CN103408670B - Application of rice transcription factor Os05g09480 gene - Google Patents
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Abstract
The invention an application of a rice transcription factor Os05g09480 gene, wherein a constitutive transcription factor is constructed by fusing a transcription factor inhibitory motif EAR, and the rice transcription factor Os05g09480 gene and a gene coding the constitutive transcription factor is transformed into crops such as rice, so that the characters of the rice grain can be improved; for example, the length and width of the single rice grain are improved. The rice transcription factor Os05g09480 gene has important theoretical value for detailed explanation of a seed development regulation and control mechanism; besides, as the grain characters of rice also can be improved in a transgenic manner, the rice transcription factor Os05g09480 gene 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 Os05g09480 gene.
Background technology
Paddy rice (Oryza sativa L.) is one of most important Three major grain crops in China and the whole world, is the staple food of world's population over half, is also the model plant of an important functional gene research.The attention of relative genetics and molecular biology research extremely investigator always, the regulation and control of transcriptional level are the important way of gene expression regulation.The research of current increasing production of rice comparatively depends on limited Rice Germplasm Resources, and traditional cross-breeding advantage weakens gradually, and Transgenic Rice technology likely excavates the potentiality that paddy rice is increased production further.
In vegitabilia, the plant that can form seed accounts for 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 serves critical effect in the accuracy controlling of genetic expression.
Rice transcription factor Os05g09480 belongs to Aux/IAA family, and Aux/IAA gene family is one of 3 genoid families making early stage response to growth hormone induction.Growth hormone induction can make the instantaneous rising of the expression amount of Aux/IAA gene. and then the expression of other genes in growth hormone signal network is regulated and controled (just regulating and controlling/negative regulation).Aux/IAA gene family is the important regulatory factor of Auxin Signal Tranducation path.Aux/IAA albumen has 4 conserved domains. and structural domain I has Transcription inhibition function.The stability of structural domain II pair of Aux/IAA albumen works. and structural domain III and IV is responsible for the dimerization of albumen.Aux/IAA albumen and transcription factor ARF(auxin response factor) family can form heterozygosis dimer.Under growth hormone effect, Aux/IAA albumen is degraded, thus transfer the expression (Dharmosiri N, Estelle M. (2004) Auxin signaling and regulated protein degradation) of growth hormone signal pathway downstream gene.The growth hormone .Aux/IAA gene that plays an important role in the growth and development process of plant is again important controlling gene in growth hormone signal transduction path.
The growth hormone signal that gene OsIAA23 in Aux/IAA family mediates plays an important role for maintaining after the embryo of paddy rice quiescent center cell.The root tissues such as root cap, side root and a crown root grow the paddy rice semidominance mutant osiaa23 of existing defects, the feature losing tip of a root quiescent center in postembryonal development is set up, side root and crown root reduced number and dysplasia, growth retardation, fertility is poor, setting percentage reduces, and root is to gravity loss for reaction.Mutant sprouts latter 5 days shoot root point disappearances, and the cell arrangement at quiescent center place is abnormal, and the meristematic cell pattern of root is abnormal, and the square section cell fission of root is abnormal.Its defect phenotype is because the stable sudden change of the second structural domain of OsIAA23 albumen causes.OsIAA23 expresses at the tip of a root QC place of paddy rice main root, side root and crown root specifically.Consistent therewith, the growth hormone signal of DR5p::GUS mark lacks at the stagnant zone of Osiaa23 mutant.Under QHB promotor controls, the phenotypic defects of Osiaa23 transfer-gen plant energy partial simulation Osiaa23 mutant.Maintain after this shows the embryo of paddy rice quiescent center cell and depend on the growth hormone signal mediated at the OsIAA23 of quiescent center.Based on the Auxin Signal Tranducation of Aux/IAA to maintaining important role (Ni Jun.et al. (2011) OsIAA23-mediated auxin signaling defines postembryonic maintenance of QC in rice.) after the embryo of the quiescent center of plant root tip.Therefore to the research of transcription factor, the molecule mechanism in theory for understanding plant seed and allelotaxis's regulation and control further provides new clue, and practice also will be provided fundamental basis for high-yield breeding of crops.
Summary of the invention
The object of this invention is to provide the application of rice transcription factor Os05g09480 gene.
In order to realize the object of the invention, first the present invention provides a kind of composing type rice transcription factor, i.e. fusion rotein EAR-Linker-Os05g09480.
Wherein, EAR is the protein motif from a section of plant transcription factor with Transcription inhibition function, its aminoacid sequence is as shown in Seq ID No.2, or this sequence is through replacing, lacking or add one or several amino acids formed aminoacid sequence with same function.
Linker is in series (such as, aminoacid sequence DPAFLYKVVPR or INYPYDVPDYAGSYPYDVPDYAAQCSGTELTSLYKKAGF etc. of GGGGG, GPPPG or Gatway vector recombination site coding) by 1 ~ 39 flexible amino acid.
The Os05g09480 related in above-mentioned fusion rotein is rice transcription factor Os05g09480, its aminoacid sequence is as shown in SEQ ID No.1, or this sequence is through replacing, lacking or add one or several amino acids formed aminoacid sequence with same function, its CDS sequence is as shown in Seq ID No.3.
The present invention also provides the gene of described composing type rice transcription factor of encoding, and under strict conditions, can with the nucleotide sequence of the nucleotide sequence hybridization of this gene.
The present invention also provides the carrier of the gene containing the described composing type rice transcription factor of coding.Described carrier is the carrier that any one bootable foreign gene is expressed in host.Preferably, described carrier is plant binary expression vector (such as, pCAMBIA1301).When described composing type rice transcription factor of the present invention being encoded gene constructed is in plant expression vector, any one strong promoter (such as, corn strong promoter Ubiquitin) or inducible promoter can be added before its transcription initiation Nucleotide.In addition, when described composing type rice transcription factor of the present invention being encoded gene constructed is in plant expression vector, enhanser can also be used, and these enhanser regions must be identical with the reading frame of encoding sequence, to guarantee the translation of whole piece sequence.
The expression vector carrying the gene of described composing type rice transcription factor of encoding imports (Weissbach in vegetable cell by using the standard biologic technological methods such as Ti-plasmids, plant viral vector, directly delivered DNA, microinjection, electroporation, 1998, Method for Plant Molecular Biology VIII, Academy Press, New York, 411-463 page; Geiserson and Corey, 1998, Plant Molecular Biology, the second edition).
The present invention also provides the engineering bacteria and clone that contain the described composing type rice transcription factor of coding.
The present invention also provides a kind of construction process of transgenic rice plant, be specially: adopt agriculture bacillus mediated method, the carrier of the gene containing the described composing type rice transcription factor of coding is proceeded in Rice Callus, transform with the AAM nutrient solution containing inductor and Agrobacterium, material after conversion through the exercise of Dual culture-screen-break up-take root-transgenic seedling and transplanting, screening transgenic rice plant.
The present invention also provides the application of the gene of described composing type rice transcription factor of encoding in improvement rice grain proterties (such as increasing Grain Length in Rice and width).
Present invention also offers the primer pair for amplifying rice transcription factor Os05g09480 gene C DS sequence, comprise forward primer F5'-CAAAAAAGCAGGCTTCATGGCTTGGAATGGCAGG-3' and reverse primer R5'-CAAGAAAGCTGGGTCTCACTCGGCAGCTGCC-3'.
The present invention further provides the application of rice transcription factor Os05g09480 gene in adjusting and controlling rice grain characters.Utilize transcription factor to suppress motif EAR and rice transcription factor Os05g09480 to construct and obtain composing type transcription factor, and by the gene transformation of the described composing type transcription factor of coding to farm crop as in paddy rice, thus improve the proterties of transgenic paddy rice seed.
Aforesaid application, its be the CDS sequence of rice transcription factor Os05g09480 gene is removed terminator codon after, be building up to the downstream that transcription factor suppresses motif EAR encoding gene (SEQ ID No.4), rice transformation (as rice varieties ' kitaake '), thus the proterties of improvement transgenic paddy rice seed.After preferably the CDS sequence of rice transcription factor Os05g09480 gene being removed terminator codon, suppressed the downstream of motif EAR encoding gene to transcription factor by Gateway system constructing.
The present invention utilizes transcription factor to suppress motif EAR and rice transcription factor Os05g09480 to construct first and obtains composing type transcription factor, and by coding described composing type transcription factor gene transformation to farm crop as in paddy rice, thus improvement rice grain proterties, such as Grain Length in Rice and width increase.For illustrating regulation and control seed development mechanism in detail, there is important theory value, and can transgenic approach be passed through, the grain type of improvement paddy rice, therefore also significant in production practice.
Accompanying drawing explanation
Fig. 1 is nEAR-hyg-asRED Vector map in the embodiment of the present invention 1.
Fig. 2 is ubi:EAR-Os05g09480 Vector map in the embodiment of the present invention 1.
Fig. 3 is that in the embodiment of the present invention 3, PCR detects EAR-Os05g09480 transgenic positive strain; Wherein, M is DNA molecular amount standard, and WT is wild rice ' kitaake ', E0533H-27 and E0533H-36 is EAR-Os05g09480 transgenic paddy rice strain.
Fig. 4 is the comparison of the phenotype length of transgenic paddy rice grain characters in the embodiment of the present invention 4; Wherein, WT is wild rice ' kitaake ', E0533H-27 and E0533H-36 is EAR-Os05g09480 transgenic paddy rice strain.
Fig. 5 is the comparison of the phenotype width of transgenic paddy rice grain characters in the embodiment of the present invention 4; Wherein, WT is wild rice ' kitaake ', E0533H-27 and E0533H-36 is EAR-Os05g09480 transgenic paddy rice strain.
Fig. 6 is the species test data statistic analysis result of transgenic paddy rice grain characters in the embodiment of the present invention 4; Wherein, WT is wild rice ' kitaake ', E0533H-27 and E0533H-36 is EAR-Os05g09480 transgenic paddy rice strain.
Fig. 7 is the collection of illustrative plates of plant binary expression vector pCambia1301-UbiN in the embodiment of the present invention 1.
Embodiment
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.If do not specialize, embodiment is experiment condition all conveniently, as Sambrook equimolecular Cloning: A Laboratory Manual (Sambrook J & Russell DW, Molecular cloning:a laboratory manual, 2001) condition of, or according to manufacturer's specification sheets advising.
The acquisition of embodiment 1 Os05g09480 gene C DS sequence and the structure of plant expression vector
The acquisition of 1 Os05g09480 gene C DS sequence
Os05g09480 gene is found in plant transcription factor database (http://rice.plantbiology.msu.edu/analyses_search_locus.shtml), according to its CDS sequences Design pcr amplification primer, forward primer F5'-CAAAAAAGCAGGCTTCATGGCTTGGAATGGCAGG-3' and reverse primer R5'-CAAGAAAGCTGGGTCTCACTCGGCAGCTGCC-3').With the wild-type Japan total cDNA of fine ' kitaake ' paddy rice for template, utilize primers F and R to carry out PCR, obtain the CDS sequence (Seq ID No.3) that Os05g09480 gene is complete.
The structure of 2 plant expression vectors
The CDS sequence of rice transcription factor Os05g09480 gene is suppressed the downstream of motif EAR encoding gene (SEQ ID No.4) to transcription factor by Gateway system constructing.
Above-mentioned PCR primer is cloned on pDONER cloning vector by 2.1
PCR is carried out according to PrimeSTAR polymeric enzymatic amplification system and response procedures.Two-wheeled PCR is comprised in this process, the primer gene primer (F and R) adding part adaptor attB joint of first round PCR, and the second template of taking turns PCR primer of the first round, and complete adaptor attB primer (attB5'adaptor:5'-GTGGGGACAAGTTTGTACAAAAAAGCAGGCTTC-3', attB3'adaptor:5'-GTGGGGACCACTTTGTACAAGAAAGCTGGGTC-3') of primer.PCR primer is cloned on pDONER cloning vector (purchased from Invitrogen), obtains the identical sequence with goal gene through order-checking qualification.
The structure of 2.2 plant expression vectors
With plant binary expression vector pCambia1301-UbiN(Fig. 7) sequence that comprises of right boundary is for frame sequence, pass through vitro recombination, ubi promoter-EAR-Gateway is expressed unit, 35S promoter-asRED expresses unit and 35S promoter-hyg expression unit constructs with it, obtain the complete sequence of carrier nEAR-hyg-asRED as shown in SEQ ID No.6, Vector map is shown in Fig. 1.
Expression vector nEAR-hyg-asRED contains Gateway recombination system, and pDONR as entry vector (Entery vector), can complete the structure of destination gene expression carrier with the plasmid of goal gene by LR reaction.
By LR reaction by the CDS sequence construct of Os05g09480 gene to its goal gene 5 ' hold be connected with on the plant expression vector nEAR-hyg-asRED of EAR encoding gene, obtain the expression vector ubi:EAR-Os05g09480 carrying described composing type rice transcription factor EAR-Linker-Os05g09480 gene of encoding, its complete sequence is as shown in SEQ ID No.5, and Vector map is shown in Fig. 2.
LR reaction system is as follows:
Spend the night in 25 DEG C of reactions.With reaction system transformation of E. coli DH5 α, screening positive clone.
The acquisition of embodiment 2 transgenic rice plant
Water intaking rice ' kitaake ' mature seed, artificial or mechanical dejacketing, selects the full bright and clean seed without bacterial plaque and is inoculated on inducing culture after sterilizing and carries out inducing culture.Selection outward appearance is good, the Rice Callus that growing ability is good is acceptor material, agrobacterium-mediated transformation is adopted to proceed in Rice Callus by ubi:EAR-Os05g09480, transform with the AAM nutrient solution being the Agrobacterium of 0.7 containing the Syringylethanone of 100 μMs and OD value, the callus soaked by conversion fluid is placed on Dual culture base and carries out Dual culture, 25 DEG C of light culture 3d are placed in screening culture medium and cultivate about 30d, and every 10d subculture once.Then transferred on division culture medium by the kanamycin-resistant callus tissue sifted out and break up about 20d, every 10d subculture once.The kanamycin-resistant callus tissue differentiating green seedling is transferred on root media and takes root, grow hardening after flourishing root system until about 7d, and calculate the conversion transgenic seedling number that obtains, obtain 40 transfer-gen plants altogether.Grown in field is transferred to after hardening 7d.
The culture medium prescription related in the present embodiment is as follows:
A large amount of+B5 trace+the NB of inducing culture: N6 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, after adjusting pH to 5.8 ~ 5.9, add plant gel 4g/L.
A large amount of+B5 trace+the NB of Dual culture base: N6 is organic+molysite+2.5mg/L2,4D+0.5g/L glutamine+0.6g/L acid hydrolyzed casein+10g/L glucose+30g/L sucrose, with water preparation, after adjusting pH to 5.2, add plant gel 4g/L.After sterilizing, about 50 DEG C add AS(Syringylethanone) 100 ~ 200 μ g/mL.
A large amount of+B5 trace+the NB of screening culture medium: N6 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, after adjusting pH to 5.8 ~ 5.9, add plant gel 4g/L.35mg/L Totomycin (purchased from Shanghai Niu Jin Bioisystech Co., Ltd) or 5mg/LBialaphos(is added purchased from Beijing Baeyer enlightening biotech company) after sterilizing.
Division culture medium: MS inorganic+MS-B5 trace+MS is organic+molysite+MS-copper cobalt mother liquor+0.05mg/L NAA+2.0mg/L Kinetin(kinetin) and+30g/L sorbyl alcohol+2g/L caseinhydrolysate+30g/L sucrose, with water preparation, after adjusting pH to 5.8 ~ 5.9, add plant gel 4g/L.
The qualification of embodiment 3 transgenic positive strain
In order to detect the T2 of the EAR-Os05g09480 transgenic paddy rice strain obtained in embodiment 2 for the process LAN situation of gene in rice strain (E0533H-27 and E0533H-36), design following primer according to carrier ubi:EAR-Os05g09480:
Forward primer F5'-GTGGGGACAAGTTTGTACAAAAAAGCAGGCTTC-3'
Reverse primer R5'-GTGGGGACCACTTTGTACAAGAAAGCTGGGTC-3'
Carrying out PCR detection, carry out agarose gel electrophoresis to amplified production, as shown in Figure 3, there is obvious specific band in result.Wherein, WT represents wild rice ' kitaake ', E0533H-27 and E0533H-36 represents EAR-Os05g09480 transgenic paddy rice strain.Containing EAR-Os05g09480 fusion gene in E0533H-27 and E0533H-36.
Embodiment 4 transgenic paddy rice phenotype analytical and species test analysis
The transgenic paddy rice strain (E0533H-27 and E0533H-36) and wild-type ' kitaake ' rice paddy seed that obtain in embodiment 2 are compared, can significantly find out phenotype, the seed of transgenic paddy rice obviously elongated, broaden (Fig. 4, Fig. 5).Species test data analysis shows (Fig. 6), and the width of transgenic paddy rice seed is significantly greater than wild rice seed.
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 (2)
1. the application of encoding gene in improvement rice grain grain length and the wide proterties of grain of composing type rice transcription factor EAR-Linker-Os05g09480, wherein, EAR is the protein motif from a section of plant transcription factor with Transcription inhibition function, and its aminoacid sequence is as shown in Seq ID No.2; Linker is in series by 1 ~ 39 flexible amino acid; Os05g09480 is rice transcription factor Os05g09480, and its aminoacid sequence is as shown in SEQ ID No.1, and the aminoacid sequence of Linker is GGGGG, GPPPG, DPAFLYKVVPR or INYPYDVPDYAGSYPYDVPDYAAQCSGTELTSLYKKAGF.
2. the application of rice transcription factor Os05g09480 gene in adjusting and controlling rice seed grain length and the wide proterties of grain, it is characterized in that, its be the CDS sequence of rice transcription factor Os05g09480 gene is removed terminator codon after, suppressed the downstream of motif EAR encoding gene to transcription factor by Gateway system constructing, rice transformation, thus the proterties of improvement transgenic paddy rice seed;
Wherein, described transcription factor suppresses the nucleotide sequence of motif EAR encoding gene as shown in SEQ ID No.4.
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| Title |
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| AltName: Full=Indoleacetic acid-induced protein 16.《NCBI》.2013,UniProtKB/Swiss-Prot: P0C127.1, GI88911332. * |
| 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);第733-739页 * |
| NCBI.RecName:Full=Auxin-responsive protein IAA16 * |
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