CN102775498A - Application of rice transcription factor Os05g41070 genes - Google Patents
Application of rice transcription factor Os05g41070 genes Download PDFInfo
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Abstract
The invention relates to an application of rice transcription factor Os05g41070 genes. The application utilizes transcription factor activation sequence motif VP64 (namely four transcription factors activation sequence motif VP16) to be fused with the rice transcription factor Os05g41070 genes to build and obtain constitutive transcription factors, and the constitutive transcription factors are converted into crops such as rice to put off the heading stage of transgenosis rice and prolong the growth period. The application has an important theoretical value on clarifying molecule mechanisms of heading regulation of the rice in detail, therefore, the growth period of the rice through a transgenosis method can be prolonged, adaptation of the rice on different ecoregions can be improved, and accordingly, an important significance on production practice can be provided.
Description
Technical field
The invention belongs to field of genetic engineering, specifically, relate to the application of rice transcription factor Os05g41070 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 extensive studies.GI-CO-FT is bloom conservative approach (Yano etc., 2000 of control paddy rice; Kojima etc., 2002; Hayama etc., 2002).Hd3a is the homologous gene of FT in the paddy rice, and it receives 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 receives the regulation and control of OsGI, and this effect is positioned at the upper reaches (Kim etc., 2007) of Ehd1.On the contrary, under the long day condition, Hd1 suppresses the expression of Hd3a and postpones paddy rice bloom (Hayama etc., 2003).Current research shows that Hd3a and 14-3-3 albumen change in the nucleus after forming complex body, combines with transcription factor FD1 to form tripolymer FAC (florigen activation complex), induces transcribing of OsMADS15, thereby causes paddy rice to bloom.
VP16 finds in the animal virus gene, has been widely applied in the plant at present, 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 domain motif, it will strengthen the function of transcription factor, and obvious phenotypes changes thereby in transfer-gen plant, occur more.
Summary of the invention
The purpose of this invention is to provide the application of rice transcription factor Os05g41070 gene.
In order to realize the object of the invention, the present invention at first provides a kind of fusion rotein, and this fusion rotein is Os05g41070-Linker-(VP16)
nOr (VP16)
n-Linker-Os05g41070; Wherein, n is>=1 integer; Linker is by 1 ~ 20 flexible amino acid be in series (for example, GGGGG, GPPPG, or Gatway carrier recombination site amino acid sequence coded DPAFLYKVVPR); VP16 is the VP16 albumen from hsv (Herpes simplex virus); Os05g41070 is rice transcription factor Os05g41070, its aminoacid sequence shown in SEQ ID No.1, or this sequence through replacement, lack or add one or several amino acids formed aminoacid sequence with same function.
Preferably, n is 4 in the aforementioned fusion rotein.Wherein, (VP16)
4Be VP64, merge the enhanser of forming by 4 VP16 domain motifs, its aminoacid sequence shown in SEQID No.2, or this sequence through replacement, lack or add one or several amino acids formed aminoacid sequence with same function.
More preferably, aforementioned fusion rotein is Os05g41070-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, said stringent condition is 65 ℃, hybridization 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.Said carrier is any carrier that can guide foreign gene in the host, to express.Preferably, said carrier be plant binary expression vector (for example, pCAMBIA1301).When encoding said fusion protein of the present invention gene constructed is in plant expression vector, can before its transcription initiation Nucleotide, add any strong promoter (for example, corn strong promoter Ubiquitin) or inducible promoter.In addition, when encoding said fusion protein of the present invention gene constructed is in plant expression vector, 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 gene of encoding said fusion protein postponing the paddy rice heading, prolonging the application in breeding time.
The present invention further provides rice transcription factor Os05g41070 the application of gene; It is that the CDS sequence of rice transcription factor Os05g41070 gene (complete translation area) is building up to the upper reaches that 4 transcription factors activate motif VP16; Transform plant, screening and final acquisition transgenic plant.
Aforesaid application; It is the upper reaches of the CDS sequence of rice transcription factor Os05g41070 gene being activated motif VP16 through Gateway system constructing to 4 transcription factor; Rice transformation (for example; Rice varieties ' kitaake '), thus postpone transgenic paddy rice heading stage, prolong breeding time.Wherein, the CDS sequence of rice transcription factor Os05g41070 gene is shown in SEQ IDNo.3, and the nucleotide sequence of 4 transcription factor activation motif VP16 is shown in SEQ ID No.4.
The expression carrier that carries encoding said fusion protein can import (Weissbach in the vegetable cell through using conventional biotechnological meanss such as Ti-plasmids, plant viral vector, directly DNA conversion, microinjection, electroporation; 1998; Method for Plant Molecular Biology VIII; Academy Press, New York, 411-463 page or leaf; Geiserson and Corey, 1998, Plant Molecular Biology, 2
NdEdition).
The present invention utilizes transcription factor to activate motif VP64 (promptly 4 transcription factors activate motif VP16) first and obtains the composing type transcription factor with rice transcription factor Os05g41070 gene fusion structure; And be transformed into farm crop; In paddy rice, thus postpone transgenic paddy rice heading stage, prolong breeding time.For the molecule mechanism of sets forth in detail paddy rice heading regulation and control, have important theory and be worth, and can pass through the transgenic means, prolong growth period duration of rice, improve the adaptive faculty of paddy rice to planted in different ecological areas, therefore significant equally aborning.
Description of drawings
Fig. 1 is a cVP64-bar-asRED carrier collection of illustrative plates in the embodiment of the invention 1.
Fig. 2 is a ubi:Os05g41070-VP64 carrier collection of illustrative plates in the embodiment of the invention 1.
Fig. 3 is the result that Western Blot detects transgenic positive rice strain in the embodiment of the invention 3; Wherein, WT is wild-type paddy rice ' kitaake ', and bzip74-VP64 is an Os05g41070-VP64 transgenic paddy rice strain system.
Fig. 4 is the phenotype analytical result of Os05g41070-VP64 transgenic paddy rice strain system in the embodiment of the invention 4; Wherein, WT is wild-type paddy rice ' kitaake ', and bzip74-VP64 is an Os05g41070-VP64 transgenic paddy rice strain system.
Embodiment
Following examples are used to explain the present invention, but are not used for limiting scope of the present invention.If do not specialize the conventional means that used technique means is well known to those skilled in the art among the embodiment, the raw materials used commercial goods that is.
The acquisition of embodiment 1 rice transcription factor Os05g41070 gene and the structure of plant expression vector
Rice transcription factor Os05g41070 gene is found in login http://rice.plantbiology.msu.edu/analyses_search_locus.shtml website, according to its sequences Design pcr amplification primer:
Os05g41070-F：5'-CAAAAAAGCAGGCTTCATGATTCAGGCAATGGCTTCGCA-3'
Os05g41070-R:5'-CAAGAAAGCTGGGTCGAAGGCGGCCGAGCTTGTTCGCCT-3'
With paddy rice ' Japan is fine ' blade is material; The TRIzol method is extracted total RNA; Reverse transcription gets cDNA; Reactions step is following: (1) adds following material successively in the PCR reaction tubes that does not contain nucleicacidase on ice: total RNA 6 μ l (0.1ng-5 μ g), Oligo (dT) 18 primers 1 μ l does not contain the ddH of nucleicacidase
2O 5 μ l place 65 ℃ of reactions of PCR appearance 5min; (2) and then add following material: 5 * reaction buffer, 4 μ l, RNase suppressor factor 1 μ l, 10mM dNTP2 μ l, M-M μ lV ThermoScript II 1 μ l, mixing gently, 45 ℃ of reaction 60min in the PCR appearance; (3) in the PCR appearance, 70 ℃ of 5min, termination reaction.With total cDNA is template, carries out pcr amplification, and amplification system is: reaction buffer 25 μ l, dNTP 4 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 Os05g41070 gene, its nucleotide sequence is 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 first round PCR is with the primer that adds part adaptor attB joint, and second template of taking turns is with the PCR product of the first round, and the complete adaptor attB primer of primer employing.The PCR product cloning to connecting on the pDONR cloning vector, is identified through order-checking to obtain and the identical sequence of goal gene.Through the LR reaction that Os05g41070 is gene constructed to plant expression vector cVP64-bar-asRED (Fig. 1), obtain carrier ubi:Os05g41070-VP64 (Fig. 2, the carrier complete sequence is shown in SEQ ID No.6).
Wherein, The building process of plant expression vector cVP64-bar-asRED is: the sequence that comprises with border, the binary expression vector pCAMBIA1300 left and right sides is a frame sequence; Pass through vitro recombination; Ubi promoter-Gateway-VP64 is expressed unit, 35S promoter-asRED express unit and 35S promoter-bar and express the unit and construct with it and obtain, the complete sequence of carrier cVP64-bar-asRED is shown in SEQ ID No.5.
The acquisition of embodiment 2 transgenic rice plants
Water intaking rice ' kitaake ' mature seed, manual work or mechanical dejacketing, the seed of selecting full bright and clean no bacterial plaque is inoculated into after sterilization and carries out inducing culture on the inducing culture.The selection outward appearance is good; The rice callus that growing ability is good is organized as acceptor material; Adopt agrobacterium-mediated transformation that ubi:Os05g41070-VP64 is changed in the rice callus tissue, Syringylethanone and the O.D. value that use contains 100 μ M is that the AAM conversion fluid of 0.7 Agrobacterium transforms, and the callus that conversion fluid was soaked places and carries out common cultivation on the common substratum; 25 ℃ of dark 3d of cultivation are placed on and cultivate about 30d on the screening culture medium, and every 10d subculture once.Then the kanamycin-resistant callus tissue that sifts out is transferred to the about 20d of differentiation on the division culture medium, every 10d subculture once.The kanamycin-resistant callus tissue that differentiates green seedling transferred on the root media take root, treat to refine seedling after about 7d grows flourishing root system, and calculate and transform the transgenic seedling number that obtains.Be transferred to grown in field behind the refining seedling 7d.Screen transgenic paddy rice with Basta, grow and begin to spray Basta behind 4 spires (1:1000 v:v), at a distance from spray in 1 day 1 time, sprays 3 times altogether.Obtain transgenic paddy rice 20 strains altogether.
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 Stimulina+30g/L sucrose with the water preparation, transfers pH to 5.8 ~ 5.9 backs to add plant gel 4g/L.
Altogether culture medium prescription is: and N6 is a large amount of+and B5 trace+NB is organic+molysite+2.5mg/L2,4D+0.5g/L Stimulina+0.6g/L acid hydrolyzed casein+10g/L glucose+30g/L sucrose with the water preparation, transfers pH to 5.2 back to add plant gel 4g/L.After the sterilization, add AS (Syringylethanone) 100 ~ 200 μ g/mL about 50 ℃.
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/L2; 4D+0.6g/L acid hydrolyzed casein+2.878g/L proline(Pro)+0.5g/L Stimulina+30g/L sucrose; With the water preparation, transfer pH to 5.8 ~ 5.9 backs to add plant gel 4g/L.The sterilization back adds 35mg/L Totomycin (available from Shanghai Niu Jin Bioisystech Co., Ltd) or 5mg/L Bialaphos (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, transfer pH to 5.8 ~ 5.9 backs to add plant gel 4g/L.
Embodiment 3Western Blot detects the expression of fusion rotein Os05g41070-VP64 in the transgenic paddy rice
For detect the Os05g41070-VP64 gene at T2 for the expression of crossing in the transgenic paddy rice; Utilize VP64 antibody that it is identified on protein level; 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 following:
An amount of sample is put into the freezing back of liquid nitrogen grind into powder, add an amount of sample-loading buffer mixing, centrifugal 10 minutes of 12000rpm; Get 5 μ l supernatant application of samples, after 30 minutes, 120V electrophoresis 60-90 minute is when the bottom of tetrabromophenol sulfonphthalein arrival gel can stop electrophoresis with 90V voltage SDS-PAGE electrophoresis; Adopt half-dried transfer method to change film behind the electrophoresis, and film is dyeed, observe and change the film effect with the ponceau staining fluid; After changeing film and finishing, film is put into the PBST solution sealing room temperature sealing that contains 5% skim-milk spent the night in 60 minutes or 4 ℃; Room temperature next anti-(VP64 antibody) was hatched 1 hour or 4 ℃ of incubated overnight, then with PBST washing 3 times, each 5 minutes; Two anti-under the room temperature (the goat-anti rabbit, available from Abmart, article No. M21002S) hatched 1 hour, then with PBST washing 3 times, each 5 minutes; On film, add substrate, make public.
Wherein, VP64 antibody is how anti-as the rabbit source that specific antigen prepares according to the synthetic polypeptide of the aminoacid sequence of VP64 by Ai Bimate biological medicine (Shanghai) Co., Ltd..
Embodiment 4 transgenic paddy rice phenotype analyticals
' kitaake ' compares with the wild-type paddy rice, and the strain of Os05g41070-VP64 transgenic paddy rice is obviously to postpone the heading stage of bzip74-VP64, 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 is the upper reaches of the CDS sequence of rice transcription factor Os05g41070 gene being activated motif VP16 through Gateway system constructing to 4 transcription factor; Rice transformation kind ' kitaake ', thus postpone transgenic paddy rice heading stage, prolong breeding time.Likewise; The CDS sequence of rice transcription factor Os05g41070 gene is activated the downstream of motif VP16 to >=1 transcription factor through the Gateway system constructing; Or activate the upper reaches of motif VP16 through 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 that prolongs breeding time.
Though, the present invention has been done detailed description in the preceding text with general explanation and specific embodiments, on basis of the present invention, can to some modifications of do or improvement, this will be apparent to those skilled in the art.Therefore, these modifications or the improvement on the basis of not departing from spirit of the present invention, made all belong to the scope that requirement of the present invention is protected.
Claims (10)
1. a fusion rotein is characterized in that, this fusion rotein is Os05g41070-Linker-(VP16)
nOr (VP16)
n-Linker-Os05g41070;
Wherein, n is >=1 integer; Linker is in series by 1 ~ 20 flexible amino acid; VP16 is the VP16 albumen from hsv (Herpes simplex virus); Os05g41070 is rice transcription factor Os05g41070.
2. fusion rotein according to claim 1 is characterized in that, n is 4.
3. fusion rotein according to claim 2 is characterized in that, this fusion rotein is Os05g41070-Linker-(VP16)
4
4. the gene of each said fusion rotein of coding claim 1-3.
5. the carrier that contains the said gene of claim 4.
6. the transgenic cell line that contains the said gene of claim 4.
7. the engineering bacteria that contains the said gene of claim 4.
8. the described gene of claim 4 is being postponed the paddy rice heading, is being prolonged the application in breeding time.
9. the application of rice transcription factor Os05g41070 gene is characterized in that, it is the upper reaches of CDS sequence construct to 4 transcription factor of rice transcription factor Os05g41070 gene being activated motif VP16, transforms plant, and screening also finally obtains transgenic plant.
10. application according to claim 9; It is characterized in that; It is that the CDS sequence of rice transcription factor Os05g41070 gene is activated the upper reaches of motif VP16 through Gateway system constructing to 4 transcription factor, rice transformation, thereby postpone transgenic paddy rice heading stage, prolong breeding time.
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| CN103255148A (en) * | 2013-04-28 | 2013-08-21 | 中国农业科学院作物科学研究所 | Application of paddy rice transcription factor Os01g18440 gene |
| CN103254314A (en) * | 2013-04-28 | 2013-08-21 | 中国农业科学院作物科学研究所 | Application of paddy rice transcription factor Os05g41070 gene |
| CN103266114A (en) * | 2013-05-10 | 2013-08-28 | 中国农业科学院作物科学研究所 | Application of rice transcription factor Os01g23760 gene |
| CN103265634A (en) * | 2013-04-28 | 2013-08-28 | 中国农业科学院作物科学研究所 | Application of rice transcription factor Os01g64730 gene |
| CN103408670A (en) * | 2013-08-23 | 2013-11-27 | 中国农业科学院作物科学研究所 | Application of rice transcription factor Os05g09480 gene |
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| CN103255148A (en) * | 2013-04-28 | 2013-08-21 | 中国农业科学院作物科学研究所 | Application of paddy rice transcription factor Os01g18440 gene |
| CN103254314A (en) * | 2013-04-28 | 2013-08-21 | 中国农业科学院作物科学研究所 | Application of paddy rice transcription factor Os05g41070 gene |
| CN103265634A (en) * | 2013-04-28 | 2013-08-28 | 中国农业科学院作物科学研究所 | Application of rice transcription factor Os01g64730 gene |
| CN103255148B (en) * | 2013-04-28 | 2014-08-13 | 中国农业科学院作物科学研究所 | Application of paddy rice transcription factor Os01g18440 gene |
| 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 |
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| CN103408670A (en) * | 2013-08-23 | 2013-11-27 | 中国农业科学院作物科学研究所 | Application of rice transcription factor Os05g09480 gene |
| CN103408670B (en) * | 2013-08-23 | 2015-02-11 | 中国农业科学院作物科学研究所 | Application of rice transcription factor Os05g09480 gene |
| CN111206038A (en) * | 2020-03-16 | 2020-05-29 | 南京林业大学 | Bambusa multiplex transcription factor BmbZIP62 gene and application thereof |
| CN111206038B (en) * | 2020-03-16 | 2021-12-07 | 南京林业大学 | Bambusa multiplex transcription factor BmbZIP62 gene and application thereof |
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