CN103265634B - Application of rice transcription factor Os01g64730 gene - Google Patents
Application of rice transcription factor Os01g64730 gene Download PDFInfo
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- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention relates to application of a rice transcription factor--the Os01g64730 gene. According to the invention, the transcription factor inhibitory motif EAR and the rice transcription factor--the Os01g64730 gene are fused to construct a constitutive transcription factor which is then transformed into a crop like rice, so a heading period of transgenic rice is advanced and a growth period of the transgenic rice is shortened. The rice transcription factor has a critical theoretical value to elaboration of molecular mechanism of heading regulation and control of the rice; through transgenic means, the growth period of the rice is shortened, adaptive capacity of the rice to different ecological zones is improved, so the rice transcription factor also has great significance in production practice.
Description
Technical field
The present invention relates to genetically engineered field, 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 is conservative approach (Yano etc., 2000 of controlling Rice Flowering; Kojima etc., 2002; Hayama etc., 2002).Hd3a is the homologous gene of FT in paddy rice, and it is subject to the induction of Type B response regulator Ehd1, and this approach is independent of Hd1 gene (Doi etc., 2004) under short day condition.OsMADS51 is subject to the regulation and control of OsGI, and this effect is positioned at the upstream (Kim etc., 2007) of Ehd1.On the contrary, under long day condition, Hd1 suppresses the expression of Hd3a and postpones Rice Flowering (Hayama etc., 2003).Current research shows, after Hd3a and 14-3-3 albumen formation complex body, proceeds in nucleus, is combined forms tripolymer FAC (florigen activation complex) with transcription factor FD1, and induction OsMADS15 transcribes, thereby makes Rice Flowering.
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 EAR functional domain motif, it will suppress the function of transcription factor, thereby in transfer-gen plant, occurs that more obvious phenotype changes.
Summary of the invention
The object of this invention is to provide the application of rice transcription factor Os01g64730 gene.
In order to realize the object of the invention, first the present invention provides a kind of fusion rotein, and this fusion rotein is Os01g64730-Linker-EAR or EAR-Linker-Os01g64730.Wherein, Linker for example, by 1～20 flexible amino acid be in series (, the aminoacid sequence DPAFLYKVVPR of GGGGG, GPPPG or Gatway carrier recombination site coding etc.).
EAR is that a section from plant transcription factor has the albumen motif of transcribing inhibit feature, and 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,
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, aforementioned fusion rotein is Os01g64730-Linker-EAR.
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 DEG C, hybridizes and wash film in the solution of 0.1 × SSPE or 0.1 × SSC, 0.1%SDS.
The present invention also provides the carrier of the gene that contains encoding said fusion protein.Described carrier is the carrier that any bootable foreign gene is expressed in host.Preferably, described carrier is plant binary expression vector (for example, pCAMBIA1301).Gene constructed in plant expression vector time by encoding said fusion protein of the present invention can be added 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 by encoding said fusion protein of the present invention, can also use enhanser, and these enhanser regions 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 gene that the present invention also provides encoding said fusion protein make Rice Heading in advance, shorten the application in breeding time.
The present invention further provides the application of rice transcription factor Os01g64730 gene, it is that the CDS sequence of rice transcription factor Os01g64730 gene (complete translation district) is building up to the upstream that transcription factor suppresses motif EAR, rice transformation, screening the final transgenic paddy rice that obtains.
Aforesaid application, it is the upstream that the CDS sequence of rice transcription factor Os01g64730 gene is suppressed to motif EAR by Gateway system constructing to transcription factor, rice transformation (as rice varieties ' kitaake '), thus the heading stage of render transgenic paddy rice in advance, shorten breeding time.Wherein, the CDS sequence of rice transcription factor Os01g64730 gene is as shown in Seq ID No.3, and transcription factor suppresses the nucleotide sequence of motif EAR as shown in Seq ID No.4.
The expression vector that carries the gene of encoding said fusion protein can be by using the conventional biotechnological means such as Ti-plasmids, plant viral vector, directly delivered DNA, microinjection, electroporation to import (Weissbach in vegetable cell; 1998; Method for Plant Molecular Biology VIII; Academy Press; New York, pp.411-463; Geiserson and Corey, 1998, Plant Molecular Biology, 2
ndedition).
The present invention utilizes first transcription factor to suppress motif EAR and rice transcription factor Os01g64730 gene fusion and builds and obtain composing type transcription factor, and is transformed into farm crop, in paddy rice, thereby the heading stage of render transgenic paddy rice in advance, shortening breeding time.For the molecule mechanism of illustrating in detail Rice Heading regulation and control, there is important theory value, and can pass through transgenosis means, shorten growth period duration of rice, improve the adaptive faculty of paddy rice to planted in different ecological areas, therefore significant equally aborning.
Brief description of the drawings
Fig. 1 is cEAR-bar-asRED carrier collection of illustrative plates in the embodiment of the present invention 1.
Fig. 2 is ubi:Os01g64730-EAR 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, real-time fluorescence quantitative PCR detects transgenic positive rice strain; Wherein, WT is wild-type paddy rice ' kitaake ', and E17-1, E17-2 and E17-3 are respectively 3 strains of Os01g64730-EAR transgenic paddy rice.
Fig. 4 is the phenotype analytical result of Os01g64730-EAR transgenic paddy rice strain in the embodiment of the present invention 4; Wherein, WT is wild-type paddy rice ' kitaake ', and E17-1, E17-2 and E17-3 are respectively 3 strains of Os01g64730-EAR transgenic paddy rice.
Fig. 5 is Os01g64730-EAR transgenic paddy rice strain heading number of days in the embodiment of the present invention 4; Wherein, WT is wild-type paddy rice ' kitaake ', and E17-1, E17-2 and E17-3 are respectively 3 strains of Os01g64730-EAR transgenic paddy rice.
Embodiment
Following examples are used for illustrating 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, finds rice transcription factor Os01g64730 gene, according to its sequences Design pcr amplification primer:
Os01g64730-F：5′-CAAAAAAGCAGGCTTCATGATGGCGTCGAGGGTG-3′
Os01g64730-R:5′-CAAGAAAGCTGGGTCCCACTCCATCGAGTTTGTTCTTC-3′
Taking rice varieties ' Japan is fine ' blade as material, TRIzol method is extracted total RNA, reverse transcription obtains cDNA, reactions steps is as follows: (1) on ice to not containing adding successively following material in the PCR reaction tubes of nuclease: (0.1ng-5 μ is g) for total RNA 6 μ l, Oligo (dT) 18 primer 1 μ l, containing the ddH of nuclease
2o 5 μ l, are placed in 65 DEG C of reaction 5min of PCR instrument; And then add following material (2): 5 × reaction buffer, 4 μ l, RNase inhibitor 1 μ l, 10mM dNTP 2 μ l, M-M μ lV ThermoScript II 1 μ l, mixes gently, 45 DEG C of reaction 60min in PCR instrument; (3) in PCR instrument, 70 DEG C of 5min, termination reaction.Taking total cDNA as template, carry out pcr amplification, amplification system is: reaction buffer 25 μ l, dNTP 4 μ l, ddH
2o 17.5 μ l, Taq archaeal dna polymerase 0.5 μ l, response procedures is 98 DEG C of 10s of warm start, 57 DEG C of 5s, 72 DEG C of 1min, after 30 circulations, 72 DEG C are extended 10min, and last 25 DEG C of reactions finish.
The CDS sequence that obtains 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.According to the requirement of Gateway clone technology, in this process, comprise two-wheeled PCR, the primer of first round PCR adopts the primer that adds part adaptor attB joint, and the PCR product of the first round for the second template of taking turns, and primer adopts complete adaptor attB primer.PCR product cloning, to pDONR cloning vector, is obtained and the identical sequence of goal gene through order-checking qualification.React gene constructed Os01g64730 to plant expression vector cEAR-bar-asRED(Fig. 1 by LR) upper, obtain carrier ubi:Os01g64730-EAR(Fig. 2, carrier complete sequence is as shown in Seq ID No.5).
Wherein, the building process of plant expression vector cEAR-bar-asRED is: the sequence comprising taking border, binary expression vector pCAMBIA1300 left and right is as frame sequence, pass through vitro recombination, ubi promoter-Gateway-EAR is expressed to unit, 35S promoter-asRED expression unit and 35S promoter-bar expression unit and construct and obtain with it, the complete sequence of carrier cEAR-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, selects the full bright and clean seed without bacterial plaque and after sterilization, is inoculated into and on inducing culture, carries out inducing culture.Selection outward appearance is good, the Rice Callus that growing ability is good is acceptor material, adopt agrobacterium-mediated transformation that ubi:Os01g64730-EAR is proceeded in Rice Callus, transform with the AAM conversion fluid that contains 100 Syringylethanones of μ M and the Agrobacterium of OD=0.7, the callus that conversion fluid was soaked is placed in and on common substratum, carries out common cultivation, 25 DEG C dark cultivates 3d and is placed on and in screening culture medium, cultivates about 30d, every 10d subculture 1 time.Then the kanamycin-resistant callus tissue sifting out is transferred to and on division culture medium, broken up about 20d, every 10d subculture 1 time.The kanamycin-resistant callus tissue that differentiates green seedling is transferred on root media and taken root, hardening after about 7d grows flourishing root system, and calculate the conversion transgenic seedling number that obtains.After hardening 7d, be transferred to grown in field.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, 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/L2,4D+0.6g/L acid hydrolyzed casein+2.878g/L proline(Pro)+0.5g/L glutamine+30g/L sucrose, with water preparation, tune 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/L2, and 4D+0.5g/L glutamine+0.6g/L acid hydrolyzed casein+10g/L glucose+30g/L sucrose, with water preparation, adds plant gel 4g/L after adjusting pH to 5.2.After sterilizing, 50 DEG C of left and right add AS(Syringylethanone) 100～200 μ g/mL.
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/L2,4D+0.6g/L acid hydrolyzed casein+2.878g/L proline(Pro)+0.5g/L glutamine+30g/L sucrose, with water preparation, tune adds plant gel 4g/L behind pH to 5.8～5.9.After sterilizing, add 35mg/L Totomycin (purchased from Shanghai Niu Jin Bioisystech Co., Ltd) or 5mg/L Bialaphos(purchased from Beijing Baeyer enlightening biotech company).
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 water preparation, tune adds plant gel 4g/L behind pH to 5.8～5.9.
Embodiment 3 real-time fluorescence quantitative PCRs detect the expression of Os01g64730-EAR in transgenic paddy rice
For detect Os01g64730-EAR T2 in transgenic paddy rice cross expression, utilize real-time fluorescence quantitative PCR to identify on transcriptional level it, through PCR detection by quantitative, result shows that the expression amount of transfer-gen plant Os01g64730 is apparently higher than wild-type (Fig. 3).
Concrete realtime fluorescent quantitative PCR experiment flow process is as follows:
Getting appropriate transgenic line and wild-type rice leaf, is cDNA according to method extraction RNA reverse transcription in embodiment 1.Carry out PCR reaction as template, response procedures is: 95 DEG C of 30s of denaturation; 95 DEG C of sex change 5s, 20s, 40 circulations are extended in 60 DEG C of annealing.The standard program of melt curve analysis: 95 DEG C, 15 seconds; 60 DEG C, 1 minute; 95 DEG C, 15 seconds; 0.5 DEG C/circulation.According to formula: relative expression quantity (RQ)=2
-△ △ CTmethod calculate relative expression quantity.Reaction system is: SYBR Premix Ex Taq (2 ×) 10.0 μ l, PCR forward primer (10 μ M) 0.4 μ l, PCR reverse primer (10 μ M), 0.4 μ l, ROX reference dyestuff (ROX Reference Dye, 50 ×) 0.4 μ l, cDNA template 1.0 μ l, ddH
2o(sterile purified water) 7.8 μ l, cumulative volume is 20.0 μ l.
Real-time fluorescence quantitative PCR the primer sequence is: bzip17-QF:5 '-GGAGGAGAACGCCAAGATGT-3 '; Bzip17-QR:5 '-GATCTCGTGCTGACGTTTTCC-3 '.
Embodiment 4 transgenic paddy rice phenotype analyticals
Compared with wild-type paddy rice ' kitaake ', obviously shift to an earlier date the heading stage of Os01g64730-EAR transgenic paddy rice strain (E17-1, E17-2 and E17-3), though wild-type is not eared, transgenic line completes heading (Fig. 4 and Fig. 5) substantially.
The embodiment more than providing is the upstream that the CDS sequence of rice transcription factor Os01g64730 gene is suppressed to motif EAR by Gateway system constructing to transcription factor, rice transformation kind ' kitaake ', thus the heading stage of render transgenic paddy rice in advance, shorten breeding time.Similarly, the CDS sequence of rice transcription factor Os01g64730 gene is suppressed to the downstream of motif EAR to transcription factor by Gateway system constructing, rice transformation kind, also can reach the heading stage of transgenic paddy rice in advance, the effect that shortens breeding time.
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 without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Claims (5)
1. a fusion rotein, is characterized in that, this fusion rotein is Os01g64730-Linker-EAR;
Wherein, the sequence of Linker is GGGGG; EAR is that a section from plant transcription factor has the albumen motif of transcribing inhibit feature, and its aminoacid sequence is as shown in Seq ID No.2; Os01g64730 is rice transcription factor Os01g64730, and its aminoacid sequence is as shown in Seq ID No.1.
2. the gene of fusion rotein described in coding claim 1.
3. contain the carrier of gene described in claim 2.
4. contain the engineering bacteria of gene described in claim 2.
Gene claimed in claim 2 at Rice Heading in advance, shorten the application in breeding time.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102775499A (en) * | 2012-08-01 | 2012-11-14 | 中国农业科学院作物科学研究所 | Application of rice transcription factor Os01g64730 gene |
| CN102775498A (en) * | 2012-08-01 | 2012-11-14 | 中国农业科学院作物科学研究所 | Application of rice transcription factor Os05g41070 genes |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102775499A (en) * | 2012-08-01 | 2012-11-14 | 中国农业科学院作物科学研究所 | Application of rice transcription factor Os01g64730 gene |
| CN102775498A (en) * | 2012-08-01 | 2012-11-14 | 中国农业科学院作物科学研究所 | Application of rice transcription factor Os05g41070 genes |
Non-Patent Citations (2)
| Title |
|---|
| Dominant repression of target genes by chimeric repressors that include the EAR motif, a repression domain, in Arabdopsis;Keiichiro Hiratsu et al.,;《The Plant Journal》;20030630;第34卷(第5期);733-739 * |
| Keiichiro Hiratsu et al.,.Dominant repression of target genes by chimeric repressors that include the EAR motif, a repression domain, in Arabdopsis.《The Plant Journal》.2003,第34卷(第5期), * |
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