CN104341522A - Application of oryza sativa transcription factor Os05g27980 gene CDS sequence - Google Patents
Application of oryza sativa transcription factor Os05g27980 gene CDS sequence Download PDFInfo
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- CN104341522A CN104341522A CN201310334913.2A CN201310334913A CN104341522A CN 104341522 A CN104341522 A CN 104341522A CN 201310334913 A CN201310334913 A CN 201310334913A CN 104341522 A CN104341522 A CN 104341522A
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Abstract
The invention relates to the application of a oryza sativa transcription factor Os05g27980 gene CDS sequence. According to the invention, a transcription factor activation motif VP64 is fused with the oryza sativa transcription factor Os05g27980, 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 width 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 genetically engineered field, specifically, relate to the application of rice transcription factor Os05g27980 gene C DS sequence.
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.
LOB(Lateral Organ Boundaries) family's transcription factor has important adjusting function in the raw tissue development in side.LOB gene is expressed at the paraxial base portion of the raw tissue in all sides formed from seedling apical meristem, and expresses at the base portion of side root.Find that there is 43 LOB domian genes in Arabidopis thaliana, be divided into 2 classifications and three subclass.LBD Gene A S2 (ASYMMETRIC LEAVES2) in Arabidopis thaliana is found in the foundation of vein network system and works, and comprises middle main lobe arteries and veins, the growth of symmetrical blading.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, wherein has 4 leucine LXLXLXL sequences.
The albumen of LOB genes encoding is that a plant is distinctive, comprises a conservative region (LOB domain), and this conservative region is present in other 42 arabidopsis thaliana proteins, is also present among other plant species simultaneously.LOB gene is Arabidopis thaliana growth needs under particular circumstances, but the Arabidopsis Mutants of disappearance LOB gene does not have obvious phenotype at the standard conditions, may there is functional redundancy phenomenon.Studies have reported that the ectopic expression of LOB gene causes the size of blade and flower, shape changes, and can cause male and female sterile.
In paddy rice, LOB gene is divided into two subspecies, and wherein in oryza sativa l. BD gene family, the formation of a member ARL1 to adventitious root primordia is necessary, and it plays vital effect in the forming process of adventive root, although the expression of ARL1 is not only confined to adventive root.These are studied as our function of further Study On Rice LOB gene family provides good reference.The growth of current LOB family to leaf has preliminary research, but to the research of seed size shape control mechanism and understanding seldom.
Summary of the invention
The object of this invention is to provide the application of rice transcription factor Os05g27980 gene C DS sequence.
In order to realize the object of the invention, first the present invention provides a kind of composing type rice transcription factor, i.e. fusion rotein (VP16)
4-Linker-Os05g27980.
Wherein, VP16 is the VP16 albumen from hsv, is merged by 4 VP16 functional domain motifs and can form a class enhanser, strengthens the function of transcription factor, thus in transfer-gen plant, occurs more obvious character mutation.(VP16) that relate in above-mentioned fusion rotein
4, namely VP64 is the fusion rotein of gap-forming with Gly-Ser by the aminoacid sequence of 4 VP16 albumen, and its aminoacid sequence and nucleotide sequence are respectively as shown in SEQ ID No.10 and SEQ ID No.4.
The Linker related in above-mentioned fusion rotein is in series by 39 flexible amino acid, and its aminoacid sequence is as shown in SEQ ID No.9, and the nucleotide sequence of this Linker that encodes is as shown in SEQ ID No.3.
The Os05g27980 related in above-mentioned fusion rotein is rice transcription factor Os05g27980, and its aminoacid sequence 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; The CDS sequence of rice transcription factor Os05g27980 gene is the nucleotide sequence shown in SEQ ID No.1.
The present invention also provides the gene of described composing type rice transcription factor of encoding, and under strict conditions, with the nucleotide sequence of this gene, the nucleotide sequence of hybridizing can occur.
The present invention also provides the carrier of gene, engineering bacteria and clone containing the described composing type rice transcription factor of coding.
The construction process of described carrier is as follows:
(1) in plant transcription factor database (http://rice.plantbiology.msu.edu/analyses_search_locus.shtml), Os05g27980 gene is found, according to its sequences Design pcr amplification primer pair, it is forward primer F:5'-CAAAAAAGCAGGCTTCATGGGCGGTGGGTC-3' and reverse primer R:5'-CAAGAAAGCTGGGTCCTACGTCCAGAGCGA-3'.
(2) with the total cDNA of wild Japanese fine ' kitaake ' paddy rice for template, utilize above-mentioned primers F and R to carry out PCR, obtain the CDS sequence that Os05g27980 gene is complete.
(3) above-mentioned PCR primer is cloned on pDONER cloning vector, obtains the identical sequence with goal gene through order-checking qualification.
(4) with plant binary expression vector pCambia1301-UbiN(Fig. 6) sequence that comprises of 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) by LR reaction by the CDS sequence construct of Os05g27980 gene to its goal gene 5 ' hold be connected with on the plant expression vector nVP64-hyg-asRED of VP64 encoding gene, obtain the expression vector ubi:VP64-Os05g27980 carrying described composing type rice transcription factor VP64-Linker-Os05g27980 gene of encoding, its complete sequence is as shown in SEQ ID No.6.
Above-mentioned expression vector 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 a kind of construction process of transgenic rice plant, be specially: adopt agriculture bacillus mediated method, above-mentioned expression vector is proceeded in Rice Callus, transform with the AAM nutrient solution 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 described composing type rice transcription factor of encoding in improvement rice grain proterties (such as increasing rice grain wide).
Present invention also offers the primer pair for amplifying rice transcription factor Os05g27980 gene C DS sequence, comprise forward primer F:5'-CAAAAAAGCAGGCTTCATGGGCGGTGGGTC-3' and reverse primer R:5'-CAAGAAAGCTGGGTCCTACGTCCAGAGCGA-3'.
The present invention further provides the application of rice transcription factor Os05g27980 gene C DS sequence in adjusting and controlling rice grain characters.Utilize transcription factor activation motif VP64(SEQ ID No.10) construct obtain composing type transcription factor with rice transcription factor Os05g27980, and by the gene transformation of the described composing type transcription factor of coding to farm crop as in paddy rice, thus the proterties of improvement transgenic paddy rice seed.
Aforesaid application is by the downstream of the CDS sequence construct of rice transcription factor Os05g27980 gene to transcription factor activation motif VP64 encoding gene (SEQ ID No.4), rice transformation, thus the proterties of improvement transgenic paddy rice seed.Preferably by the downstream of the CDS sequence of rice transcription factor Os05g27980 gene by Gateway system transcription factor activation motif VP64 encoding gene.
The present invention utilizes transcription factor activation motif VP64(i.e. 4 transcription factor activation motif VP16 first) construct obtain composing type transcription factor with rice transcription factor Os05g27980, and by coding described composing type transcription factor gene transformation to farm crop as in paddy rice, thus improvement rice grain proterties, such as rice grain width increases.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-Os05g27980 Vector map in the embodiment of the present invention 1.
Fig. 3 is that in the embodiment of the present invention 3, PCR detects VP64-Os05g27980 transgenic positive strain, and wherein WT is wild rice ' kitaake ', and V2605H-01, V2605H-11 are VP64-Os05g27980 transgenic paddy rice strain.
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 rice ' kitaake ', and V2605H-01, V2605H-11 are VP64-Os05g27980 transgenic paddy rice strain.
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 rice ' kitaake ', and V2605H-01, V2605H-11 are VP64-Os05g27980 transgenic paddy rice strain.
Fig. 6 is pCambia1301-UbiN Vector map in the embodiment of the present invention 1.
Embodiment
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.If do not specialize, the conventional means that technique means used in embodiment is well known to those skilled in the art, is raw materials usedly commercial goods.
Embodiment 1
The acquisition of Os05g27980 gene C DS sequence and the structure of plant expression vector
The acquisition of 1.Os05g27980 gene C DS sequence
Os05g27980 gene is found in plant transcription factor database (http://rice.plantbiology.msu.edu/analyses_search_locus.shtml), its nucleotide sequence is as shown in SEQ ID No.11, according to its sequences Design pcr amplification primer, forward primer F:5'-CAAAAAAGCAGGCTTCATGGGCGGTGGGTC-3' and reverse primer R:5'-CAAGAAAGCTGGGTCCTACGTCCAGAGCGA-3').With the wild-type Japan total cDNA of fine ' kitaake ' paddy rice for template, utilize primers F and R to carry out PCR, obtain the CDS sequence (as shown in SEQ ID No.1) that Os05g27980 gene is complete.
2. the structure of plant expression vector
By the CDS sequence of rice transcription factor Os05g27980 gene by the downstream of Gateway system constructing to 4 transcription factor activation motif VP16 encoding genes (as shown in SEQ ID No.4).
2.1 above-mentioned PCR primer is cloned on pDONER cloning vector
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 (F and R) adding part adaptor attB joint of first round PCR, and the second template of taking turns PCR primer of the first round, and complete adaptor attB primer (attB5'adaptor:5'-GTGGGGACAAGTTTGTACAAAAAAGCAGGCTTC-3', attB3'adaptor:5'-GTGGGGACCACTTTGTACAAGAAAGCTGGGTC-3') of primer.PCR primer is cloned on pDONER cloning vector (purchased from Invitrogen), obtains the identical sequence with goal gene through order-checking qualification.
The structure of 2.2 plant expression vectors
With plant binary expression vector pCambia1301-UbiN(Fig. 6) sequence that comprises of 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, obtain the complete sequence of carrier nVP64-hyg-asRED as shown in SEQ ID No.5, Vector map is shown in Fig. 1.
Expression vector nVP64-hyg-asRED contains Gateway recombination system, and pDONR as entry vector (Entery vector), can complete the structure of destination gene expression carrier with the plasmid of goal gene by LR reaction.
By LR reaction by the CDS sequence construct of Os05g27980 gene to its goal gene 5 ' hold be connected with on the plant expression vector nVP64-hyg-asRED of VP64 encoding gene, obtain the expression vector ubi:VP64-Os05g27980 carrying described composing type rice transcription factor VP64-Linker-Os05g27980 gene of encoding, its complete sequence is as shown in SEQ ID No.6, and Vector map is shown in Fig. 2.
LR reaction system is as follows:
Spend the night in 25 DEG C of reactions.With reaction system transformation of E. coli DH5 α, screening positive clone.
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-Os05g27980, transform with the AAM nutrient solution being the Agrobacterium of 0.7 containing the Syringylethanone of 100 μMs and OD 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.Obtain 45 strain transgenic seedlings.
The culture medium prescription related in the present embodiment is as follows:
A large amount of+B5 trace+the NB of inducing culture: N6 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 water preparation, after adjusting pH to 5.8 ~ 5.9, add plant gel 4g/L.
A large amount of+B5 trace+the NB of Dual culture base: N6 is organic+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.
A large amount of+B5 trace+the NB of screening culture medium: N6 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 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.
Division culture medium: MS inorganic+MS-B5 trace+MS is organic+molysite+MS-copper cobalt mother liquor+0.05mg/L NAA+2.0mg/L Kinetin(kinetin) and+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 detect embodiment 2 obtain VP64-Os05g27980 transgenic paddy rice strain in ubi:VP64-Os05g27980 gene at T2 for the process LAN situation in transgenic paddy rice (V2605H-01, V2605H-11), the present embodiment designs primer (forward primer: 5'-GTGGGGACAAGTTTGTACAAAAAAGCAGGCTTC-3' and reverse primer: 5'-GTGGGGACCACTTTGTACAAGAAAGCTGGGTC-3') at carrier and goal gene joint and carries out PCR detection, obtains obvious specific band.
As seen from Figure 3, the stripe size amplified is at more than 500bp, and primer designs at carrier and goal gene joint, the clip size amplified and goal gene (603bp) basically identical.Therefore can illustrate, containing VP64-Os05g27980 fusion gene in transgenic paddy rice strain V2605H-01, V2605H-11 that embodiment 2 obtains.
Embodiment 4 transgenic paddy rice phenotype analytical and species test analysis
The transgenic paddy rice strain V2605H-01, the V2605H-11 that embodiment 2 are obtained and wild rice seed compare, and can significantly find out phenotype, find that the seed of transgenic line obviously broadens (Fig. 4).Species test data analysis shows (Fig. 5), and the width of the VP64-Os05g27980 transgenic paddy rice seed that the present invention obtains significantly is 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 composing type rice transcription factor, is characterized in that, it is fusion rotein (VP16)
4-Linker-Os05g27980;
Wherein, VP16 is the VP16 albumen from hsv, (VP16)
4be be the fusion rotein of gap-forming with Gly-Ser by the aminoacid sequence of 4 VP16 albumen, its aminoacid sequence is as shown in SEQ ID No.10; Linker is in series by 39 flexible amino acid, and its aminoacid sequence is as shown in SEQ ID No.9; Os05g27980 is rice transcription factor Os05g27980, and its aminoacid sequence 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.
2. the gene of composing type rice transcription factor described in coding claim 1.
3. the carrier containing gene described in claim 2.
4. the engineering bacteria containing gene described in claim 2.
5. the construction process of a transgenic rice plant, it is characterized in that, adopt agriculture bacillus mediated method, carrier according to claim 3 is proceeded in Rice Callus, transform with the AAM nutrient solution 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.
6. the application of gene according to claim 2 in improvement rice grain proterties.
7. for the primer pair of amplifying rice transcription factor Os05g27980 gene C DS sequence, it is characterized in that, comprise forward primer F:5'-CAAAAAAGCAGGCTTCATGGGCGGTGGGTC-3' and reverse primer R:5'-CAAGAAAGCTGGGTCCTACGTCCAGAGCGA-3';
Wherein, the CDS sequence of rice transcription factor Os05g27980 gene is:
Nucleotide sequence shown in SEQ ID No.1.
8. the application of rice transcription factor Os05g27980 gene C DS sequence in adjusting and controlling rice grain characters.
9. application according to claim 8, it is characterized in that, it utilizes transcription factor activation motif VP64 and rice transcription factor Os05g27980 to construct to obtain composing type transcription factor, and by the gene transformation paddy rice of the described composing type transcription factor of coding, thus the proterties of improvement transgenic paddy rice seed;
Wherein, the aminoacid sequence of described transcription factor activation motif VP64 is as shown in SEQ ID No.10.
10. application according to claim 8, it is characterized in that, it is by the CDS sequence of rice transcription factor Os05g27980 gene by the downstream of Gateway system constructing to transcription factor activation motif VP64 encoding gene, rice transformation, thus the proterties of improvement transgenic paddy rice seed;
Wherein, the nucleotide sequence of described transcription factor activation motif VP64 encoding gene is as shown in SEQ ID No.4.
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