening, educate 10～60min, frequently mixing in 65 ℃.
2) with isopyknic 24: 1 chloroform/octanols or chloroform/primary isoamyl alcohol extracting homogenate, put upside down and make abundant mixing, in 4 ℃, the centrifugal 5min of 7500g reclaims the upper strata water.
3) the upper strata of reclaiming mutually in 65 ℃ CTAB/NaCl solution of adding 1/10 volume, put upside down mixing.
4) with isopyknic chloroform/octanol extracting, mixing, centrifugal, reclaim the upper strata water.
5) add isopyknic CTAB precipitated liquid, put upside down mixing.
6) in 4 ℃, the centrifugal 5min of 500g.
7) shift out supernatant, resuspended if precipitation is difficult to the resuspended precipitation of TE damping fluid of high salt, in 65 ℃ of incubation 30min, repeat until all or most of resolution of precipitates.
8) the isopropanol precipitating nucleic acid of adding 0.6 volume, abundant mixing is in 4 ℃ of 7500g, centrifugal 15min.
9) with 80% washing with alcohol throw out, drying is with resuspended (the every gram parent material 0.1～0.5mL) of the least possible TE damping fluid.
Utilize Prime Star DNA Polymerase whole promoter DNA sequence amplification out:
Utilize the Prime Star DNA Polymerase of Takara company, the amplification OsRTS3 promoter sequence take the genomic dna of wild-type Nipponbare as template (diluting 20 times).Reaction system is as follows:
PrimeSTAR HS DNA Polymerase(2.5U/μl)0.2μl。
In the enterprising performing PCR amplification of PTC200 type PCR instrument, reaction conditions is as follows:
1)94℃ 2min，
2)94℃ 10s
3)62-58℃ Ramp 2℃/s
4)72℃ 3min
5) return 2), circulate 30 times
6)72℃ 7min
7) 4 ℃ of preservations.
After the purified and PstI/NcoI enzyme of PCR product is cut on 0.8% sepharose electrophoresis and rubber tapping reclaim the purpose band, the dna fragmentation that reclaims is cut through Xba I/Nco I enzyme and is inserted the pCAMBIAl300-GUSplus carrier (Fig. 1) of cutting through Xba I/Nco I enzyme equally, connect the product thermal shock and transform the bacillus coli DH 5 alpha competent cell, coated plate is cultivated.Some bacterium colonies of picking are at random cut through shaking bacterium, extracting plasmid, Xba I/Nco I enzyme, electrophoresis detection, and picking can cut out the positive monoclonal order-checking of correct length dna fragmentation.The correct clone who identifies cuts through Xba I/EcoR I enzyme, and the fragment that cuts out is connected to the pBI101.1-GUS plus carrier (Fig. 1) of cutting through Xba I/EcoR I enzyme equally, connects the product thermal shock and transforms the bacillus coli DH 5 alpha competent cell, and coated plate is cultivated.Some bacterium colonies of picking are at random cut through shaking bacterium, extracting plasmid, PstI/SacI enzyme, electrophoresis detection, and picking can cut out the positive mono-clonal of bacterial strain of correct length dna fragmentation.Enzyme is cut and is accredited as positive mono-clonal bacterium liquid and expands and take out plasmid after numerous, is the OsRTS3P-GUSplus plasmid.This plasmid electric shock is transformed into Agrobacterium EHA105 competent cell.This agrobacterium strains is used for Transgenic Rice.
The carrier of using in the above-mentioned experimentation is according to common carrier to come according to the ordinary method transformation.The method of GUS plus sequence on the pCAMBIA1305.1 with PCR amplified, and upstream primer 5 ' termination has added BamH I and Kpn I enzyme cut-grafting head, and downstream primer 5 ' termination has added Sac I enzyme cut-grafting head.The PCR product is connected among the pBI101.3 that cuts with same enzyme after cutting with BamH I/Sac I enzyme, and the carrier of acquisition is called pBI101.1-GUS plus.
The primer sequence is:
GUS plus-up AACGGATCCACGGTACCATGGTAGATCTGAGGGTAAATTTC
GUS plus-low ACGGAGCTCTCACACGTGATGGTGATGGTGATGG
PBI101.1-GUS plus carrier cuts out the fragment that contains gus gene with Hind III/EcoR I, is connected in the pCAMBIA1300 carrier of cutting with same enzyme, and the carrier of gained is called pCAMBIA1300-GUSplus.
Embodiment 2,
Mature seed of rice is placed on the mature embryo inducing culture through shelling with after disinfecting, and cultivates for 3 weeks in 28 ℃ of illumination boxs.The embryo callus that picking divides naturally (faint yellow, it is spherical that densification is) is inserted in the subculture medium, and succeeding transfer culture in 28 ℃ of illumination boxs obtains rice callus.Rice callus infects, cultivates altogether, screens containing the antibiotic substratum of G418 through the Agrobacterium of OsRTS3P-GUSplus Plasmid Transformation.Callus that can normal growth obtains transfer-gen plant, transfer-gen plant through breaking up, take root, practicing transplantation of seedlings.The rice transformation system of Agrobacterium (EHA105) mediation is mainly used on the method basis of people (1994) reports such as Hiei and is optimized.The transgenic seedling offspring who obtains with the dyeing of GUS dye liquor after as shown in Figure 3.
Show that the GUS of OsRTS3 gene promoter driving is at tip of a root quiescent center specifically expressing.
Comparative Examples 1,
Cauliflower mosaic virus 35 S promoter is plant transgene promotor commonly used.The OsRTS3P-GUSplus plasmid that uses among the embodiment 2 namely contains 35S promoter, is used for starting the G418 resistant gene.35S promoter can guarantee that the G418 resistant gene expresses in a organized way in the institute that comprises callus, thus guarantee to change the callus of OsRTS3P-GUSplus plasmid over to and break up after plant can survive containing under the culture condition of G418.If use 35S promoter to substitute the OsRTS3 promotor, can cause equally foreign gene (such as GUS) in nearly all tissue, to be expressed, thereby lose tissue specificity.
At last, it is also to be noted that what more than enumerate only is several specific embodiments of the present invention.Obviously, the invention is not restricted to above embodiment, many distortion can also be arranged.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention all should be thought protection scope of the present invention.
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| CN103695422B (en) * | 2013-12-05 | 2016-04-06 | 浙江省农业科学院 | Paddy rice root tip specific expression promoter Pro-Os04g24469 and application thereof |
| CN105087591B (en) * | 2015-09-23 | 2017-11-21 | 安徽省农业科学院水稻研究所 | Paddy rice root tip specific expression promoter POsRo3 |
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