CN104341516B - The application of rice transcription factor Os05g46780 gene C DS sequences - Google Patents
The application of rice transcription factor Os05g46780 gene C DS sequences Download PDFInfo
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Abstract
The present invention relates to the application of rice transcription factor Os05g46780 gene C DS sequences, it is to construct to obtain composing type transcription factor using transcription factor activation motif VP64 and rice transcription factor Os05g46780, and the genetic transformation of the composing type transcription factor will be encoded into crops such as rice, so as to improve rice grain character, such as increase rice grain width.There is important theory value for elaborating regulation and control seed development mechanism, and the grain type of rice by transgenic approach, can be improved, therefore be also of great significance in production practices.
Description
Technical field
The present invention relates to genetic engineering field, specifically, is related to rice transcription factor Os05g46780 gene C DS sequences
Application.
Background technology
Rice (Oryza sativa L.) is one of most important Three major grain crops in China and the whole world, is the world one
The staple food of more than half population, and the model plant of an important functional gene research.Relative science of heredity and molecule
Always by the attention of researcher, the regulation and control of transcriptional level are the important ways of gene expression regulation for biological study.Current water
The research of rice volume increase relatively depends on limited Rice Germplasm Resources, and traditional crossbreeding advantage gradually weakens, and rice
Transgenic technology is possible to excavate the potentiality that rice further increases production.
In plant kingdom, the plant that can form seed accounts for more than 2/3rds of plant total, as important breeding
Organ, seed also provide food source for people at the same time, and 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 play the role of in the accuracy controlling of gene expression it is critical.
In recent years, the molecular genetic regulatory mechanism research in relation to seed size character, has achieved certain progress, such as 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 the serine carboxypeptidase of an adjusting seed size,
It is the positive regulating factor for controlling seed size, being overexpressed GS5 can make rice grain substantially become larger；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
Variation, which can make cane become dwarfing, influences 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 as knot
It can increase seed length and weight after closing the repressor overexpression of the bHLH of DNA.
FHA transcription factors take part in the growth and development many aspects of biology.Analyzed from structure, typical FHA domains
It is made of about 55-75 amino acid, wherein including three highly conserved regions separated by interval region.FHA family at present
Researchs of the race transcription factor Os05g46780 to seed size shape control mechanism and understanding is seldom, therefore the transcription factor is ground
Study carefully, provide new clue to further understand the molecule mechanism of vegetable seeds and allelotaxis's regulation and control in theory, putting into practice
On will also provide fundamental basis for high-yield breeding of crops.
The content of the invention
The object of the present invention is to provide the application of rice transcription factor Os05g46780 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-Os05g46780。
Wherein, VP16 is the VP16 albumen from herpes simplex virus, and 4 VP16 functional domain motifs are merged can
A kind of enhancer is formed, strengthens the function of transcription factor, so as to occur more obvious character mutation in transfer-gen plant.It is above-mentioned
Involved 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 4.
Linker involved 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.
Os05g46780 involved in above-mentioned fusion protein is rice transcription factor Os05g46780, its amino acid sequence is such as
Shown in SEQ ID No.2, or the sequence through replace, lack or add it is one or several it is amino acids formed have the function of it is equal
Amino acid sequence；The CDS sequences of rice transcription factor Os05g46780 genes are as shown in SEQ ID No.11.
, can be with this present invention also provides the gene for encoding the composing type rice transcription factor, and under strict conditions
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 Os05g46780 genes are found in), according to its CDS sequence design PCR amplification primer pair, it is just
To primers F 5 '-CAAAAAAGCAGGCTTCGGCGGTGGAGCGGAGGGAGCATT-3 ' and reverse primer R5 '-
CAAGAAAGCTGGGTCTCATGAAGCAAAACATACTCCCGGC-3′。
（2）Using the total cDNA of fine ' kitaake ' rice of wild Japanese as template, PCR is carried out using above-mentioned primers F and R, is obtained
The complete CDS sequences of Os05g46780 genes.
（3）Above-mentioned PCR product is cloned on pDONER cloning vectors, is 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 includes is skeleton sequence
Row, by vitro recombination, ubi promoter-VP64-Gateway expression units, 35S promoter-asRED are expressed single
Member and 35S promoter-hyg expression units construct therewith, obtain the complete sequence such as SEQ of carrier nVP64-hyg-asRED
Shown in ID No.5.
（5）5 ' ends of the CDS sequence constructs of Os05g46780 genes to its target gene are connected with by VP64 by LR reactions
On the plant expression vector nVP64-hyg-asRED of encoding gene, acquisition carries the coding composing type rice transcription factor
The expression vector ubi of VP64-Linker-Os05g46780 genes：VP64-Os05g46780, its complete sequence such as SEQ ID No.6
It is shown.
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, the second 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, is converted with the AAM nutrient solutions containing derivant and Agrobacterium, after conversion
Material pass through co-cultivation-screening-and break up-take root-exercise and transplanting of transgenic seedling, screening transgenic rice plant.
The present invention also provides encode the gene of the composing type rice transcription factor in improvement rice grain character（Such as increase
Add rice grain wide and mass of 1000 kernel）In application.
Present invention also offers the primer pair for amplifying rice transcription factor Os05g46780 gene C DS sequences, including
Forward primer F5 '-CAAAAAAGCAGGCTTCGGCGGTGGAGCGGAGGGAGCATT-3 ' and reverse primer R5 '-
CAAGAAAGCTGGGTCTCATGAAGCAAAACATACTCCCGGC-3′。
The present invention further provides rice transcription factor Os05g46780 gene C DS sequences in adjusting and controlling rice grain characters
Application.Utilize transcription factor activation motif VP64（SEQ ID No.10）Constructed with rice transcription factor Os05g46780
Obtain composing type transcription factor, and the genetic transformation of the composing type transcription factor will be encoded into crops such as rice, so that
Improve the character of transgenic paddy rice seed.
Foregoing application, it is to swash the CDS sequence constructs of rice transcription factor Os05g46780 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 Os05g46780 genes are passed through into Gateway system transcription factor activation motifs VP64
The downstream of encoding gene.
The present invention utilizes transcription factor activation motif VP64 first（That is 4 transcription factor activation motif VP16）Turn with rice
Record factor Os05g46780 constructs to obtain composing type transcription factor, and the gene for encoding the composing type transcription factor is turned
Change into crops such as rice, so that rice grain character is improved, such as the increase of rice grain width.For elaborating regulation and control
Seed development mechanism has important theory value, and by transgenic approach, can improve the grain type of rice, therefore in life
Also it is of great significance in production practice.
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-Os05g46780 Vector maps.
Fig. 3 is the result that PCR detects VP64-Os05g46780 transgenic paddy rice positive strains in the embodiment of the present invention 3；Its
In, M is wild rice ' kitaake ' for DNA Marker, WT, and V2260H-06 and V2260H-62 are VP64-
Os05g46780 transgenic paddy rice strains.
Fig. 4 is the comparison of the phenotype width of 4 transgenic rice grain character of the embodiment of the present invention；Wherein, WT is wild
Type rice ' kitaake ', V2260H-06 and V2260H-62 are transgenic paddy rice strain.
Fig. 5 is the data statistic analysis result of 4 transgenic rice grain character of the embodiment of the present invention；Wherein, WT is open country
Raw type rice ' kitaake ', V2260H-06 and V2260H-62 are transgenic paddy rice strain.
Fig. 6 is the collection of illustrative plates of plant binary expression vector pCambia1301-UbiN in the embodiment of the present invention 1.
Embodiment
Following embodiments are used to illustrate the present invention, but are not limited to the scope of the present invention.Unless otherwise specified, embodiment
According to conventional laboratory conditions, such as Sambrook equimolecular Cloning: A Laboratory Manuals（New York:Gold Spring Harbor
Laboratory Press,1989）, or the condition according to manufacturer's specification suggestion.
The acquisition of 1 Os05g46780 gene C DS sequences of embodiment and the structure of plant expression vector
The acquisition of 1 Os05g46780 gene C DS sequences
In plant transcription factor database（http://rice.plantbiology.msu.edu/analyses_
search_locus.shtml）In find Os05g46780 genes, according to its CDS sequence design PCR amplification primer, forward primer
F 5 '-CAAAAAAGCAGGCTTCGGCGGTGGAGCGGAGGGAGCATT-3 ' and reverse primer R 5 '-
CAAGAAAGCTGGGTCTCATGAAGCAAAACATACTCCCGGC-3′）.It is total with wild type Nipponbare ' kitaake ' rice
CDNA is template, carries out PCR using primers F and R, obtains the complete CDS sequences of Os05g46780 genes（Seq ID No.1）.
The structure of 2 plant expression vectors
The CDS sequences of rice transcription factor Os05g46780 genes are passed through into Gateway system constructings to 4 transcription factors
Activate motif VP16 encoding genes（SEQ ID No.4）Downstream.
2.1 are cloned into above-mentioned PCR product on pDONER cloning vectors
PCR is carried out according to PrimeSTAR polymeric enzymatic amplifications system and response procedures.Comprising two-wheeled PCR during this, first
Take turns the primer gene primer for adding part adaptor attB connectors of PCR（F and R）, and the template of the second wheel is with the first round
PCR product, and the complete adaptor attB primers of primer（attB 5′adaptor：5′-
The adaptor of GTGGGGACAAGTTTGTACAAAAAAGCAGGCTTC-3 ', attB 3 '：5′-
GTGGGGACCACTTTGTACAAGAAAGCTGGGTC-3′）.PCR product 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 includes is frame sequence,
By vitro recombination, by ubi promoter-VP64-Gateway expression units, 35S promoter-asRED expression units and
35S promoter-hyg expression units construct 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.
Expression vector nVP64-hyg-asRED contains Gateway recombination systems, and plasmids of the pDONR with target gene is made
For entry vector（Entery vector）, the structure that can complete destination gene expression carrier is reacted by LR.
5 ' ends of the CDS sequence constructs of Os05g46780 genes to its target gene are connected with by VP64 codings by LR reactions
On the plant expression vector nVP64-hyg-asRED of gene, acquisition carries the coding composing type rice transcription factor VP64-
The expression vector ubi of Linker-Os05g46780 genes：VP64-Os05g46780, its complete sequence as shown in SEQ ID No.6,
Vector map is shown in Fig. 2.
LR reaction systems are as follows：
In 25 DEG C of reactions overnight.Bacillus coli DH 5 alpha, screening positive clone are converted with reaction system.
The acquisition of 2 transgenic rice plant of embodiment
Water intaking rice ' kitaake ' mature seed, manually or mechanically shells, the seed for selecting full bright and clean no bacterial plaque is sterilized
It is inoculated into afterwards on inducing culture and carries out Fiber differentiation.Selection appearance is good, and the good Rice Callus of growing power is acceptor
Material, using agrobacterium-mediated transformation by ubi：VP64-Os05g46780 is transferred in Rice Callus, with the second containing 100 μM
Acyl syringone and OD values are converted for the AAM nutrient solutions of 0.7 Agrobacterium, and the callus that conversion fluid was soaked is placed in altogether
Co-cultured on culture medium, 25 DEG C of light culture 3d, which are placed on screening and culturing medium, cultivates about 30d, per 10d subcultures once.Then
The kanamycin-resistant callus tissue sifted out is transferred on differential medium and breaks up about 20d, per 10d subcultures once.Green seedling will be differentiated
Kanamycin-resistant callus tissue, which is transferred on root media, takes root, the hardening after about 7d grows flourishing root system, and calculates conversion and obtain transgenosis
Seedling number, obtains 65 transfer-gen plants altogether.Grown in field is transferred to after hardening 7d.
Culture medium prescription involved 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
Casein+2.878g/L proline+0.5g/L glutamine+30g/L sucrose is solved, 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
Casein+2.878g/L proline+0.5g/L glutamine+30g/L sucrose is solved, 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
Plant gel 4g/L is added after 5.8~5.9.
The identification of 3 transgenic positive strain of embodiment
To detect ubi：VP64-Os05g46780 genes in T2 for the overexpression situation in transgenic paddy rice, according to carrier
ubi：VP64-Os05g46780 designs primer（Forward primer 5 '-GTGGGGACAAGTTTGTACAAAAAAGCAGGCTTC-3 ' and
Reverse primer 5 '-GTGGGGACCACTTTGTACAAGAAAGCTGGGTC-3 '）PCR detections are carried out, agar is carried out to amplified production
, there is obvious specific band in sugared gel electrophoresis.Wherein, WT represent wild rice ' kitaake ', V2260H-06 and
V2260H-62 represents VP64-Os05g46780 transgenic paddy rice strains（Fig. 3）.
4 transgenic paddy rice phenotypic analysis of embodiment and species test analysis
Transgenosis and wild type ' kitaake ' rice paddy seed are compared, it is apparent that transgenosis from phenotype
The seed of rice substantially broadens（Fig. 4）.Species test data analysis shows（Fig. 5）, the width noticeably greater than open country of transgenic paddy rice seed
Raw type rice 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. increase the encoding gene of the composing type rice transcription factor of transgenic paddy rice Kernel-Width in improvement rice grain character
In application, wherein it is described improvement rice grain character refer to increase rice grain width；
Wherein, the composing type rice transcription factor of the increase transgenic paddy rice Kernel-Width is fusion protein (VP16)4-
Linker-Os05g46780；Wherein VP16 is the VP16 albumen from herpes simplex virus, (VP16)4It is by 4 VP16 albumen
The fusion protein that is formed using Gly-Ser as interval of amino acid sequence, its amino acid sequence is as shown in SEQ ID No.10；
Linker is in series by 39 flexible amino acids, its amino acid sequence is as shown in SEQ ID No.9；Os05g46780 is rice
Transcription factor Os05g46780.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102775499A (en) * | 2012-08-01 | 2012-11-14 | 中国农业科学院作物科学研究所 | Application of rice transcription factor Os01g64730 gene |
| 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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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102775499A (en) * | 2012-08-01 | 2012-11-14 | 中国农业科学院作物科学研究所 | Application of rice transcription factor Os01g64730 gene |
| 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 |
|---|
| EEE64570.1;Yu,J.等;《GenBank》;20090205;全文 * |
| 水稻功能基因图位克隆研究进展;童继平等;《中国生物工程杂志》;20121231;第32卷(第3期);全文 * |
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