CN103421120B - Application of rice transcription factor Os11g02540 genes - Google Patents
Application of rice transcription factor Os11g02540 genes Download PDFInfo
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- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention relates to the application of rice transcription factor Os11g02540 genes. The application is achieved by using transcription factors to restrain fusion and construction of sequence motif EAR and the rice transcription factor Os11g02540 genes to obtain constituted type transcription factors, and the genes encoding the constituted type transcription factors are transformed to crops like rice so as to reform rice grain characters, for instant, to increase the widths of rice grains. The application has important theoretical value in illuminating seed development mechanism regulation and control in detail, and can change the grain type of the rice through the genetically modified method. Therefore, the application also 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 Os11g02540 gene.
Background technology
Paddy rice (Oryza sativa L.) is one of most important Three major grain crops of 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.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.The research of current increasing production of rice depends on limited Rice Germplasm Resources, and traditional cross-breeding advantage weakens gradually, and Transgenic Rice technology is likely excavated the further potentiality of volume increase of paddy rice.
In vegitabilia, the plant that can form seed accounts for the 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 has played critical effect in the accuracy controlling of genetic expression.
Rice transcription factor Os11g02540 is a member of WRKY family, and WRKY albumen is the important transcription factor family of a class, because of the WRKY structural domain that contains high conservative, gains the name, and is mainly present in plant.WRKY structural domain is about 60 amino acid, and it has WRKYGQ (K/E) K motif near N-end, after with having 1 C2H2 or C2HC zinc to refer to type structure.According to the difference of WRKY structural domain basic structural feature, paddy rice WRKY family can be divided into 3 large classes, and they participate in regulating rice growth and biotic and abiotic stress to reply by the signal pathway of regulation and control growth regulatory substance mediation widely.By bioinformatics method, in 2 subspecies of Indica round-grained rice, predict more than 100 WRKY genes at present, their wide participations of evidence are replied biology or abiotic stress and the adjusting of the process such as grow, and relate to the signal path (Eulgem T.et al. (2000) The WRKY superfamily of plant transcription factors) of the multiple growth regulatory substance mediation such as growth hormone (IAA), Plant hormones regulators,gibberellins (GA), dormin (ABA), Whitfield's ointment (SA) and jasmonic (JA).
The difference of gene expression pattern has embodied the difference on their biological functions, and the expression of the induction WRKY gene that multiple biology and the abiotic stress factor can be in various degree, illustrates that WRKY albumen plays an important role in paddy rice defense response.In addition, the change of part WRKY gene expression dose, has also affected generation, the growth and old and feeble of rice organ.Overexpression WRKY family gene OsWRKY31 has improved the resistance of transgenic line to rice blast, but formation and the elongation of lateral root are suppressed simultaneously, showing as lateral root reduces and shortens, be that plant height is downgraded heading stage, root reduced number, analysis found in expression plant that the early stage responsive genes of growth hormone was constructive expression as OsIAA4 and OsCrl1, and to external source high density IBA, NAA and 2, the Reduced susceptibility of 4-D, illustrate that overexpression OsWRKY31 has changed response or the transhipment of IAA, this shows that OsWRKY31 participates in the conduction of defensive raction signal and the conduction of growth hormone response signal of paddy rice simultaneously, regulate paddy rice to the disease resistance of rice blast with to the response of growth hormone (Zhang J.et al. (2008) Constitutive expression of pathogen inducible OsWRKY31enhances disease resistance and affects rootgrowth and auxin response in transgenic rice plants.).WRKY transcription factor gene OsWRKY89 is processed and ultraviolet-B radiation induced strong by methyl jasmonate.OsWRKY89 is positioned nucleus, and 67 amino acid of its C end are essential to transcriptional activity, and there is enhancement in the leucine zipper region of N end to transcriptional activity.Cross expression OsWRKY89 and cause vine growth and development early growth slow, and internode shortens.OsWRKY89 crosses expression strain leaf surface waxes precipitation to be increased, and stem's lignifying increases, and phenolic compound content reduces.In addition, cross expression OsWRKY89 and forced SA level, strengthened the resistance of paddy rice to Pyricularia oryzae, white backed planthopper and ultraviolet-B radiation.Therefore, OsWRKY89 play an important role in biotic and abiotic stress is replied (Wang H H.et al. (2007) Overexpression of rice WRKY89enhances ultraviolet B tolerance and disease resistance in rice plants.).WRKY transcription factor gene OsWRKY78 has expression in all sense organ, and extending, stem expression is the highest.OsWRKY78RNAi plant shows as semi-dwarf mutant, and particle shape diminishes, and it is the same with wild-type to cross expression plant.Its T-DNA phenotype and RNAi are similar.Rice matter does not have considerable change, and endosperm starch crystalline structure in RNAi plant changes a little, this shows, OsWRKY78 may play and regulate stem to extend and the process (Zhang C Q.et al. (2011) The WRKY transcription factor OsWRKY78regulates stem elongation and seed development in rice.) of seed development.Therefore the research to transcription factor, provides new clue for further understanding the molecule mechanism of plant seed and allelotaxis's regulation and control in theory, in practice, also will provide 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 Os11g02540 gene.
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 Os11g02540-Linker-EAR.
Wherein, EAR is for having the albumen motif of transcribing inhibit feature from a section of plant transcription factor, its aminoacid sequence is as 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.
Linker for example, by 1～11 flexible amino acid be in series (, the aminoacid sequence DPAFLYKVVPR of GGGGG, GPPPG or Gatway carrier recombination site coding or PRHHFVQESWV etc.).
The Os11g02540 relating in above-mentioned fusion rotein is rice transcription factor Os11g02540, its aminoacid sequence is as shown in SEQ ID No.1, or this sequence is through replacing, lacking or add one or several amino acids formed aminoacid sequence with same function, its CDS sequence is as shown in Seq ID No.3.
The present invention also provides the gene of the described composing type rice transcription factor of coding, and under stringent condition, can with the nucleotide sequence of the nucleotide sequence hybridization of this gene.
The present invention also provides the carrier of the gene that contains the described composing type rice transcription factor of encoding.The carrier that described carrier is expressed in host for any bootable foreign gene.Preferably, described carrier be plant binary expression vector (for example, pCAMBIA1301).Gene constructed in plant expression vector time at described composing type rice transcription factor that the present invention is encoded can be added any strong promoter (for example, corn strong promoter Ubiquitin) or inducible promoter before its transcription initiation Nucleotide.In addition, gene constructed in plant expression vector time at described composing type rice transcription factor that the present invention is encoded, can also use enhanser, and these enhanser regions must be identical with the reading frame of encoding sequence, to guarantee the translation of whole piece sequence.
The expression vector that carries the gene of the described composing type rice transcription factor of coding can be by being used the conventional biotechnological means such as Ti-plasmids, plant viral vector, directly delivered DNA, microinjection, electroporation to import (Weissbach in vegetable cell, 1998, Method for Plant Molecular Biology VIII, Academy Press, New York, 411-463 page; Geiserson and Corey, 1998, Plant Molecular Biology, second edition).
The present invention also provides engineering bacteria and the clone that contains the described composing type rice transcription factor of encoding.
The present invention also provides a kind of construction process of transgenic rice plant, be specially: adopt agriculture bacillus mediated method, the carrier of the gene that contains the described composing type rice transcription factor of encoding is proceeded in Rice Callus, with the AAM nutrient solution containing inductor and Agrobacterium, transform, material after conversion through cultivating altogether-screen-break up-take root-exercise and the transplanting of transgenic seedling, screening transgenic rice plant.
For example, application in improvement rice grain proterties (increasing rice grain width) of the gene that the present invention also provides the described composing type rice transcription factor of coding.
The present invention also provides the primer pair for amplifying rice transcription factor Os11g02540 gene C DS sequence, comprises forward primer F5'-CAAAAAAGCAGGCTTCATGGAGGAGGCCTACTG-3' and reverse primer R5'-CAAGAAAGCTGGGTCTTAAGTGAGGAAATGGGTG-3'.
The present invention further provides the application of rice transcription factor Os11g02540 gene in adjusting and controlling rice grain characters.Utilize transcription factor to suppress motif EAR and construct and obtain composing type transcription factor with rice transcription factor Os11g02540, and by the gene transformation of the described composing type transcription factor of coding to farm crop as in paddy rice, thereby improve the proterties of transgenic paddy rice seed.
Aforesaid application, its be the CDS sequence of rice transcription factor Os11g02540 gene is removed to terminator codon after, be building up to the upstream that transcription factor suppresses motif EAR encoding gene (SEQ ID No.4), rice transformation (as rice varieties ' kitaake '), thereby the proterties of improvement transgenic paddy rice seed.After preferably the CDS sequence of rice transcription factor Os11g02540 gene being removed to terminator codon, by Gateway system constructing, to transcription factor, suppress the upstream of motif EAR encoding gene.
The present invention utilizes first transcription factor inhibition motif EAR and rice transcription factor Os11g02540 to construct and obtains composing type transcription factor, and by coding described composing type transcription factor gene transformation to farm crop as in paddy rice, thereby improvement rice grain proterties, for example rice grain width increases.For illustrating in detail regulation and control seed development mechanism, there is important theory value, and can pass through transgenosis means, the grain type of improvement paddy rice, therefore also significant in production practice.
Accompanying drawing explanation
Fig. 1 is cEAR-bar-asRED carrier collection of illustrative plates in the embodiment of the present invention 1.
Fig. 2 is ubi:Os11g02540-EAR carrier collection of illustrative plates in the embodiment of the present invention 1.
Fig. 3 is that in the embodiment of the present invention 3, PCR detects EAR-Os11g02540 transgenic positive strain; Wherein, M is DNA molecular amount standard, and WT is wild-type paddy rice ' kitaake ', and E0182-20 and E0182-24 are Os11g02540-EAR 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-type paddy rice ' kitaake ', and E0182-20 and E0182-24 are Os11g02540-EAR transgenic paddy rice strain.
Fig. 5 is the species test data statistic analysis result of transgenic paddy rice grain characters in the embodiment of the present invention 4; Wherein, WT is wild-type paddy rice ' kitaake ', and E0182-20 and E0182-24 are Os11g02540-EAR 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 examples are used for illustrating the present invention, but are not used for limiting the scope of the invention.If do not specialize, embodiment is all according to normal experiment condition, as Sambrook equimolecular cloning experimentation handbook (Sambrook J & Russell DW, Molecular cloning:a laboratory manual, 2001), or according to the condition of manufacturer specification sheets suggestion.
The acquisition of embodiment 1Os11g02540 gene C DS sequence and the structure of plant expression vector
The acquisition of 1Os11g02540 gene C DS sequence
In plant transcription factor database (http://rice.plantbiology.msu.edu/analyses_search_locus.shtml), find Os11g02540 gene, according to its CDS sequences Design pcr amplification primer, forward primer F5'-CAAAAAAGCAGGCTTCATGGAGGAGGCCTACTG-3' and reverse primer R5'-CAAGAAAGCTGGGTCTTAAGTGAGGAAATGGGTG-3').The wild-type Japan total cDNA of fine ' kitaake ' paddy rice of take is template, utilizes primers F and R to carry out PCR, obtains the CDS sequence (Seq ID No.3) of Os11g02540 gene complete.
The structure of 2 plant expression vectors
The CDS sequence of rice transcription factor Os11g02540 gene is suppressed to the upstream of motif EAR encoding gene (SEQ ID No.4) to transcription factor by Gateway system constructing.
2.1 by above-mentioned PCR product cloning to pDONER cloning vector
According to PrimeSTAR polymeric enzymatic amplification system and response procedures, carry out PCR.In this process, comprise two-wheeled PCR, the gene primer (F and R) that adds part adaptor attB joint for the primer of first round PCR, and the PCR product of the first round for the second template of taking turns, and complete adaptor attB primer (attB5'adaptor:5'-GTGGGGACAAGTTTGTACAAAAAAGCAGGCTTC-3', attB3'adaptor:5'-GTGGGGACCACTTTGTACAAGAAAGCTGGGTC-3') for primer.PCR product cloning is upper to pDONER cloning vector (purchased from Invitrogen), through order-checking, identify and obtain and the identical sequence of goal gene.
The structure of 2.2 plant expression vectors
Take plant binary expression vector pCambia1301-UbiN(Fig. 6) sequence that comprises of border, left and right is frame sequence, pass through vitro recombination, ubi promoter-Gateway-EAR is expressed to unit, 35S promoter-asRED expression unit and 35S promoter-bar expression unit to construct with it, obtain the complete sequence of carrier cEAR-bar-asRED as shown in SEQ ID No.6, Fig. 1 is shown in by carrier collection of illustrative plates.
Expression vector nEAR-hyg-asRED contains Gateway recombination system, and pDONR as entry vector (Entery vector), reacts the structure that can complete destination gene expression carrier with the plasmid of goal gene by LR.
By LR, reacting the 3 ' end to its goal gene by the CDS sequence construct of Os11g02540 gene is connected with on the plant expression vector cEAR-bar-asRED of EAR encoding gene, acquisition carries the expression vector ubi:Os11g02540-EAR of the described composing type rice transcription factor Os11g02540-Linker-EAR gene of coding, its complete sequence is as shown in SEQ ID No.5, and Fig. 2 is shown in by carrier collection of illustrative plates.
LR reaction system is as follows:
In 25 ℃ of reactions, spend the night.By reaction system, transform bacillus coli DH 5 alpha, screening positive clone.
The acquisition of embodiment 2 transgenic rice plants
Water intaking rice ' kitaake ' mature seed, artificial or mechanical dejacketing, selects the full bright and clean seed without bacterial plaque and after sterilization, is inoculated into and on inducing culture, carries out inducing culture.Selection outward appearance is good, the Rice Callus that growing ability is good is acceptor material, adopt agrobacterium-mediated transformation that ubi:EAR-Os11g02540 is proceeded in Rice Callus, the AAM nutrient solution of the Agrobacterium that is 0.7 by the Syringylethanone that contains 100 μ M and OD value transforms, the callus that conversion fluid was soaked is placed in and on common substratum, carries out common cultivation, 25 ℃ of dark cultivation 3d are placed in screening culture medium and cultivate about 30d, and every 10d subculture once.Then the kanamycin-resistant callus tissue sifting out is transferred on division culture medium and broken up about 20d, every 10d subculture once.The kanamycin-resistant callus tissue that differentiates green seedling is transferred on root media and taken root, hardening after about 7d grows flourishing root system, and calculate the conversion transgenic seedling number that obtains, obtain altogether 30 transfer-gen plants.After hardening 7d, be transferred to grown in field.
The culture medium prescription relating in the present embodiment is as follows:
Inducing culture: 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 water preparation, tune adds plant gel 4g/L behind pH to 5.8～5.9.
Altogether substratum: N6 a large amount of+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 water preparation, adds plant gel 4g/L after adjusting pH to 5.2.After sterilizing, 50 ℃ of left and right add AS(Syringylethanone) 100～200 μ g/mL.
Screening culture medium: 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 water preparation, tune adds plant gel 4g/L behind pH to 5.8～5.9.After sterilizing, add 35mg/L Totomycin (purchased from Shanghai Niu Jin Bioisystech Co., Ltd) or 5mg/LBialaphos(purchased from Beijing Baeyer enlightening biotech company).
Division culture medium: 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 water preparation, tune adds plant gel 4g/L behind pH to 5.8～5.9.
The evaluation of embodiment 3 transgenic positive strains
In order to detect the expression excessively of the middle gene of T2Dai rice strain (E0182-20 and E0182-24) of the Os11g02540-EAR transgenic paddy rice strain obtaining in embodiment 2, according to carrier ubi:Os11g02540-EAR, design following primer:
Forward primer F5'-GTGGGGACAAGTTTGTACAAAAAAGCAGGCTTC-3'
Reverse primer R5'-GTGGGGACCACTTTGTACAAGAAAGCTGGGTC-3'
Carrying out PCR detection, amplified production is carried out to agarose gel electrophoresis, as shown in Figure 3, there is obvious specific band in result.Wherein, WT represents wild-type paddy rice ' kitaake ', and E0182-20 and E0182-24 represent Os11g02540-EAR transgenic paddy rice strain.In E0182-20 and E0182-24, contain Os11g02540-EAR fusion gene.
Embodiment 4 transgenic paddy rice phenotype analyticals and species test analysis
The transgenic paddy rice strain (E0182-20 and E0182-24) obtaining in embodiment 2 and wild-type ' kitaake ' rice paddy seed are compared, phenotype, can significantly find out, the seed of transgenic paddy rice obviously broaden (Fig. 4).Species test data analysis shows (Fig. 5), and the width of transgenic paddy rice seed is significantly greater than wild-type rice grain.
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, all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (9)
1. a composing type rice transcription factor, is characterized in that, it is fusion rotein Os11g02540-Linker-EAR;
Wherein, EAR is for having the albumen motif of transcribing inhibit feature from a section of plant transcription factor, and its aminoacid sequence is as shown in Seq ID No.2; Linker is in series by 1～11 flexible amino acid; Os11g02540 is rice transcription factor Os11g02540, and its aminoacid sequence is as shown in SEQ ID No.1.
2. composing type rice transcription factor according to claim 1, is characterized in that, the aminoacid sequence of Linker is GGGGG, GPPPG, DPAFLYKVVPR or PRHHFVQESWV.
3. the gene of composing type rice transcription factor described in the claim 1 or 2 of encoding.
4. the carrier that contains gene described in claim 3.
5. the engineering bacteria that contains 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 claimed in claim 4 is proceeded in Rice Callus, with the AAM nutrient solution containing inductor and Agrobacterium, transform, material after conversion through cultivating altogether-screen-break up-take root-exercise and the transplanting of transgenic seedling, screening transgenic rice plant.
7. the application of gene claimed in claim 3 in improvement rice grain width.
8. the application of rice transcription factor Os11g02540 gene in improvement rice grain width, wherein, the CDS sequence of rice transcription factor Os11g02540 gene is as shown in Seq ID No.3.
9. application according to claim 8, it is characterized in that, its be the CDS sequence of rice transcription factor Os11g02540 gene is removed to terminator codon after, by Gateway system constructing, to transcription factor, suppress the upstream of motif EAR encoding gene, rice transformation, thereby the width of improvement transgenic paddy rice seed;
Wherein, the nucleotide sequence that described transcription factor suppresses motif EAR encoding gene is as shown in SEQ ID No.4.
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| 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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| 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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| ccession number:NM_001072124, Oryza sativa Japonica Group Os11g0117600 (Os11g0117600) mRNA, complete cds;Tanaka,T. et al.;《Genbank》;20100608;feature和origin部分 * |
| Tanaka,T. et al..ccession number:NM_001072124, Oryza sativa Japonica Group Os11g0117600 (Os11g0117600) mRNA, complete cds.《Genbank》.2010,feature和origin部分. * |
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