CN104211812A - Application of rice transcription factor Os02g49370 gene - Google Patents

Abstract

The invention relates to an application of a rice (oryza sativa l.) transcription factor Os02g49370 gene. According to the application, transcription factor activated sequence motifs VP64 is utilized to be fused with the rice transcription factor Os02g49370 gene to construct a combined type transcription factor, and the combined type transcription factor is transformed into crops such as rice, so as to improve rice grain traits; for example, the rice grain length and width are increased. The application has an important theoretical value in illuminating regulation and control of a seed development mechanism in detail and also can improve the rice grain type through a genetically modified method, so that the application also has important significance in production practices.

Description

The application of rice transcription factor Os02g49370 gene

Technical field

The present invention relates to genetically engineered field, specifically, relate to the application of rice transcription factor Os02g49370 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.

In recent years, about the molecular genetic regulatory mechanism of seed size proterties is studied, achieve certain progress, as: investigators utilize QTL to located some seed size genes involveds, wherein GS3 encodes a membranin, is the negative regulatory factor of seed size and organ size; The research such as Zhang Qifa finds that GS5 encodes one and regulates the serine carboxypeptidase of seed size, and be the positive regulating factor controlling seed size, process LAN GS5 can make rice grain obviously become large; Transcription factor plays very important effect in regulation and control seed size, and OsWRKY78 can regulate the elongation of rice stem and the growth of seed, and OsWRKY78 mutant can make cane become dwarfing affects Grain Development; Helix-loop-helix protein (bHLH) class transcription factor is by regulating and controlling the size of the effect length grain of lemma and glumelle cell; PGL1 bHLH protein (bHLH) heterodimer can increase seed length and weight as after the sub-process LAN of suppression of the bHLH in conjunction with DNA.

VP64 is that 4 VP16 functional domain motifs merge composition, is a class enhanser.VP16 finds in animal virus gene, has now been widely applied in plant, is mainly used in the research of the transcriptional control of plant gene.Transcription factor effect in vivo can be divided into two kinds substantially: a kind of is transcriptional enhancer, and another kind is Transcription inhibition.After transcription factor and VP16 functional domain motif merge, it will strengthen the function of transcription factor, thus in transfer-gen plant, occur more obvious character mutation.

Homeoboxes (homeodomain protein) HB family transcription factor is in animal kingdom first in fruit bat, and plant is found first in Arabidopis thaliana.There is more than 300 member in HB family, take part in biological many aspects of growing.Analyze from structure, Homeoboxes has DNA recognition site, comprises a series of structural domains combined with DNA such as TATA.Current HB family transcription factor Os02g49370 is to the research of seed size shape control mechanism and be familiar with seldom, and therefore the research of this transcription factor is in theory for the molecule mechanism understanding plant seed and allelotaxis's regulation and control further provides new clue; 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 Os02g49370 gene.

In order to realize object of the present invention, the present invention provide firstly a kind of fusion rotein, and described fusion rotein is (VP16) ₄-Linker-Os02g49370;

Wherein, Linker is in series by 39 flexible amino acid, and its sequence is as shown in SEQ ID No.9, and its nucleotide sequence is as shown in SEQ ID No.3; VP16 is the VP16 albumen from hsv; Os02g49370 is rice transcription factor Os02g49370.

The aminoacid sequence of described rice transcription factor Os02g49370 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.

Wherein, (VP16) ₄i.e. VP64, merge with Gly Ser two acids apart the enhanser formed by 4 VP16 functional domain motifs (Asp Ala Leu Asp Asp Phe Asp Leu Asp Met Leu), its sequence is as shown in SEQ ID No.10, and its nucleotide sequence is as shown in SEQ ID No.4.

The present invention also provides the gene of encoding said fusion protein, and under strict conditions, can with the nucleotide sequence of the nucleotide sequence hybridization of this gene; Wherein, described stringent condition is 65 DEG C, 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 containing encoding said fusion protein.

The construction process of described carrier is as follows:

(1) in plant transcription factor database, Os02g49370 gene is found, according to its sequences Design pcr amplification primer pair, it is forward primer F:5 '-CAAAAAAGCAGGCT TCATGGAGGCCGGCTACCC-3 ' and reverse primer R:5 '-CAAGAAAGCTGGGTC CTTGTATCCGAACGGATGCTG-3 ';

(2) with the total cDNA of the fine paddy rice of wild Japanese for template, carry out PCR and obtain Os02g49370 complete sequence;

(3) PCR primer is cloned on connection pDONER cloning vector, obtains the identical sequence with goal gene through order-checking qualification;

(4) sequence comprised with binary expression vector right boundary is for frame sequence, pass through vitro recombination, ubi promoter-VP64-Gateway is expressed unit, 35S promoter-asRED expresses unit and 35S promoter-hyg expression unit constructs with it, obtains the complete sequence of carrier nVP64-hyg-asRED as shown in SEQ ID No.5;

(5) reacting by LR N end Os02g49370 being building up to its order ground gene has merged on the plant expression vector nVP64-hyg-asRED of VP64 label, and obtain carrier ubi:VP64-Os02g49370, carrier complete sequence is as shown in SEQ ID NO.6.

The expression vector carrying the gene of encoding said fusion protein 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, 2nd Edition).

The present invention also provides the engineering bacteria of the gene containing encoding said fusion protein.

The present invention also provides a kind of construction process of transgenic rice plant, be specially, adopt agriculture bacillus mediated method, aforementioned bearer is proceeded in Rice Callus, transform with the AAM conversion fluid 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 encoding said fusion protein in improvement rice grain proterties (as increased the wide and thousand seed weight of paddy rice grain length, grain)

Present invention also offers the primer pair for amplifying rice transcription factor Os02g49370 gene, it is forward primer F:5 '-CAAAAAAGCAGGCTTCATGGAGGCCGG CTACCC-3 ' and reverse primer R:5 '-CAAGAAAGCTGGGTC CTTGTATCC GAACGGATGCTG-3 '.

The present invention also provides the application of rice transcription factor Os02g49370 gene in adjusting and controlling rice grain characters further.

Aforesaid application is by the CDS sequence of rice transcription factor Os02g49370 gene by the downstream of Gateway system constructing to 4 transcription factor activation motif VP16, rice transformation, thus the proterties of improvement transgenic paddy rice seed.

The present invention utilizes transcription factor activation motif VP64(i.e. 4 transcription factor activation motif VP16 first) build obtain composing type transcription factor with rice transcription factor Os02g49370 gene fusion, and be transformed in paddy rice, thus improvement rice grain proterties, 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 nVP64-hyg-asRED Vector map in the embodiment of the present invention 1.

Fig. 2 is ubi:VP64-Os02g49370 Vector map in the embodiment of the present invention 1.

Fig. 3 is that Western blot of the present invention detects VP64-Os02g49370 transgenic positive strain, and wherein WT is wild rice ' kitaake ', and V0476H-05, V0476H-12 are VP64-Os02g49370 transgenic paddy rice strain.

Fig. 4 is the comparison of the phenotype length of transgenic line grain characters of the present invention, and wherein WT is wild rice ' kitaake ', and V0476H-05, V0476H-12 are transgenic paddy rice strain.

Fig. 5 is the comparison of the phenotype width of transgenic line grain characters of the present invention, and wherein WT is wild rice ' kitaake ', and V0476H-05, V0476H-12 are transgenic paddy rice strain.

Fig. 6 is transgenic line grain characters data statistic analysis of the present invention, and wherein WT is wild rice ' kitaake ', and V0476H-05, V0476H-12 are transgenic paddy rice strain.

Embodiment

Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.

The separation of embodiment 1Os02g49370 gene and plant expression vector construction

Os02g49370 gene is found in plant transcription factor database, according to its sequences Design pcr amplification primer, according to its sequences Design pcr amplification primer (F1:5 '-CAAAAAAGCAGGCTTCATGGAGGCCGGCTACCC-3 ' and reverse primer R1:5 '-CAAGAAAGCTGGGTC CTTGTATCCGAACGGATGCTG-3 ').With the total cDNA of the wild-type fine paddy rice of Japan for template, carry out PCR and obtain Os02g49370 complete sequence, its nucleotide sequence is as shown in SEQ ID NO.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 (F1 and R1) adding part adaptor attB joint of first round PCR, and the second template of taking turns PCR primer of the first round, and the complete adaptor attB primer (attB5 ' adaptor:5 '-GTGGGGACAAGTTTG TACAAAAAAGCAGGCTTC-3 ', attB3 ' adaptor:5 '-GTGGGGACCAC TTTGTACAAGAAAGCTGGGTC-3 ') of primer.PCR primer is cloned into and connects on pDONER cloning vector (purchased from Invitrogen), obtain the identical sequence with goal gene through order-checking qualification.By LR reaction, Os02g49370 is building up to nVP64-hyg-asRED(Fig. 1, carrier complete sequence is as shown in SEQ ID No.5) on, obtain carrier ubi:VP64-Os02g49370(Fig. 2, carrier complete sequence is as shown in SEQ ID No.6).

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:VP64-Os02g49370, transform with the AAM conversion fluid being the Agrobacterium of 0.7 containing the Syringylethanone of 100 μMs and O.D. 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 calculating conversion institute obtains transgenic seedling number.Grown in field is transferred to after hardening 7d.

Wherein, inducing culture based formulas is: a large amount of+B5 trace+NB of N6 is organic+and 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, adds plant gel 4g/L after adjusting pH to 5.8 ~ 5.9.

Dual culture based formulas is: a large amount of+B5 trace+NB of N6 is organic+and 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.

Screening and culturing based formulas is: a large amount of+B5 trace+NB of N6 is organic+and 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) is added after sterilizing.

Differentiation culture based formulas is: MS inorganic+MS-B5 trace+MS is organic+and 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, after adjusting pH to 5.8 ~ 5.9, add plant gel 4g/L.

The qualification of embodiment 3 transgenic positive strain

For detecting ubi:VP64-Os02g49370 gene at T2 for the process LAN situation in transgenic paddy rice, VP64 antibody is utilized to identify on protein level it, through SDS-PAGE protein electrophoresis → immunoblotting analysis → Immunofluorescence Reactions, Western Blot qualification result shows that transfer-gen plant exists target protein, the band (Fig. 3) and wild-type is not mixed out.

Concrete Western Blot experiment flow is as follows:

Appropriate amount of sample is put into the freezing rear grind into powder of liquid nitrogen, add the mixing of appropriate sample-loading buffer, 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, the bottom arriving gel when tetrabromophenol sulfonphthalein can stop electrophoresis; Adopt half-dried transfer method transferring film after electrophoresis, and with ponceau staining fluid, film is dyeed, observe transferring film effect; After transferring film, film is put into containing 5% skim-milk PBST solution close room temperature close within 60 minutes or 4 DEG C, spend the night; Under room temperature, primary antibodie (VP64 antibody) is hatched 1 hour or 4 DEG C of overnight incubation, then washs 3 times with PBST, each 5 minutes; Two anti-under room temperature (goat-anti rabbit, purchased from Abmart, article No. M21002S) hatch 1 hour, then wash 3 times with PBST, each 5 minutes; Film adds substrate, exposes.

Wherein, VP64 antibody is that the rabbit source of being prepared as specific antigen according to the aminoacid sequence improvement on synthesis of VP64 by Ai Bimate biological medicine (Shanghai) Co., Ltd. resists more.

Embodiment 4 transgenic paddy rice phenotype analytical and species test analysis

Transgenosis and wild rice seed are compared, can significantly find out phenotype, find transgenic line seed obviously elongated, broaden (Fig. 4, Fig. 5).Species test data analysis shows (Fig. 6), and the length and width of transgenic paddy rice seed is all 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 (10)

1. a fusion rotein, is characterized in that, described fusion rotein is (VP16) ₄-Linker-Os02g49370;

Wherein, Linker is in series by 39 flexible amino acid; VP16 is the VP16 albumen from hsv; Os02g49370 is rice transcription factor Os02g49370.

2. fusion rotein as claimed in claim 1, it is characterized in that, the aminoacid sequence of described rice transcription factor Os02g49370 is as shown in SEQ ID No.2.

3. the gene of fusion rotein described in any one of coding claim 1-2.

4. the carrier containing gene described in claim 3.

5. the engineering bacteria containing gene described in claim 3.

6. the construction process of a transgenic rice plant, it is characterized in that, adopt agriculture bacillus mediated method, carrier according to claim 4 is proceeded in Rice Callus, transform with the AAM conversion fluid 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.

7. the application of gene according to claim 3 in improvement rice grain proterties.

8., for the primer pair of amplifying rice transcription factor Os02g49370 gene, it is forward primer F:5 '-CAAAAAAGCAGGCTTCATGGAGGCCGGCTACCC-3 ' and reverse primer R:5 '-CAAGAAAGCTGGGTC CTTGTATCCGAACGGAT GCTG-3 '.

9. the application of rice transcription factor Os02g49370 gene in adjusting and controlling rice grain characters.

10. apply as claimed in claim 9, it is characterized in that, it is by the CDS sequence of rice transcription factor Os02g49370 gene by the downstream of Gateway system constructing to 4 transcription factor activation motif VP16, rice transformation, thus the proterties of improvement transgenic paddy rice seed.