CN104341510B - The application of rice transcription factor Os04g55590.1 gene C DS sequences - Google Patents
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Abstract
The present invention relates to the application of rice transcription factor Os04g55590.1 gene C DS sequences, it is to construct to obtain composing type transcription factor using transcription factor activation motif VP64 and rice transcription factor Os04g55590.1, and the genetic transformation of the composing type transcription factor will be encoded into crops such as paddy rice, so as to improve rice grain character, for example, increase rice grain width.There is important theory value for elaborating regulation and control seed development mechanism, and the grain type of paddy rice by transgenic approach, can be improved, thus it is also significant in production practices.
Description
Technical field
The present invention relates to genetic engineering field, specifically, it is related to rice transcription factor Os04g55590.1 gene C DS sequences
The application of row.
Background technology
Paddy rice (Oryza sativa L.) is one of most important Three major grain crops of China and the whole world, is the world one
The staple food of more than half population, is also the model plant of an important functional gene research.Relative science of heredity and molecule
The biological study extremely attention of researcher always, the regulation and control of transcriptional level are the important ways of gene expression regulation.Current water
The research of rice volume increase relatively depends on limited Rice Germplasm Resources, and traditional crossbreeding advantage gradually weakens, and paddy rice
Transgenic technology is possible to excavate the potentiality that paddy rice is further increased production.
In plant kingdom, the plant that can form seed accounts for more than 2/3rds of plant total, is used as important breeding
Organ, seed also provides food source for people simultaneously, and paddy rice is exactly important representative therein, and seed source is in the embryo of after fertilization
Pearl.For the angle of molecular biology, the development and sprouting of seed are a gene expression orderly, selective
Journey.And transcription factor serves critical effect in the accuracy controlling of gene expression.
In recent years, studied about the molecular genetic regulatory mechanism of seed size character, and achieved certain progress, for example, ground
Study carefully personnel and located some seed size related genes using QTL, wherein GS3 encodes a memebrane protein, is seed size and device
The negative regulatory factor of official's size；Zhang Qifa etc.（Yibo Li,Chuchuan Fan,QIfa Zhang.et al.(2011)
Natural variation in GS5plays an important role in regulating grain size and
yield in rice.Nature Gennetics）Research finds that GS5 encodes the serine carboxypeptidase of a regulation seed size,
It is the positive regulating factor for controlling seed size, being overexpressed GS5 can make rice grain substantially become big；Transcription factor is big in regulation and control seed
Small aspect plays very important effect, and OsWRKY78 can adjust the elongation of rice stem and the development of seed, and OsWRKY78 dashes forward
Variant can make cane become dwarfing influence Grain Development；Helix-loop-helix protein (bHLH) class transcription factor can be by regulating and controlling lemma
With the size of the effect length grain of glumelle cell；PGL1 bHLH proteins (bHLH）Heterodimer is used as knot
It can increase seed length and weight after the repressor overexpression for the bHLH for closing DNA.
Homeoboxes (homeodomain protein) HB family's transcription factors are in the animal kingdom first in drosophila, and plant is being intended
It is found first in southern mustard.There is more than 300 member in HB families, take part in many aspects of growing of biology.Divide from structure
Analysis, Homeoboxes possesses DNA recognition sites, the domain that a series of including TATA etc. and DNA is combined.Current HB families transcription
Researchs and understanding of the factor Os04g55590.1 to seed size shape control mechanism are seldom, therefore the research of the transcription factor exists
The molecule mechanism for theoretically further understanding vegetable seeds and allelotaxis's regulation and control provides new clue；Also will in practice
Provided fundamental basis for high-yield breeding of crops.
The content of the invention
It is an object of the invention to provide the application of rice transcription factor Os04g55590.1 gene C DS sequences.
In order to realize the object of the invention, present invention firstly provides a kind of composing type rice transcription factor, i.e. fusion protein
（VP16）4-Linker-Os04g55590.1。
Wherein, VP16 is the VP16 albumen from herpes simplex virus, and 4 VP16 functional domain motifs are merged can
A class enhancer is constituted, strengthens the function of transcription factor, so as to occur more obvious character mutation in transfer-gen plant.It is above-mentioned
It is related in fusion protein（VP16）4, i.e. VP64 is to be formed by the amino acid sequence of 4 VP16 albumen by interval of Gly-Ser
Fusion protein, its amino acid sequence and nucleotide sequence are respectively as shown in SEQ ID No.10 and SEQ ID No.4.
The Linker being related in above-mentioned fusion protein is in series by 39 flexible amino acids, its amino acid sequence such as SEQ
Shown in ID No.9, the nucleotide sequence of the Linker is encoded as shown in SEQ ID No.3.
The Os04g55590.1 being related in above-mentioned fusion protein is rice transcription factor Os04g55590.1, its amino acid sequence
Row are as shown in SEQ ID No.2, or the sequence is one or several amino acids formed with equal work(through replacing, lacking or add
The amino acid sequence of energy；The CDS sequences of rice transcription factor Os04g55590.1 genes are：Nucleosides shown in SEQ ID No.1
Acid sequence.
The present invention also provides the gene of the coding composing type rice transcription factor, and under strict conditions, can be with this
The nucleotide sequence that the nucleotide sequence of gene hybridizes.
The present invention also provides carrier, engineering bacteria and cell containing the gene for encoding the composing type rice transcription factor
System.
The construction method of the carrier is as follows：
（1）In plant transcription factor database（http://rice.plantbiology.msu.edu/analyses_
search_locus.shtml）In find Os04g55590.1 genes, according to its sequences Design pcr amplification primer thing pair, it is just
To primers F：5 '-CAAAAAAGCAGGCTTCATGAGGCTTCACCATCTGC-3 ' and reverse primer R：5′-
CAAGAAAGCTGGGTCAGCTTTTCCCTGGGGATGC-3′。
（2）Using the total cDNA of fine ' kitaake ' paddy rice of wild Japanese as template, performing PCR is entered using above-mentioned primers F and R, is obtained
The complete CDS sequences of Os04g55590.1 genes.
（3）Above-mentioned PCR primer is cloned on pDONER cloning vectors, obtained and the complete phase of target gene through sequencing identification
Same sequence.
（4）With plant binary expression vector pCambia1301-UbiN（Fig. 6）The sequence that right boundary is included is skeleton sequence
Row, by vitro recombination, express single by ubi promoter-VP64-Gateway expression units, 35S promoter-asRED
Member and 35S promoter-hyg expression units are constructed therewith, obtain carrier nVP64-hyg-asRED complete sequence such as SEQ
Shown in ID No.5.
（5）Reacted by LR and be connected with 5 ' ends of the CDS sequence constructs of Os04g55590.1 genes to its target gene
On the plant expression vector nVP64-hyg-asRED of VP64 encoding genes, acquisition carries the coding composing type paddy rice transcription
The expression vector ubi of factor Ⅴ P64-Linker-Os04g55590.1 genes：VP64-Os04g55590.1, its complete sequence such as SEQ
Shown in ID No.6.
Above-mentioned expression vector can be by using Ti-plasmids, plant viral vector, directly delivered DNA, microinjection, electroporation etc.
Standard biologic technical method is imported in plant cell（Weissbach, 1998, Method for Plant Molecular
Biology VIII, Academy Press, New York, the 411-463 pages；Geiserson and Corey, 1998, Plant
Molecular Biology, 2nd Edition）.
The present invention also provides a kind of construction method of transgenic rice plant, is specially：Using agriculture bacillus mediated method,
Above-mentioned expression vector is transferred in Rice Callus, converted with the AAM nutrient solutions containing derivant and Agrobacterium, after conversion
Material break up-take root-exercise and transplanting of transgenic seedling, screening transgenic rice plant by co-cultivation-screening-.
The present invention also provides the gene of the coding composing type rice transcription factor in improvement rice grain character（For example increase
The grain of rice that adds water is wide and mass of 1000 kernel）In application.
Present invention also offers the primer pair for amplifying rice transcription factor Os04g55590.1 gene C DS sequences, bag
Include forward primer F：5 '-CAAAAAAGCAGGCTTCATGAGGCTTCACCATCTGC-3 ' and reverse primer R：5′-
CAAGAAAGCTGGGTCAGCTTTTCCCTGGGGATGC-3′。
The present invention further provides rice transcription factor Os04g55590.1 gene C DS sequences in adjusting and controlling rice grain characters
In application.Utilize transcription factor activation motif VP64（SEQ ID No.10）Merged with rice transcription factor Os04g55590.1
Structure obtains composing type transcription factor, and by the genetic transformation of the coding composing type transcription factor into crops such as paddy rice,
So as to improve the character of transgenic paddy rice seed.
Foregoing application, is to swash the CDS sequence constructs of rice transcription factor Os04g55590.1 genes to transcription factor
Motif VP64 encoding genes living（SEQ ID No.4）Downstream, rice transformation, so as to improve the character of transgenic paddy rice seed.
It is preferred that the CDS sequences of rice transcription factor Os04g55590.1 genes are passed through into Gateway system transcription factor activation motifs
The downstream of VP64 encoding genes.
The present invention utilizes transcription factor activation motif VP64 first（I.e. 4 transcription factor activation motif VP16）Turn with paddy rice
Record factor Os04g55590.1, which is constructed, obtains composing type transcription factor, and the gene that will encode the composing type transcription factor
It is transformed into crops such as paddy rice, so that rice grain character is improved, such as rice grain width increase.For elaborating tune
Control seed development mechanism has important theory value, and can be by transgenic approach, the grain type of improvement paddy rice, therefore
It is also significant in production practices.
Brief description of the drawings
Fig. 1 is nVP64-hyg-asRED Vector maps in the embodiment of the present invention 1.
Fig. 2 is ubi in the embodiment of the present invention 1：VP64-Os04g55590.1 Vector maps.
Fig. 3 is the positive strain of PCR detection VP64-Os04g55590.1 transgenic paddy rices in the embodiment of the present invention 3；Wherein,
WT is wild rice ' kitaake ', and V2478H-42, V2478H-43 are VP64-Os04g55590.1 transgenic paddy rice strains.
Fig. 4 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 type
Paddy rice ' kitaake ', V2478H-42, V2478H-43 are VP64-Os04g55590.1 transgenic paddy rice strains.
Fig. 5 is the data statistic analysis result of transgenic paddy rice grain characters in the embodiment of the present invention 4；Wherein WT is wild
Type paddy rice ' kitaake ', V2478H-42, V2478H-43 are VP64-Os04g55590.1 transgenic paddy rice strains.
Fig. 6 is pCambia1301-UbiN Vector maps in the embodiment of the present invention 1.
Fig. 7 is in the embodiment of the present invention 1
GCS-2*FLAG+en35s-AsRed-pCambia1301-UbiN Vector maps.
Embodiment
Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.Unless otherwise specified, embodiment
In the conventional meanses that are well known to those skilled in the art of used technological means, raw materials used is commercial goods.
The acquisition of embodiment 1Os04g55590.1 gene C DS sequences and the structure of plant expression vector
1Os04g55590.1 the acquisition of gene C DS sequences
In plant transcription factor database（http://rice.plantbiology.msu.edu/analyses_
search_locus.shtml）In find Os04g55590.1 genes, according to its sequences Design pcr amplification primer thing, forward primer
F：5 '-CAAAAAAGCAGGCTTCATGAGGCTTCACCATCTGC-3 ' and reverse primer R：5′-
CAAGAAAGCTGGGTCAGCTTTTCCCTGGGGATGC-3′）.Using the total cDNA of wild type Nipponbare ' kitaake ' paddy rice as mould
Plate, performing PCR is entered using primers F and R, obtains the complete CDS sequences of Os04g55590.1 genes（As shown in SEQ ID No.1）.
The structure of 2 plant expression vectors
By the CDS sequences of rice transcription factor Os04g55590.1 genes by Gateway system constructings to 4 transcription because
Son activation motif VP16 encoding genes（As shown in SEQ ID No.4）Downstream.
2.1 are cloned into above-mentioned PCR primer on pDONER cloning vectors
Enter performing PCR according to PrimeSTAR polymeric enzymatic amplifications system and response procedures.Two-wheeled PCR, first are included during this
Take turns the PCR primer gene primer for adding part adaptor attB joints（F and R）, and the template of the second wheel is with the first round
PCR primer, and primer is with complete adaptor attB primers（attB5′adaptor：5′-
GTGGGGACAAGTTTGTACAAAAAAGCAGGCTTC-3 ', attB3 ' adaptor：5′-
GTGGGGACCACTTTGTACAAGAAAGCTGGGTC-3′）.PCR primer is cloned into pDONER cloning vectors（It is purchased from
Invitrogen）On, obtained and the identical sequence of target gene through sequencing identification.
The structure of 2.2 plant expression vectors
With plant binary expression vector pCambia1301-UbiN（Fig. 6）The sequence that right boundary is included is frame sequence,
By vitro recombination, by ubi promoter-VP64-Gateway expression units, 35S promoter-asRED expression units and
35S promoter-hyg expression units are constructed therewith, obtain carrier nVP64-hyg-asRED complete sequence such as SEQ ID
Shown in No.5, Vector map is shown in Fig. 1.
Reacted by LR and 5 ' ends of the CDS sequence constructs of Os04g55590.1 genes to its target gene are connected with VP64 volumes
On the plant expression vector nVP64-hyg-asRED of code gene, acquisition carries the coding composing type rice transcription factor
The expression vector ubi of VP64-Linker-Os04g55590.1 genes：VP64-Os04g55590.1, its complete sequence such as SEQ ID
Shown in No.6, Vector map is shown in Fig. 2.
Using carrier pCambia1301-UbiN as skeleton (Fig. 6), the sequence between SpeI and PstI has changed GCS-2* into
FLAG, has changed the sequence between HindIII and BstEII into 35senhancer+P35s-AsRed, completes GCS-2*FLAG+
En35s-AsRed-pCambia1301-UbiN structure（Fig. 7）.The end of synthesis 5 ' and 3 ' carries the double-strand of KpnI restriction enzyme sites
DNA sequence dna VP64-2*HA.Fragment VP64-2*HA is inserted into by carrier by KpnI sites（Fig. 1）, finally obtain expression vector
nVP64-HYG-AsRed-pCambial1301-Ubi.Target gene is building up on plant expression vector by LR reactions.
Expression vector contains Gateway recombination systems, and plasmids of the pDONR with target gene is used as entry vector
（Entery vector）, the structure of destination gene expression carrier can be completed by LR reactions.
LR reaction systems：
25 DEG C of reactions are stayed overnight.Reaction solution converts bacillus coli DH 5 alpha, screening positive clone.
The acquisition of the transgenic rice plant of embodiment 2
Water intaking rice ' kitaake ' mature seed, manually or mechanically shells, selects the full bright and clean seed without bacterial plaque sterilized
It is inoculated into afterwards on inducing culture and carries out Fiber differentiation.Select outward appearance good, the good Rice Callus of growing power is acceptor
Material, using agrobacterium-mediated transformation by ubi：VP64-Os04g55590.1 is transferred in Rice Callus, with containing 100 μM
Acetosyringone and OD values are converted for the AAM nutrient solutions of 0.7 Agrobacterium, and the callus that conversion fluid soaked is placed in
Co-culture and co-cultured on base, be placed in after 25 DEG C of light culture 3d on screening and culturing medium and cultivate about 30d, per 10d subcultures once.So
The kanamycin-resistant callus tissue sifted out is transferred on differential medium afterwards and breaks up about 20d, per 10d subcultures once.Green seedling will be differentiated
Kanamycin-resistant callus tissue be transferred on root media and take root, the hardening after about 7d grows flourishing root system, and calculate conversion and obtain and turn base
Because of seedling number.Grown in field is transferred to after hardening 7d.Obtain 50 plants of transgenic seedlings.
The culture medium prescription being related in the present embodiment is as follows：
Inducing culture：A large amount of micro+the NB of+B5 of N6 are organic+molysite+copper cobalt mother liquor+2.5mg/L2,4D+0.6g/L sour waters
Casein+2.878g/L proline+0.5g/L glutamine+30g/L sucrose is solved, is prepared with water, adjusts and adds after pH to 5.8~5.9
Enter plant gel 4g/L.
Co-culture base：A large amount of micro+the NB of+B5 of N6 are organic+molysite+2.5mg/L2,4D+0.5g/L glutamine+0.6g/L
Acid hydrolyzed casein+10g/L glucose+30g/L sucrose, is prepared with water, adjusts and plant gel 4g/L is added after pH to 5.2.Sterilizing
Afterwards, 50 DEG C or so addition AS（Acetosyringone）100~200 μ g/mL.
Screening and culturing medium：A large amount of micro+the NB of+B5 of N6 are organic+molysite+copper cobalt mother liquor+2.5mg/L2,4D+0.6g/L sour waters
Casein+2.878g/L proline+0.5g/L glutamine+30g/L sucrose is solved, is prepared with water, adjusts and adds after pH to 5.8~5.9
Enter plant gel 4g/L.35mg/L hygromycin is added after sterilizing（Purchased from Shanghai Niu Jin Bioisystech Co., Ltd）.
Differential medium：Micro+the MS of the inorganic+MS-B5 of MS are organic+molysite+MS- copper cobalt mother liquor+0.05mg/L NAA+
2.0mg/L Kinetin（Kinetin）+ 30g/L sorbierite+2g/L caseinhydrolysate+30g/L sucrose, is prepared with water, adjusts pH extremely
Plant gel 4g/L is added after 5.8~5.9.
The identification of the transgenic positive strain of embodiment 3
Ubi in the VP64-Os04g55590.1 transgenic paddy rice strains obtained for detection embodiment 2：VP64-
Os04g55590.1 genes are in T2 for transgenic paddy rice（V2478H-42、V2478H-43）In overexpression situation, the present embodiment
Primer is designed in carrier and target gene joint（Forward primer：5′-GTGGGGACAAGTTTGTACAAAAAAGCAGGCTTC-
3 ' and reverse primer：5′-GTGGGGACCACTTTGTACAAGAAAGCTGGGTC-3′）Enter performing PCR detection, obtained obvious
Specific band.As seen from Figure 3, the stripe size amplified is that and primer is in carrier and target gene joint less than 750bp
Place's design, the clip size and target gene amplified（711bp）It is basically identical.It can be said that it is bright, what embodiment 2 was obtained
Contain VP64-Os04g55590.1 fusions in transgenic paddy rice strain V2478H-42, V2478H-43.
The transgenic paddy rice phenotypic analysis of embodiment 4 and species test analysis
Transgenic paddy rice strain V2478H-42, V2478H-43 and wild rice seed that embodiment 2 is obtained are carried out
Compare, can significantly find out from phenotype, it is found that the seed of transgenic line substantially broadens（Fig. 4）.Species test data analysis table
It is bright（Fig. 5）, the width of VP64-Os04g55590.1 transgenic paddy rice seeds that the present invention is obtained is noticeably greater than wild rice seed
Grain.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, 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. fusion protein (VP16)4Application of-Linker-Os04g55590.1 the encoding gene in improvement rice grain grain is wide,
Characterized in that, VP16 is the VP16 albumen from herpes simplex virus, (VP16)4It is by the amino acid sequence of 4 VP16 albumen
The fusion protein formed by interval of Gly-Ser is arranged, its amino acid sequence is as shown in SEQ ID No.10；Linker is soft by 39
Acidic amino acid is in series, and its amino acid sequence is as shown in SEQ ID No.9；Os04g55590.1 is rice transcription factor
Os04g55590.1, its amino acid sequence is as shown in SEQ ID No.2.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102816243A (en) * | 2012-08-03 | 2012-12-12 | 中国农业科学院作物科学研究所 | Application of rice transcription factor Os06g08400 genes |
| CN103214581A (en) * | 2013-03-29 | 2013-07-24 | 中国农业科学院作物科学研究所 | Application of synthetic transcription factor VP64-Os03g57670 |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102816243A (en) * | 2012-08-03 | 2012-12-12 | 中国农业科学院作物科学研究所 | Application of rice transcription factor Os06g08400 genes |
| CN103214581A (en) * | 2013-03-29 | 2013-07-24 | 中国农业科学院作物科学研究所 | Application of synthetic transcription factor VP64-Os03g57670 |
Non-Patent Citations (2)
| Title |
|---|
| Expression of a Rice Homeobox Gene Causes Altered Morphology of Transgenic Plants;Makoto Matsuoka et al.,;《The Plant Cell》;19930930;第5卷;1039-1048 * |
| http://signal.salk.edu/cgi-bin/RiceGE5?JOB=TEXT&TYPE=GENE&QUERY=Os04g55590，Code Os04g55590;Salk Institute Genomic Analysis Laboratory;《RiceGE:Genome Express Database》;20101006;1-2 * |
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