CN103214581A - Application of synthetic transcription factor VP64-Os03g57670 - Google Patents
Application of synthetic transcription factor VP64-Os03g57670 Download PDFInfo
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Abstract
The invention provides an application of a synthetic transcription factor VP64-Os03g57670. The synthetic transcription factor is constructed by fusing a transcription factor activated sequence motif VP64 (namely four transcription factor activated sequence motifs VP16) and paddy transcription factor Os03g57670 gene, and is converted to the paddy, so as to change the grain characteristic of paddy, so that the transgenic paddy seed is remarkably shorter, wider, and lighter in thousand seed weight, and paddy blade is crimped, therefore, the paddy has a better market application prospect.
Description
Technical field
The invention belongs to field of genetic engineering, particularly, relate to the application of rice transcription factor Os03g57670 gene and the application of synthesis type transcription factor VP64-Os03g57670.
Background technology
Paddy rice (Oryza sativa L.) is China and whole world important crops, also be the model plant of an important function gene studies and the staple food of the world's 1/2 above population, relative genetics and molecular biology research be extremely investigator's attention always, and the regulation and control of transcriptional level are the important way of gene expression regulation.From molecular biological angle, growth and development of plant be one orderly, optionally genetic expression process.And transcription factor has played critical effect in the accuracy controlling of gene.
LOB(Lateral Organ Boundaries) family's transcription factor has important adjusting function in the adnation tissue development.The LOB gene is expressed at the paraxial base portion of all adnation tissues that form from the meristem of seedling top, and expresses at the base portion of lateral root.Finding in the Arabidopis thaliana has 43 LOB domian genes, is divided into 2 classifications and three subclass.LBD gene A S2 (ASYMMETRIC LEAVES2) in the Arabidopis thaliana is found in the foundation of vein network system and works, main lobe arteries and veins in comprising, the growth of symmetrical blading.The LBD gene family has about 100 amino acid whose conservative regions, and 4 conservative halfcystine great majority are intended, and southern mustard LBD gene protein sequence comprises a coiling coil structures, and 4 leucine LXLXLXL sequences are wherein arranged.
The albumen of LOB genes encoding is a plant specific, comprises a conservative region (LOB domain), and this conservative region is present in other 42 arabidopsis thaliana proteins, also is present among the other plant species simultaneously.The LOB gene is the Arabidopis thaliana growth needs under particular environment, but the arabidopsis mutant body that lacks the LOB gene does not have obvious phenotypes under standard conditions, may have the functional redundancy phenomenon.There is the ectopic expression of research report LOB gene to cause size, the shape of blade and flower to change, can causes male and female sterile.
The LOB gene is divided into two subspecies in the paddy rice, and wherein a member ARL1 is necessary to the formation of adventitious root primordia in the oryza sativa l. BD gene family, and it plays crucial effects in the forming process of adventive root, though the expression of ARL1 not only is confined to adventive root.These researchs provide good reference for the function that we further study oryza sativa l. OB gene family.At present there is preliminary research in LOB family to the growth of leaf, but to seed size shape Study of Mechanism and understanding seldom.
Expressing transcription factor by simple mistake is the approach commonly used that changes the transgenic line proterties.Yet because a little less than some transcription factor effect, the simple expression excessively often can not cause transgene receptor to show significant proterties change.VP64 is that 4 VP16 transcriptional activation function territory motifs merge composition, is a class enhanser.VP16 finds in the animal virus gene, now has been applied to widely in the research of transcribing control that is mainly used in plant gene in the plant.After transcription factor and the fusion of VP16 functional domain motif, it will strengthen the activated gene expressive function of transcription factor, occurs more obvious phenotype easily and change in transfer-gen plant.
Summary of the invention
The object of the present invention is to provide the application of rice transcription factor Os03g57670 gene and the application of synthesis type transcription factor VP64-Os03g57670.The Os03g57670 gene belongs to LOB family.
In order to realize the object of the invention, the present invention at first provides a kind of fusion rotein, and this fusion rotein is (VP16)
n-Linker-Os03g57670; Wherein, n is 〉=1 integer; Linker is in series by 1～50 flexible amino acid.The aminoacid sequence of linker is INYPYDVPDYAGSYPYDVPDYAAQCSGTELTSLYKKAGF in the embodiment of the invention, and its nucleotide sequence is SEQ ID No.3; VP16 is the VP16 albumen from hsv (Herpes simplex virus); Os03g57670 is rice transcription factor Os03g57670, its CDS consecutive nucleotides sequence is shown in SEQ ID No.1, its aminoacid sequence is shown in SEQ ID No.2, or this sequence is through replacing, lack or adding one or several amino acids formed aminoacid sequence with same function.
The invention provides the primer of the Os03g57670 gene that is used to increase,
It comprises forward primer F1:5'-CAAAAAAGCAGGCTTC ATGTCCCCCGGTGAGGAGGACGTGG-3' and reverse primer R1:5'-CAAGAAAGCTGGGTC AGGAAACGAGAATTCAGATGCGAAG-3'.
Preferably, n is 4 in the aforementioned fusion rotein.
Wherein, (VP16)
4Be VP64, merge the enhanser of forming by 4 VP16 functional domain motifs, its nucleotide sequence is shown in SEQID No.4, or this sequence is through replacing, lack or add the nucleotide sequence with same function of one or several base formation.
More preferably, aforementioned fusion rotein is (VP16)
4-Linker-Os03g57670.
The present invention also provides the gene of encoding said fusion protein, and under stringent condition, can with the nucleotide sequence of the nucleotide sequence hybridization of this gene; Wherein, described stringent condition is 65 ℃, hybridization 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 that contains encoding said fusion protein.The carrier that described carrier is expressed in the host for any bootable foreign gene.Preferably, described carrier be plant binary expression vector (for example, pCAMBIA1301).When encoding said fusion protein of the present invention gene constructed is in plant expression vector, can before its transcription initiation Nucleotide, add any strong promoter (for example, corn strong promoter Ubiquitin) or inducible promoter.In addition, when encoding said fusion protein of the present invention gene constructed is in plant expression vector, can also use enhanser, and these enhanser zones must be identical with the reading frame of encoding sequence, to guarantee the translation of whole piece sequence.
The present invention also provides the transgenic cell line of the gene that contains encoding said fusion protein.
The present invention also provides the engineering bacteria of the gene that contains encoding said fusion protein.
The present invention also provides the gene of encoding said fusion protein postponing the paddy rice heading, prolonging the application in breeding time.
The present invention further provides the application of rice transcription factor Os03g57670 gene, it is that the CDS sequence of rice transcription factor Os03g57670 gene (complete translation district) is building up to the upstream that 4 transcription factors activate motif VP16, transform plant, screening and final acquisition transgenic plant.The CDS sequence is shown in SEQ ID NO.1.
Aforesaid application, it is the upstream of the CDS sequence of rice transcription factor Os03g57670 gene being activated motif VP16 by Gateway system constructing to 4 transcription factor, rice transformation (for example, rice varieties ' kitaake '), thus postpone transgenic paddy rice heading stage, prolong breeding time.Wherein, the CDS sequence of rice transcription factor Os03g57670 gene is shown in SEQ IDNo.3, and 4 transcription factors activate the nucleotide sequence of motif VP16 shown in SEQ ID No.4.
The expression carrier that carries encoding said fusion protein can import (Weissbach in the vegetable cell by using conventional biotechnological meanss such as Ti-plasmids, plant viral vector, directly DNA conversion, microinjection, electroporation, 1998, Method for Plant Molecular Biology VIII, Academy Press, New York, the 411-463 page or leaf; Geiserson and Corey, 1998, Plant Molecular Biology, 2
NdEdition).
The present invention utilizes transcription factor to activate i.e. 4 the transcription factors activation motif VP16 of motif VP64(first) obtain the composing type transcription factor with rice transcription factor Os03g57670 gene fusion structure,
The present invention clones the CDS sequence of an oryza sativa l. OB transcription factor family gene Os03g57670 first, and (its nucleotide sequence is shown in SEQ ID NO.1, and the cDNA that VP64 activates motif and this gene merged, made up VP64-Os03g57670 synthesis type transcription factor.And be transformed into farm crop, in paddy rice, find that by rice conversion this synthesis type transcription factor can cause the remarkable change of rice grain proterties, improved the ability of coercing in the high salt adverse circumstance of arid of transgenic paddy rice, and the render transgenic rice paddy seed obviously shortens, broadens, thickening, thousand seed weight reduce, and has market using value preferably.This research provides new clue for the molecule mechanism of in theory further understanding plant seed and allelotaxis regulation and control, and provides a feasible way for high-yield breeding of crops in practice.
Description of drawings
Fig. 1 is a nVP64-hyg-asRED carrier collection of illustrative plates among the embodiment 1.
Fig. 2 is a ubi:VP64-Os01g63510 carrier collection of illustrative plates among the embodiment 1.
Fig. 3 is for detecting the PCR result of transgenic positive rice strain, and wherein, WT is wild-type paddy rice ' kitaake ', and UBI:2602 is a VP64-Os03g57670 transgenic paddy rice strain system.
Fig. 4 is the phenotype that transgenosis VP64-Os03g57670 rice strain changes grain characters, and wherein WT is wild-type paddy rice ' kitaake ', and TF2602 is a VP64-Os03g57670 transgenic paddy rice strain system.
Fig. 5 is the phenotype of transgenosis VP64-Os03g57670 rice strain leaf rolling, and wherein WT is wild-type paddy rice ' kitaake ', and TF2602-7, TF2602-10 are VP64-Os03g57670 transgenic paddy rice strain system.Wherein Fig. 5 a, Fig. 5 b, Fig. 5 c are respectively the comparison of seed width, length and size.
Fig. 6 binds fruit for Western blot detects the transgenic positive strain, and wherein WT is wild-type paddy rice ' kitaake ', and 2602-7,2602-10 are VP64-Os03g57670 transgenic paddy rice strain system.
(4 * VP16) activate the analysis of statistical data of rice grain length proterties species test in the Os03g57670 transgenic paddy rice strain system to Fig. 7 for VP64 among the present invention, the length that demonstrates the transgenic line seed shortens, wherein WT is wild-type paddy rice ' kitaake ', and TF2602-7, TF2602-10 are VP64-Os03g57670 transgenic paddy rice strain system.
(4 * VP16) activate the analysis of statistical data of rice grain proterties width species test in the Os03g57670 transgenic paddy rice strain system to Fig. 8, and the width that demonstrates the transgenic line seed increases, and illustrates same Fig. 7 for VP64 among the present invention.
(4 * VP16) activate the analysis of statistical data of rice grain thousand seed weight in the Os03g57670 transgenic paddy rice strain system to Fig. 9, demonstrate the proterties result of the thousand seed weight minimizing of transgenic line seed, illustrate same Fig. 7 for VP64 among the present invention.
Embodiment
Following examples are used to illustrate the present invention, but are not used for limiting the scope of the invention.Without departing from the spirit and substance of the case in the present invention, modification or replacement to the inventive method, step or condition are done all belong to scope of the present invention.
If do not specialize, used chemical reagent is conventional commercial reagent among the embodiment, the conventional means that used technique means is well known to those skilled in the art among the embodiment.
The separation and the plant expression vector construction of embodiment 1Os03g57670 gene
In plant transcription factor database (http://planttfdb.cbi.edu.cn/index.php sp=Osj), find the Os03g57670 gene, according to its sequences Design pcr amplification primer (forward primer F1:5'-CAAAAAAGCAGGCTTCATGTCCCCCGGTGAGGAGGACGTGG-3' and reverse primer R1:
5'-CAAGAAAGCTGGGTCAGGAAACGAGAATTCAGATGCGAAG-3'。) be template with the total cDNA of the fine paddy rice of wild Japanese, carry out the CDS sequence that PCR obtains the Os03g57670 gene, its nucleotide sequence is shown in SEQ ID NO.1.
Carry out PCR according to PrimeSTAR polymeric enzymatic amplification system and response procedures.Comprise two-wheeled PCR in this process, the primer of the first round PCR gene primer (F1 and R1) that adds part adaptor attB joint, and second template of the taking turns PCR product of the first round, and primer is with complete adaptor attB primer (attB5'adaptor:5'-GTGGGGACAAGTTTGTACAAAAAAGC AGGCTTC-3', attB3'adaptor:5'-GTGGGGACCACTTTGTACAAGAAA GCTGGGTC-3').The PCR product cloning to connecting on the pDONER cloning vector (available from Invitrogen), is identified through order-checking to obtain and the identical sequence of goal gene.Obtain and the identical sequence of goal gene through the order-checking evaluation.By the LR reaction Os03g57670 is building up to cVP64-hyg-asRED(Fig. 1, the carrier complete sequence is shown in SEQ ID No.5) on, obtain carrier ubi:VP64-Os03g57670(Fig. 2, the carrier complete sequence is shown in SEQ ID No.6).Wherein, the building process of plant expression vector cVP64-hyg-asRED is: the sequence that comprises with border, the binary expression vector pCAMBIA1300 left and right sides is a frame sequence, by vitro recombination, ubi promoter-Gateway-VP64 is expressed unit, 35S promoter-asRED express unit and 35S promoter-hyg and express the unit and construct with it and obtain.
The acquisition of embodiment 2 transgenic rice plants
Water intaking rice ' kitaake ' mature seed, artificial or mechanical dejacketing, the seed of selecting full bright and clean no bacterial plaque is inoculated into after sterilization and carries out inducing culture on the inducing culture.The selection outward appearance is good, the rice callus that growing ability is good is organized as acceptor material, adopt agrobacterium-mediated transformation that ubi:VP64-Os03g57670 is changed in the rice callus tissue, the AAM conversion fluid that with Syringylethanone that contains 100 μ M and O.D. value is 0.7 Agrobacterium transforms, the callus that conversion fluid was soaked places and carries out common cultivation on the common substratum, 25 ℃ of dark 3d of cultivation are placed on and cultivate about 30d on the screening culture medium, and every 10d subculture once.Then the kanamycin-resistant callus tissue that sifts out is transferred to the about 20d of differentiation on the division culture medium, every 10d subculture once.The kanamycin-resistant callus tissue that differentiates green seedling transferred on the root media take root, treat that about 7d grows hardening behind the flourishing root system, and calculate and transform the transgenic seedling number that obtains.Be transferred to grown in field behind the hardening 7d.
Wherein, the inducing culture based formulas is: and N6 is a large amount of+and B5 trace+NB 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 the water preparation, transfers pH to 5.8～5.9 backs to add plant gel 4g/L.
Altogether culture medium prescription is: and N6 is a large amount of+and B5 trace+NB is organic+molysite+2.5mg/L2,4D+0.5g/L glutamine+0.6g/L acid hydrolyzed casein+10g/L glucose+30g/L sucrose with the water preparation, transfers pH to 5.2 back to add plant gel 4g/L.After the sterilization, add the AS(Syringylethanone about 50 ℃) 100～200 μ g/mL.
The screening and culturing based formulas is: and N6 is a large amount of+and B5 trace+NB 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 the water preparation, transfer pH to 5.8～5.9 backs to add plant gel 4g/L.The sterilization back adds 35mg/L Totomycin (available from Shanghai Niu Jin Bioisystech Co., Ltd).
The differentiation culture based formulas is: and MS is inorganic+and MS-B5 trace+MS is organic+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 the water preparation, transfer pH to 5.8 back to add plant gel 4g/L.The evaluation of embodiment 3 transgenic paddy rices
Whether be integrated into oryza sativa genomic dna in order to detect the Os03g57670 gene, extracted the transgenic paddy rice leaf DNA of wild-type (' kitaake ') and the present invention preparation, the a pair of primer (VP64-1F:5'-ATGGACGCGCTGGACGATTT-3' of plant expression vector design from the gene two ends, 2602-R:5'-AGGAAACGAGAATTCAG-3') carry out PCR and identify transfer-gen plant according to PrimeSTAR polymeric enzymatic amplification system and response procedures, found that by PCR, transgenic paddy rice all amplifies purpose band (780bp), and do not have this purpose band in the wild-type paddy rice, the results are shown in Figure 3.Preliminary definite Os03g57670 gene is inserted in the rice genome by agrobacterium mediation method, and the PCR product is a target gene sequences through the sequencing result analysis.
For detect the ubi:VP64-Os03g57670 gene at T2 for the expression of crossing in the transgenic paddy rice, utilize VP64 antibody that it is identified on protein level, through the SDS-PAGE protein electrophoresis → immune marking → Immunofluorescence Reactions, Western Blot qualification result shows that there is target protein in transfer-gen plant, the band (Fig. 4) and wild-type is not mixed out.
Concrete Western Blot experiment flow is as follows:
An amount of sample is put into the freezing back of liquid nitrogen grind into powder, add an amount of sample-loading buffer mixing, centrifugal 10 minutes of 12000rpm; Get 5 μ l supernatant application of samples, after 30 minutes, 120V electrophoresis 60-90 minute is when the bottom of tetrabromophenol sulfonphthalein arrival gel can stop electrophoresis with 90V voltage SDS-PAGE electrophoresis; Adopt half-dried transfer method to change film behind the electrophoresis, and film is dyeed, observe and change the film effect with the ponceau staining fluid; After changeing film and finishing, film is put into the PBST solution sealing room temperature sealing that contains 5% skim-milk spent the night in 60 minutes or 4 ℃; Room temperature next anti-(VP64 antibody) was hatched 1 hour or 4 ℃ of overnight incubation, then with PBST washing 3 times, each 5 minutes; Two anti-under the room temperature (the goat-anti rabbit, available from Abmart, article No. M21002S) hatched 1 hour, then with PBST washing 3 times, each 5 minutes; On film, add substrate, expose.
Wherein, VP64 antibody is how anti-as the rabbit source that specific antigen prepares according to the synthetic polypeptide of the aminoacid sequence of VP64 by Ai Bimate biological medicine (Shanghai) Co., Ltd..
Embodiment 4 transgenic paddy rice phenotype analyticals and species test analysis:
Transgenic paddy rice seed and wild-type (' kitaake ') rice paddy seed that the present invention is obtained compare, and can significantly find out that transgenic seed significantly shortens, broadens, thousand seed weight reduces on the phenotype, see Fig. 5 a, Fig. 5 b, Fig. 5 c, Fig. 7-9.Data analysis result by species test can find out significantly that the transgenic paddy rice seed proterties compares with wild-type and have extremely significant difference and change.Fig. 6 shows that transgenic paddy rice leaf rolling phenomenon that the present invention obtains is obviously in the rice leaf of wild-type contrast.
Though 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 all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (10)
1. a fusion rotein is characterized in that, this fusion rotein is Os03g57670-Linker-(VP16)
nOr (VP16)
n-Linker-Os03g57670; Wherein, n is 〉=1 integer; Linker is in series by 1～50 flexible amino acid; VP16 is the VP16 albumen from hsv (Herpes simplex virus); Os03g57670 is rice transcription factor Os03g57670.
2. fusion rotein according to claim 1 is characterized in that, n is 4.
3. fusion rotein according to claim 2 is characterized in that, this fusion rotein is (VP16)
4-Linker-Os03g57670.
4. the gene of each described fusion rotein of coding claim 1-3.
5. the carrier that contains the described gene of claim 4.
6. the transgenic cell line that contains the described gene of claim 4.
7. the engineering bacteria that contains the described gene of claim 4.
8. the described gene of claim 4 is being postponed the paddy rice heading, is being prolonged the application in breeding time.
9. the application of rice transcription factor Os03g57670 gene, it is characterized in that, it is the upstream of CDS sequence construct to 4 transcription factor of rice transcription factor Os03g57670 gene being activated motif VP16, transform plant, screening and final acquisition transgenic plant, described CDS sequence is shown in SEQ ID NO.1.
10. application according to claim 9, it is characterized in that, it is the upstream of the CDS sequence of rice transcription factor Os03g57670 gene being activated motif VP16 by Gateway system constructing to 4 transcription factor, rice transformation, thereby the render transgenic rice grain broadens and shortens, and rice leaf is curled.
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| CN104341507A (en) * | 2013-07-31 | 2015-02-11 | 中国农业科学院作物科学研究所 | Application of oryza sativa transcription factor Os01g62514.1 gene CDS sequence |
| CN104341510B (en) * | 2013-07-31 | 2017-09-05 | 中国农业科学院作物科学研究所 | The application of rice transcription factor Os04g55590.1 gene C DS sequences |
| CN104341522A (en) * | 2013-08-02 | 2015-02-11 | 中国农业科学院作物科学研究所 | Application of oryza sativa transcription factor Os05g27980 gene CDS sequence |
| CN104341524A (en) * | 2013-08-02 | 2015-02-11 | 中国农业科学院作物科学研究所 | Application of oryza sativa transcription factor Os03g55990 gene CDS sequence |
| CN104341527A (en) * | 2013-08-09 | 2015-02-11 | 中国农业科学院作物科学研究所 | Application of CDS (coding sequence) of gene for coding oryza sativa L. transcription factor Os03g12350 |
| CN104341527B (en) * | 2013-08-09 | 2017-09-05 | 中国农业科学院作物科学研究所 | The application of rice transcription factor Os03g12350 gene C DS sequences |
| CN113430210A (en) * | 2021-06-09 | 2021-09-24 | 吉林大学 | Corn side organ boundary protein gene ZmLBD24 and application |
| CN114807221A (en) * | 2022-04-28 | 2022-07-29 | 宁波大学科学技术学院 | Mutant, protein and expression vector of rice root elongation regulatory gene OsDRP1C and application thereof |
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