CN106244600A - The oryza officinalis Oo92082 gene of leaf spot bacteria abduction delivering and specific amplification primer thereof - Google Patents
The oryza officinalis Oo92082 gene of leaf spot bacteria abduction delivering and specific amplification primer thereof Download PDFInfo
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Abstract
The present invention discloses oryza officinalis Oo92082 gene and the specific amplification primer thereof of a kind of leaf spot bacteria abduction delivering, also discloses this gene and is coerced the expression analysis primer of different time points at oryza officinalis by leaf spot bacteria.This gene is in 0h, 24h, 48h, 72h and 96h of leaf spot bacteria PXO99 and C5 Stress treatment, and relative expression quantity (RQ) all reaches summit at 48h, and wherein PXO99 coerces rear RQ is more than 16 times of 0h, and it is more than 85 times of 0h that C5 coerces rear RQ.This gene shows as up-regulated expression after inoculation leaf spot bacteria, annotated to nucleic acid with albumen database, does not obtains any functional annotation, is the new gene of a direct participation oryza officinalis bacterial blight-resisting answering process.This gene be successfully separated clone, lay a good foundation with oryza officinalis bacterial blight-resisting mechanism for furtheing investigate this gene.
Description
Technical field
The invention belongs to biomolecule clone technology field, be specifically related to the oryza officinalis of leaf spot bacteria abduction delivering
Oo92082 gene, analyzes primer and specific amplification primer based on this gene order design expression.Utilize expression analysis
The phase of the different time points that primer can be coerced by leaf spot bacteria at oryza officinalis by this gene of Real-time pcr analysis
To expression；Utilize design specific amplification primer can from oryza officinalis this gene of separating clone, or from other plant
The homologous genes of middle separating clone oryza officinalis Oo92082 gene.
Background technology
By rice Xanthomonas campestris Oryza sativa L. pvs oryzae and oryzicola (Xanthomonas oryzae pv.oryzae, Xoo) cause white
Leaf blight (Bacterial Blight, BB) is one of disease the most serious in world's Rice Production.Cultivate disease-resistant variety, separation
Clone's disease-resistant gene assistant breeding is to prevent and treat bacterial blight of rice method most economical, effective.Although some bacterial blight-resisting bases
Because by the application technology such as molecular marking supplementary breeding and transgenic, accelerating the process of paddy disease-resistant breeding, but existing greatly
The application in rice breeding of the part bacterial leaf spot resistance gene nevertheless suffers from the biggest restriction, and that cultivates carries disease-resistant master
The disease-resistant material of effect gene, owing to evolution and the variation of pathogen biological strain cause its original resistant gene gradually to lose
Resistance.Therefore separating clone can mediate lasting and without race-specific resistance disease-resistant related gene, it is possible to creates wide
Spectrum and lasting resistant material, have great significance to bacterial blight-resisting breeding.Oryza officinalis (Oryza officinalis
Wall.) many Common Diseases and adverse circumstances etc. all being had resistance and toleration, its resistant material recall rate and resistance rank are
In various wild rices the highest, either in natural environment or in wild rice garden, oryza officinalis is seldom fallen ill, because of
This, is by excavating the new gene of high resistance to hoja blanca in oryza officinalis, and the therefrom disease-resistant key gene of clone and separate, to Oryza sativa L.
Bacterial blight-resisting breeding is significant.
The object of study of transcript profile order-checking (RNA Sequence) is that specific cells can be transcribed under a certain functional status
The summation of all RNA out, broadly includes messenger RNA, ribosomal RNA, transfer RNA and non-coding RNA, refers to institute in the narrow sense
There is the set of mRNA.Transcript profile sequencing technologies can obtain a certain species particular organization or organ the most rapidly in a certain state
Under nearly all transcript sequence information, in recent years, the technology path being consequently formed is widely used in gene structure, gene
Function, the searching research field such as new gene, analysis of gene differential expression, and obtain remarkable effect.
The difference expression gene of the oryza officinalis transcript profile order-checking that the present invention coerces from leaf spot bacteria obtains one
Contig_92082 gene, is analyzed by differential expression level, and this gene is under bacterial leaf-blight strong pathogenic bacterium PXO99 and C5 coerces
Expression than do not connect bacterium process matched group high；Contig_92082 gene is annotated to numbers such as NR, GO and KEGG
According to storehouse, do not obtain any functional annotation, and the aminoacid sequence that this gene maximum open reading frame (ORF) encodes is through BLASTp ratio
Right, there is no any homology.Through above-mentioned analysis, thus it is speculated that Contig_92082 gene is that a direct participation oryza officinalis is anti-white
The new gene of leaf blight answering process, by its named oryza officinalis Oo92082 gene.
Summary of the invention
The present invention seeks to overcome in existing cultivated rice original Bacterial blight resistance gene due to pathogen biological strain
Constantly variation and gradually lose resistance, thus lack defect and the deficiency of the new gene of bacterial blight-resisting, it is provided that a kind of new white
The oryza officinalis Oo92082 gene of leaf spoting bacteria abduction delivering, this gene is relevant to bacterial leaf spot resistance；Also provide for a kind of point
The expression primer of the different time points that analysis oryza officinalis Oo92082 gene is coerced by leaf spot bacteria at oryza officinalis
And method, a kind of specific amplified oryza officinalis Oo92082 gene open reading frame primer and method, thus for entering
One step separating clone Bacterial blight resistance gene from oryza officinalis lays the foundation.
Oryza officinalis Oo92082 gene provided by the present invention is the oryza officinalis by coercing from leaf spot bacteria
The difference expression gene of transcript profile sequencing analysis screens difference is the most notable, the gene of numbered Contig_92082,
This gene expression under leaf spot bacteria is coerced is higher than the matched group not connecing bacterium process；This numbering gene is annotated
To data bases such as NR, GO and KEGG, do not obtain any functional annotation；And the ammonia that this gene maximum open reading frame (ORF) encodes
Base acid sequence, through BLASTp comparison, does not has any homology.Through above-mentioned analysis, thus it is speculated that Contig_92082 gene be one directly
Participate in the new gene of oryza officinalis bacterial blight-resisting answering process, by its named oryza officinalis Oo92082 gene.This medicine
May be used for analyzing this gene by wild Oo92082 gene order and coerced different time points at oryza officinalis by leaf spot bacteria
Expression, and for this gene of separating clone provide sequence information.
Technical scheme is as follows:
1. an oryza officinalis Oo92082 gene for leaf spot bacteria abduction delivering, its nucleotide sequence such as SEQ ID
Shown in NO:1.
2. the oryza officinalis Oo92082 gene of a kind of leaf spot bacteria abduction delivering described in technical scheme 1 is medicinal
The expression that wild rice is coerced different time points by leaf spot bacteria analyzes primer, and described expression analyzes primer by upstream
Primer and downstream primer composition, described expression analyze primer forward primer sequence as shown in SEQ ID NO:3, described table
Reach the downstream primer sequence of horizontal analysis primer as shown in SEQ ID NO:4.
3. the oryza officinalis of a kind of leaf spot bacteria abduction delivering described in a separating clone technical scheme 1
The specific amplification primer of Oo92082 gene open reading frame, described specific amplification primer is made up of forward primer and downstream primer,
The forward primer sequence of described specific amplification primer as shown in SEQ ID NO:5, the downstream primer sequence of described specific amplification primer
Row are as shown in SEQ ID NO:6.
Sequence table shown in SEQ ID NO:1 is the nucleotide sequence of oryza officinalis Oo92082 gene.It is by being subject to
Leaf spot bacteria coerces what lower oryza officinalis transcript profile sequencing analysis obtained.
In sequence table shown in SEQ ID NO:2 be by SEQ ID NO:1 design specific amplification primer, from medicinal wild
The nucleotide sequence of the open reading frame of the oryza officinalis Oo92082 gene that separating clone obtains in rice.
In sequence table shown in SEQ ID NO:3 be oryza officinalis Oo92082 gene at oryza officinalis by bacterial leaf spot
Pathogenic bacteria coerces the base sequence of the forward primer of the expression analysis primer of different time points.
In sequence table shown in SEQ ID NO:4 be oryza officinalis Oo92082 gene at oryza officinalis by bacterial leaf spot
Pathogenic bacteria coerces the base sequence of the downstream primer of the expression analysis primer of different time points.
Sequence table shown in SEQ ID NO:5 is separating clone oryza officinalis Oo92082 gene open reading frame
The base sequence of the forward primer of specific amplification primer.
Sequence table shown in SEQ ID NO:6 is separating clone oryza officinalis Oo92082 gene open reading frame
The base sequence of the downstream primer of specific amplification primer.
Sequence table shown in SEQ ID NO:7 is the amino of oryza officinalis Oo92082 gene open reading frame codes
Acid sequence.
The present invention provides the oryza officinalis Oo92082 gene of leaf spot bacteria abduction delivering first, utilizes this gene
Sequential design oryza officinalis Oo92082 gene is coerced the expression water of different time points at oryza officinalis by leaf spot bacteria
Divide analysis primer and the specific amplification primer of separating clone oryza officinalis Oo92082 gene open reading frame equally.
The expression that the present invention provides is utilized to analyze primer, the oryza officinalis by Real-time pcr analysis
Oo92082 gene 24h, 48h, 72h, 96h medicinal after not connecing the matched group of leaf spot bacteria (0h) and connecing leaf spot bacteria
The expression situation of 5 time points in wild rice blade, finds that oryza officinalis Oo92082 gene is in inoculation bacterial leaf-blight
Show as up-regulated expression after bacterium, and in these 5 time points, this oryza officinalis Oo92082 gene is at leaf spot bacteria physiology
Microspecies PXO99 (hereinafter referred to as: PXO99) and leaf spot bacteria biological strain C5 (hereinafter referred to as: C5) coerce rear relative expression quantity
(RQ value) occur summit all at 48h, after wherein PXO99 coerces, the relative expression quantity (RQ value) at 48h time point is comparison
More than 16 times of group, C5 is more than 85 times of matched group at the relative expression quantity (RQ value) of 48h time point after coercing, and illustrates: medicinal
When wild rice is coerced by leaf spot bacteria, the expression of this oryza officinalis Oo92082 gene progressively strengthens, expression
Raise, in 5 time points of detection, reach the highest at 48h relative expression quantity, therefore, this oryza officinalis Oo92082 gene
Belong to the oryza officinalis inducible expression of gene by bacterial leaf-blight abduction delivering, its mediation oryza officinalis bacterial leaf-blight immunity
Reaction, shows: the oryza officinalis Oo92082 gene obtained from oryza officinalis is the base directly related with bacterial blight-resisting
Cause, is new Bacterial blight resistance gene.
Utilize specific amplification primer of the present invention, expand medicinal from the oryza officinalis that leaf spot bacteria is coerced by PCR
Wild rice Oo92082 gene, PCR primer detects through 1.5% agargel electrophoresis, the band (Fig. 2) of amplification to about bp more than 500,
Stripe size is close with expection gene stripe size (512bp).This gene band is carried out glue and reclaims rear clone to pClone007
On carrier, obtaining SEQ ID NO:2 sequence through order-checking, SEQ ID NO:2 sequence is carried out with transcribing sequencing sequence SEQ ID NO:1
Homology comparison, finds that the SEQ ID NO:2 sequence of separating clone is consistent with SEQ ID NO:1 sequence, shows: by this special expansion
Increase primer and can expand oryza officinalis Oo92082 gene from oryza officinalis.The one-tenth of oryza officinalis Oo92082 gene
Merit separating clone and the base sequence information of offer, resist with oryza officinalis for further investigation oryza officinalis Oo92082 gene
Bacterial leaf-blight mechanism is laid a good foundation, is provided theoretical foundation.
Accompanying drawing explanation
Fig. 1 is that oryza officinalis Oo92082 gene is not inoculating matched group 0h and the inoculation bacterial leaf-blight of leaf spot bacteria
Relative expression quantity in the oryza officinalis blade of each time point of bacterium 24h, 48h, 72h, 96h.0h: with ddH2O replaces bacterium solution to connect
Plant and process time point；24h, 48h, 72h, 96h: inoculation PXO99 and C5 processes time point respectively.Abscissa express time, vertical seat
Mark represents oryza officinalis Oo92082 gene relative expression quantity in oryza officinalis blade, and in figure, black strip represents: strong
Pathogenic bacterium PXO99 (Philippiiies race 6) connects bacterium process group, strong to oryza officinalis pathogenecity；Twill strip represents: cause a disease by force
Bacterium C5 (sick representative strain is forced in paddy area in the southern part of China) connects bacterium process group, and causing a disease oryza officinalis can be more weak.
Fig. 2 is that the specific amplification primer utilizing oryza officinalis Oo92082 gene order SEQ ID NO:1 to design is from Yunnan
In oryza officinalis, amplification is to the agarose gel electrophoresis figure of oryza officinalis Oo92082 gene open reading frame.M in Fig. 2:
D2000DNA Marker；Swimming lane 1 is the oryza officinalis of the specific amplification primer amplification utilizing SEQ ID NO:1 to design
Oo92082 gene open reading frame genetic fragment, it is contemplated that size 512bp.Described specific amplification primer is by forward primer and downstream
Primer forms, and the forward primer sequence of described specific amplification primer is as shown in SEQ ID NO:5, under described specific amplification primer
Trip primer sequence is as shown in SEQ ID NO:6.
Detailed description of the invention
Experiment material: oryza officinalis picks up from Chinese yunnan and saves.
Leaf spot bacteria biological strain PXO99 and C5 is by KUNMING INST OF BOTANY CAS
Molecule room preserves.Described leaf spot bacteria biological strain PXO99 " uses Molecular Marker Assisted Selection Technology at non-patent literature
Improve the research of 9311 bacterial leaf spot resistances " (Zhou Yuan flies, Molecular Plant Breeding, 2003, volume 1, the 3rd phase, 343-
Page 349) open, described leaf spot bacteria biological strain C5 is at non-patent literature " southwest rice varieties leaf white to Oryza sativa L.
The Analysis of Resistance of rot " and (Wang Yongji etc., China's agronomy circular, 2011,27 (27): 260-264)) open, the applicant ensures
From present patent application day, provide described leaf spot bacteria biological strain PXO99 to the public in 20 years and leaf spot bacteria physiology is little
Kind of C5, the applicant contact address: Xue Yun road, Wuhua District, Kunming, Yunnan Province 9, Yunnan Agriculatural Academy's biotechnology with
Research of fruit germplasm resource institute, postcode: 650223.
Above-mentioned leaf spot bacteria biological strain PXO99 and leaf spot bacteria biological strain C5 is all to utilize it to invade Oryza sativa L.
Dye ability thus cause rice leaf formed bacterial leaf-blight scab general character, therefore, there is the various white leaf of same pathogenecity
Withered Biological Strains of The Pest is attained by the experiment effect of the present invention.Each experiment reagent of following example is commercially available.
Following example without specified otherwise for conventional method.
Embodiment 1 oryza officinalis Oo92082 gene is coerced different time points at oryza officinalis by leaf spot bacteria
Expression is analyzed
(1) extraction of total serum IgE
The extraction of oryza officinalis total serum IgE usesPlant Mini Kit extracts, genome
Removal useRNase-Free DNase set test kit is carried out.All of RNA extraction step is the completeest
Becoming, all of centrifugation step completes on the standard small centrifuge of 20-25 DEG C, and centrifuging temperature is not less than 20 DEG C, white to inoculation
The sample of leaf spoting bacteria and do not connect the sample of leaf spot bacteria and all operate by following steps:
A, inoculate Yunnan of the same race oryza officinalis respectively with leaf spot bacteria biological strain PXO99 and C5, connect after bacterium processes
Every 24h, i.e. 24h, 48h, 72h, 96h collects blade, simultaneously with ddH2O replaces bacterium solution to inoculate oryza officinalis of the same race, as
The comparison of 0h.
B, weigh the oryza officinalis vegetable material less than 50mg, be placed at once in the mortar processed, add liquid nitrogen
It is ground.By powder fast transfer to RNase-free, and through the 2mL centrifuge tube of liquid nitrogen cooling, notice that material to keep
Freezing state.
C, adding 500 μ L Buffer RLT (1mL Buffer RLT adds 10 μ L beta-mercaptoethanols), vortex mixes.
D, solute is transferred in the QIAshredder spin column with 2mL collecting pipe, maximum (top) speed
14000 × g is centrifuged 2min.Supernatant in collecting pipe is transferred in new 2mL centrifuge tube.
E, add the dehydrated alcohol of 0.5 times of volume in supernatant, mix gently.
F, sample (about 650 μ L) is transferred in the RNeasy spin column with 2mL collecting pipe.Close the lid gently
Son, >=8000 × g is centrifuged 15s, outwells waste liquid in collecting pipe.
G, add in 700 μ LBuffer RW1 to RNeasy spin column, close the lid gently, >=8000 × g from
Heart 15s cleans the filter membrane on pillar, outwells waste liquid.
H, add in 500 μ L Buffer RPE to RNeasy spin column, close the lid gently, >=8000 × g from
Heart 15s cleans the filter membrane on pillar, outwells waste liquid.
I, add in 500 μ L Buffer RPE to RNeasy spin column, close the lid gently, >=8000 × g from
Heart 2min cleans the filter membrane on pillar, outwells waste liquid.
K, RNeasy spin column is placed in new 1.5mL centrifuge tube, directly adds on the film of spin column
Entering 30～50 μ L RNase-free water, close the lid gently, >=8000 × g is centrifuged 1min, eluted rna.
L, the agarose gel electrophoresis of 1.0% carry out the detection of RNA integrity, and normal RNA sample passes through gel electrophoresis
Can run out of 3 band, the sedimentation coefficient of 3 band 28S, 18S and 5S respectively, wherein 28S is the brightest, and 5S is the most weak.With
RNA purity is detected by NanoDrop 2000 ultramicrospectrophotometer, with two ratios of OD260/280 and OD260/230
As index, when OD260/280 is between 1.9～2.1, and OD260/230 is between 2.0～2.5, then it is assumed that the purity of RNA
High.
(2) synthesis of cDNA the first chain
CDNA uses PrimeScriptTMRT reagent with gDNA Eraser test kit is carried out, and concrete operations are such as
Under:
A, the removal reaction of genomic DNA.5×gDNA Eraser Buffer、gDNA Eraser、RNase Free
dH2O is immediately placed on ice after at room temperature thawing, and prepares the removal genome reactant shown in table 1 in 250 μ L PCR pipe
System:
Genome reaction system removed by table 1
After reactant liquor prepares, it is placed in PCR instrument, 42 DEG C hatches 2min, is subsequently placed on ice.
B, reverse transcription reaction.Inverse transcription reaction liquid is prepared on ice.For ensureing the accuracy of reactant liquor preparation, carry out every
During reaction, prepare Master Mix by the amount of stoichiometric number+1, be dispensed into the most again in each reaction tube.
Table 2 reverse transcription reaction system
After reverse transcription Compound mixed solution is good, it is placed in PCR instrument, 37 DEG C are reacted 15min, 85 DEG C of 5sec.React rear short
Temporarily centrifugal, be stored in-20 DEG C standby.
(3) Real-time PCR system is set up and response procedures
Differential expression after the oryza officinalis transcript profile order-checking that A, the leaf spot bacteria obtained from previous experiments are coerced
Gene analysis, it is thus achieved that difference is the most notable, the gene of numbered Contig_92082, and this numbering gene is annotated to NR, GO
With KEGG data base, do not obtain any functional annotation；The aminoacid sequence of coding, through BLASTp comparison, does not has any homology,
This, as shown in SEQ ID NO:7, therefore, is derived from oryza officinalis by the aminoacid sequence of Contig_92082 gene code
The Contig_92082 unnamed gene of transcript profile is oryza officinalis Oo92082 gene, SEQ in its nucleotide sequence such as sequence table
Shown in ID NO:1.
B, according to the sequence SEQ ID NO:1 of oryza officinalis Oo92082 gene obtained, utilize biosoftware
Primer5.0 design expression analyzes primer, and this expression is analyzed primer and is made up of forward primer and downstream primer, described
Expression analyzes the base sequence of forward primer of primer as shown in SEQ ID NO:3 in sequence table, described expression
Analyze the base sequence of downstream primer of primer as shown in SEQ ID NO:4 in sequence table.This expression is analyzed primer and is sent China
The synthesis of big genome company.
C, the SYBR Premix Ex Taq II (Tli RnaseH Plus) of employing TaKaRa carry out Real-time PCR
Reaction system is prepared, and uses Applied Biosystems QuantStudio 6&7 real-time fluorescence quantitative PCR instrument to carry out Real-
Time PCR response procedures.
Component as shown in table 3 prepares PCR reactant liquor on ice:
Table 3 Real-time PCR reaction system
Real-time PCR response procedures: 95 DEG C of denaturations 30sec；95 DEG C of 30sec, 60 DEG C of 34sec, 40 circulations；Molten
Solution curve: 95 DEG C of 15sec, 60 1min, 95 DEG C of 15sec.
(4) interpretation
By Real-time round pcr, oryza officinalis Oo92082 gene (SEQ ID NO:1) is not being connect bacterium process
Matched group and respectively inoculate leaf spot bacteria biological strain PXO99 and C5 each time point (0h, 24h, 48h, 72h, 96h)
Relative expression quantity be analyzed, its differential expression level has two kinds of situations, and one is that oryza officinalis Oo92082 gene is connecing
Planting relative expression quantity in the Yunnan oryza officinalis of PXO99 and C5 all high than matched group, this situation shows oryza officinalis
Oo92082 gene belongs to leaf spot bacteria inducible expression of gene, simultaneously by analyzing two biological strains of PXO99 and C5 to medicine
By the difference of wild rice Oo92082 Primary structure amount, the medicinal wild of oryza officinalis Oo92082 gene mediated can be analyzed
Raw bacterial blight of rice immunoreation expression variation tendency in early days.
Interpretation of result: oryza officinalis Oo92082 gene do not connect bacterium matched group (0h) and connect leaf spot bacteria (24h,
48h, 72h, 96h) oryza officinalis blade in the relative expression quantity of 5 time points show, this oryza officinalis Oo92082
Gene shows as up-regulated expression (shown in Fig. 1) after inoculation leaf spot bacteria.In these 5 time points, oryza officinalis
There is the equal of summit in Oo92082 gene relative expression quantity (RQ value) after leaf spot bacteria PXO99 and C5 biological strain coerce
At 48h, after wherein PXO99 coerces, the relative expression quantity (RQ value) at 48h time point is more than 16 times of matched group, C5 coerce after
The relative expression quantity (RQ value) of 48h time point is more than 85 times of matched group.Illustrate that this oryza officinalis Oo92082 gene is medicinal
When wild rice is coerced by leaf spot bacteria, the expression of this oryza officinalis Oo92082 gene progressively strengthens, expression
Raising, in 5 time points of detection, the relative expression quantity at this gene of 48h reaches the highest.According to this interpretation of result, medicinal
Wild rice Oo92082 gene belongs to bacterial leaf-blight in the oryza officinalis of Yunnan and coerces the inducible expression of gene of expression, and it is situated between
Lead the reaction of oryza officinalis bacterial leaf-blight early immune.Show the oryza officinalis obtained from the oryza officinalis of Yunnan
Oo92082 gene is the gene directly related with bacterial blight-resisting, is new Bacterial blight resistance gene.
Oryza officinalis Oo92082 gene order SEQ ID NO:1 is for analyzing this gene at Yunnan oryza officinalis by white
Leaf spoting bacteria is coerced the expression of rear different time points and is provided sequence information, for excavating new resisting in the oryza officinalis of Yunnan
Ospc gene provides theoretical foundation.
The separating clone of embodiment 2 oryza officinalis Oo92082 gene
(1) the oryza officinalis Oo92082 gene order SEQ ID NO:1 obtained according to early stage transcriptome analysis, utilizes
Biosoftware Primer5.0 devises the specific amplification primer of oryza officinalis Oo92082 gene open reading frame, this medicinal wild
The specific amplification primer of raw rice Oo92082 gene open reading frame is made up of forward primer and downstream primer, described medicinal wild
SEQ ID NO in the base sequence such as sequence table of the forward primer of the specific amplification primer of rice Oo92082 gene open reading frame:
Shown in 5, the base sequence of the downstream primer of the specific amplification primer of described oryza officinalis Oo92082 gene open reading frame is such as
In sequence table shown in SEQ ID NO:6.The specific amplification primer of this oryza officinalis Oo92082 gene open reading frame send Hua Da
Genome company synthesizes.
(2) using the cDNA in embodiment 1 step (2) as PCR reaction template, the medicine of amplification leaf spot bacteria abduction delivering
With wild rice Oo92082 gene, use the high-fidelity of TaKaRaMax DNA Polymerase carries out purpose base
The amplification of cause, the component as shown in table 4 prepares PCR amplification system on ice:
Table 4 oryza officinalis Oo92082 gene PCR amplification system
PCR reaction condition: 98 DEG C of denaturations 2min；98 DEG C of 10sec, anneal 5sec, and 72 DEG C extend 10sec, 35 circulations；
72 DEG C extend 10min.
(3) PCR primer is through 1.5% agarose gel electrophoresis, by the electrophoretic band with the expection close size of size 512bp
(Fig. 2) the SanPrep pillar DNA glue using Sangon Biotech (Shanghai) Co., Ltd. reclaims test kit and reclaims,
Concrete operations are as follows:
A. under uviol lamp will with expection the close size of size 512bp electrophoretic band (Fig. 2) cut, put into 1.5mL from
In heart pipe, weigh；Weight according to blob of viscose and concentration, by every 100mg agarose (as glue is then supplemented to water less than 100mg
Ratio 100mg) adding 300～600 μ L Buffer B2 adds Buffer B2；
B., centrifuge tube is placed in 50 DEG C of water-baths 5～10min, and period constantly shakes, until blob of viscose dissolves completely；
C. adding 500 μ L Wash solution, room temperature in adsorption column, 9000 × g is centrifuged 30sec, outwells waste liquid, will
Adsorption column is put in same collecting pipe；Repeat this step once；
D. suction attached column and collecting pipe being put into centrifuge 9000 × g and be centrifuged 1min, it remains in Wash solution
Ethanol volatilization dry；
F. add the Elution buffer of 30 μ L to adsorption column film central authorities, room temperature is placed 2min, 9000 × g and is centrifuged 1min
Eluted dna, eluting obtains DNA solution.
(4) oryza officinalis Oo92082 gene and the connection of pClone007
The DNA solution that the present embodiment 2 step (3) F obtains reclaims after test kit reclaims must return through SanPrep pillar DNA glue
The section of taking up, uses the pClone007Blunt Simple Vector Kit of TSINAKE will reclaim fragment and pClone007 carrier
It is attached.Component as shown in table 5 prepares linked system on ice:
Table 5 oryza officinalis Oo92082 gene and the linked system of pClone007
After mixing, of short duration centrifugal, it is placed in room temperature 5min.
(5) what step (4) connected product converts bacillus coli DH 5 alpha competent cell by thermal shock, and concrete operations are such as
Under:
A. step (4) 10 μ L connection product is all joined in 100 μ L DH5 α competent cells, mix gently with rifle head
30min is placed on ice after even；Join in 100 μ L DH5 α competent cells as negative control using 1 μ L sterilized water simultaneously；
B.42 thermal shock 60sec in DEG C water-bath.The coldest 2～3min (forbidding shake) it are immediately placed on after thermal shock；
C. in often pipe, add 700 μ L LB fluid medium (without any antibiotic), mix rear 37 DEG C of 180rpm concussion
Cultivate 1h, make antibacterial restore normal growth state, and the antibiotics resistance gene of expression plasmid coding；
D. room temperature, 4000rpm is centrifuged 5min, discards 600 μ L of supernatant liquid, hangs equal bacterium solution gently with rifle head, takes 100 μ L bacterium solution
Coat on the LB flat board containing 50mg/L Amp antibiotic；
F.37 DEG C inversion light culture is overnight, until growing bacterium colony.
(6) oryza officinalis Oo92082 gene and the screening of pClone007 carrier recon
A. there are picking list bacterium colony the flat board of bacterium colony, the LB liquid culture of inoculation 50mg/L Amp from above-mentioned steps (5) length
In base, 37 DEG C of shaking table shaken cultivation 12-16h；
B. take 1 μ L bacterium solution and carry out PCR detection, it is determined whether be oryza officinalis Oo92082 gene and pClone007 carrier
Recon, PCR reaction system and PCR response procedures are by embodiment 2 step (2)；
C. detect through PCR, send Shenzhen Hua Da Gene science to have the recon of described recovery fragment with pClone007 carrier
Limit company checks order, and the nucleotide sequence of this recovery fragment is as shown in SEQ ID NO:2；The nucleotide sequence of fragment will be reclaimed
As SEQ ID NO:2 compares with transcript profile sequencing sequence SEQ ID NO:1.
(7) interpretation of result: utilize specific amplification primer of the present invention from the Yunnan oryza officinalis that leaf spot bacteria is coerced
The nucleotide sequence of separating clone recovery fragment as shown in SEQ ID NO:2, it is understood that there may be 3 kinds of situations: one is that amplification is to pre-
Phase gene stripe size 512bp, two is that gene band is arrived in amplification, but gene stripe size is different from expection size (512bp), three
It is that amplification is less than gene band.By amplification to gene band reclaim rear clone on pClone007 carrier through glue, after order-checking with
Transcript profile sequencing sequence SEQ ID NO:1 compares, and confirms correct sequence.
Interpretation of result: utilize specific amplification primer of the present invention, by PCR from the oryza officinalis that leaf spot bacteria is coerced
The DNA fragmentation that amplification is arrived, PCR primer detects through 1.5% agargel electrophoresis, band (Fig. 2 swimming lane of amplification to about bp more than 500
1), stripe size is close with expection gene stripe size (512bp).This gene band carries out glue recovery rear clone arrive
On pClone007 carrier, through order-checking obtain SEQ ID NO:2 sequence, SEQ ID NO:2 sequence with transcribe sequencing sequence SEQ ID
NO:1 carries out homology comparison, finds that the SEQ ID NO:2 sequence of separating clone is consistent with SEQ ID NO:1 sequence, SEQ ID
Sequence shown in NO:2 is the sequence of the open reading frame of sequence shown in SEQ ID NO:1, i.e. expands with specific amplification primer of the present invention
Increase to DNA fragmentation be really oryza officinalis Oo92082 gene, show: can be from medicinal wild by special primer of the present invention
In raw rice, amplification is to its nucleotide sequence oryza officinalis Oo92082 gene as shown in SEQ ID NO:1.Oryza officinalis
Oo92082 gene be successfully separated clone, lay the foundation for this gene functional research follow-up.
Sequence shown in the SEQ ID NO:1 of oryza officinalis Oo92082 gene is subject at oryza officinalis for analyzing this gene
Leaf spot bacteria coerce rear different time points expression and therefrom this gene of separating clone provide sequence information, for excavate
Disease-resistant gene new in the oryza officinalis of Yunnan provides theoretical foundation.
The present invention is described in detail to have used general explanation and specific embodiments thereof, but at this
On the basis of bright, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, exist
Without departing from modifications or improvements on the basis of spirit of the present invention, belong to the scope of protection of present invention.
<110>KUNMING INST OF BOTANY CAS
<120>the oryza officinalis Oo92082 gene of leaf spot bacteria abduction delivering and specific amplification primer thereof
<130> /
<160> 7
<170> PatentIn version 3.3
<210> 1
<211> 708
<212> DNA
<213>oryza officinalis (Oryza officinalis Wall.)
<220>
<221>start codon
<222> (42)..(44)
<220>
<221>open reading frame
<222> (42)..(293)
<220>
<221>termination codon
<222> (291)..(293)
<400> 1
tttttttttt gacatgttat caatgcattt atatttgaca tatgatgtat ccttacaaaa 60
atggcaatgg taagaaaaaa ttacattacc aagaagacta ttcaacgata atagtgatcc 120
atgatactct tataagcaag tggtattaca agtgccccgt taatcttcag gtttccaaca 180
taaattcagg taacccgatt acagatcttg tttcccaaac taaaatcgga aacaggcacg 240
cacaaactta cgcaaagaaa aaaagaaaac caaacaaaca tactagtacg tgattatctc 300
gttgtcaagg cctcctttga aactgaggaa ttttgtgaga atctttccaa aactaaattc 360
tctcaaaatt cctgcatttc aaagaagatt tcgcccatct ctcttctacc ccaggaatgc 420
gaagaagatc gcgcatccca gcagccacat ggcatggtaa aaccccatga tggccagcac 480
gtgagagttc atcaccggcg cggtggagct caggatgagg atcccgccgg cggcgtacag 540
catcaggagc acgccggaag gtcgtcgccg gccgccgccg ccgccgactc gcgctcctgc 600
aggctccatc ctgtggtagc tagtacacct gccggccacc tagtgctggg atgaagtgat 660
gaggttttct ctgattagtt gatcagcaca gggtgagtag tccgcccc 708
<210> 2
<211> 512
<212> DNA
<213>oryza officinalis (Oryza officinalis Wall.)
<220>
<221>start codon
<222> (1)..(3)
<220>
<221>open reading frame
<222> (1)..(252)
<220>
<221>termination codon
<222> (250)..(252)
<400> 2
atgatgtatc cttacaaaaa tggcaatggt aagaaaaaat tacattacca agaagactat 60
tcaacgataa tagtgatcca tgatactctt ataagcaagt ggtattacaa gtgccccgtt 120
aatcttcagg tttccaacat aaattcaggt aacccgatta cagatcttgt ttcccaaact 180
aaaatcggaa acaggcacgc acaaacttac gcaaagaaaa aaagaaaacc aaacaaacat 240
actagtacgt gattatctcg ttgtcaaggc ctcctttgaa actgaggaat tttgtgagaa 300
tctttccaaa actaaattct ctcaaaattc ctgcatttca aagaagattt cgcccatctc 360
tcttctaccc caggaatgcg aagaagatcg cgcatcccag cagccacatg gcatggtaaa 420
accccatgat ggccagcacg tgagagttca tcaccggcgc ggtggagctc aggatgagga 480
tcccgccggc ggcgtacagc atcaggagca cg 512
<210> 3
<211> 25
<212> DNA
<213>oryza officinalis (Oryza officinalis Wall.)
<400> 3
gtatccttac aaaaatggca atggt 25
<210> 4
<211> 19
<212> DNA
<213>oryza officinalis (Oryza officinalis Wall.)
<400> 4
gcgtgcctgt ttccgattt 19
<210> 5
<211> 18
<212> DNA
<213>oryza officinalis (Oryza officinalis Wall.)
<400> 5
atgatgtatc cttacaaa 18
<210> 6
<211> 17
<212> DNA
<213>oryza officinalis (Oryza officinalis Wall.)
<400> 6
cgtgctcctg atgctgt 17
<210> 7
<211> 83
<212> PRT
<213>oryza officinalis (Oryza officinalis Wall.)
<400> 7
Met Met Tyr Pro Tyr Lys Asn Gly Asn Gly Lys Lys Lys Leu His Tyr
1 5 10 15
Gln Glu Asp Tyr Ser Thr Ile Ile Val Ile His Asp Thr Leu Ile Ser
20 25 30
Lys Trp Tyr Tyr Lys Cys Pro Val Asn Leu Gln Val Ser Asn Ile Asn
35 40 45
Ser Gly Asn Pro Ile Thr Asp Leu Val Ser Gln Thr Lys Ile Gly Asn
50 55 60
Arg His Ala Gln Thr Tyr Ala Lys Lys Lys Arg Lys Pro Asn Lys His
65 70 75 80
Thr Ser Thr
Claims (3)
1. an oryza officinalis Oo92082 gene for leaf spot bacteria abduction delivering, its nucleotide sequence such as SEQ ID NO:1
Shown in.
2. the oryza officinalis Oo92082 gene of a kind of leaf spot bacteria abduction delivering described in claim 1 is medicinal wild
The expression that rice is coerced different time points by leaf spot bacteria analyzes primer, and described expression analyzes primer by forward primer
With downstream primer form, described expression analyze primer forward primer sequence as shown in SEQ ID NO:3, described expression water
Divide the downstream primer sequence of analysis primer equally as shown in SEQ IDNO:4.
3. the oryza officinalis Oo92082 base of a kind of leaf spot bacteria abduction delivering described in a separating clone claim 1
Because of the specific amplification primer of open reading frame, described specific amplification primer is made up of forward primer and downstream primer, described special
The forward primer sequence of amplimer as shown in SEQ ID NO:5, the downstream primer sequence such as SEQ of described specific amplification primer
Shown in ID NO:6.
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| CN102660777A (en) * | 2012-04-28 | 2012-09-12 | 云南省农业科学院生物技术与种质资源研究所 | Method for constructing suppression subtractive hybridization (SSH) library of oryza rufipogon threatened by bacterial blight germs |
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