CN102775499B - Application of rice transcription factor Os01g64730 gene - Google Patents
Application of rice transcription factor Os01g64730 gene Download PDFInfo
- Publication number
- CN102775499B CN102775499B CN2012102726216A CN201210272621A CN102775499B CN 102775499 B CN102775499 B CN 102775499B CN 2012102726216 A CN2012102726216 A CN 2012102726216A CN 201210272621 A CN201210272621 A CN 201210272621A CN 102775499 B CN102775499 B CN 102775499B
- Authority
- CN
- China
- Prior art keywords
- rice
- transcription factor
- gene
- os01g64730
- transgenic
- 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
Landscapes
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention relates to an application of rice transcription factor Os01g64730 gene. A transcription factor activation motif VP64 (consisting of four transcription factor activation motifs VP16) and a rice transcription factor Os01g64730 gene are fused to build a combined-type transcription factor which is transformed in crops such as rice, so as to delay heading period of transgenic rice and prolong growth period. An important theoretical value is provided to the detailed description of molecular mechanism of rice heading control; and meanwhile, with the adoption of the transgenic means, the rice growth period is prolonged and the adaptation of rice to different ecotopes is enhanced, so the gene also has an important significance in production practices.
Description
Technical field
The invention belongs to field of genetic engineering, specifically, relate to the application of rice transcription factor Os01g64730 gene.
Background technology
In long day plant (LDP) Arabidopis thaliana (Baurle and Dean, 2006; Imaizumi and Kay, 2006) and short day plant (SDP) paddy rice (Izawa, 2007; Tuji etc., 2008) in, the signal pathway of blooming has obtained research widely.GI-CO-FT controls Rice Flowering conservative approach (Yano etc., 2000; Kojima etc., 2002; Hayama etc., 2002).Hd3a is the homologous gene of FT in paddy rice, and it is subject to inducing of a B-type response regulator Ehd1, and this approach is independent of Hd1 gene (Doi etc., 2004) under the short day condition.OsMADS51 is subjected to the regulation and control of OsGI, and this effect is positioned at the upstream (Kim etc., 2007) of Ehd1.On the contrary, under the long day condition, Hd1 suppresses the expression of Hd3a and postpones Rice Flowering (Hayama etc., 2003).Current research shows, changes in nucleus after Hd3a and 14-3-3 albumen formation complex body, is combined with transcription factor FD1 and forms tripolymer FAC (florigen activation complex), induces transcribing of OsMADS15, thereby causes Rice Flowering.
VP16 finds in the animal virus gene, now be widely applied in plant, is mainly used in the research of transcriptional control of plant gene.Transcription factor effect in vivo can be divided into two kinds substantially: a kind of is transcriptional enhancer, another kind of for transcribing inhibition.After transcription factor and the fusion of VP16 functional domain motif, it will strengthen the function of transcription factor, thereby occurs that in transfer-gen plant more obvious phenotype changes.
Summary of the invention
The purpose of this invention is to provide the application of rice transcription factor Os01g64730 gene.
In order to realize the object of the invention, at first the present invention provides a kind of fusion rotein, and this fusion rotein is Os01g64730-Linker-(VP16)
nOr (VP16)
n-Linker-Os01g64730; Wherein, n is 〉=1 integer; Linker is by 1 ~ 20 flexible amino acid be in series (for example, GGGGG, GPPPG, or the aminoacid sequence DPAFLYKVVPR of Gatway carrier recombination site coding); VP16 is the VP16 albumen from hsv (Herpes simplex virus); Os01g64730 is rice transcription factor Os01g64730, and its aminoacid sequence is as shown in SEQ ID No.1, or this sequence is through replacing, lack or adding one or several amino acids formed aminoacid sequence with same function.
Preferably, in aforementioned fusion rotein, n is 4.Wherein, (VP16)
4Be VP64, by 4 VP16 functional domain motifs, merge the enhanser that forms, its aminoacid sequence is as shown in SEQ ID No.2, or this sequence is through replacing, lack or adding one or several amino acids formed aminoacid sequence with same function.
More preferably, aforementioned fusion rotein is Os01g64730-Linker-(VP16)
4
The present invention also provides the gene of encoding said fusion protein, and under stringent condition, can with the nucleotide sequence of the nucleotide sequence hybridization of this gene; Wherein, described stringent condition is 65 ℃, hybridizes in the solution of 0.1 * SSPE or 0.1 * SSC, 0.1%SDS and washes film.
The present invention also provides the carrier of the gene that contains encoding said fusion protein.The carrier that described carrier is expressed in the host for any bootable foreign gene.Preferably, described carrier be plant binary expression vector (for example, pCAMBIA1301).Gene constructed in plant expression vector time the with encoding said fusion protein of the present invention, can add any strong promoter (for example, corn strong promoter Ubiquitin) or inducible promoter before its transcription initiation Nucleotide.In addition, gene constructed in plant expression vector time the with encoding said fusion protein of the present invention, can also use enhanser, and these enhanser zones must be identical with the reading frame of encoding sequence, to guarantee the translation of whole piece sequence.
The present invention also provides the transgenic cell line of the gene that contains encoding said fusion protein.
The present invention also provides the engineering bacteria of the gene that contains encoding said fusion protein.
The present invention also provides the application of gene in postponing Rice Heading, prolong growth period of encoding said fusion protein.
The present invention further provides the application of rice transcription factor Os01g64730 gene, it is the CDS sequence of rice transcription factor Os01g64730 gene (complete translation district) to be building up to 4 transcription factors activate the upstream of motif VP16, conversion of plant, screening and final acquisition transgenic plant.
Aforesaid application, it is the CDS sequence of rice transcription factor Os01g64730 gene to be activated the upstream of motif VP16 by Gateway system constructing to 4 transcription factor, rice transformation (for example, rice varieties ' kitaake '), thus postpone heading stage, the prolong growth period of transgenic paddy rice.Wherein, the CDS sequence of rice transcription factor Os01g64730 gene is as shown in SEQ ID No.3, and 4 transcription factors activate the nucleotide sequence of motif VP16 as shown in SEQ ID No.4.
The expression vector that carries the gene of encoding said fusion protein can import (Weissbach in vegetable cell by using the conventional biotechnological means such as Ti-plasmids, plant viral vector, directly delivered DNA, microinjection, electroporation, 1998, Method for Plant Molecular Biology VIII, Academy Press, New York, the 411-463 page; Geiserson and Corey, 1998, Plant Molecular Biology, 2nd Edition).
The present invention utilizes transcription factor to activate i.e. 4 the transcription factors activation motif VP16 of motif VP64(first) build and obtain the composing type transcription factor with rice transcription factor Os01g64730 gene fusion, and be transformed into farm crop, in paddy rice, thus heading stage, the prolong growth period of postponement transgenic paddy rice.Molecule mechanism for illustrating in detail the Rice Heading regulation and control, have important theory value, and can pass through the transgenosis means, extends growth period duration of rice, improves the adaptive faculty of paddy rice to planted in different ecological areas, and is therefore significant equally aborning.
Description of drawings
Fig. 1 is cVP64-bar-asRED carrier collection of illustrative plates in the embodiment of the present invention 1.
Fig. 2 is ubi:cVP64-bar-asRED carrier collection of illustrative plates in the embodiment of the present invention 1.
Fig. 3 is the result that in the embodiment of the present invention 3, Western Blot detects transgenic positive rice strain; Wherein, WT is wild-type paddy rice ' kitaake ', and bzip17-VP64-1, bzip17-VP64-2 and bzip17-VP64-3 are respectively 3 strains of Os01g64730-VP64 transgenic paddy rice.
Fig. 4 is the phenotype analytical result of Os01g64730-VP64 transgenic paddy rice strain in the embodiment of the present invention 4; Wherein, WT is wild-type paddy rice ' kitaake ', and VP64-bzip17-1, VP64-bzip17-2 and VP64-bzip17-3 are respectively 3 strains of Os01g64730-VP64 transgenic paddy rice.
Embodiment
Following examples are used for explanation the present invention, but are not used for limiting the scope of the invention.If do not specialize, the conventional means that in embodiment, technique means used is well known to those skilled in the art, the raw materials used commercial goods that is.
The acquisition of embodiment 1 rice transcription factor Os01g64730 gene and the structure of plant expression vector
Login http://rice.plantbiology.msu.edu/analyses_search_locus.shtml website, find rice transcription factor Os01g64730 gene, according to its sequences Design pcr amplification primer:
Os01g64730-F：5'-CAAAAAAGCAGGCTTCATGATGGCGTCGAGGGTG-3'
Os01g64730-R:5'-CAAGAAAGCTGGGTCCCACTCCATCGAGTTTGTTCTTC-3'
Take paddy rice ' Japan is fine ' blade as material, the TRIzol method is extracted total RNA, reverse transcription obtains cDNA, reactions steps is as follows: (1) on ice to adding successively following material in the PCR reaction tubes that does not contain nuclease: total RNA 6 μ l (0.1ng-5 μ g), Oligo (dT) 18 primer 1 μ l, do not contain the ddH of nuclease
2O 5 μ l, be placed in 65 ℃ of reaction 5min of PCR instrument; (2) and then add following material: 5 * reaction buffer, 4 μ l, RNase inhibitor 1 μ l, 10mM dNTP2 μ l, M-M μ lV ThermoScript II 1 μ l, mix gently, 45 ℃ of reaction 60min in the PCR instrument; (3) in the PCR instrument, 70 ℃ of 5min, termination reaction.Take total cDNA as template, carry out pcr amplification, amplification system is: reaction buffer 25 μ l, dNTP4 μ l, ddH
2O17.5 μ l, Taq archaeal dna polymerase 0.5 μ l, response procedures are 98 ℃ of 10s of warm start, 57 ℃ of 5s, and 72 ℃ of 1min, after 30 circulations, 72 ℃ are extended 10min, and last 25 ℃ of reactions finish.
Obtain the CDS sequence of rice transcription factor Os01g64730 gene, its nucleotide sequence is as shown in SEQ ID No.3.Carry out PCR according to PrimeSTAR polymeric enzymatic amplification system and response procedures.Requirement according to the Gateway clone technology, comprise two-wheeled PCR in this process, the primer of the first round PCR primer that adds part adaptor attB joint, and the second template of taking turns PCR product of the first round, and primer adopts complete adaptor attB primer.The PCR product cloning, to connecting on the pDONR cloning vector, is identified and obtained and the identical sequence of goal gene through order-checking.React that by LR Os01g64730 is gene constructed to plant expression vector cVP64-bar-asRED(Fig. 1) on, obtain carrier ubi:Os01g64730-VP64(Fig. 2, the carrier complete sequence is as shown in SEQ ID No.5).
Wherein, the building process of plant expression vector cVP64-bar-asRED is: the sequence that comprises take border, binary expression vector pCAMBIA1300 left and right is as frame sequence, pass through vitro recombination, ubi promoter-Gateway-VP64 is expressed unit, 35S promoter-asRED expression unit and 35S promoter-bar expression unit construct and obtain with it, the complete sequence of carrier cVP64-bar-asRED is as shown in SEQ ID No.6.
The acquisition of embodiment 2 transgenic rice plants
Water intaking rice ' kitaake ' mature seed, artificial or mechanical dejacketing, select full bright and clean seed without bacterial plaque and be inoculated on inducing culture and carry out inducing culture after sterilization.The selection outward appearance is good, the Rice Callus that growing ability is good is acceptor material, adopt agrobacterium-mediated transformation that ubi:Os01g64730-VP64 is changed in Rice Callus, the AAM conversion fluid that with the Syringylethanone that contains 100 μ M and O.D. value is 0.7 Agrobacterium transforms, the callus that conversion fluid was soaked is placed on common substratum and carries out common cultivation, 25 ℃ of dark 3d of cultivation are placed on screening culture medium and cultivate approximately 30d, and every 10d subculture once.The kanamycin-resistant callus tissue that then will sift out is transferred on division culture medium and is broken up approximately 20d, and every 10d subculture once.The kanamycin-resistant callus tissue that differentiates green seedling is transferred on root media and taken root, hardening after approximately 7d grows flourishing root system, and calculate the conversion transgenic seedling number that obtains.Be transferred to grown in field after hardening 7d.With Basta screening transgenic paddy rice, start to spray Basta (1:1000, v:v) after growing 4 spires,, every spray in 1 day 1 time, spray altogether 3 times.Obtain altogether transgenic paddy rice 42 strains.
Wherein, the inducing culture based formulas is: and N6 is a large amount of+and B5 trace+NB is organic+molysite+copper cobalt mother liquor+2.5mg/L 2,4D+0.6g/L acid hydrolyzed casein+2.878g/L proline(Pro)+0.5g/L glutamine+30g/L sucrose, with the water preparation, accent adds plant gel 4g/L behind pH to 5.8 ~ 5.9.
Altogether culture medium prescription is: N6 is a large amount of+B5 trace+NB is organic+molysite+2.5mg/L 2, and 4D+0.5g/L glutamine+0.6g/L acid hydrolyzed casein+10g/L glucose+30g/L sucrose, with the water preparation, adds plant gel 4g/L after transferring pH to 5.2.After sterilizing, 50 ℃ of left and right add the AS(Syringylethanone) 100 ~ 200 μ g/mL.
The screening and culturing based formulas is: and N6 is a large amount of+and B5 trace+NB is organic+molysite+copper cobalt mother liquor+2.5mg/L 2,4D+0.6g/L acid hydrolyzed casein+2.878g/L proline(Pro)+0.5g/L glutamine+30g/L sucrose, with the water preparation, accent adds plant gel 4g/L behind pH to 5.8 ~ 5.9.Add 35mg/L Totomycin (available from Shanghai Niu Jin Bioisystech Co., Ltd) or 5mg/L Bialaphos(after sterilizing available from Beijing Baeyer enlightening biotech company).
The differentiation culture based formulas is: and MS is inorganic+and MS-B5 trace+MS is organic+molysite+MS-copper cobalt mother liquor+0.05mg/L NAA+2.0mg/L Kinetin(kinetin)+30g/L sorbyl alcohol+2g/L caseinhydrolysate+30g/L sucrose, with the water preparation, accent adds plant gel 4g/L behind pH to 5.8 ~ 5.9.
Embodiment 3 Western Blot detect the expression of fusion rotein Os01g64730-VP64 in transgenic paddy rice
For detect the Os01g64730-VP64 gene at T2 for the expression of crossing in transgenic paddy rice, utilize VP64 antibody to identify on protein level it, through the SDS-PAGE protein electrophoresis → immune marking → Immunofluorescence Reactions, Western Blot qualification result shows that there is target protein in transfer-gen plant, the band (Fig. 3) and wild-type is not mixed out.
Concrete Western Blot experiment flow is as follows:
Appropriate sample is put into the freezing rear grind into powder of liquid nitrogen, add appropriate sample-loading buffer to mix, centrifugal 10 minutes of 12000rpm; Get 5 μ l supernatant application of samples, after 30 minutes, 120V electrophoresis 60-90 minute, when the bottom of tetrabromophenol sulfonphthalein arrival gel gets final product stop electrophoresis with 90V voltage SDS-PAGE electrophoresis; Adopt half-dried transfer method transferring film after electrophoresis, and with the ponceau staining fluid, film is dyeed, observe the transferring film effect; After transferring film is complete, film is put into the PBST solution sealing room temperature sealing that contains 5% skim-milk spent the night in 60 minutes or 4 ℃; Primary antibodie under room temperature (VP64 antibody) was hatched 1 hour or 4 ℃ of overnight incubation, then with PBST washing 3 times, each 5 minutes; Two anti-under room temperature (the goat-anti rabbit, available from Abmart, article No. M21002S) hatched 1 hour, then with PBST washing 3 times, each 5 minutes; Add substrate on film, expose.
Wherein, VP64 antibody is how anti-as the rabbit source that specific antigen prepares by Ai Bimate biological medicine (Shanghai) Co., Ltd. polypeptide synthetic according to the aminoacid sequence of VP64.
Embodiment 4 transgenic paddy rice phenotype analyticals
' kitaake ' compares with the wild-type paddy rice, obviously postpone the heading stage of Os01g64730-VP64 transgenic paddy rice strain (bzip17-VP64-1, bzip17-VP64-2 and bzip17-VP64-3), though wild-type is eared, the sword-like leave of transgenic line is not extracted (Fig. 4) yet fully out.
The embodiment that more than provides activates the CDS sequence of rice transcription factor Os01g64730 gene the upstream of motif VP16 by Gateway system constructing to 4 transcription factor, rice transformation kind ' kitaake ', thereby heading stage, the prolong growth period of postponement transgenic paddy rice.Similarly, the CDS sequence of rice transcription factor Os01g64730 gene is activated the downstream of motif VP16 to 〉=1 transcription factor by the Gateway system constructing, or activate the upstream of motif VP16 by Gateway system constructing to 1 ~ 3 or 4 above transcription factors, the rice transformation kind, also can reach the heading stage of postponing transgenic paddy rice, the effect of prolong growth period.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements, all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (1)
1. the application of rice transcription factor Os01g64730 gene, it is characterized in that, it is the CDS sequence of rice transcription factor Os01g64730 gene to be activated the upstream of motif VP16 by Gateway system constructing to 4 transcription factor, rice transformation, thereby heading stage, the prolong growth period of postponement transgenic paddy rice; Wherein, the aminoacid sequence of rice transcription factor Os01g64730 is as shown in SEQ ID No.1, and 4 transcription factors activate the aminoacid sequence of motif VP16 as shown in SEQ ID No.2.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012102726216A CN102775499B (en) | 2012-08-01 | 2012-08-01 | Application of rice transcription factor Os01g64730 gene |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012102726216A CN102775499B (en) | 2012-08-01 | 2012-08-01 | Application of rice transcription factor Os01g64730 gene |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102775499A CN102775499A (en) | 2012-11-14 |
| CN102775499B true CN102775499B (en) | 2013-11-13 |
Family
ID=47120627
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2012102726216A Active CN102775499B (en) | 2012-08-01 | 2012-08-01 | Application of rice transcription factor Os01g64730 gene |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102775499B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103421119B (en) * | 2013-08-23 | 2015-03-18 | 中国农业科学院作物科学研究所 | Application of rice transcription factor Os05g41450 genes |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103224563B (en) * | 2013-03-29 | 2014-09-03 | 中国农业科学院作物科学研究所 | Application of synthetic transcription factor VP64-Os01g63510 |
| CN103214581B (en) * | 2013-03-29 | 2014-12-10 | 中国农业科学院作物科学研究所 | Application of synthetic transcription factor VP64-Os03g57670 |
| CN103265634B (en) * | 2013-04-28 | 2014-11-26 | 中国农业科学院作物科学研究所 | Application of rice transcription factor Os01g64730 gene |
| CN103319603A (en) * | 2013-06-09 | 2013-09-25 | 中国农业科学院作物科学研究所 | Application of rice transcription factor Os01g64730 gene |
| CN104341516B (en) * | 2013-07-31 | 2018-04-27 | 中国农业科学院作物科学研究所 | The application of rice transcription factor Os05g46780 gene C DS sequences |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001000815A1 (en) * | 1999-05-28 | 2001-01-04 | Sangamo Biosciences, Inc. | Molecular switches |
| JP2009219397A (en) * | 2008-03-14 | 2009-10-01 | Institute Of Physical & Chemical Research | Induction method for plant cell wall |
| CN102250952A (en) * | 2011-06-17 | 2011-11-23 | 中国人民解放军军事医学科学院生物工程研究所 | Vector for mediating high-efficiency expression of exogenous gene in cells of mammal and use thereof |
-
2012
- 2012-08-01 CN CN2012102726216A patent/CN102775499B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001000815A1 (en) * | 1999-05-28 | 2001-01-04 | Sangamo Biosciences, Inc. | Molecular switches |
| JP2009219397A (en) * | 2008-03-14 | 2009-10-01 | Institute Of Physical & Chemical Research | Induction method for plant cell wall |
| CN102250952A (en) * | 2011-06-17 | 2011-11-23 | 中国人民解放军军事医学科学院生物工程研究所 | Vector for mediating high-efficiency expression of exogenous gene in cells of mammal and use thereof |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103421119B (en) * | 2013-08-23 | 2015-03-18 | 中国农业科学院作物科学研究所 | Application of rice transcription factor Os05g41450 genes |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102775499A (en) | 2012-11-14 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102816243B (en) | Application of rice transcription factor Os06g08400 genes | |
| CN103214581B (en) | Application of synthetic transcription factor VP64-Os03g57670 | |
| CN102786599B (en) | Application of rice transcription factor Os05g39950 gene | |
| CN102775499B (en) | Application of rice transcription factor Os01g64730 gene | |
| CN102775498B (en) | Application of rice transcription factor Os05g41070 genes | |
| CN103224563B (en) | Application of synthetic transcription factor VP64-Os01g63510 | |
| CN101628941B (en) | Bovine lactoferrin antibacterial peptide fusion protein, coding gene and application thereof | |
| Sasaki et al. | Transient expression of tobacco BBF1-related Dof proteins, BBF2 and BBF3, upregulates genes involved in virus resistance and pathogen defense | |
| CN102021179B (en) | Application of rice gene KT484 to improvement of stress tolerance performance of plants | |
| CN106674338A (en) | Application of stress resistance-related protein to regulation and control on stress resistance of plants | |
| CN103254314A (en) | Application of paddy rice transcription factor Os05g41070 gene | |
| CN102226184A (en) | Method for cultivating transgenic nitrogen-fixing plants | |
| CN104211809A (en) | Application of synthetic transcription factor VP64-linker-Os03g11370 to improve paddy rice grain properties | |
| CN103421119B (en) | Application of rice transcription factor Os05g41450 genes | |
| CN103265634B (en) | Application of rice transcription factor Os01g64730 gene | |
| CN103319603A (en) | Application of rice transcription factor Os01g64730 gene | |
| Ito et al. | Radial axis differentiation in a globular embryo is marked by HAZ1, a PHD-finger homeobox gene of rice | |
| CN101987867A (en) | Ethylene receptor NTHK1 interactive protein relevant to plant stress tolerance as well as coding gene and application thereof | |
| CN103421121A (en) | Application of rice transcription factor Os02g07780 genes | |
| CN106674339A (en) | Application of protein to regulation and control of plant adverse resistance | |
| CN104211812A (en) | Application of rice transcription factor Os02g49370 gene | |
| CN104341514A (en) | Application of oryza sativa transcription factor Os03g05590.1 gene CDS sequence | |
| CN102226190B (en) | Method for cultivating transgenic hardy plants | |
| CN103614385A (en) | Application of gene KT525 to improvement of stress tolerance of plants | |
| CN102373187A (en) | Plant stress tolerance-associated protein and encoding gene and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |