CN102732535B - Application of histone demethylase gene OsJ5 in raising resistance of paddy rice - Google Patents

Abstract

The invention belongs to the technical field of paddy rice gene engineering and specifically relates to an application of a paddy rice histone demethylase gene OsJ5, which can raise disease resistance of bacterial blight, in paddy rice's disease resistance genetic improvement. Genetically modified rice of OsJ5 overexpression can be obtained by an Agrobacterium-mediated transgenic method. Transgenic plant begins to gradually show ghost spot at tillering stage. Bacterial leaf blight is inoculated at booting stage and it is found that the transgenic plant is more resistant to diseases than a control wild-type plant. It is found through histone modification of the detected transgenic plant that histone H3K36 methylation and H3K9 trimethylation levels of the transgenic plant are obviously lower than those of the wild type. It shows that the histone demethylase gene OsJ5 of the present invention can regulate and control paddy rice's histone methylation modification and raise resistance of paddy rice to bacterial leaf blight.

Description

The application of histone demethylase gene OsJ5 in improving the paddy rice resistance

Technical field

The invention belongs to technical field of rice gene engineering.Relevant with the transgenic paddy rice field.Be specifically related to separating clone, functional verification and the application of a paddy rice histone demethylase gene OsJ5.Described gene is relevant with raising paddy rice resistance.

Background technology

The expression of eukaryotic gene is subjected to the inside and outside multi-level adjusting of nucleus, presents multistage regulation and control: comprise genetic regulation (genetic regulation) and epigenetic regulation and control (epigenetic regulation).Epigenetic refers to directly not to be subjected to the gene of dna sequence dna control to take place to express or the variation of function and phenomenon that can heredity, its essence is that the dynamic change of chromatin Structure is to the influence of genetic expression, because it does not relate to the change of gene order, not meeting the mode of inheritance of Mendelian inheritance, is a kind of brand-new genetic mechanism.Common epigenetic phenomenon comprises: (Levenson JM and Sweatt JD.Epigenetic mechanisms in memory formation.Nat Rev Neurosci such as dna methylation, histone modification, karyomit(e) are reinvented, genetic imprinting, x chromosome inactivation, RNA regulation and control, transposable element, 2005,6:108-118).

Cell each reaction of making of environment to external world all will be regulated some expression of gene, and this nearly all can relate to the variation of chromatin activity, and the variation of chromatin activity often realizes by modifying histone.The aminoterminal modification of histone comprises phosphorylation, ubiquitinization, methylates/demethylation, acetylize/deacetylation etc.; these combinations of modifying the various marks that produce have just formed " Histone Code " (Jenuwein T and Allis CD.Translating the histone code.Science of so-called adjusting chromatin Structure and genomic expression; 2001,293:1074-1080).Wherein the modification that methylates of histone plays an important role to growth and development of plant.Histone demethylase (Histone demethylase, HDM) be class I histone modifying enzyme (the Klose et al. that found in recent years, JmjC-domain-containingproteins and histone demethylation.Nat Rev Genet.2006,7:715-727) can remove the methyl of the histone afterbody of being modified by methylating, with the functions reversed of ZNFN3A1 (HMT).The gene that contains Jumonji C (JmjC) structural domain is a newfound genoid with histone demethylase function in the mankind, and has locus specificity (Klose et al., Regulation of histone methylation by demethylimination and demethylation.Nature reviews.2007,8:307-318.).Along with going deep into of research, more and more the member of this proteinoid is found, and function is also more and more abundanter.In the plant, to jumonji family function report the earliest be EARLY FLOWERING 6 (ELF6) and RELATIVE OF EARLY FLOWERING 6 (REF6) (the Noh et al. that the regulation and control Arabidopis thaliana delivered in 2004 is bloomed, Divergent Roles of a Pair of Homologous Jumonji/Zinc-Finger-Class Transcription Factor Proteins in the Regulation of Arabidopsis Flowering Time.The Plant Cell.2004,16:2601-2613.).The gene JMJ706 of paddy rice confirms to have the activity of removing H3K9 dimethylization and trimethylammoniumization, its mutant presents the unusual of the variation of floral shape and seed endosperm, experimental results show that the histone modification level that it may be by the relevant gene M ADS47 of regulation and control flower development and DH1 and impel its expression cause flower development unusual (Sun and Zhou.Rice jmjC domain-containing geneJMJ706 encodes H3K9 demethylase required for floral organ development.PNAS.2008,36:13679-13684).20 jumonji genes are arranged in the rice genome, the function of other gene is all unknown except JMJ706, we just more have reason to believe like this, in occupation of consequence, the research of relevant fermentation also has certain scientific research and using value to this genoid in the epigenetic of paddy rice is regulated and control and grown.

Summary of the invention

The objective of the invention is to clone and identify a kind of new paddy rice histone demethylase gene OsJ5 and proteins encoded thereof.By rice transformation itself, cultivate a kind of transgenic paddy rice that can improve the paddy rice resistance.

The present invention is achieved through the following technical solutions:

The applicant clone obtains paddy rice histone demethylase gene OsJ5, and its nucleotide sequence is shown in 123-3983 among the SEQ NO:1.Histone of the present invention goes the encoding gene (SEQ ID NO:1) of first enzyme gene OsJ5 to derive from paddy rice, and by 4145 based compositions, the protein coding sequence of supposition has 3861 bases, is that sequence 1 is formed to 3983 bit bases from 5 ' end the 123rd bit base.The complete encoding sequence (Coding sequence) of this gene is building up to overexpression vector PU1301 (see figure 1), obtain directly to change paddy rice over to behind the conversion carrier pU1301-OsJ5, transfer-gen plant begins to engender the cape horn fever spot in a minute evil phase, inoculates bacterial leaf-blight in boot stage and finds that transfer-gen plant is than the disease-resistant many (see figure 3)s of contrast wild-type.Detect the histone modification situation of transfer-gen plant and find that the histone H 3 K36 trimethylammoniumization of transfer-gen plant and H3K93 trimethylammonium level will be starkly lower than the wild-type (see figure 6).Show that histone demethylase gene OsJ5 of the present invention can the histone methylated modification of adjusting and controlling rice and improve paddy rice to the resistance of bacterial leaf-blight.

The concrete operations step is as follows:

(1) obtains the nucleotide sequence of OsJ5 gene (accession number AK068952) from ncbi database (http://www.ncbi.nlm.nih.gov), right according to this sequences Design primer, the segment area of amplifying specific (see shown in the SEQ ID NO:1 by its nucleotide sequence, wherein 123-3983bp is the coding region), the right dna sequence dna of described primer is as follows:

OXJ5-F (forward primer): 5 '---GGGGGTACCATGAGGCCCTCGCCGCCTCC---3 '

OXJ5-R (reverse primer): 5 '---GGGGGATCCTCAACTCTTTGCTTTCTTCTTCT------3 '

(2) amplified fragments in (1) is connected to the pU1301 (see figure 1), obtains conversion carrier PU1301-OsJ5.

(3) utilize agriculture bacillus mediated transgenic method (Hiei Y et.al., Efficient transformation ofrice (Oryza sativaL.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA.PlantJ.1994,6:271-282) with spending 11 (from Institute of Crop Science, Chinese Academy of Agricultural Science) in the described carrier importing paddy rice acceptor, obtain transformed plant;

(4) identify positive transfer-gen plant by Northern Blot methods analyst, and preliminary observation statistics transfer-gen plant phenotype;

(5) utilize the endogenous OsJ5 gene expression pattern of RT-PCR methods analyst paddy rice;

(6) utilize realtime-PCR to analyze Expression of Related Genes in the transfer-gen plant;

(7) analyze transfer-gen plant histone modification situation by Western Blot;

(8) transfer-gen plant inoculation bacterial leaf spot bacterial strain PX099, and carry out the resistance statistics.

Advantage of the present invention is as follows:

The function of histone demethylase gene OsJ5 of the present invention in improving the paddy rice resistance at home and abroad do not appeared in the newspapers, and the research of histone demethylase function and apparent regulation and control are played important meaning to the research of paddy rice resistance.The report of histone H 3 K36 trimethylammonium enzyme gene is not also arranged in paddy rice at present, and OsJ5 of the present invention can remove the function of histone H 3 K36 trimethylammoniumization to the important biological significance that is supplemented with of histone modification enzyme gene molecule function.Overexpression OsJ5 transfer-gen plant can improve paddy rice to the resistance of bacterial leaf-blight, and this research to rice bacterial blight resistance has important directive significance, can disclose disease-resistant mechanism at molecular level.And can lay a good foundation for further being applied to the variety of crops improvement, significant to ensureing Chinese grain security.

Description of drawings

Sequence table SEQ ID NO 1: the nucleotide sequence and the corresponding amino acid sequence that are the present invention's paddy rice histone demethylase of cloning.The sequence total length is 4145bp, and wherein the 123-3983bp zone is the coding region.

Fig. 1: be the overexpression carrier PU1301 synoptic diagram that the present invention makes up.

Fig. 2: be that the OsJ5 gene detects for the expression amount in the overexpression transfer-gen plant at T1.In spend 11 negative contrasts, among the figure: 1-1,2-5,3-9,4-2 are the transgenic positive plant, 2-2 is the negative plant of transgenosis;

Fig. 3: the form phenotype and the phenotype of transfer-gen plant inoculation bacterial leaf spot bacterial strain PXO99 after 15 days that are OsJ5 gene overexpression rotaring gene plant blade.Fig. 3 A is the overexpression transfer-gen plant phenotypic map of blade during boot stage under field conditions (factors); Fig. 3 B is that the overexpression transfer-gen plant is inoculated the blade phenotype of bacterial leaf spot bacterial strain PXO99 after 15 days in boot stage.In spend 11 negative contrasts, among the figure: 1-1,2-5,3-9,4-2 are the blade that the phenotype plant is arranged, 2-2 is the blade that transgenosis does not have the negative plant of phenotype.

Fig. 4: OsJ5 expression of gene pattern.Be the expression pattern of OsJ5 gene in rice callus, bud, root, stem, young fringe, sword-like leave, spire among the figure, actin is confidential reference items.

Fig. 5: the expression of hAT and HSR201 in the OsJ5 gene overexpression rotaring gene plant blade.Fig. 5 A is the relative expression's level of hAT in the overexpression transfer-gen plant; Fig. 5 B is the relative expression's level of HSR201 in the overexpression transfer-gen plant.In spend 11 for control material, among the figure: 1-1,2-5,4-2 be the genetically modified T1 of OsJ5 gene overexpression for material, actin is confidential reference items;

Fig. 6: the histone modification of OsJ5 gene overexpression transfer-gen plant.The primary antibodie of Western Blot experiment usefulness is respectively H3 antibody, trimethylammonium H3K9 antibody, trimethylammonium H3K36 antibody; Spend during the albumen that detects is respectively 11 and OsJ5 gene overexpression transgenosis T1 for the histone of material 2-5.

Fig. 7: transfer-gen plant inoculation bacterial leaf spot bacterial strain PXO99 state of an illness statistics.Adopt leaf-cutting method inoculation bacterial leaf spot pathogenic bacteria PXO99 (Philippines fungus strain physiological strain) boot stage, the leaf of 5 full extension of every strain inoculation is inoculated back 15 days investigation scab length, weighs the state of an illness according to lesion area (scab length/sick leaf length).In spend 11 to be contrast, 1-22 be OsJ5 overexpression transgenosis T1 for material, upright letters mark be isolated transgenic positive plant, the underscore mark be transgenosis feminine gender plant;

Embodiment

Embodiment 1 OsJ5 gene fragment clone

To gene (the accession number AK068952 that wants required for the present invention, http://www.ncbi.nlm.nih.gov/nucleotide/32978976), main by the RT-PCR method (referring to J. Sa nurse Brooker, EF is the Ritchie not, T Manny A Disi work, Huang Peitang, Wang Jiaxi etc. translate, molecular cloning experiment guide (third edition), Beijing, Science Press, 2002 editions) increasing obtains one section special sequence of OsJ5, concrete steps are: first extracting is from the RNA of plant leaf in boot stage, and RNA extracting reagent is the Trizol extraction agent box (the concrete operations step is seen the test kit specification sheets) of Invitrogen company; The step of synthetic cDNA first chain of reverse transcription is as follows among the RT-PCR: 1. join mixed solution 1: total RNA 2 μ g, DNAseI2u, 10x DNAseI buffer 1 μ l, add DEPC (diethylpyrocarbonate, the strongly inhibited agent of RNA enzyme) handles water (0.01%DEPC) to 10 μ l, behind the mixing mixed solution 1 is placed 20 minutes to remove DNA at 37 ℃, 2. place 70 ℃ of water-bath temperature to bathe 10 minutes to remove DNAse I activity mixed solution 1 after 20 minutes, placed then 5 minutes on ice, 3. the oligo (dT) that adds 1 μ l, 500 μ g/ml in the mixed solution 1,4. the mixed solution 1 with cooling places 70 ℃ of water-bath temperature to bathe immediately 10 minutes, thoroughly to make the RNA sex change, placed then 5 minutes on ice, 5. join mixed solution 2: mixed solution 110 μ l, 5x first strand buffer 4 μ l, 0.1M DTT (mercaptoethanol) 2 μ l, 10mM dNTP mixture1.5 μ l, DEPC handles water 0.5 μ l, ThermoScript II 2 μ l, behind the mixing mixed solution 2 placed in 42 ℃ of water-baths temperature to bathe 1.5 hours, 6. place 90 ℃ to do bath 3 minutes mixed solution 2 after reacting end, 7.-20 final product is reacted in a ℃ preservation, and the reagent of using in the reaction is all available from Invitrogen company; The full length cDNA sequence of the OsJ5 gene of announcing according to ncbi database (http://www.ncbi.nlm.nih.gov) then, design special primer pcr amplification specific fragment.The system that PCR uses is 20 μ l, specifically joins method to be: the cDNA first chain template 1 μ l, 10xPCR buffer 2 μ l, 10mM dNTP 1.6 μ l, 2.5mMMg ²⁺1.5 μ l, each 0.4 μ l of OXJ5-F and OXJ5-R primer, TAQ enzyme 0.2 μ l adds water to 20 μ l (used PCR buffer, dNTP, Mg ²⁺, rTAQ enzyme etc. is all available from precious biotechnology Dalian company limited).The PCR reaction conditions is as follows: 1. 94 ℃ 4 minutes, 2. 94 ℃ 30 seconds, 3. 60 ℃ 30 seconds, 4. 72 ℃ 4 minutes, 5. from 2.-4. circulating 30 times, 6. 72 ℃ 7 minutes, 7. 4 ℃ of preservations.At last amplified production being connected to T/A cloning vector pGEMT-vector (available from Pu Luomaige (Beijing) Bioisystech Co., Ltd, i.e. U.S. Promega company) goes up with T7 and SP6 primer sequence verification.The primer that is used for cloning the full length fragment of OsJ5 is:

OXJ5-F (forward primer): 5 '---GGGGGTACCATGAGGCCCTCGCCGCCTCC---3 '

OXJ5-R (reverse primer): 5 '---GGGGGATCCTCAACTCTTTGCTTTCTTCTTCT---3 '

The full length cDNA sequence of the OsJ5 gene that finally obtains is shown in 123-3983 position among the sequence table SEQ ID NO:1.

The structure of embodiment 2 overexpression vector PU1301-OsJ5

Concrete steps are as follows:

1) the T/A clone that will have OsJ5 fragment (shown in 123-3983 position among the sequence table SEQ ID NO:1) cuts with KpnI and BamHI enzyme, reclaim target stripe, be connected with the expression vector plasmid PU1301 (see figure 1) that the BamHI enzyme is cut with KpnI, pU1301 is the plant genetic conversion carrier pCAMBIA1301 (Sun etc. that use always in the world, 2004, Xa26, a gene conferring resistance to Xanthomonas oryzae pv.oryzae in rice, encoding a LRR receptor kinase-like protein.Plant Journal.37:517-527) transforming on the basis, is the agriculture bacillus mediated genetic transformation carrier that carries the corn ubiquitin promoter with composing type and overexpression feature.The pCAMBIA1301 carrier is so kind as to give by Australian CAMBIA laboratory (Center for the Application of Molecular Biology to International Agriculture).Restriction endonuclease KpnI and BamHI be connected employed T4 ligase enzyme all available from precious biotechnology Dalian company limited, the product description that usage and consumption provide according to the said firm;

2) (electric conversion instrument is eppendorf company product by the electric method that transforms to connect product, applied voltage is 1800v, working method is seen the instrument specification sheets) import intestinal bacteria DH10B (available from Pu Luomaige (Beijing) Bioisystech Co., Ltd, be U.S. Promega company), (LA fills a prescription referring to J. Sa nurse Brooker at the LA that contains 250ppm kantlex (available from Luo Shi biotech firm product), EF is the Ritchie not, T Manny A Disi work, Huang Peitang, Wang Jiaxi etc. translate, molecular cloning experiment guide (third edition), Science Press, 2002 editions) be coated with ware on the LA resistance substratum and cultivate;

3) single bacterium colony of growing on the LA resistance substratum is inoculated in the 10ml centrifuge tube of sterilization at Bechtop, adds the LB resistance substratum that 3ml contains the 250ppm kantlex in the pipe in advance, cultivated 16-18 hour at 37 ℃ of shaking tables then.According to (" molecular cloning experiment guide ", J. Sa nurse Brooker and D.W. Russell are outstanding, Huang Peitang etc. translate, Science Press, 2002) described method extracting plasmid, cut the detection with PCR with KpnI and BamHI enzyme, obtain positive overexpression plasmid vector with its called after PU1301-OsJ5 according to the size of inserting fragment;

4) the overexpression vector PU1301-OsJ5 of step 3) is undertaken by the method for the method (voltage parameter of reference and use is according to present embodiment step 2) of electricity conversion) import in Agrobacterium EHA105 (available from the Australian CAMBIA laboratory) bacterial strain.With the bacterial strain called after T-OsJ5 after transforming;

Positive and the expression amount detection of the conversion of embodiment 3 double base Ti-plasmids carriers and transfer-gen plant:

Concrete steps are as follows:

1) T-OsJ5 is transformed to paddy rice acceptor kind " in spend 11 " (Institute of Crop Science, Chinese Academy of Agricultural Science), method for transformation is with reference to people's reported method such as Hiei (Hiei etc., Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA.Plant J, 1994,6:271-282) carry out.The T0 that obtains is for transfer-gen plant called after OsJ5-n, n=1 wherein, 2,3 ... represent genetically modified different family.

2) get T0 for the total DNA of transformed plant blade extracting, the DNA method for extracting is CTAB method (Zhang etc., genetic diversity and differentiation of indica an japonica rice detected by RFLP analysis, 1992, Theor Appl Genet, 83,495-499).With PCR method T0 is carried out positive detection for transformed plant with the Totomycin primer then.The sequence of Totomycin primer is as follows: Hn-F 5 '-agaagaagatgttggcgacct-3 ', Hn-R 5 '-gtcctgcgggtaaatagctg-3 ' (it is synthetic that worker's biotechnology company limited is given birth in Shanghai).PCR reaction cumulative volume is 20 μ l, specifically joins method and is: template 100ng, 10xPCR buffer 2 μ l, 10mM dNTP 1.6 μ l, 2.5mM Mg ²⁺1.5 μ l, each 0.4 μ l of left and right primer (Hn-F and Hn-R), TAQ enzyme 0.2 μ l adds deionized water to 20 μ l (used PCR buffer, dNTP, Mg ²⁺, rTAQ enzyme etc. is all available from precious biotechnology Dalian company limited).The PCR reaction conditions is as follows: 1. 94 ℃ 4 minutes, 2. 94 ℃ 30 seconds, 3. 56 ℃ 30 seconds, 4. 72 ℃ 2.5 minutes, 5. from 2.-4. circulating 32 times, 6. 72 ℃ 7 minutes, 7. 4 ℃ of preservations.The PCR product is electrophoresis detection on 1% sepharose.Because hygromycin gene is peculiar by conversion carrier, the transfer-gen plant that can amplify the special band of hygromycin gene like this is positive plant.T0 is received seed (T1 generation) for positive plant, for field planting and the proterties investigation in T1 generation are prepared.

3) in order to detect the expression amount of target gene in the transfer-gen plant, the applicant adopts the method for Northern-blot that transgenosis T1 has been carried out expression analysis for plant.Test used total RNA from the rice leaf in tillering phase, RNA extracting reagent is the Trizol extraction agent box (the concrete operations step is seen the test kit specification sheets) of Invitrogen company; It is 15-20 μ g that Northern changes the film applied sample amount, probe mark adopts the method for random primer labelling, and employed isotopic label is a-32P-Dctp, film first prehybridization about 3 hours in hybrid pipe, add isotope-labeled probe then, continue hybridization 12 hours; Hybridization with 2 * sodium citrate buffer solution (SSC), with Hybond membrane rinsing 10 minutes, uses same film washing liquid 65 ℃ of rinsings under 0.1% sodium lauryl sulphate (SDS) normal temperature after finishing then, and each two minutes, till signal value is suitable.Place clean filter paper to dry film after washing film, wrap with preservative film, read the result with FUJIFILMFLA5100 scanning at last in compressing tablet 4-6 hour with phosphorus screen (Phosphorimage, FUJI, Japan) then.

In the present embodiment, the method that obtains transfer-gen plant adopts agriculture bacillus mediated genetic transforming method, and (document is referring to Hiei et al., Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA.Plant J, 1994,6:271-282) Bao Dao key step, the reagent of substratum and use is mainly with reference to Hua Zhong Agriculture University's granted patent (patent No. ZL200710053552.9, the title of invention: the separating clone of rice wide compatibility gene S 5 and application; Patent disclosure day: on June 18th, 2008; Publication number: CN101200725; License day: on 04 21st, 2010), the step of the embodiment of the invention and the difference of above-mentioned document are: (resistance screening) method is cultivated in callus washing and selection.The present invention should the stage operation steps be:

(1) washs the paddy rice callus to cannot see Agrobacterium with sterilized water.

(2) callus is immersed in the sterilized water that contains 400mg/L Pyocianil (CN) 30 minutes.

(3) this callus is transferred on the good filter paper of sterilization, makes this callus not be with water.

(4) shift callus to selecting to select 2-3 time each 2 weeks (concentration of Totomycin is 400 mg/litre for the first time, and the concentration of Totomycin is 250 mg/litre after reaching for the second time) on the substratum.

Final the present invention obtains independent transgenic paddy rice overexpression material T0 generation 10 strains altogether.

Embodiment 4 T1 investigate and expression analysis for proterties:

1) T0 of the present invention being received seed (T1 generation) kind for positive plant (being the plant that the OsJ5 gene expression amount rises) goes into the land for growing field crops and carries out phenotypic evaluation.Found that the transgenic positive plant all has phenotype in various degree, be embodied in blade appearance cape horn fever spot (as shown in Figure 3) in various degree.

2) in order to detect Expression of Related Genes amount in the transfer-gen plant, we have carried out expression analysis with quantifying PCR method to transfer-gen plant.Test used total RNA from the blade of plant in boot stage, RNA extracting reagent is the Trizol extraction agent box (the concrete operations step is seen the test kit specification sheets) of Invitrogen company; The step of synthetic cDNA first chain of reverse transcription is as follows among the RT-PCR: 1. join mixed solution 1: total RNA 2 μ g, DNAseI 2u, 10x DNAseI buffer 1 μ l, add DEPC (diethylpyrocarbonate, the strongly inhibited agent of RNA enzyme) handles water (0.01%DEPC) to 10 μ l, behind the mixing mixed solution 1 is placed 20 minutes to remove DNA at 37 ℃, 2. place 70 ℃ of water-bath temperature to bathe 10 minutes to remove DNAse I activity mixed solution 1 after 20 minutes, placed then 5 minutes on ice, 3. the oligo (dT) that adds 1 μ l, 500 μ g/ml in the mixed solution 1,4. the mixed solution 1 with cooling places 70 ℃ of water-bath temperature to bathe immediately 10 minutes, thoroughly to make the RNA sex change, placed then 5 minutes on ice, 5. join mixed solution 2: mixed solution 1 10 μ l, 5x first strand buffer 4 μ l, 0.1M DTT (mercaptoethanol) 2 μ l, 10mMdNTP mixture 1.5 μ l, DEPC handles water 0.5 μ l, ThermoScript II 2 μ l, behind the mixing mixed solution 2 placed in 42 ℃ of water-baths temperature to bathe 1.5 hours, 6. place 90 ℃ to do bath 3 minutes mixed solution 2 after reacting end, 7.-20 final product is reacted in a ℃ preservation, and the reagent of using in the reaction is all available from Invitrogen company; The system that RT-PCR uses is 20 μ l, specifically joins method to be: the cDNA first chain template 1 μ l, 10xPCRbuffer 2 μ l, 10mM dNTP 1.6 μ l, 2.5mM Mg ²⁺1.5 μ l, each 0.4 μ l of left and right primer, TAQ enzyme 0.2 μ l adds water to 20 μ l (used PCR buffer, dNTP, Mg ²⁺, rTAQ enzyme etc. is all available from precious biotechnology Dalian company limited).The PCR reaction conditions is as follows: 1. 94 ℃ 2 minutes, 2. 94 ℃ 1 minute, 3. 56 ℃ 1 minute, 4. 72 ℃ 1 minute, 5. from 2.-4. circulating 30 times, 6. 72 ℃ 7 minutes, 7. 4 ℃ of preservations.The product of reverse transcription carries out quantitative PCR, and the system of its reaction is 25 μ l, wherein contains left and right sides primer (following listed hAT-F and the hAT-R of 1.5 μ l reverse transcription products, 0.25M; HSR201-F and HSR201-R; Actin-F and Actin-R) and the SYBR Green mixture (Applied Biosystems) of 12.5 μ l.Be reflected on the 7500real-time quantitative PCR instrument (Applied Biosystems) and carry out, response procedures carries out according to the operational manual that Applied Biosystems provides, paddy rice actin gene (accession number AK060893.1http: //www.ncbi.nlm.nih.gov/nuccore/32970911) as the confidential reference items in the reaction.All primers react 40 circulations all 58 ℃ of annealing, and each sample arranges three repetitions, carry out equilibration with the actin expression amount and handle.The result shows, cloned genes OsJ5 of the present invention can activate retrotransposon hAT (accession number AL606658, http://www.ncbi.nlm.nih.gov/nucleotide/57834145) expresses, also can cause the increase (as shown in Figure 5) of programmed death (PCD) genes involved HSR201 (accession number AK065433, http://www.ncbi.nlm.nih.gov/nucleotide/32975451) expression amount.

The nucleotide sequence of the primer that quantitative PCR is used is as follows:

Realtime?hAT-F：5’-AATGGTTGGCAAGGATCGC-3’，

Realtime?hAT-R：5’-TCCTCGCGTAATTGCTGCAA-3’；

Realtime?HSR201-F：5’-TTGTGGCGTGAAAAACCGTA-3’，

Realtime?HSR201-R：5’-TTGGCGGACTAACAGTCGGTA-3’；

Realtime?Actin-F?5’-TGTATGCCAGTGGTCGTACCA-3’，

Realtime?Actin-R?5’-CCAGCAAGGTCGAGACGAA-3’；

Embodiment 5 detects the expression pattern of paddy rice native gene OsJ5

Be material with rice varieties " in spend 11 ", respectively at callus, bud, root, stem, young fringe, sword-like leave and spire sampling.Carry out reverse transcription after extracting total RNA by the method among the embodiment 4, the product of reverse transcription carries out sxemiquantitative PCR and detects gene at the expression level at each position of plant.The result shows that the OsJ5 gene is constitutive expression, but in young fringe and blade expression amount higher (result as shown in Figure 4).

The protein function of embodiment 6 OsJ5 genes detects

In order to determine the effect of OsJ5 albumen on histone modification, the applicant is by extracting the histone of OsJ5 transgenosis and wild-type, (method is with reference to Huang et al. to utilize the commercialization antibody (seeing below described) of histone modification class to do Western Blot experiment, Down-regulation of a Silent Information Regulator2-related gene, OsSRT1, induces DNA fragmentation and cell death in rice.Plant Physiol, 2007,144:1508-1519.).Concrete operations are: get the after planting wild-type about 40 days and genetically modified blade 4-5 sheet respectively, put into after the liquid nitrogen grinding 3 times of volumes the histone extraction buffer (10mM Tris-HCl, pH 7.5; 2mMEDTA; 0.25M HCl; 5mM 2-mercaptoethanol; 0.2mM abundant mixing phenylmethylsulfonyl fluoride (PMSF)), through 12000g, 4 ℃ after centrifugal 10 minutes, draw supernatant in another new centrifuge tube, by volume adding trichoroacetic acid(TCA) to final concentration is 25%, 17000g, 4 ℃ centrifugal 30 minutes, abandon supernatant, precipitation is with washing with acetone three times, be dissolved in after drying sample-loading buffer (62.5mM Tris-HCl, pH 6.8; 2%SDS; 25% glycerine (glycerol); 0.01% tetrabromophenol sulfonphthalein (Bromophenol Blue); (among the β-mercaptoethanol), be that 15% SDS-PAGE electrophoresis can detect extraction rate was acquired through concentration, electrophoresis adopts the Mini-PROTEAN3 electrophoresis chamber system of Bio-Rad company to 10% beta-mercaptoethanol, operates according to its specification sheets.

Carry out the western detection with extracting good histone, western blot changes the Mini Trans-Blot Cell transfer groove system that film uses Bio-Rad company, according to its specification sheets, histone is gone on the Hybond-P pvdf membrane of Amersham company, with PBS damping fluid (the NaCl 137mmol/L that contains 2% (mass volume ratio) bovin serum albumin (BSA) for preparing, KCl 2.7mmol/L, Na ₂HPO ₄4.3mmol/L, KH ₂PO ₄1.4mmol/L pH 7.5) membrane closure more than two hours, is added different antibody (shown in the back) incubated at room about two hours with volume ratio then at 1: 10000.Used antibody is: available from the anti-H3K9 trimethylammoniumization (article No.: ab8898 of Abcam company, the green strong bio tech ltd agency of Shanghai water chestnut) antibody and anti-H3K36 trimethylammoniumization (article No.: ab9050, the green strong bio tech ltd agency of Shanghai water chestnut) antibody and anti-H3 (article No.: ab1791, the green strong bio tech ltd agency of Shanghai water chestnut) antibody.After outwelling primary antibodie, wash film three times with the PBS damping fluid, each 15 minutes, add is that the goat-anti rabbit two of HRP mark of dilution in 1: 10000 is anti-(available from SouthernBiotech company by volume again, must win bio tech ltd (Beijing) agency), incubated at room 1-2 hour, wash film three times with the PBS damping fluid then, each 15 minutes, use SuperSingnalPico (available from Pierce company) test kit by specification working method to carry out the X-ray film developing, through scanner scanning X-ray sheet post analysis result.

Experimental result shows that OsJ5 has histone demethylation function, is a histone demethylase, mainly acts on H3K9 and H3K36 trimethylammonium site (as shown in Figure 6).

The resistance of embodiment 7 OsJ5 transfer-gen plants is identified

Whether have resistance against diseases in order to study OsJ5 overexpression transfer-gen plant, transgenosis T1 is inoculated bacterial leaf spot pathogenic bacteria PXO99 (Philippines fungus strain physiological strain is from the International Rice Research Institute) for plant.The cultivation of inoculation bacterial leaf spot bacterium and concentration modulation are with reference to (Lin et al. such as Lin, Identification and mapping of a new gene for bacterial blight resistance in rice based on RFLP makers.Phytopathology, 1996,86:1156-1159), (component and preparation method: 300g peeling potato slices boils the bacterial leaf-blight bacterial strain of activation at potato culture, add 0.5g Ca (NO3) 2 after the filtered through gauze, 2gNaH2PO4,15g sucrose, the 5g peptone, the 20g agar powder; Adjust pH is to 6.8-7.0 after adding water to 1L) 28 ℃ of following the cultivations 2～3 days in inclined-plane, sterilized water is diluted to the concentration (turbidimetry) of about 9 * 109/mL, adopt leaf-cutting method (Sun et al. boot stage, A gene conferring resistance to Xanthomonas oryzae pv.oryzae in rice, encodes an LRR receptor kinase-like protein.Plant J, 2004,37:517-5272004) inoculation bacterial leaf spot pathogenic bacteria (Philippines fungus strain physiological strain, PXO99), the leaf of 5 full extension of every strain inoculation, inoculate back 15 days investigation scab length, weigh the state of an illness according to lesion area (scab length/sick leaf length), enquiry data is carried out T test Analysis significance (P＜0.05).

Experimental result shows that (seeing Fig. 2 and Fig. 7) OsJ5 gene overexpression of the present invention transfer-gen plant is stronger than spending 11 (wild-types) in the contrast to the resistance of bacterial leaf spot bacterium, proves that the present invention clone's OsJ5 gene has played keying action in the raising paddy rice to bacterial leaf spot bacterium resistance.

Claims (2)

1. the gene of a paddy rice histone demethylase gene OsJ5 to the application in the bacterial leaf spot pathogenic bacteria resistance, is characterized in that the nucleotide sequence of this gene is shown in sequence table SEQ ID NO:1 at adjusting and controlling rice.

2. the gene of a paddy rice histone demethylase gene OsJ5 to the application in the bacterial leaf spot pathogenic bacteria resistance, is characterized in that the amino acid sequence coded of this gene is shown in sequence table SEQ ID NO:2 at adjusting and controlling rice.

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