CN104341519B - Application of oryza sativa transcription factor Os01g45090.1 gene CDS sequence - Google Patents
Application of oryza sativa transcription factor Os01g45090.1 gene CDS sequence Download PDFInfo
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- CN104341519B CN104341519B CN201310329917.1A CN201310329917A CN104341519B CN 104341519 B CN104341519 B CN 104341519B CN 201310329917 A CN201310329917 A CN 201310329917A CN 104341519 B CN104341519 B CN 104341519B
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Abstract
The invention relates to the application of a oryza sativa transcription factor Os01g45090.1 gene CDS sequence. According to the invention, a transcription factor activation motif VP64 is fused with the oryza sativa transcription factor Os01g45090.1, such that a constitutive transcription factor is constructed; the gene coding the constitutive transcription factor is transferred to a crop such as oryza sativa, such that oryza sativa grain traits can be improved. For example, oryza sativa grain length can be increased. The invention has an important theoretical value in detailed explanation of seed development mechanism. Also, oryza sativa grain type can be improved through transgenic means. Therefore, the application provided by the invention also has important significance in production practices.
Description
Technical field
The present invention relates to genetic engineering field, specifically, it is related to rice transcription factor Os01g45090.1 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 a model plant for 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, used as important breeding
Organ, simultaneously also for people provide food source, paddy rice is exactly important representative therein to seed, and seed source is in the embryo of after fertilization
Pearl.From 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, the molecular genetic regulatory mechanism about seed size proterties was studied, and had achieved certain progress, for example, grind
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 GS5 plays an important role in regulating grain size and
yield in rice.Nature Gennetics）Research finds that GS5 encodes a serine carboxypeptidase for regulation seed size,
It is the positive regulating factor for controlling seed size, overexpression 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
Can increase seed length and weight after the repressor overexpression of the bHLH for closing DNA.
MYB albumen is the protein containing MYB domains, and MYB domains are one and contain 52 DNA integrated structures of amino acid
Domain.In c-MYB, this domain has three repetitions（R1, R2, R3）, and each not exclusively repetition uses spiral-spiral shell
The configuration of rotation-turn-helix is come in the major groove for inserting target DNA.Topmost MYB albumen has two not exclusively to repeat in plant
（R2, R3）.Additionally, it has also been found that there is three MYB albumen of repetition in plant, and comprise only the MYB albumen in MYB domains.MYB
GAP-associated protein GAP has many functions, such as combines telomeric sequence, used as transcription factor, the tune of involved in plant phenylpropyl alcohol alkanes secondary metabolism
Section, participates in cell differentiation, the developmental regulation of morphogenesis, response of the involved in plant to hormone, the biological and non-life of involved in plant
Thing resistance etc..Current MYB families transcription factor Os01g45090.1 is to paddy rice photomorphogenesis and hormone response regulatory mechanism
Research and understanding are little, therefore the research of the transcription factor is to further understand vegetable seeds photomorphogenesis and swash in theory
The molecule mechanism of plain response regulation and control provides new clue；To also be provided fundamental basis for high-yield breeding of crops in practice.
The content of the invention
It is an object of the invention to provide the application of rice transcription factor Os01g45090.1 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-Os01g45090.1。
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 formed by interval of Gly-Ser by 4 amino acid sequences of VP16 albumen
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 Os01g45090.1 being related in above-mentioned fusion protein is rice transcription factor Os01g45090.1, 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 Os01g45090.1 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 the cell of the gene containing the coding 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 Os01g45090.1 genes, according to its sequences Design pcr amplification primer thing pair, it is just
To primers F：5 '-CAAAAAAGCAGGCTTCATGGAATCAGAATCAGAACA-3 ' and reverse primer R：5′-
CAAGAAAGCTGGGTCCACCACGTCTGAAACCG-3′。
（2）With the total cDNA of fine ' kitaake ' paddy rice of wild Japanese as template, performing PCR is entered using above-mentioned primers F and R, obtained
The complete CDS sequences of Os01g45090.1 genes.
（3）Above-mentioned PCR primer is cloned on pDONER cloning vectors, is obtained and the complete phase of genes of interest through sequencing identification
Same sequence.
（4）With plant binary expression vector pCambia1301-UbiN（Fig. 7）The sequence that right boundary is included is skeleton sequence
Row, it is by vitro recombination, ubi promoter-VP64-Gateway expression units, 35S promoter-asRED expression is single
Unit and 35S promoter-hyg expression units are constructed therewith, obtain the complete sequence such as SEQ of carrier nVP64-hyg-asRED
Shown in ID No.5.
（5）Reacted by LR and be connected with 5 ' ends of the CDS sequence constructs of Os01g45090.1 genes to its genes of interest
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-Os01g45090.1 genes：VP64-Os01g45090.1, 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, specially：Using agriculture bacillus mediated method,
Above-mentioned expression vector is transferred in Rice Callus, is converted with the AAM nutrient solutions containing derivant and Agrobacterium, after conversion
Material by co-culturing-screen-break up-take root-exercise and transplanting of transgenic seedling, screening transgenic rice plant.
The present invention also provides the gene of the coding composing type rice transcription factor in improvement rice grain proterties（For example increase
The grain of rice that adds water is long and mass of 1000 kernel）In application.
Present invention also offers the primer pair for amplifying rice transcription factor Os01g45090.1 gene C DS sequences, bag
Include forward primer F：5 '-CAAAAAAGCAGGCTTCATGGAATCAGAATCAGAACA-3 ' and reverse primer R：5′-
CAAGAAAGCTGGGTCCACCACGTCTGAAACCG-3′。
The present invention further provides rice transcription factor Os01g45090.1 gene C DS sequences in adjusting and controlling rice grain characters
In application.Using transcription factor activation motif VP64（SEQ ID No.10）Merged with rice transcription factor Os01g45090.1
Structure obtains composing type transcription factor, and will encode the genetic transformation of the composing type transcription factor in crops such as paddy rice,
So as to improve the proterties of transgenic paddy rice seed.
Foregoing application, is that the CDS sequence constructs of rice transcription factor Os01g45090.1 genes are swashed to transcription factor
Motif VP64 encoding genes living（SEQ ID No.4）Downstream, rice transformation, so as to improve the proterties of transgenic paddy rice seed.
It is preferred that the CDS sequences of rice transcription factor Os01g45090.1 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 Os01g45090.1 is constructed and is obtained composing type transcription factor, and will encode the gene of the composing type transcription factor
It is transformed into crops such as paddy rice, so as to improve rice grain proterties, for example Grain Length in Rice increases.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-Os01g45090.1 Vector maps.
Fig. 3 is PCR detection VP64-Os01g45090.1 transgenic positive strains in the embodiment of the present invention 3, and wherein WT is open country
Raw type paddy rice ' kitaake ', V2011H-27, V2011H-30 are VP64-Os01g45090.1 transgenic paddy rice strains.
Fig. 4 is the comparing of the phenotype length of transgenic paddy rice grain characters in the embodiment of the present invention 4；Wherein WT is wild type
Paddy rice ' kitaake ', V2011H-27, V2011H-30 are VP64-Os01g45090.1 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 ', V2011H-27, V2011H-30 are VP64-Os01g45090.1 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.
Specific embodiment
Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.If not specializing, embodiment
In the conventional meanses that are well known to those skilled in the art of technological means used, it is raw materials used to be commercial goods.
Embodiment 1
The acquisition of Os01g45090.1 gene C DS sequences and the structure of plant expression vector
The acquisition of 1.Os01g45090.1 gene C DS sequences
In plant transcription factor database（http://rice.plantbiology.msu.edu/analyses_
search_locus.shtml）In find Os01g45090.1 genes, its nucleotide sequence as shown in SEQ ID No.11, according to
Its sequences Design pcr amplification primer thing, forward primer F：5 '-CAAAAAAGCAGGCTTCATGGAATCAGAATCAGAACA-3 ' and
Reverse primer R：5′-CAAGAAAGCTGGGTCCACCACGTCTGAAACCG-3′）.With wild type Nipponbare ' kitaake ' paddy rice
Total cDNA is template, and performing PCR is entered using primers F and R, obtains the complete CDS sequences of Os01g45090.1 genes（Such as SEQ ID
Shown in No.1）.
2. the structure of plant expression vector
By the CDS sequences of rice transcription factor Os01g45090.1 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 above-mentioned PCR primer is cloned on pDONER cloning vectors
Enter performing PCR according to PrimeSTAR polymeric enzymatic amplifications system and response procedures.Comprising two-wheeled PCR, first during this
Take turns the primer gene primer for adding part adaptor attB joints of PCR（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 genes of interest 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 the complete sequence such as SEQ ID of carrier nVP64-hyg-asRED
Shown in No.5, Vector map is shown in Fig. 1.
Reacted by LR and 5 ' ends of the CDS sequence constructs of Os01g45090.1 genes to its genes of interest 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-Os01g45090.1 genes：VP64-Os01g45090.1, its complete sequence such as SEQ ID
Shown in No.6, Vector map is shown in Fig. 2.
With 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+
The structure of en35s-AsRed-pCambia1301-UbiN（Fig. 7）.The end of synthesis 5 ' and 3 ' the double-strand with 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.Genes of interest is reacted by LR and is building up on plant expression vector.
Expression vector contains Gateway recombination systems, and plasmids of the pDONR with genes of interest is used as entry vector
（Entery vector）, being reacted by LR can complete the structure of destination gene expression carrier.
LR reaction systems：
25 DEG C of reactions are 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, and selects the full bright and clean seed without bacterial plaque sterilized
Being inoculated into afterwards carries out Fiber differentiation on inducing culture.Selection outward appearance is good, and the good Rice Callus of growing power are acceptor
Material, using agrobacterium-mediated transformation by ubi：VP64-Os01g45090.1 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 on screening and culturing medium after 25 DEG C of light culture 3d and cultivate about 30d, per 10d subcultures once.So
The kanamycin-resistant callus tissue that will be sifted out afterwards is transferred on differential medium 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 30 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/L 2,4D+0.6g/L sour waters
Solution casein+2.878g/L proline+0.5g/L glutamine+30g/L sucrose, is prepared with water, is added after adjusting 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/L 2,4D+0.5g/L glutamine+0.6g/L
Acid hydrolyzed casein+10g/L glucose+30g/L sucrose, is prepared with water, and plant gel 4g/L is added after adjusting 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/L 2,4D+0.6g/L sour waters
Solution casein+2.878g/L proline+0.5g/L glutamine+30g/L sucrose, is prepared with water, is added after adjusting 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
5.8 plant gel 4g/L is added after~5.9.
The identification of the transgenic positive strain of embodiment 3
Ubi in the VP64-Os01g45090.1 transgenic paddy rice strains obtained for detection embodiment 2：VP64-
Os01g45090.1 genes are in T2 for transgenic paddy rice（V2011H-27、V2011H-30）In overexpression situation, the present embodiment
Primer is designed in carrier and genes of interest 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 for amplifying is in more than 500bp, and primer is in carrier and genes of interest joint
Place's design, the clip size and genes of interest for amplifying（687bp）It is basically identical.It can be said that it is bright, what embodiment 2 was obtained
Contain VP64-Os01g45090.1 fusions in transgenic paddy rice strain V2011H-27, V2011H-30.
The transgenic paddy rice phenotypic analysis of embodiment 4 and species test are analyzed
Transgenic paddy rice strain V2011H-27, V2011H-30 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 is substantially elongated（Fig. 4）.Species test data analysis table
It is bright（Fig. 5）, the length of VP64-Os01g45090.1 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 (6)
1. a kind of composing type rice transcription factor for improveing rice grain grain length, it is characterised in that it is fusion protein
(VP16)4-Linker-Os01g45090.1；
Wherein, VP16 is the VP16 albumen from herpes simplex virus, (VP16)4Be by 4 amino acid sequences of VP16 albumen with
Gly-Ser is the fusion protein that interval is formed, and its amino acid sequence is as shown in SEQ ID No.10；Linker is by 39 flexible ammonia
Base acid is in series, and its amino acid sequence is as shown in SEQ ID No.9；Os01g45090.1 is rice transcription factor
Os01g45090.1, its amino acid sequence is as shown in SEQ ID No.2.
2. the gene of composing type rice transcription factor described in claim 1 is encoded.
3. the carrier of gene described in claim 2 is contained.
4. the engineering bacteria of gene described in claim 2 is contained.
5. a kind of construction method of transgenic rice plant, it is characterised in that agriculture bacillus mediated method is used, by claim
Carrier described in 3 is transferred in Rice Callus, is converted with the AAM nutrient solutions containing derivant and Agrobacterium, after conversion
Material by co-culturing-screen-break up-take root-exercise and transplanting of transgenic seedling, screening transgenic rice plant.
6. application of the gene described in claim 2 in rice grain grain length is improved.
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|---|---|---|---|---|
| CN102016032A (en) * | 2008-03-04 | 2011-04-13 | 丰田自动车株式会社 | Genes that increase the production of oil in plants, and method of using the same |
| 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 |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102016032A (en) * | 2008-03-04 | 2011-04-13 | 丰田自动车株式会社 | Genes that increase the production of oil in plants, and method of using the same |
| 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 |
Non-Patent Citations (2)
| Title |
|---|
| Accession ID：AP004367，Oryza sativa Japonica Group genomic DNA, chromosome1, PAC clone:P0696E01;Sasaki, .T，etc.;《NCBI GenBank数据库》;20080216;全文 * |
| 水稻MYB cDNA的克隆和表达分析;陈俊等;《植物生理与分子生物学学报》;20021231;第28卷(第4期);第267页第2-3段，第272-273页最后1段 * |
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