CN102786599B - Application of rice transcription factor Os05g39950 gene - Google Patents
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Abstract
The invention relates to application of rice transcription factor Os05g39950 gene. According to the application, a transcription factor activation sequence motif VP64 (four transcription factor activation sequence motifs VP16) and the rice transcription factor Os05g39950 gene are fused and built to obtain a forming transcription factor, and the forming transcription factor is converted into crops such as rice, so the rice grain property is improved, for example, the rice grain is obviously shortened, widened and thickened, and the grain weight is reduced. The important theoretical value is realized on the detail clarification of seed development mechanism regulation and control, and the rice grain form can be improved through a transgenosis measure, so the important significance is also realized in the production practice.
Description
Technical field
The invention belongs to field of genetic engineering, specifically, relate to the application of rice transcription factor Os05g39950 gene.
Background technology
Paddy rice (Oryza sativa L.) is one of most important food crop of depending on for existence of the mankind, also be the pattern species of an important functional gene research, relative genetics and molecular biology research receive much attention always, wherein, the regulation and control of transcriptional level are the important way of gene expression regulation.In vegitabilia, the plant that can form seed accounts for the more than 2/3rds of plant total, and as important organ of multiplication, seed is simultaneously also for people provide food source, and paddy rice is exactly important representative wherein, and seed source is in the ovule of after fertilization.From molecular biological angle, the Development and germination of seed be one orderly, optionally genetic expression process.And transcription factor has played critical effect in the accuracy controlling of gene betwixt.In model plant Arabidopis thaliana, a lot of transcription factors have all affected seed and allelotaxis; some members of YABBY class guard (Golz and Hudson leaf, floral organ and ovule from the function aspect the decision of axial cell characteristic; 1998); the dormancy of the gene of bZIP class, Dof gene and seed and sprouting relevant (Jakoby etc., 2002; Gualberti etc., 2002), TTG2 (Garcia etc., 2005), haiku Mutants (Garcia etc., 2003) .BIGPETALp, (Szecsi etc., 2006), BIG BROTHER(Disch etc., 2006), DA1(Yunhai Li etc., 2008), DAGG3(Yunhai Li etc., 2012).Recently in paddy rice, also in succession report the research report that seed is relevant with allelotaxis, (Lin etc., 1995; Huang etc., 1997; Redona and Mackill, 1998; Kubo etc., 2001; Xing etc., 2001; Thomson etc., 2003; Yan etc., 2003; Li etc., 2004; Rabiei etc., 2004; Tian etc., 2005; Yamagishi etc., 2002; Xu etc., 2002; Wan etc., 2006,2008; Xie etc., 2006; Yoon etc., 2006; Tan etc., 2000; Lei etc., 2008; Bai etc., 2010; Lin and Wu; 2003), plant seed and allelotaxis's regulation and control are emerging research fields, at present to the research of its regulatory mechanism and be familiar with lessly, therefore to the research of transcription factor, for further understanding the molecule mechanism of plant seed and allelotaxis's regulation and control, new approaches are provided in theory; In practice, also will provide fundamental basis for high-yield breeding of crops.
VP16 finds in animal virus gene, has now been 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 in transfer-gen plant, occurs that more obvious phenotype changes.
First OVATE albumen be in the news in tomato, and in the gene of this albumen of encoding, the point mutation meeting of certain specific site causes its afunction, and tomato shape becomes pyriform from circle, also can affect the g and D of blade and flower in the time of this gene of overexpression.By inference, itself does not control the shape difference of fruit OVATE albumen, and it may be that a plant growth suppresses to regulate albumen (Liu etc., 2002).Genomics research shows, in paddy rice and arabidopsis gene group, also there are (Ku etc. in this albumen, 2000,2001), this gene of overexpression in Arabidopis thaliana, demonstrate growing of hypoevolutism, blade ,Hua He angle fruit and be suppressed, petal sepal presents oval and silicle isophenous changes (Hackbusch etc., 2005).In recent years, in relevant Arabidopis thaliana OVATE protein family, the research of gene is in the news in succession, AtOFP1 is that a transcription factor suppresses son, regulate and control a kind of Plant hormones regulators,gibberellins synthase gene expression that cell extends of controlling, its expression excessively can reduce cell length, thereby the phenotype that its hypocotyl, cotyledon, blade, floral organ, angle is really showed all shorten changes, AtOFP2 also has similar phenotype (wang etc., 2007) to AtOFP7 simultaneously.Yet but have no the relevant report of OVATE albumen regulatory mechanism in paddy rice.
Summary of the invention
The object of this invention is to provide the application of rice transcription factor Os05g39950 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 (VP16)
n-Linker-Os05g39950; Wherein, the integer that n is>=1; Linker for example, by 1 ~ 20 flexible amino acid be in series (, GGGGG, GPPPG, or the aminoacid sequence DPAFLYKVVPR of Gatway carrier recombination site coding); VP16 is the VP16 albumen from hsv (Herpes simplex virus); Os05g39950 is rice transcription factor Os05g39950, 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.
The present invention also provides a kind of primer for amplifying rice transcription factor Os05g39950 gene, and it comprises forward primer F:5'-CAAAAAAGCAGGCTTCATGTTGTCCAGCGAACCAGG-3' and reverse primer R:5'-CAAGAAAGCTGGGTCTGGCATGACGCCACAGG-3'.Os05g39950 is that reported first is separated and obtain the OVATE protein family gene of Function Identification from paddy rice.
Preferably, in aforementioned fusion rotein, n is 4, and aforementioned fusion rotein is (VP16)
4-Linker-Os05g39950.Wherein, (VP16)
4be VP64, by 4 VP16 functional domain motifs, merge the enhanser forming, 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.
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 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.The carrier that described carrier is expressed in host for any bootable foreign gene.Preferably, described carrier be 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 application of the gene that the present invention also provides encoding said fusion protein in improvement rice grain proterties (as changed rice grain grain type, increase grain heavy).
The present invention further provides the application of rice transcription factor Os05g39950 gene, it is that the CDS sequence of rice transcription factor Os05g39950 gene (complete translation district) is building up to the downstream that 4 transcription factors activate motif VP16, conversion of plant, screening final acquisition transgenic plant.
Aforesaid application, it is the CDS sequence of rice transcription factor Os05g39950 gene to be activated to the downstream of motif VP16 by Gateway system constructing to 4 transcription factor, rice transformation (for example, rice varieties ' kitaake '), thus the proterties of improvement transgenic paddy rice seed (as changed rice grain grain type, increase grain heavy).Wherein, the CDS sequence of rice transcription factor Os05g39950 gene is as shown in SEQ ID No.3, and the nucleotide sequence of 4 transcription factor activation motif VP16 is as shown in SEQ ID No.4.
The expression vector that carries the gene of encoding said fusion protein can be by being used 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, 411-463 page; Geiserson and Corey, 1998, Plant Molecular Biology, 2
ndedition).
The present invention utilizes transcription factor to activate i.e. 4 the transcription factors activation motif VP16 of motif VP64(first) build and obtain composing type transcription factor with rice transcription factor Os05g39950 gene fusion, and be transformed into farm crop, in paddy rice, thereby improvement rice grain proterties, as Grain Length in Rice shortens, and width, thickness and grain heavily decline.For illustrating in detail regulation and control seed development mechanism, there is important theory value, and can pass through transgenosis means, the grain type of improvement paddy rice, therefore also significant in production practice.
Accompanying drawing explanation
Fig. 1 is nVP64-bar-asRED carrier collection of illustrative plates in the embodiment of the present invention 1.
Fig. 2 is ubi:VP64-Os05g39950 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, PCR detects transgenic positive rice strain; Wherein, WT is wild-type paddy rice ' kitaake ', and UBI:Os05g39950 is VP64-Os05g39950 transgenic paddy rice strain.
Fig. 4 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 Os05g39950-OX-1 and Os05g39950-OX-2 are VP64-Os05g39950 transgenic paddy rice strain.
Fig. 5 is VP64-Os05g39950 transgenic paddy rice seed grain type analysis result in the embodiment of the present invention 4; Wherein, WT is wild-type paddy rice ' kitaake ', and UBI:Os05g39950 is VP64-Os05g39950 transgenic paddy rice strain.
Fig. 6 is the species test analysis of statistical data result of VP64-Os05g39950 transgenic paddy rice grain characters in the embodiment of the present invention 4.
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 Os05g39950 gene and the structure of plant expression vector
Login http://rice.plantbiology.msu.edu/analyses_search_locus.shtml website, finds rice transcription factor Os05g39950 gene, according to its sequences Design pcr amplification primer:
Os05g39950-F：5'-CAAAAAAGCAGGCTTCATGTTGTCCAGCGAACCAGG-3'
Os05g39950-R：5'-CAAGAAAGCTGGGTCTGGCATGACGCCACAGG-3'
Paddy rice ' Japan the is fine ' blade of take is 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: total RNA 6 μ l (0.1ng-5 μ g), Oligo (dT) 18 primer 1 μ l, containing the ddH of nuclease
2o 5 μ l, are placed in 65 ℃ of reaction 5min of PCR instrument; And then add following material (2): 5 * reaction buffer, 4 μ l, RNase inhibitor 1 μ l, 10mM dNTP2 μ l, M-M μ lV ThermoScript II 1 μ l, mixes gently, 45 ℃ of reaction 60min in PCR instrument; (3) in PCR instrument, 70 ℃ of 5min, termination reaction.Take total cDNA as template, carry out pcr amplification, 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, template 1 μ l, each 0.5 μ l of up/down trip primer.Response procedures is 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.
The CDS sequence that obtains rice transcription factor Os05g39950 gene, its nucleotide sequence is as shown in SEQ ID No.3.According to PrimeSTAR polymeric enzymatic amplification system and response procedures, carry out PCR.According to the requirement of Gateway clone technology, in this process, comprise two-wheeled PCR, the primer that adds part adaptor attB joint for the primer of first round PCR, 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 connecting on pDONR cloning vector, is identified and obtained and the identical sequence of goal gene through order-checking.By LR, react that Os05g39950 is gene constructed to plant expression vector nVP64-bar-asRED(Fig. 1) upper, obtain carrier ubi:VP64-Os05g39950(Fig. 2, carrier complete sequence is as shown in SEQ ID No.6).
Wherein, the building process of plant expression vector nVP64-bar-asRED is: the sequence that border, binary expression vector pCAMBIA1300 left and right comprises of take is frame sequence, pass through vitro recombination, ubi promoter-VP64-Gateway 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 nVP64-bar-asRED is as shown in SEQ ID No.5.
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:Os05g39950-VP64 is proceeded in Rice Callus, the AAM conversion fluid of the Agrobacterium that is 0.7 by the Syringylethanone that contains 100 μ M and O.D. value transforms, the callus that conversion fluid was soaked is placed in and on common substratum, carries out common cultivation, 25 ℃ of dark cultivation 3d are placed in screening culture medium and cultivate about 30d, and every 10d subculture once.Then the kanamycin-resistant callus tissue sifting out is transferred on division culture medium and broken up about 20d, every 10d subculture once.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 40 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/L 2,4D+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/L 2, 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 ℃ 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/L 2,4D+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.
The evaluation of embodiment 3 Os05g39950-VP64 transgenic paddy rices
For detecting the CDS sequence of Os05g39950 gene, whether be integrated in oryza sativa genomic dna, first adopt CTAB method to extract respectively wild-type and the total DNA of transgenic paddy rice blade, design detects primer sequence, upstream primer: 5'-ATGGACGCGCTGGACGATTT-3', downstream primer: 5'-TCACTTGTCATCGTCGTCCTTGTAG-3 ', carries out PCR, and amplification system is: reaction buffer 5 μ l, dNTP 1 μ l, ddH
2o 2.2 μ l, Taq archaeal dna polymerase 0.2 μ l, each 0.3 μ l of upstream and downstream primer, DNA profiling 1 μ l. reaction conditions is 96 ℃ of 5min of warm start; 95 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 2min, totally 32 circulations; 72 ℃ are extended 10min, and last 25 ℃ of reactions finish.Identify transfer-gen plant, amplified production is carried out to agarose gel electrophoresis, electrophoresis result shows, transgenic paddy rice all amplifies object band, and in wild-type paddy rice, do not amplify object band (Fig. 3), the CDS sequence of preliminary definite Os05g39950 gene is inserted in rice genome by agriculture bacillus mediated method, and PCR product is after order-checking, and sequencing result is as shown in SEQ ID No.3.
Because over-express vector is with FLAG label, in transgenic paddy rice, will merge with target protein, therefore utilize this FLAG antibody can identify the protein expression situation of goal gene in transgenic paddy rice, through the SDS-PAGE protein electrophoresis → immune marking → Immunofluorescence Reactions, Western Blot qualification result shows that transfer-gen plant exists target protein, the band (Fig. 4) 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, with 90V voltage SDS-PAGE electrophoresis, after 30 minutes, 120V electrophoresis 60-90 minute, when the bottom of tetrabromophenol sulfonphthalein arrival gel can stop electrophoresis; After electrophoresis, adopt half-dried transfer method transferring film, and with ponceau staining fluid, film is dyeed, observe transferring film effect; After transferring film, film is put into PBST solution containing 5% skim-milk and seal room temperature sealing and spend the night for 60 minutes or 4 ℃; Under room temperature, add primary antibodie (FLAG antibody, purchased from Abmart, article No. M20008L) to hatch 1 hour or 4 ℃ of overnight incubation, then with PBST washing 3 times, each 5 minutes; Under room temperature, add two anti-(sheep anti mouse, purchased from Abmart, article No. M21001S) to hatch 1 hour, then with PBST washing 3 times, each 5 minutes; On film, add substrate, expose.
Compare with wild-type paddy rice ' kitaake ', the seed of VP64-Os05g39950 transgenic paddy rice obviously shortens, broadens, thickening, grain heavily decline (Fig. 5).According to species test data results, can obviously find out that above-mentioned 4 proterties of VP64-Os05g39950 transgenic paddy rice seed are compared with wild-type significant difference (Fig. 6).
The embodiment more than providing activates the CDS sequence of rice transcription factor Os05g39950 gene the downstream of motif VP16 by Gateway system constructing to 4 transcription factor, rice transformation kind ' kitaake ', thereby improvement rice grain proterties, as Grain Length in Rice shortens, grain heavily declines, and width, thickness increase.Similarly, the CDS sequence of rice transcription factor Os05g39950 gene is activated to the downstream of motif VP16 by Gateway system constructing to 1 ~ 3 or 4 above transcription factors, rice transformation kind, also can reach the effect of improvement rice grain proterties.
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 (8)
1. a fusion rotein, is characterized in that, this fusion rotein is VP64-Linker-Os05g39950;
Wherein, Linker is in series by 1～20 flexible amino acid; The aminoacid sequence of VP64 is as shown in SEQ ID No.2; Os05g39950 is rice transcription factor Os05g39950, and its aminoacid sequence is as shown in SEQ ID No.1.
2. the gene of fusion rotein described in the claim 1 of encoding.
3. the carrier that contains gene described in claim 2.
4. the engineering bacteria that contains gene described in claim 2.
5. the application of gene claimed in claim 2 in improvement rice grain proterties.
6. the application of rice transcription factor Os05g39950 gene in adjusting and controlling rice grain characters, the aminoacid sequence of described rice transcription factor Os05g39950 is as shown in SEQ ID No.1.
7. application according to claim 6, is characterized in that, it is the CDS sequence construct to 4 of a rice transcription factor Os05g39950 gene transcription factor to be activated to the downstream of motif VP16, conversion of plant, screening final acquisition transgenic plant.
8. application according to claim 6, it is characterized in that, it is the CDS sequence of rice transcription factor Os05g39950 gene to be activated to the downstream of motif VP16 by Gateway system constructing to 4 transcription factor, rice transformation, thereby the proterties of improvement transgenic paddy rice seed.
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| CN103243114B (en) * | 2013-03-29 | 2014-11-26 | 中国农业科学院作物科学研究所 | Fusion gene, protein expressed by same and application thereof |
| 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 |
| CN103266114B (en) * | 2013-05-10 | 2014-07-30 | 中国农业科学院作物科学研究所 | Application of rice transcription factor Os01g23760 gene |
| CN104341523B (en) * | 2013-08-02 | 2017-05-17 | 中国农业科学院作物科学研究所 | Application of oryza sativa transcription factor Os03g33090 gene CDS sequence |
| CN103421120B (en) * | 2013-08-23 | 2014-11-05 | 中国农业科学院作物科学研究所 | Application of rice transcription factor Os11g02540 genes |
| CN103820480B (en) * | 2014-03-09 | 2015-12-02 | 吉林大学 | The application of a kind of rice transcription factor in improvement rice high yield proterties |
| CN110305218A (en) * | 2018-03-22 | 2019-10-08 | 江苏师范大学 | The application of arabidopsis transcription factor at3g46080 gene |
| CN114752605B (en) * | 2022-05-27 | 2023-07-21 | 扬州大学 | Rice OsOFP22 s Gene and method for increasing grain length, thousand grain weight and improving amylose content of rice by using same |
| CN115894646B (en) * | 2022-09-02 | 2023-10-27 | 扬州大学 | OsJDG1 gene and application thereof in regulation of rice grain type and thousand grain weight |
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| CN100362104C (en) * | 2004-12-21 | 2008-01-16 | 华中农业大学 | Using gene of transcriptional factor OSNACX of paddy to increase drought resistance and salt tolerant abilities of plants |
| CN1800407A (en) * | 2005-12-23 | 2006-07-12 | 上海瑞丰农业科技公司 | Construction of chloroplast transgenic expression vector capable of regulating expression |
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