CN101875690A - Rice gene OsWRKY78 and application thereof - Google Patents

Abstract

The invention relates to a gene OsWRKY78 for the rice transcription factor WRKY and the application thereof, belonging to the technical field of the agriculture. The rice transcription factor WRKY78 has the amino acid sequence of SEQ ID NO: 2, and the gene OsWRKY78 for coding the rice transcription factor WRKY78 has the nucleotide sequence of SEQ ID NO: 1. The transgenic rice with the RNA-interfered genes OsWRKY78 is short, has straight sword leaves and contains high chlorophyll because the genes OsWRKY78 control the growth and the development of the rice. The gene expression can be changed by the genetic engineering method and other methods so that the morphology of the rice can be changed to some extent and the excellent and new rice variety can be cultured.

Description

Rice gene OsWRKY 78 and application thereof

Technical field the present invention relates to paddy rice WRKY class transcription factor gene OsWRKY78 and application thereof.

Background technology

Plant has formed complicated and exquisite regulation mechanism during evolution to adapt to the polytropy of external environment.By the perception that internal and external environment is changed, cause the adjustment of related gene expression by a series of signal transduction pathway, finally give plant in physiological suitability.In numerous adaptability mechanism, the transcriptional regulatory of genetic expression plays an important role.The gene expression regulation of transcriptional level be by trans-acting factor be transcription factor (Transcription factor) with cis-acting elements (cis-element) between specific recognition, combine and activate or suppress Expression of Related Genes.Transcription factor can also be by the regulation and control of the interaction between protein-protein Expression of Related Genes (Jos é Luis Riechmann etc., 2000) in addition.The DNA of transcription factor has determined it and cis-acting elements bonded specificity in conjunction with the territory, and transcription regulatory region has determined it that genetic expression is risen to activate or restraining effect.According to the difference of DNA in conjunction with the territory, transcription factor is divided into numerous families.In recent years, be cloned into a large amount of transcription factors from plant, they participate in all respects of regulating growth of plants and the defense response of various biology and abiotic stress.WRKY class transcription factor is wherein playing an important role aspect growth and development of plant and the various stress response.

Form from amino acid, one forms (Schwechheimer etc., 1998) by DNA land, transcription regulatory region (comprising active region or inhibitory area), oligomerization site and these 4 functional areas of nuclear localization signal transcription factor.DNA binding domains (DNA-binding domain) is the structural domain of transcription factor most critical, is transcription factor identification DNA cis-acting elements and one section amino acid preface of bonded with it.There are WRKY (Eulgem etc., 2000), AP2/EREBP (Riechmann etc., 1998) etc. in more typical DNA land in the plant transcription factor.Under one situation, transcription factor has only a DNA in conjunction with the territory or have only one to be responsible for and the combining of DNA element, have two WRKY structural domains as I group membership in the WRKY protein family, but the WRKY structural domain that has only C-terminal (C-end) has dna binding activity (Eulgem etc., 2000); Transcription regulatory region (transcription regulation domain) comprises transcriptional activation domain (transcription activation domain) and transcribes two kinds of inhibitory areas (transcription repression domain) they have determined which kind of function is transcription factor carry out; Oligomerization site (oligomerization site) is that different transcription factors are so as to interactional functional domain takes place; (nuclear localizaion signal NLS) is the zone of being rich in arginine and lysine residue in the transcription factor to nuclear localization signal, and the control transcription factor enters nucleus (Raikhel etc., 1992) from tenuigenin.

The present WRKY genoid of identifying, wide participation growth and development of plant regulation and control, disease resistance response, degeneration-resistant border coerce etc.For example, Arabidopis thaliana AtWRKY22 and AtWRKY29 are positioned at the downstream of mapk kinase cascade reaction, and can activate the resistance reaction (Asai etc., 2002) to bacterium and fungal disease; Arabidopis thaliana AtWRKY33 and tobacco NtWRKY12 all can participate in PRl expression of gene (Andreasson etc., 2005 in the disease-resistant approach of Whitfield's ointment; Verk etc., 2008); Some WRKY genes of overexpression can significantly improve resistance (Li etc., 2004 of plant to germ as AtWRKY70, NtWRKY12, OsWRKY13 etc.; Jing etc., 2009; Marcel etc. 2008; Shimono etc., 2007; Qiua waits 2009).In addition, some WYKY transcription factors also play a part negative regulation in disease-resistant, have all participated in negative regulation (NoellieJournot-Catalino etc., 2006 of disease-resistant process as Arabidopis thaliana AtWRKY11 and rice OsWRKY 45-1, OsWRKY62; Tao etc., 2009; Peng etc., 2009).

The abiotic stress of plant comprises wound, low temperature, arid, salt damage etc.In plant, identified at present the WRKY transcription factor that a collection of participation this respect is replied.Discover, the AtWRKY11 in the Arabidopis thaliana, 15,22,33,44,53,60 and tobacco in WIZZ and the CaWRKY2 in the capsicum participated in wound and replied (Chcong etc., 2002; Hara etc., 2000; Oh etc., 2006); Barley HvWRKY38 is not only by cold-peace arid institute abduction delivering, and discovery overexpression in paspalum notatum (Paspalum notatum Flugge) can strengthen its drought tolerance (Xin Xiong etc., 2009); OsWRKY45 in the paddy rice not only can improve the resistance of paddy rice to rice blast, and overexpression can improve the resistance (Qiu etc., 2008) of Arabidopis thaliana to salt and arid in Arabidopis thaliana; Overexpression rice Os WRKY11 and OsWRKY89 can improve the drought-enduring of plant and UV resistant radiating ability (Wu etc., 2009 respectively; Wang etc., 2007).

Aspect the regulation and control that participate in growing, Arabidopis thaliana TTG2 (Transparent Testa Glabra2) is that first is by WRKY gene (Johnson etc., 2002) of being identified involved in plant fine hair body and planting the skin developmental regulation; Arabidopis thaliana WRKY gene M IN13 (AtWRKY10) has participated in formation regulation and control (Luo etc., 2005 of seed size; Zhou Y etc., 2009); The regulation and control of Plant hormones regulators,gibberellins signal transduction when rice Os WRKY51 and OsWRKY71 have participated in seed germination (Xie etc., 2006); In addition, the WRKY transcription factor also wide participation The Plant Senescence (as OsWRKY4,23,82) and metabolic regulation aspect (Liu etc., 2008 such as (as HvWRKY46,38); Jing etc., 2009; Sun etc., 2003,2005; Xie etc., 2007).

Paddy rice is one of most important food crop of China, also is the pattern species of grass research, has crucial biology and economics and is worth.Finishing of rice genome order-checking to having laid a good foundation in full genome, identifying with research WRKY gene.The WRKY family member is numerous, some obtained achievements in research show at present, the WRKY genoid of having identified has almost participated in all respects of regulating growth of plants, metabolism, resistance, as biology and abiotic stress and somatic embryo developments such as disease, arid, high salt, low temperature, epidermal hair forms or the variation of biochemical component etc.By will be on a brand-new level to the research of these Physiology and biochemistry approach and function being the high yield of crop, high anti-and select excellent providing fundamental basis.To the structure of WRKY transcription factor and the Analysis and Identification of function, it is one of important content of illustrating gene expression regulation mechanism under various conditions, disclose between the transcription factor and and DNA between interactional concrete mechanism, thereby the expression that just can control specific gene artificially provides strong instrument for breeding research.

Summary of the invention

The objective of the invention is in the separating clone paddy rice one with the WRKY genoid OsWRKY78 and the application thereof of growing relevant.

A paddy rice WRKY class transcription factor provided by the present invention, name is called WRKY78, is the protein with one of following amino acid residue sequences: 1) the SEQ ID No:2 in the sequence table; 2) with the amino acid residue sequence of SEQ ID No:2 in the sequence table through the replacement of one or several amino-acid residue/or disappearance and/or protein of adding, perhaps its function is equivalent to the subfragment of sequence shown in the SEQ ID No2.

Wherein, SEQ ID No:2 is made up of 618 amino-acid residues, and its iso-electric point is 5.93, and molecular weight is 66.16KD.Structural analysis shows that this albumen contains two conservative WRKY structural domains (WRKYGQK), lays respectively at the 241st to 247 of aminoterminal and 415 to 421 amino acids residues.In addition, this albumen also contains two Zinc finger domains (CX4CX22HXH, " X " represents any Nucleotide), is positioned at aminoterminal the 261st to 291 and 435 to the 466 amino acids residues of SEQ ID No:2.The the 375th to 378 of aminoterminal at SEQ ID No:2 also contains a nuclear localization signal.This albumen belongs to the Ia group of WRKY family.

The cDNA of above-mentioned OsWRKY78 gene can have one of following nucleotide sequence: the 1) nucleotide sequence of SEQ ID No:1 in the sequence table; 2) SEQ ID No:1 protein sequence corresponding DNA sequences in the protein sequence table; 3) with sequence table in the cDNA sequence of SEQ ID No:1 have 90% above homology, and the identical function protein DNA sequence of encoding.

The invention also discloses the application of OsWRKY78 gene in adjusting and controlling rice grows.The OsWRKY78 gene is imported rice plant, and the preparation transgenic paddy rice can be improved the form of paddy rice, cultivates the new rice variety of the good plant type of tool.

The recombinant expression vector, transgenic cell line and the engineering bacteria that contain gene of the present invention all belong to protection scope of the present invention.Can utilize the OsWRKY78 gene of having cloned to make probe, from cDNA library and genomic library, screen gene of the present invention or homologous gene, also can utilize PCR (polymerase chain reaction) technology, amplification obtains OsWRKY78 gene of the present invention or homologous gene sequence from genome, mRNA and cDNA.Experiment showed, that rice gene OsWRKY 78 of the present invention expresses the highlyest in the stem stalk of normal water rice plants and blade, secondly is in the seed of growing.In addition, OsWRKY78 handles low temperature, hormone and salt certain response.The RNA interference transfer-gen plant of rice gene OsWRKY 78 of the present invention becomes short, sword-like leave upright, chlorophyll content improves, and shows that OsWRKY78 participates in growing of adjusting and controlling rice.Therefore, can change this expression of gene by methods such as genetically engineereds, the form of improvement paddy rice is to a certain extent cultivated the new rice variety of the good plant type of tool.

Description of drawings

Fig. 1 is that relative expression quantity Fig. 2 that real-time quantitative RT-PCR detects OsWRKY78 is that Subcellular Localization figure Fig. 3 that OsWRKY78-GFP merges albumen is that OsWRKY78RNAi carrier (A) and Overexpression vector (B) schematic diagram Fig. 4 are that the RT-PCR analysis chart Fig. 5 of genes of interest in the OsWRKY78RNAi transgenic rice plant is that RT-PCR analysis chart 6 that OsWRKY78 crosses genes of interest in expression (OEX) transgenic rice plant is that form feature figure Fig. 7 of OsWRKY78RNAi rice plant (A) and stem stalk (B) is that aspect graph Fig. 8 of OsWRKY78RNAi paddy rice seed grain is the absorption spectrum of OsWRKY78RNAi rice plant (C8580 and C8589) and unconverted contrast (WT) leaf chlorophyll.

Embodiment

By following example the invention is further described with the definition and and unrestricted.By example, the scientific research personnel can have clearer understanding to the present invention, can make certain change and modification to the present invention on this basis, to obtain different research effects.

Embodiment 1:OsWRKY78 gene sequencing and the aminoacid sequence of clone according to the barley WRKY class transcription factor SUSIBA2 albumen of having delivered (being called HvWRKY46 again), search for by the BLASTP comparison in NCBI (http://www.ncbi.nlm.nih.gov/) website, in paddy rice, found the homologous gene of SUSIBA2, locus is Os07g39480 (Os07g0583700), naming rule according to (2005) such as Xie, be called OsWRKY78 (Gene bank Accession NO:AK070537), OsWRKY70 or OsWRKY59 etc. are otherwise known as in other research reports.Inquiry in rice genome lexical or textual analysis database (http://rice.plantbiology.msu.edu/index.shtml) finds that this gene is positioned on fine the 7th karyomit(e) of paddy rice order-checking kind Japan, contains 6 exons.According to prediction, this full length gene encoding sequence (CDS) is 1857bp, long respectively 47bp of its 5 ' UTR and 3 ' UTR and 425bp (sequence table SEQ ID No:1).One of this genes encoding contains the polypeptide (sequence table SEQ ID No:2) of 618 amino-acid residues.Analyze the constructional feature of this polypeptide on the UniProtKB/Swiss-Prot website, show that its iso-electric point is 5.93, molecular weight is 66.16KD.Structural analysis shows that also this albumen contains two conservative WRKY structural domains (WRKYGQK) and two Zinc finger domains (CX4CX22HXH, " X " represents any Nucleotide).According to the principle of classification of (2005) such as Xie, the OsWRKY78 transcription factor belongs to Ia group in the WRKY superfamily.

According to the sequences Design primer of the Genbank number of landing AK070537 (drawing) from Japanese Rice Genome Resource and as template with STF1-1 (5-CA GGATCCGCAGCAATGGCCGATTCGCCAAA-3, underscore are depicted as the BamHI restriction enzyme site) and STF 1-2 (5-TA GAGCTCTCACGGACCCATGACTAA-3, underscore are depicted as the SacI restriction enzyme site) OsWRKY78 full length gene cDNA sequence is used to increase.According to the sequences Design primer of the Genbank number of landing BK005212 (drawing) from national cara gene and as template with W9P-F (5-GT AAGCTTCTCGCCTCCGAAACAACATG-3, underscore are depicted as the HindIII restriction enzyme site) and W9P-R (5-AT CCATGGCTGCGGTTGCGCTGCGCTGCGCT-3, underscore are depicted as the NcoI restriction enzyme site) OsWRKY78 gene promoter partial sequence is used to increase.Full length coding region sequence (CDS) and 5 ' upstream promoter region sequence (ATG upstream 2.0kb) that method by PCR increases from corresponding clone and obtained the OsWRKY78 gene, and by being cloned into respectively on pMD18-T (available from the TaKaRa company) carrier.Positive colony is served marine life Engineering Co., Ltd order-checking, and cloned sequence is through confirming to be used for after errorless the various vector constructions and the functional study of subsequent embodiment.

The expression characteristic 1 of embodiment 20sWRKY78, the expression analysis of OsWRKY78 transcriptional level are chosen the tissue that the fine difference of japonica rice variety Japan is grown period, with cold phenol method extracted total RNA.With plant tissue grind into powder in liquid nitrogen, add the 5ml extraction buffer (50mmol/l Tris-HCl pH9.0,20mmol/l EDTA, 1%SDS) and the saturated phenol (precooling) of isopyknic Tris damping fluid, at abundant mixing 10min on ice.In 4000rpm, 4 ℃ of centrifugal 20min, draw the upper strata water then, add the ice-cold dehydrated alcohol of two volumes, mixing leaves standstill 30min on ice.Behind the recentrifuge, abandon supernatant, precipitation is washed once with 70% cold ethanol, puts then and dries precipitation under the room temperature naturally, uses 1ml frozen water dissolution precipitation again, and adds the 8mol/l LiCl of 1/3 volume, spends the night in 4 ℃ of placements behind the mixing.Second day, at 4000rpm, 4 ℃ of centrifugal 10min, after washing twice of RNA precipitation with 70% ice-cold ethanol, at room temperature dry, be dissolved in then in an amount of water, again extractive total RNA is carried out purifying with the RNeasy Plant Mini test kit of Qiagen company, use DNaseI (RNase free) to remove the DNA among total RNA again, be dissolved in the sterilized water.Total RNA of purifying measures concentration and purity on Eppendorf Biophotometer spectrophotometer, and is settled to 1 μ g/1 μ l, again with the quality of denaturing formaldehyde total RNA that detected through gel electrophoresis is carried.The first chain cDNA's is synthetic according to the SuperScript of Invitrogen company ^TMWorking specification and reagent that First-Strand Synthesis System test kit provides, from each sample, get the total RNA of 5 μ g, the dNTP MixOligo (dT) that adds 1 μ l respectively, complement to 10 μ l with RNase-free water, in 65 ℃ of heat denatured, in cooling fast on ice, add 2 μ l, 10 * buffer, 4 μ l MgCl2,2 μ l DTT and 1 μ l RNaseOut then successively behind the 5min, behind 42 ℃ of water-bath 2min, add 1 μ l SuperScript again ^TMThe II ThermoScript II is again at 42 ℃ of water-bath 50min, at last 70 ℃ of heating 15min termination reactions.In cooled on ice, and add 1 μ l RNase H again and remove residual RNA after the termination reaction.Each sample is equipped with the contrast that does not add ThermoScript II in the experiment, and whole process is provided with contrast (NTC, the no-template control) Monitoring systems that does not add template and pollutes.Increase with primer WRT-F (5-ATAAAGGGCGTCACAATCAC-3) and WRT-R (5-GAACAGGCTCAATCAT ACCAG-3), on Eppendorf Master cycler fast PCR instrument, carry out.Response procedures is as follows: 95 ℃, 5min; 95 ℃, 50sec, 52 ℃, 50sec, 72 ℃, 25sec, (15,20,25,30) individual circulation, 72 ℃, 10min.PCR product 180bp, the agarose gel electrophoresis 1.5%, imaging analysis.Protogene OsActin1 (ACT-F:5-GGCATCACACCTTCTACAACGA-3 in the paddy rice; ACT-R:5-ACACCATCACCA GAGTCCAACA-3) as confidential reference items, to estimate the consistence of total RNA.Detected the expression characterization of OsWRKY78 by sxemiquantitative RT-PCR.The result shows that OsWRKY78 has expression at each tissue and the different development stage thereof of rice plant, but exists than evident difference at different tissues or same organizing between different development stage.Wherein, the transcriptional level in stem is the highest, secondly is blade.

Expression amount for further quantitative analysis OsWRKY78 has carried out the fluorescence real-time quantitative PCR analysis on PRISMTM 7700 SequenceDetector of ABI company.Fluorescence real-time quantitative RT-PCR is reflected in the 96 hole PCR plates and carries out, and utilizes primer WRT-F and WRT-R to increase, and the interior protogene OsActin1 of paddy rice is as confidential reference items simultaneously.The reaction system of 20 μ l comprises: and 10 μ l, 2 * SYBR Green PCR Master Mix (Applied Biosystems, USA), the first chain cDNA template of 5 ' and 3 ' each 1 μ l of end primer (3 μ mol/l) and 1ng.Reaction conditions is as follows: 50 ℃, and 2min; 95 ℃, 10min; 95 ℃, 15s; 60 ℃, 1min; Totally 40 circulations.The result shows that the relative expression quantity of OsWRKY78 gene in stem is the highest, is leaf secondly, and certain expression is also arranged in root and the leaf sheath, and this and above-mentioned sxemiquantitative RT-PCR analytical results are more consistent.Find also that simultaneously it is high that the relative expression quantity in the more newborn blade of mature leaf is wanted, the stem that is extending is than the expression amount height (Fig. 1) that extends in the stem.

2, the Subcellular Localization of OsWRKY78-GFP fusion rotein by primer to STF1-1 (5-CA GGATCCGCAGCAATGGCCGATTCGCCAAA-3, underscore are depicted as the BamHI restriction enzyme site)/STF1-5 (5-TA TCTAGAThe TCACGGACCCATGACTAA-3 underscore is depicted as the XbaI enzyme cutting site), with the clone AK070537 that contains the goal gene full length cDNA sequence is template amplification OsWRKY78 gene coded sequence, be connected into the pMD18-T carrier, after the sequence verification, double digestion (NcoI and XbaI) also reclaims this fragment, the pMD18-T carrier that contains OsWRKY78CDS with process double digestion (BamHI and SacI) links to each other subsequently, contains the total length CDS of new restriction enzyme site (BamHI/XbaI) with structure.Meanwhile, be template with carrier pMON30049 (containing green fluorescent protein GFP encoding sequence), with primer GFP-F (5-CA available from Monsanto company TCTAGAACCATGGGCAAGGGCGAG-3, underscore are depicted as the XbaI enzyme cutting site)/GFP-R (5-GT GAGCTCACTTGTAGAGTTCATCCA-3, underscore are depicted as the SacI restriction enzyme site) amplify green fluorescent protein (GFP) complete encoding sequence (CDS), be connected into the pMD18-T carrier, sequence verification.Double digestion (XbaI and SacI) contains the pMD18-T carrier of GFP, reclaims fragment and is connected into the carrier through the reorganization pMD18-T of double digestion, finishes the fusion of two sections sequences.Downcut this fragment by BamHI and SacI at last and be connected among the binary vector p3003 (this laboratory is purchased and built preservation), cut and the method Screening and Identification positive colony of PCR, make up called after OsWRKY78::GFP through enzyme.Simultaneously in contrast with plasmid Ubi::GFP.

Select the fresh scale leaf in onion the inside to be cut into small pieces (2cm length and width), place on the MS substratum.The internal surface (one side that will bombard) that makes the onion scale leaf up.Putting dark culturing spends the night.Get an amount of bronze with 50? twice of the aseptic washing of l.With 50? the l sterilized water is resuspended.Add 5 successively? l plasmid DNA (1? g/? l), 15? l 2.5M CaCl ₂With 7? l 0.1M spermidine (whenever add equally, 2-3s can vibrate), vibration 3min, 10m on ice.Centrifugal 10～the 20s of 10000rpm abandons supernatant.Add 80? the l dehydrated alcohol, it is resuspended to vibrate, and the centrifugal 10～20s of 10000rpm abandons supernatant.With 10? l dehydrated alcohol constant volume.Resuspended stand-by.After the good DNA of parcel bombarded into onion epidermis cell, continue to put after dark culturing spends the night, go up at Confocal laser-scanning microscope (Carl Zeiss LSM510) and observe onion epidermis cell.There are some researches show, when protein molecular weight surpasses 50kDa, just can not be by physical diffusion effect turnover nucleopore.The molecular weight of GFP own is less than 50kDa, can freely pass through nucleopore, and OsWRKY78 albumen self molecular weight is 66.16kDa, so the WRKY78::GFP fusion rotein can not enter nucleopore by physical action, only just can enter in the nucleus under specific nuclear localization signal peptide effect.Experimental result shows, only observes green fluorescence in the onion epidermis cell of conversion OsWRKY78::GFP fusion gene in nucleus; And only contain in the cell of GFP gene, the disperse of green fluorescence signal is in each position of cell (Fig. 2).This result has proved that OsWRKY78 albumen is positioned in the nucleus.Can infer that in view of the above OsWRKY78 albumen is the trans-acting factor of regulate gene expression, in nucleus, work.

The acquisition of embodiment 3OsWRKY78 transgenic paddy rice and the structure of identifying the 1.OsWRKY78-RNAi carrier.

By ncbi database ( Http:// www.ncbi.nlm.nih.gov/) in do homology comparison, one section distinguished sequence having selected 420bp in OsWRKY78 gene the 3rd exon is as the purpose fragment that makes up interference vector.Primer STF1-F (the 5-CC that contains specific restriction enzyme site by design ACTAGTACTGTAGGCTTCTGCTATC-3, underscore are depicted as the SpeI restriction enzyme site) and STF1-R (5-CA GGATCCAGTAGTTGCCACACCATCAT-3, underscore is depicted as the BamHI restriction enzyme site) this section sequence amplification is come out, utilize glue to reclaim test kit behind the electrophoresis and reclaim the purpose fragment, and be cloned on the pMD18-T carrier, confirm through order-checking errorless after, contain segmental plasmid of purpose (pMD18-T) and intermediate carrier p1022 (this laboratory is purchased and built preservation) with double digestion (SpeI and BamHI) digestion through what increase.Enzyme is cut product behind electrophoresis, reclaims the carrier p1022 of purpose fragment and incision, connects two fragments by ligase enzyme, and imports in the bacillus coli DH 5 alpha.After selecting positive colony and identifying, the plasmid identified of double digestion (BglII and XbaI) once more with after purpose fragment that double digestion (SpeI and BamHI) is crossed links to each other once more, imports equally and increases in the bacillus coli DH 5 alpha and evaluation subsequently.The positive colony of identifying is again through link to each other with the binary vector p3003 that cuts through same enzyme behind BamHI and the SacI double digestion (the carrier synoptic diagram is seen Fig. 3 A), cut and the method Screening and Identification positive colony of PCR through intestinal bacteria amplification back and by enzyme, and in the importing Agrobacterium.

2.OsWRKY78 the structure of overexpression carrier contains the pMD18-T carrier of OsWRKY78 total length CDS by double digestion (BamHI and SacI), reclaim the purpose fragment, and be connected with the binary vector p3003 that contains corn ubiquitin gene Ubi promotor (2.0kb long) that cuts through same enzyme (the carrier synoptic diagram is seen Fig. 3 B), after importing the intestinal bacteria amplification, cut and the method Screening and Identification positive colony of PCR by enzyme, the extracting plasmid imports carrier in the Agrobacterium.

The evaluation of transgenic paddy rice with the carrier that builds by agriculture bacillus mediated rice conversion program (Liu Qiaoquan etc., 1998), import to japonica rice variety Japan fine in, by series of selection and cultivation, each carrier has all obtained to surpass 20 independent transformant at last.At first adopted excised leaf hygromycin resistance detection method (Liu Qiaoquan etc., 2001) that transgenic paddy rice is made preliminary screening.T0 is shown that for the detected result of transfer-gen plant the blade of most transgenic rice plants can both keep fresh green in containing the detection liquid of Totomycin, illustrate that thus hygromycin gene can both normal expression in most of rice plants.Further utilize Totomycin universal primer HP1/HP2 that these rice plants are carried out pcr amplification, prove that hygromycin resistance detects the male plant, PCR identifies still positive.Analysis above comprehensive, the regenerated transgenic rice plant overwhelming majority is positive.Through the 2-3 selection in generation, screening has obtained more stable a plurality of transgenic lines of isozygotying, to be used for follow-up analysis.RNAi transfer-gen plant called after C8580, C8589, C8620, overexpression transfer-gen plant called after C8640, C8648, C8754.

For whether the expression of goal gene in the clear and definite transfer-gen plant changes, the total RNA of transgenic rice plant has been carried out sxemiquantitative RT-PCR analyzed.Select part T2 or T3 for isozygotying transfer-gen plant, choose the prematurity seed 30-40 grain of blooming back 15 days, extract the total RNA of endosperm, described reverse transcription becomes the first chain cDNA according to materials and methods, carries out RT-PCR with primer WRT-F that crosses over intron and WRT-R and analyzes.The result shows, (wild type WT) compares, the expression of OsWRKY78 obviously descend (Fig. 4) in 3 RNAi transfer-gen plants (C) with normal unconverted parent, obviously raise (Fig. 5) and in overexpression transfer-gen plant (OEX), express, more consistent with expected result.

Embodiment 4 transgenic regulation Os WRKY78 express the 1. transgenic paddy rice economical character analyses that influence that rice plant growth is grown the above-mentioned RNAi or the overexpression transgenic paddy rice seedling replanting of seed selection are gone into the field, there is no obvious phenotypes difference in vegetative growth phase and unconverted contrast.But in the heading later stage, find that nearly all T0 can not extract out fully for the ear stem of RNAi plant, exist bag fringe phenomenon, and the overexpression transfer-gen plant can be eared normally.With " Invest, Then Investigate " the T1 of transgenic paddy rice and the economical character in T2 generation, find that the RNAi plant compares with unconverted parent, the height that plant push up from the face of land to the sword-like leave does not have difference, but to the obviously change short (Fig. 6 A) of height on fringe top, is utmost point significant difference.Further analyze find the RNAi plant fall one and fall that length shortens (Fig. 6 B) between two stipes, compare with negative control system with the parent and also show extremely significant difference, analyze and caused bag fringe phenomenon thus.The grain characters analysis revealed, RNAi transfer-gen plant paddy grain husk shell color and luster wants dark than the parent, but length and width, thick not difference but brown rice grain length degree obviously shorten (Fig. 7), and width and thickness aspect do not change.These results show that the OsWRKY78 gene plays important regulation in the growth course of the elongation of the growth of rice plant especially stem stalk and seed.

2. transgenic paddy rice photosynthetic pigments absorption spectroanalysis proterties investigation shows, OsWRKY78-RNAi plant sword-like leave is more strong than parent's contrast, and is upright, and leaf look dark (Fig. 6 A).And the overexpression plant, plant height there is no change, but blade is opposite with the interference plant, deliquescing a little, and do not stand upright.Therefore at full heading time, get a up-to-date complete leaf, take by weighing the fresh blade 0.1g of rice plant, be cut into the long fragment of 2mm, add 80% acetone, at 25mL volumetric flask constant volume.Behind dark place lixiviate 48h, vat liquor has been measured the absorption spectrum of photosynthetic pigments respectively with Ultrospec2000 type ultraviolet-visible spectrophotometer.The result shows with the parent and compares, 2 independently the absorption value of RNAi plant leaf photosynthetic pigments under each light wave length all than parent height (Fig. 8), illustrate and disturb the chlorophyll content of plant leaf raising to be arranged, show that also the OsWRKY78 gene participates in the regulation and control of each side in the paddy rice growth course simultaneously than the parent.

Claims (4)

1. paddy rice WRKY class transcription factor WRKY78, its aminoacid sequence is shown in SEQ ID NO:2.

2. one kind by the described transcription factor WRKY78 deutero-of claim 1 protein, it is characterized in that, its aminoacid sequence is to replace in the aminoacid sequence of SEQ ID NO:2, lack or add one to ten amino-acid residue, and has the identity function with WRKY78.

3. the gene OsWRKY78 of the WRKY78 that encodes, its nucleotide sequence is shown in SEQ ID NO:1.

4. the application of OsWRKY78 gene in adjusting and controlling rice grows shown in the claim 3.

Images