CN104911261A - Method for researching oryza sativa and pathogen interaction mode - Google Patents
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Abstract
The invention discloses a method for researching an oryza sativa and pathogen interaction mode, and belongs to the field of plant biotechnology. The method for researching the interaction mode of oryza sativa in response to pathogen infection through integrated application of a transtriptome, a proteome and a miRNA group comprises the concrete steps: A, respectively taking oryza sativa leaf blade total RNAs, Small RNAs and total proteins of a disease-resistant material and a susceptible material at different time points before and after pathogen inoculation; B, respectively carrying out differential-expression spectrum analysis, differential proteomics analysis and miRNA identification and target gene analysis; and C, integrating and associating differential genes, proteins and miRNA target genes identified by the three ways, to reveal disease-resistant and susceptible reaction paths activated or inhibited in the oryza sativa and pathogen interaction process, so as to relatively systematically illuminate an oryza sativa-magnaporthe oryzae interaction mechanism. The method can quickly and effectively screen candidate genes associated with oryza sativa disease resistance.
Description
Technical field
The invention belongs to plant biotechnology field, being specifically related to a kind of method of Study On Rice and pathogen cooperating type, is more specifically the method based on transcript profile, protein group (iTRAQ) and miRNA group joint study paddy rice-Pyricularia oryzae Coupling effects.
Background technology
Paddy rice (Oryza Sativa) is one of important in the world food crop, supports the population that the whole world is more than half.Infecting by Pyricularia oryzae (Magnaporthe oryzae) rice blast caused, is the important limiting factor in Rice Production, and it has, and velocity of propagation is fast, occurrence scope wide, endanger the features such as serious.Along with in succession completing of paddy rice and the plan of Pyricularia oryzae genome sequencing, the Coupling effects research of paddy rice-Pyricularia oryzae has become the modular system of plant-pathogen Coupling effects research.
The mutual work of paddy rice and Pyricularia oryzae is a very complicated and dynamic process, relate to numerous gene expression and regulation and the continuous signal the formed process of conducting step by step, mainly comprise three phases: the generation of signaling molecule and identification, have the plant hormone class signals such as JA, SA and ET usually, Ca
2+, the Equations of The Second Kind courier class signal such as active oxygen (ROS) and nitrogen protoxide (nitric oxide, NO), the signaling molecules such as the small-molecule peptides such as gsh (glutathione, GSH), lipid and carbohydrate; The conduction of Intracellular signals, as cross-film gtp binding protein, intercellular Ca
2+phosphorylation/the dephosphorylation of passage, calcium-dependent protein kinase CDPK, the cascade of mitogen-activated protein kinase MAPK and the protein of phosphatase catalytic; The unlatching of defense response and the generation of systemic acquired resistance.And each conduction all can produce corresponding biochemical reaction, thus play corresponding physiological function, complete specific biological effect.
Forefathers have adopted multiple method to carry out the difference group that infects of paddy rice response rice blast respectively and have studied, but are all single transcript profile or the research of proteomics.Wherein, biochip technology mainly discloses the change of mRNA level in-site, this technology of simple dependence can not disclose the concrete Coupling effects of plant and pathogen objective reality, and proteomic techniques also great majority adopt based on 2D electrophoretic separation and Mass Spectrometric Identification combined utilization, belong to the qualitative examination that the protein for a cell or tissue carries out, only can determine the identity of protein but final final conclusion can not be provided to the function of protein.Although Different Proteomics significantly can overcome the limitation of differential transcription group, but in fact the expression change of albumen and the expression of gene change very not consistent, this may be caused by the montage of mRNA, translational control or the factor such as post translational processing and protein degradation, this can affect truly illustrating the Coupling effects of plant and pathogen undoubtedly, and the result of proteomics and transcription group then can well organically be united by the qualification of miRNA.Therefore, single technique and method is adopted to go the reaction of research rice blast disease resistance, be difficult to the molecular resistance mechanism of real its complexity of announcement, and fully utilize transcription group, analysis that quantitative proteomics (iTRAQ) carries out difference group, both unmatched places further by miRNA carry out analyzing determining post-transcriptional level or protein translation level whether regulated and controled cause, be expected to disclose the concrete Coupling effects of paddy rice-rice blast really to illustrate the molecule mechanism of plant disease-resistant further comparatively comprehensively.
Summary of the invention
The object of the invention is to overcome the shortcoming that exists in above-mentioned prior art with not enough, provide a kind of method of Study On Rice and pathogen (Pyricularia oryzae) cooperating type, the inaccurate problem of the research effect in order to overcome existing monotechnics.
Object of the present invention is achieved through the following technical solutions: a kind of method of Study On Rice and pathogen cooperating type, the method of the cooperating type that integrated application transcript profile, protein group (iTRAQ) and miRNA group Study On Rice response pathogen infects, its concrete steps comprise:
A. the rice leaf total serum IgE of disease-resistant material and the susceptible material inoculating different time points before and after pathogen, Small RNA and total protein is not got;
B. carry out differential expression spectrum analysis respectively, Different Proteomics (iTRAQ) analyzes and miRNA identifies and target gene analysis;
C. three kinds of methods are identified differential gene, albumen, miRNA target gene carry out integrating, a resistant, susceptible reaction path that the institute that associates to disclose in paddy rice and pathogen Interaction activates or suppresses, thus can the Coupling effects of illustrating paddy rice-Pyricularia oryzae of comparatively system.
Pathogen described in steps A is preferably Pyricularia oryzae.
Before and after inoculation Pyricularia oryzae described in steps A, different time points is preferably 0h and 24h two time points.
The concrete steps of steps A comprise: get the disease-resistant material of 0h and 24h two different time points and the blade of susceptible material before and after inoculation Pyricularia oryzae respectively, liquid nitrogen grinding, centrifugal, cracking process, and carry out the preparation of total serum IgE, total protein and Small RNA and corresponding quality examination respectively.
The concrete steps of the differential expression spectrum analysis described in step B comprise: use
the RNA of RNA Clean-up (MN) purification step A gained also carries out synthesis, the purifying of double-strand cDNA; Then in-vitro transcription synthesizing biotinylated mark cRNA, carries out the fragmentation process of cRNA after purifying cRNA; Use
hybridization Oven 640 is by test materials cRNA and chip hybridization; Pass through
fluidics Station 450 carries out wash-out, dyeing; Use
scanner3000 scans chip, uses
operating Software Version1.4 software analysis chip hybridization image, is converted into numerary signal picture signal, and corrects and standardization with dChip software (http://www.dchip.org); Each probe compares to determine differential expression genes involved between different time points and different treatment.
The concrete steps that Different Proteomics (iTRAQ) described in step B is analyzed comprise: the protein example of steps A gained is carried out further FASP enzymolysis, peptide segment mark, SCX classification and LC-MS mass spectroscopy, further data checking storehouse, quantitative analysis are carried out to the Mass Spectrometric Identification result of gained, carry out GO cluster analysis, choose fold differences >1.2 or <0.8, the albumen of p-value<0.05 is further analyzed and tentatively determines differential expression associated protein.
The concrete steps of the miRNA qualification described in step B and target gene analysis thereof comprise: be connected with 5 ' adaptor, 3 ' adaptor the 18-30nt small RNA that steps A reclaims, and carry out reverse transcription; Reverse transcription product is in Solexa High-throughput Sequencer (Illumina, USA) check order, check order Solexa the 35nt sequence of gained, by removing joint, go inferior quality, depollute, statistical series length and distributed process complete primary analyses, the sequence obtained primary analyses further makes classification annotation, obtains each component and expression amount information that comprise in sample.MicroRNA target prediction is carried out to obtain the target gene information that may regulate and control to difference miRNA, and Go classification and KEGG path analysis are carried out to target gene.
The concrete steps of step C comprise: carry out the association analysis of three to the target gene of the differential gene of differential expression spectrum qualification, the differential protein of Different Proteomics qualification and miRNA prediction, machine-processed to disclose the complicated molecule that paddy rice and Pyricularia oryzae do mutually comparatively all sidedly.
Differential expression described in step C composes the differential gene identified to be fold differences the be gene of more than 2 times.
The differential protein that Different Proteomics described in step C is identified to be fold differences the be albumen of more than 1.2 times.
The present invention has following advantage and effect relative to prior art:
The present invention more completely can identify the differential gene/albumen participated in paddy rice-Pyricularia oryzae Interaction, and the embodiment of the present invention utilizes the method to identify the genes/proteins changed in some paddy rice response rice blast infection processs; The inventive method provides a method to deep announcement paddy rice-rice blast Coupling effects and uses for reference; Cultivate on Varieties Resistant To Rice Blast utilizing transgenic technology and there is important reference value.Its advantage is: 1. can screen the candidate gene be associated with paddy disease-resistant fast and effectively; 2. the present invention well utilizes these high-throughputs such as the degree of depth order-checking of biochip technology, iTRAQ technology and miRNA, accurately group to learn research means and studies the candidate gene/albumen relevant to rice anti-rice blast; 3. can be widely used in the proterties such as plant disease-resistant research and development and in conjunction with transgenic technology to cultivate the strong new variety of plant of disease resistance.
Accompanying drawing explanation
The GO functional annotation of the part variation gene of Fig. 1 differential expression spectrum qualification is analyzed; Wherein note:
Cell components (1-cell, 2-cellular portions, 3-film, 4-cell outskirt, 5-cell outer zone portions, 6-polymer complex, 7-membranin, 8-organoid, 9-organoid part, 10-synthetic enzyme, 11-synthetic enzyme part) (12-is anti-oxidant for molecular function, 13-combines, 14-catalytic activity, 15-electron carrier, 16-enzyme regulates, 17-metallochaperone, 18-transport activity, 19-nutrition stores, 20-structural molecule, 21-transcriptional regulatory, 22-translational regulation, 23-transports, biological pathway (the formation of 24-anatomical structure, 25-biological adhesion, 26-biological regulation, 27-cell components occurs, 28-cell components tissue, 29-cellular processes, 30-is dead, 31-growth course, the foundation of 32-location, 33-grows, 34-immunologic process, 35-locates, 36-moves, 37-metabolic process, 38-many kinds of bioprocesss, 39-multicellular organism process, 40-pigmentation, 41-reproduction, 42, reproductive process, 43-stress reaction, 44-periodic process)
The GO functional annotation of the part variation albumen of Fig. 2 iTRAQ differential proteomics qualification is analyzed; Wherein note:
Cell components (1-cell, 2-cellular portions, 3-film, 4-cell outskirt, 5-cell outer zone portions, 6-polymer complex, 7-membranin, 8-organoid, 9-organoid part) (10-is anti-oxidant for molecular function, 11-combines, 12-catalytic activity, 13-structural molecule, 14-transcriptional regulatory, 15-transports, biological pathway (the formation of 16-anatomical structure, 17-biological regulation, 18-cell components occurs, 19-cell components tissue, 20-cellular processes, 21-is dead, 22-growth course, the foundation of 23-location, 24-grows, 25-immunologic process, 26-locates, 27-metabolic process, 28-many kinds of bioprocesss, 29-multicellular organism process, 30-pigmentation, 31-reproduction, 32, reproductive process, 33-stress reaction)
The GO functional annotation of the part target gene of Fig. 3 miRNA group regulation and control is analyzed; Wherein note:
Cell components (1-cell, 2-cellular portions, 3-cell outer zone portions, 4-polymer complex, 5-membranin, 6-organoid, 7-organoid part) (8-combines molecular function, 9-catalytic activity, 10-transport activity, 11-structural molecule, 12-transcriptional regulatory, 13-transports, biological pathway (14-biological regulation, 15-cell components tissue, 16-cellular processes, 17-is dead, 18-growth course, the foundation of 19-location, 20-grows, 21-locates, 22-metabolic process, 23-multicellular organism process, 24-pigmentation, 25-reproduction, 26, reproductive process, 27-stress reaction)
Fig. 4 three identifies the association of the differential protein (or gene) obtained;
Fig. 5 based on the biosynthetic pathway analysis of the jasmonate (JA) of three kinds of methods, wherein note: square-shaped frame is the differential gene expression (| log2Ratio| >=1) identified in this research; Triangle frame is the target gene of the known and candidate miRNA identified in this research; Oval frame is the protein utilizing iTRAQ to identify in this research.
Embodiment
Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited thereto.Various raw material used in the present invention and items of equipment are conventional commercial product, all can be bought by market and directly obtain.
Embodiment 1
A kind of method based on transcript profile, protein group (iTRAQ) and miRNA group joint study paddy rice-Pyricularia oryzae Coupling effects
One, differential expression spectrum analysis.
1. sample preparation
Paddy rice (H4), in two soft account for (ZE) grow to 3-4 leaf phase inoculation rice blast fungi isolates GD0193, attached simulation inoculation is contrast; Gather rice leaf respectively after inoculation 0h and 24h, put into cryopreservation tube, liquid nitrogen flash freezer is placed on-80 DEG C of Ultralow Temperature Freezers and saves backup; The material that each subjects takes full backup is simultaneously verified for qRT-PCR.
Use
reagent (Invitrogen, USA) extracts total serum IgE: take about 100mg leaf tissue, and liquid nitrogen grinding, to Powdered, transferred in 2.0mL centrifuge tube, added 1mL
reagent, vibration mixing; 15-30 DEG C of incubation 5min, every 1mL Trizol adds 0.2mL chloroform, leaves standstill 2-3min after vibration 15s; 4 DEG C, the centrifugal 15min of 12000rpm, moves to upper water mutually in new 1.5mL centrifuge tube, adds 0.6mL Virahol, and room temperature places 10min; 4 DEG C, the centrifugal 10min of 12000rpm, abandons supernatant; Add 1mL 70% washing with alcohol, oscillation sample, then 4 DEG C, the centrifugal 15min of 12,000rpm; Remove supernatant, dry air RNA sample; Add 200 μ L DEPC water dissolution, then carry out the precipitation of RNA further: add 1/10 volume 3M NaAc pH5.2 and 2.5 times of volume dehydrated alcohol mixing, place minimum 1h, 4 DEG C of centrifugal 20min of 12000g for-20 DEG C; 80% washing with alcohol precipitates 2 times, and dry air precipitates; Then DEPC process water again dissolution precipitation carry out RNA concentration and purity testing.
Prepared by 2.cDNA
Utilize SuperScript
iIrT carries out the synthesis of Article 1 cDNA chain, then carries out the synthesis of Article 2 cDNA chain, and with phenol chloroform to its purifying, alcohol settling; With reference to the cRNA of BioArray High Yield RNA Transcript Labeling kit synthesizing biotinylated mark, and purify in-vitro transcription product with QIAGEN RNeasy Columns, further alcohol settling, purity and Concentration Testing, and hybridize with fragmentation cRNA.Hybridization conditions is as shown in table 1.
Equilibrium at room temperature probe before hybridization, 99 DEG C, 5min, adds the moistening chip of appropriate volume 1 × Hybridization Buffer by well, 45 DEG C, 60rpm prehybridization chip 10min, 45 DEG C of centrifugal 5min of maximum speed, take out Buffer, add the hybridization solution that equal-volume is handled well from chip, 45 DEG C, 60rpm hybridization hybrid chip 16h; Further wash-out, dyeing utilizing are carried out to chip
scanner 3000 scans chip, uses Gene
operating Software Version1.4 software analysis chip hybridization image, is converted into numerary signal picture signal, and corrects and standardization with dChip software (http://www.dchip.org).
Table 1 hybridizes system condition
3. data analysis
Each probe compares between different time points and different treatment.With Clutster3.0 software, detected signal value effective in each sample is carried out cluster analysis, adopt Hierachical, Average linkage, K-means algorithm carries out cluster, the repeatability of analysis chip, for the gene filtered out, utilize the genome database (http://www.capitalbio.com/zh-hans/support/MAS) of molecular function annotation system MAS3.0, the TIGR accession number according to each gene finds corresponding functional annotation.According to fluorescence intensity ratio, same inoculation process differential gene of (0h and 24h) between different times is screened respectively.Select differential expression twice gene (i.e. log
2ratio>=1 or log
2ratio≤-1) be the probe of significant difference, after inoculation rice blast fungi isolates GD0193,1837 rise probes and 2476 downward probes detected altogether.The impact of eliminating simulation process contrast, the special induction of energy 345 gene upregulation that infect of bacterial strain GD0193 are expressed and 215 downward expression.Carry out simple and easy GO classification (as Fig. 1) to these difference expression genes, result shows most of differential gene and mainly participates in the biological procedures such as endogenous irritant reaction, signal transduction, biostimulation reaction, protein modified, cytodifferentiation, electron transmission, cell catabolism.Wherein endogenous irritant reaction and signal transduction account for 14% and 13% respectively, abiotic irritant reaction and Stress responses account for 8% and 10% respectively, biostimulation, biosynthesizing and protein modifiedly account for 6%, 9% and 8% respectively, lipid metabolism and amino acid and derivatives metabolism all account for 5% and 7%, and other biological process is all below 5%.
Two, Different Proteomics (iTRAQ) is analyzed.
1. the extraction of leaves total protein
Water intaking rice tissue sample, proceeds in 50ml centrifuge tube with liquid nitrogen grinding powdered, adds 25ml extracting solution-20 DEG C precipitation 1h.Centrifugal 10000rpm, 45min, remove supernatant.Dry air.Add SDT buffer (4%SDS, 1mM DTT, 150mM Tris-HCl pH8.0) by 10:1, vortex mixes, boiling water bath 5min.Ultrasonication (80w, ultrasonic 10s, interval 15s, totally 10 times) boiling water bath 5min, centrifuging and taking supernatant, BCA method quantification of protein, separately gets about 20 μ g protein examples and carries out 12.5%SDS-PAGE electrophoresis and carry out coomassie brilliant blue staining.
The preparation of 2.iTRAQ sample
Get 400 μ g samples, add DTT, boiling water bath 5min, is cooled to room temperature.Add 200 μ l UA buffer (8M Urea, 150mM Tris-HCl, pH8.0) mixings, proceed to 30kd ultra-filtration centrifuge tube, centrifugal 14000g, 15min; Add 200 μ l UA buffer centrifugal 14000g, 15min, abandon filtrate.Add 100 μ l IAA (50mM IAA in UA), 600rpm vibrates 1min, lucifuge room temperature 30min, centrifugal 14000g, 10min.Add 100 μ l UA buffer, centrifugal 14000g, 10min repeat 2 times.Add 100 μ l Dissolution buffer, centrifugal 14000g, 10min repeat 2 times.Add 40 μ l Trypsin buffer (4 μ g Trypsin in 40 μ l Dissolution buffer), 600rpm vibrates 1min, 37 DEG C of 16-18h.Renew collection tube, centrifugal 14000g 10min, gets filtrate, OD
280peptide section is quantitative.Get each group of sample about 90 μ g respectively, according to AB company test kit: iTRAQ Reagent-8plex Multiplex Kit (AB SCIEX) specification sheets marks.By all peptide section mixing after mark, adopt AKTA Purifier 100 (GE Healthcare) instrument to carry out the pre-classification of SCX, collect percolation and wash-out fraction about 30 parts, often organize sample according to SCX color atlas and be merged into 10 parts, C after freeze-drying
18cartridge (Sigma) desalination.
3. Mass Spectrometric Identification and data analysis
Every increment product adopt to be received up-flow speed HPLC liquid phase systems Easy nLC and is separated.Damping fluid: A liquid is 0.1% aqueous formic acid, B liquid is 0.1% formic acid acetonitrile solution (acetonitrile is 84%).Chromatographic column balances with the A liquid of 95%.Sample is loaded to loading post Thermo scientific EASY column (2cm*100 μm of 5 μm of-C18) by automatic sampler, post Thermo scientific EASY column (75 μm of * 100mm, 3 μm of-C18) is separated by analysis again, and flow velocity is 250nl/min.Related fluid phase gradient is as follows: 0min-100min, B linear gradient is from 0% to 35%; 100min-108min, B linear gradient is from 35% to 100%; 108min-120min, B liquid maintains 100%.Every increment product carry out mass spectroscopy with Q-Exactive mass spectrograph (Thermo Finnigan) after capillary high performance liquid chromatography is separated.Analyze duration: 120min, detection mode: positive ion, precursor scans scope: 300-1800m/z, first mass spectrometric resolving power: 70,000at m/z200, AGC target:3e6, one-level Maximum IT:10ms, Number of scan ranges:1, Dynamic exclusion:40.0s.The mass-charge ratio of the fragment of polypeptide and polypeptide gathers according to following method: each full scan (full scan) gathers 10 fragment patterns stored (MS2scan) afterwards, MS2Activation Type:HCD, Isolation window:2m/z, second order ms resolving power: 17500at m/z 200, Microscans:1, secondary Maximum IT:60ms, Normalized collision energy:30eV, Underfill ratio:0.1%.Raw data through noise reduction, go the step such as isotropic substance to obtain peak Figure List.Set up reference protein sequence library simultaneously, carry out the qualification of peptide section and protein, namely the method for qualification uses special protein identification software Mascot2.2 to search for reference database.The database that this test uses is uniprot_oryza.fasta.Result filtration parameter is: Peptide FDR≤0.01.Proteome Discoverer1.3 software is adopted to carry out quantitative analysis to peptide section report quasi-molecular ions intensity level.Differential protein carries out the analysis of GO functional annotation and checking
Application Mascot software carries out expression amount calculating to sample room differentially expressed protein, when same albumen sample room relative abundance close to 1 time, show that the expression amount of this albumen is not remarkable in sample room difference, and when protein abundance ratio reaches more than 1.2, and when statistical test P-value is less than 0.05, think that this albumen is the differential protein of two sample rooms.After 24h is infected in sterilized water process and Pyricularia oryzae, quite (309 ~ 322), up-regulated expression is 137 ~ 153 to each group differentially expressed protein quantity, under be adjusted to 161 ~ 177.
In order to excavate the expression of the differential protein of Pyricularia oryzae induction further, the online drawing tool of Venny is utilized to find after carrying out common factor comparison differential protein between each process, after rice blast infects 24h, the albumen that ZE varietY specificity is expressed has 62 (28 rises, 34 downwards), the albumen of the common differential expression of ZE and H4 has 31 (15 rises, 16 downwards), H4 kind then has 88 differentially expressed proteins (raise 50, lower 38).The further GO annotation carried out these differential proteins is analyzed and is found: these differentially expressed protein significant enrichments are at physiological process, response stimulation, biotic or abiotic stress etc., metabolism etc.
Three, miRNA qualification and target gene analysis thereof.
The separation of 1.Small RNA, recovery and order-checking
RNA sample is added isopyknic 8M urea sample-loading buffer, sex change 5min in 80 DEG C of water-baths, is put into rapidly more than 2min on ice, uses microsyringe loading; After 500V electrophoresis 1.5h, under ultraviolet lamp, the microRNA corresponding to 18-30nt is scaled off, transfers to the centrifuge tube of 1.5mL; Add 1mL 0.3M sodium acetate (pH5.2) and 40U Rnasin, 16-25 DEG C of vibration 2h; 4 DEG C, the centrifugal 10min of 12000rpm.Collect supernatant, and in glue, add 0.3M NaAc (pH5.2), 16-25 DEG C of vibration 2h; 4 DEG C, the centrifugal 10min of 12000rpm.Collect supernatant, and in glue, add 0.3M NaAc (pH5.2), 16-25 DEG C of shaken overnight; 4 DEG C, the centrifugal 10min of 12000rpm.Collect supernatant, transfer in 1.5ml centrifuge tube, often pipe 0.33ml; Add the dehydrated alcohol of 2 μ l liver starch and 3 times of volumes ,-80 DEG C of precipitations 20min or-20 DEG C of precipitates overnight; 4 DEG C, the centrifugal 30min of 14000rpm.Remove supernatant, DEPC water dissolution RNA, dilution RNA to 300 μ l; Add 33 μ l 3M sodium acetate (pH5.2), 900 μ l dehydrated alcohols, 2 μ l liver starch mixings, in-80 DEG C of precipitations 20min or-20 DEG C of precipitates overnight; 4 DEG C, the centrifugal 30min of 14000rpm, removes supernatant, and 70% washing with alcohol RNA precipitation, dry air, adds 40 μ L DEPC water and 1 μ l RNasin dissolves RNA, and-80 DEG C save backup.By Fig. 2 flow process, build small RNA sequencing library.The 18-30nt small RNA reclaimed is connected with 5 ' adaptor, 3 ' adaptor, and carries out reverse transcription; Reverse transcription product checks order in Solexa High-throughput Sequencer (Illumina, USA), is completed by Hua Da gene (Shenzhen).
2. the bioinformatic analysis of sequencing result.
Check order Solexa the 35nt sequence of gained, by removing joint, go inferior quality, depollute, the process such as statistical series length and distribution completes primary analyses.The sequence obtained by primary analyses makes classification annotation, obtains each component and expression amount information that comprise in sample.The data in order-checking acquisition 4 small RNA libraries, its statistics is in table 2.A library (ZE-CK), B library (H4-CK), C library (ZE GD0193), D library (H4-GD0193) obtain the high quality sequenced fragments of 10521573,14885325,12439284,13644512 respectively; Remove without 3 ' terminal sequence, without insertion sequence, 5 ' end connector pollution etc., 8749036,12157795,9937360,11138187clean reads can obtain respectively in A, B, C, D 4 libraries.Clean reads is distributed between 10-30nt, mainly based on 21nt, 24nt.
Table 2 Small RNA sequencing data output statistics
Small RNA composition between library all shows and to a certain degree makes a variation between different rice strains, between different treatment.Small RNA composition that is anti-, sense material all shows larger difference under control treatment or pathogenic bacterium inducing, show to resist, feel storeroom phenotypic difference etc. under normal growth situation small rna expression equally also can be caused to make a variation, between the library of (A, B) and pathogenic bacteria process between the library that 79216 (3.15%) and 97743 (10.73%) small RNA sequences are respectively control treatment, (C, D) is common; But, show the most significant difference between library to be produced by the two soft libraries (A, C) that account in pathogenic bacterium inducing, only have 64782 (2.72%) small RNA sequences between A, C library, and A library has small RNA sequence the abundantest in all libraries; On the contrary, between H4 library, the variation of (B, D) is minimum, and namely the variation of the disease-resistant strain small RNA composition of pathogenic bacterium inducing is minimum, and they have the identical sequence of 123609 (11.94%).Therefore, the express spectra of different small RNA should be relevant to the rice blast resistance difference between rice strain.In order to obtain the known miRNA relevant to pathogenic bacterium inducing, this research by the library after Pyricularia oryzae GD0193 process with contrast library and compare, and use log respectively
2-ratio compares the relative expression quantity difference condition of these miRNA in different library.ZE_GD0193 and ZE_CK relatively after, have 21 and 28 miRNA expression amount after Pyricularia oryzae infects significantly to raise in ZE and H4 respectively, have 102 and 13 miRNA expression amount after Pyricularia oryzae infects significantly to reduce respectively.
By in 4 libraries totally 296 miRNA be submitted in psRNATarget website, be with reference to background with MSU7.0,149 target genes detected altogether, wherein multiple miRNA family is unanimously targeted to same gene because of the mature sequence of its member, also there is the multiple gene of single miRNA member's target.As miR156 family target SBP-box gene family, miR164 target 3 NAC transcription factor and OsFBX145.Carry out function prediction discovery to these target genes, except transcriptional control, these genes also participate in substance metabolism, signal transduction, stress response, the vital processes such as electron transmission (as Fig. 3).
Four, the association of disease-resistant relevant difference expression gene, albumen and miRNA.
These disease-resistant relevant difference expression gene, albumen and miRNA are integrated, carry out correlation analysis, as seen from Figure 4, in paddy rice and Pyricularia oryzae Interaction, differential gene is consistent in transcript profile level with protein group level majority, but also some performance inconsistent, may to be exactly miRNA cause transcribing the regulation and control in (to mRNA) or translation skill (to protein) in inconsistent place.
In the complex process that paddy rice and Pyricularia oryzae are done mutually, the generation of resistance signal molecule and intracellular signaling play vital effect.And in these signaling molecules, plant hormone then can form an important disease-resistant regulated and control network according to the interaction between hormon, wherein, Whitfield's ointment (SA), jasmonic (JA) and ethene (ET) are closely related with the disease resistance of paddy rice.In the difference expression gene that this research is induced at Pyricularia oryzae and differentially expressed protein identification and analysis, also find OsHI-LOX and OsAOS2 up-regulated expression, and the change (rise multiple) on transcriptional level is much larger than the change (rise multiple) in translation skill.MiRNA has likely participated in the expression of OsHI-LOX and OsAOS2, and then the biosynthesizing speed of control JA.And there are some researches show that the increase of JA content within blink is just enough to activate the JA response gene in downstream, and the experiment of the overexpression of AOS gene also proves to compare constitutive promoter CaMV35S, inducible promoter PBZ1 more can embody the impact that AOS gene pairs JA synthesizes.
AOS and HPL gene then belongs to CYP74 family, the substrate 13-HPOT that catalysis is common, and then generates JA and GLVs respectively, causes GLVs and JA two pathways metabolisms both to there is competitive relation and also there is the relation of mutually promoting.In addition research shows that AOS gene silencing can reduce the accumulation of JA, and increase the release of GLVs, reticent HPL gene then has contrary result.And in this research, also identify the candidate miRNA that target gene is respectively OsAOS2 and OsHPL3, infer that miRNA can by regulating and controlling the expression of AOS and HPL gene respectively, and then the equilibrium relationship of control GLVs and JA two pathways metabolisms, and the expression that (as Pyricularia oryzae infects) induces different miRNA is coerced in different outside, GLVs and JA two pathways metabolisms are tilted to certain direction, activates the defense system in downstream.
Also multiple difference expression gene relevant to plant hormone (as OsAOS2, OsJMT1 and OsACS2 etc.) is identified in research, differentially expressed protein (OsHI-LOX, OsPLD α and OsACX1 etc.), and microRNA target prediction is the candidate miRNA of plant hormone (as OsPLD β 1, OsHPL3 and OsEIL2 etc.).And miRNA take part in signal transduction and the metabolism of various plants hormone also to have many research to confirm at present, target as miR319 is the mRNA of transcription factor TCP, TCP by the key gene LOX2 of regulation and control JA biosynthesizing process thus controls the biosynthesizing of JA; MiR164 can degrade the mRNA of transcription factor NAC1, thus lowers AUX acknowledge signal; OsmiR393 overexpression has lowered the homogenic expression of acceptor TIR1/AFB family of AUX.
This research is for jasmonic JA biosynthetic pathway, by by the difference expression gene of Pyricularia oryzae stress-inducing, disease-resistant relevant albumen and candidate miRNA organic combination (Fig. 5), from transcriptional level (mRNA), miRNA regulation and control level (miRNA) and translation (Protein) level, paddy rice is participated in the Resistant reaction of Pyricularia oryzae to JA.
The research clearly can disclose the concrete Coupling effects of paddy rice-Pyricularia oryzae, and provides important reference to the disease resistance mechanisms of deeply illustrating blast resistant gene, for the scientific and effective breeding for disease resistance that instructs provides important guiding further
Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; change, the modification done under other any does not deviate from spirit of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (10)
1. a method for Study On Rice and pathogen cooperating type, is characterized in that: the method for the cooperating type that integrated application transcript profile, protein group and miRNA group Study On Rice response pathogen infects, and its concrete steps comprise:
A. the rice leaf total serum IgE of disease-resistant material and the susceptible material inoculating different time points before and after pathogen, Small RNA and total protein is not got;
B. differential expression spectrum analysis, Differential proteomic analysis and miRNA qualification and target gene analysis thereof is carried out respectively;
C. three kinds of methods are identified differential gene, albumen, miRNA target gene carry out integrating, a resistant, susceptible reaction path that the institute that associates to disclose in paddy rice and pathogen Interaction activates or suppresses, thus can the Coupling effects of illustrating paddy rice-Pyricularia oryzae of comparatively system.
2. the method for Study On Rice according to claim 1 and pathogen cooperating type, is characterized in that: the pathogen described in steps A is Pyricularia oryzae.
3. the method for Study On Rice according to claim 1 and pathogen cooperating type, is characterized in that:
Before and after inoculation Pyricularia oryzae described in steps A, different time points is 0h and 24h two time points.
4. the method for the Study On Rice according to any one of claims 1 to 3 and pathogen cooperating type, is characterized in that: the concrete steps of steps A comprise:
Get the disease-resistant material of 0h and 24h two different time points and the blade of susceptible material before and after inoculation Pyricularia oryzae respectively, liquid nitrogen grinding, centrifugal, cracking process, and carry out the preparation of total serum IgE, total protein and Small RNA and corresponding quality examination respectively.
5. the method for Study On Rice according to claim 4 and pathogen cooperating type, is characterized in that: the concrete steps of the differential expression spectrum analysis described in step B comprise:
Use the RNA of NucleoSpin RNAClean-up purification step A gained and carry out synthesis, the purifying of double-strand cDNA; Then in-vitro transcription synthesizing biotinylated mark cRNA, carries out the fragmentation process of cRNA after purifying cRNA; Use Affymetrix Hybridization Oven 640 by test materials cRNA and chip hybridization; Wash-out, dyeing is carried out by Affymetrix Fluidics Station 450; AffymetrixGeneChip Scanner3000 is used to scan chip, with Affymetrix GeneChip Operating SoftwareVersion1.4 software analysis chip hybridization image, picture signal is converted into numerary signal, and corrects and standardization with dChip software; Each probe compares to determine differential expression genes involved between different time points and different treatment.
6. the method for Study On Rice according to claim 4 and pathogen cooperating type, it is characterized in that: the concrete steps of the Differential proteomic analysis described in step B comprise: the protein example of steps A gained is carried out further FASP enzymolysis, peptide segment mark, SCX classification and LC-MS mass spectroscopy, further data checking storehouse is carried out to the Mass Spectrometric Identification result of gained, quantitative analysis, carry out GO cluster analysis, choose fold differences >1.2 or <0.8, the albumen of p-value<0.05 is further analyzed tentatively determines differential expression associated protein.
7. the method for Study On Rice according to claim 4 and pathogen cooperating type, it is characterized in that: the concrete steps of the miRNA qualification described in step B and target gene analysis thereof comprise: be connected with 5 ' adaptor, 3 ' adaptor the 18-30nt small RNA that steps A reclaims, and carry out reverse transcription; Reverse transcription product checks order in Solexa High-throughput Sequencer, check order Solexa the 35nt sequence of gained, by removing joint, go inferior quality, depollute, statistical series length and distributed process complete primary analyses, the sequence obtained primary analyses further makes classification annotation, obtains each component and expression amount information that comprise in sample; MicroRNA target prediction is carried out to obtain the target gene information that may regulate and control to difference miRNA, and Go classification and KEGG path analysis are carried out to target gene.
8. the method for Study On Rice according to claim 4 and pathogen cooperating type, it is characterized in that: the concrete steps of step C comprise: the association analysis of three is carried out to the target gene of the differential gene of differential expression spectrum qualification, the differential protein of Different Proteomics qualification and miRNA prediction, machine-processed to disclose the complicated molecule that paddy rice and Pyricularia oryzae do mutually comparatively all sidedly.
9. the method for Study On Rice according to claim 8 and pathogen cooperating type, is characterized in that: the differential expression described in step C composes the differential gene identified to be fold differences the be gene of more than 2 times.
10. the method for Study On Rice according to claim 8 and pathogen cooperating type, is characterized in that: the differential protein that the Different Proteomics described in step C is identified to be fold differences the be albumen of more than 1.2 times.
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