CN104341525A - Application of CDS (coding sequence) of gene for coding oryza sativa L. transcription factor Os02g47744 - Google Patents
Application of CDS (coding sequence) of gene for coding oryza sativa L. transcription factor Os02g47744 Download PDFInfo
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Abstract
The invention relates to an application of a CDS (coding sequence) of a gene for coding an oryza sativa L. transcription factor Os02g47744. An activation motif VP64 of the transcription factor is fused with the oryza sativa L. transcription factor Os02g47744 to construct a constitutive transcription factor and the gene for coding the constitutive transcription factor is transformed into crops, such as oryza sativa L., thus improving the traits of rice grains, for example, the rice grain width is increased. The gene has important theoretical value for expounding the seed development regulating mechanism and the grain shape of rice can be improved by means of transgenosis, so that the gene has important significance in production practice.
Description
Technical field
The present invention relates to genetically engineered field, specifically, relate to the application of rice transcription factor Os02g47744 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.
The output of cereal crop depends on the size of its seed to a great extent, and therefore the gene regulating of rice grain proterties is important field of research.MYB albumen is a class transcription factor maximum in plant materials, regulate and control many in plant materials polygenicly to transcribe, combined by the cis-acting elements (cis-acting element) in MYB structural domain and promotor, regulate and control the expression of its Target genes, in plant growth and development process, play very important effect.Complete MYB forms primarily of 3 part-structures: DNA binding domains (DNA binding domain, DBD), transcriptional activation domain (transactivation domain, and negative regulator region (negative regulatory domain, NRD) TAD).Wherein, DNA calmodulin binding domain CaM (DBD) is the structural domain of MYB most critical, usually containing the incomplete repetition factor of 3 class, represent with R1, R2 and R3, each repetition factor contains 51 ~ 53 aminoacid sequences, containing a tryptophan residue or other hydrophobic groupings in 18 amino acid, these aminoacid sequences form spiral one corner one spiral (HTH) structure, form the core texture of 3 dimensions centered by tryptophan residue.
Molecular hybridization confirms the myb transcription factor that in floral organ, ubiquity is specific expressed, comprise MYB26 and MYBl03 in Cruciferae Arabidopis thaliana, NtMYBASl and NtMYBAS2 in Solanaceae tobacco (Nic-otiana tabacum), the ZmMYBP2 in Gramineae corn (Zea mays).Gene mutation analysis discloses the function of MYB class transcription factor in pollen development process, comprise the form and programmed cell death (programmed cell death thereof that control tapetum, PCD), the dynamic change of callose, the transport of photosynthate and anther dehiscence etc., the MYB class functional transcription factor that pollen development is relevant is abnormal all likely causes part even holandry sterile (male sterility).
Myb transcription factor plays very important effect in pollen development process.In recent years, MYB functional genome research shows, in plant During Microsporogenesis and microgametophyte generating process, myb transcription factor is by regulating the expression of correlation function gene, and then causes a series of biochemical reaction and physiological change, promotes that microsporocyte develops into the pollen normally can educated.If there is myb transcription factor abnormal expression in whole transcriptional control, gene silencing or overexpression, all can affect the normal development of flower pesticide, forms cavity shape, lopsided pollen, cause the sterile even holandry of partial male sterile.In Plant Pollen Development process, myb transcription factor is expressed by regulation and control MST family gene and then is regulated the transport of photosynthate.(the Zhang H such as Zhang, Liang W Q.et al. (2010) Carbon starved anther encodes a MYB domain protein that regulates sugar partitioning required for rice pollen development.) clone the specific expressed CSA of rice tapetum (carbon starved anther) gene, this gene belongs to R2R3-MYB transcription factor, in csa rice mutant tapetum, the transcriptional level of MST8 significantly declines, because photosynthate can not be transported in flower pesticide, sufficient nutrient is not had to ensure normally carrying out of pollen development, so that there is cavity shape pollen.
Myb transcription factor is expressed under the regulation and control of environmental factor, hormone and other transcription factors, and the target gene of regulation and control myb transcription factor is transcribed further, thus reaches the effect controlling pollen development process.For MYB80/UNDEAD, tapetum PCD is formed for functional pollen has very important effect, suppresses or postpones PCD and cause tapetum loose and vacuolization, make it normally can not complete physiological function, cause plant to occur male sterile.In a word, controlling rice grain growth is a complex process, relate to the cooperation control of polygene many approach, the gene that current discovery regulates and controls this proterties is few, the molecule mechanism of regulation and control Grain Development is not yet illustrated, therefore, find and control gene that grain characters grows and new excavation means to crop improvement and to improve crop yield significant.
Summary of the invention
The object of this invention is to provide the application of rice transcription factor Os02g47744 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-Os02g47744.
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 Os02g47744 related in above-mentioned fusion rotein is rice transcription factor Os02g47744, 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 Os02g47744 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), Os02g47744 gene is found, according to its sequences Design pcr amplification primer pair, it is forward primer F:5'-CAAAAAAGCAGGCTTCATGAGTTCATCCTGGACAAC-3' and reverse primer R:5'-CAAGAAAGCTGGGTCTCACTGAAAGTTGTGGTAC-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 Os02g47744 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 Os02g47744 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-Os02g47744 carrying described composing type rice transcription factor VP64-Linker-Os02g47744 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 increase rice grain wide and thousand seed weight).
Present invention also offers the primer pair for amplifying rice transcription factor Os02g47744 gene C DS sequence, comprise forward primer F:5'-CAAAAAAGCAGGCTTCATGAGTTCATCCTGGACAAC-3' and reverse primer R:5'-CAAGAAAGCTGGGTCTCACTGAAAGTTGTGGTAC-3'.
The present invention further provides the application of rice transcription factor Os02g47744 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 Os02g47744, 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 Os02g47744 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 Os02g47744 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 Os02g47744, 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-Os02g47744 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-Os02g47744 transgenic positive strain, and wherein WT is wild rice ' kitaake ', and V2155H-11, V2155H-35 are VP64-Os02g47744 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 V2155H-11, V2155H-35 are VP64-Os02g47744 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 V2155H-11, V2155H-35 are VP64-Os02g47744 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 Os02g47744 gene C DS sequence and the structure of plant expression vector
The acquisition of 1Os02g47744 gene C DS sequence
Os02g47744 gene is found in plant transcription factor database (http://rice.plantbiology.msu.edu/analyses_search_locus.shtml), according to its sequences Design pcr amplification primer, forward primer F:5'-CAAAAAAGCAGGCTTCATGAGTTCATCCTGGACAAC-3' and reverse primer R:5'-CAAGAAAGCTGGGTCTCACTGAAAGTTGTGGTAC-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 Os02g47744 gene is complete.
The structure of 2 plant expression vectors
By the CDS sequence of rice transcription factor Os02g47744 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 the complete adaptor attB primer (attB5'adaptor:5'-GTGGGGACAAGTTTG TACAAAAAAGCAGGCTTC-3', attB3'adaptor:5'-GTGGGGACCAC TTTGTACAAGAAAGCTGGGTC-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-Os02g47744, 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 40 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+30 g/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/LNAA+2.0mg/LKinetin(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-Os02g47744 transgenic paddy rice strain in ubi:VP64-Os02g47744 gene at T2 for the process LAN situation in transgenic paddy rice (V2155H-11, V2155H-35), 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 250bp, and primer designs at carrier and goal gene joint, the clip size amplified and goal gene (306bp) basically identical.Therefore can illustrate, containing VP64-Os02g47744 fusion gene in transgenic paddy rice strain V2155H-11, V2155H-35 that embodiment 2 obtains.
Embodiment 4 transgenic paddy rice phenotype analytical and species test analysis
The transgenic paddy rice strain V2155H-11, the V2155H-35 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 wide of VP64-Os02g47744 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-Os02g47744;
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; Os02g47744 is rice transcription factor Os02g47744, 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 Os02g47744 gene C DS sequence, it is characterized in that, comprise forward primer F:5'-CAAAAAAGCAGGCTTCATGAG TTCATCCTGGACAAC-3' and reverse primer R:5'-CAAGAAAGCTGGGT CTCACTGAAAGTTGTGGTAC-3';
Wherein, the CDS sequence of rice transcription factor Os02g47744 gene is:
Nucleotide sequence shown in SEQ ID No.1.
8. the application of rice transcription factor Os02g47744 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 Os02g47744 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 Os02g47744 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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| CN111499709A (en) * | 2020-05-19 | 2020-08-07 | 中国农业大学 | RGN1 protein related to grain number per ear of rice as well as encoding gene and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111499709A (en) * | 2020-05-19 | 2020-08-07 | 中国农业大学 | RGN1 protein related to grain number per ear of rice as well as encoding gene and application thereof |
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