CN106676114A - Oryza sativa gene OsUEP3 and application of disease resistance regulation function - Google Patents
Oryza sativa gene OsUEP3 and application of disease resistance regulation function Download PDFInfo
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Abstract
The invention discloses oryza sativa gene OsUEP3. The open reading frame of the gene is 390 bp in length, and the gene encodes a ubiquitin extension protein and comprises 129 amino acids. Ubiquitin molecules of 76 amino acids are arranged at the N terminal of the protein, and 60S ribosomal protein L40e of 53 amino acids are arranged at the C terminal of the protein. The regulation effect of the gene on the bacterial disease resistance of oryza sativa is illuminated for the first time by constructing an overexpression transgenic oryza sativa of the gene and analyzing disease resistance. The resistance of the overexpression transgenic oryza sativa plants to Xanthomonas oryzae strains PXO99 and PXO112 is improved remarkably, so that the positive regulation function of the gene for the Xanthomonas oryzae resistance is revealed, and the regulation function has broad spectrum for different strains. The oryza sativa gene OsUEP3 is a novel gene resource suitable for creating and breeding novel oryza sativa materials and novel varieties with broad-spectrum resistance to Xanthomonas oryzae and other bacterial diseases.
Description
Technical field
The invention belongs to biological technical field, is related to the disease-resistant adjusting function analysis of a paddy gene (OsUEP3) and its answers
With.
Background technology
1st, Gene Clone in Plant
Methods of plant gene cloning has many kinds, increasing for the plant species completed with genome sequence determination, is based on
The methods of plant gene cloning of database sequence is increasingly widely applied.The method operating procedure is mainly included based on guarantor
The design of primers of sequence is kept, purpose plant tissue RNA is extracted, reverse transcription-polymerase chain reaction (RT-PCR), PCR primer
With the connection of carrier, the Bacterial Transformation of connection product, plasmid extraction, enzyme action inspection, sequencing analysis etc..
2nd, gene expression in plants control and gene function analysis technology
The function of plant gene is performed by gene expression and protein accumulation.Therefore, gene function is generally by comparing
Phenotype (Phenotype) or Function situation in the case of unconventional expression and the normal expression of analysis gene come judge with it is bright
Really.The unconventional expression of gene includes two big class of the expression higher than normal level and the expression less than normal level.Higher than normal
The expression of level is mainly overexpression/overexpression (Over-expression), mainly by connecting a strong promoter driving
The mode of destination gene expression is reaching.Less than normal level expression mainly by RNA interference (RNA interfering, surpass
Expression), the mode such as gene knockout (Knock-out) to be reaching.Overexpression contains same tract by building and converting one
Section rightabout inserts hairpin structure that an intron or sequence that other are not expressed formed to realize, is as a result genes of interest
The reduction of expression.Gene knockout (Knock-out) is then long non-by one is inserted in the genes of interest in Plant Genome
Plant sequence cuts off the modes such as genes of interest to reach, and is as a result nearly fully completely suppressing for destination gene expression.
Gene overexpression plant and/or overexpression plant, knock out mutants body are built, compares phenotype and character and the open country of these plant
The difference of raw type/normal plant, the regulatory function of the gene pairss character that can just have a definite purpose.
3rd, plant transgenic technology
For exogenous gene is imported purpose plant, so as to obtain the transgenic plant of foreign gene-carrying.Including gene
Marksmanship, agrobacterium-mediated transformation etc..Agrobacterium-mediated transformation includes for genes of interest being cloned into plant expression vector, and expression vector is to agriculture
The conversion of bacillus, carries infection of the Agrobacterium of expression vector to purpose plant tissue, the regeneration of transfer-gen plant and plant
The step such as middle gene transformation and the detection and analysis of expression.
4th, disease resistance of plant detection and analysis technology
The phytopathy original includes the polytypes such as funguses, oomycetes, antibacterial, bacterium substance, virus and nematicide.Different type cause of disease
The inoculation method of thing is different.For funguses, can produce it is conidial, generally with conidial suspension spray inoculation plant
Thing.Do not produce it is conidial, then with mycelia block etc. be inoculated with plant.For oomycetes, typically first culture produces a large amount of Sporangiums, and
Low- temperature culture is allowed to discharge zoospore afterwards, is finally inoculated with plant with zoospore suspension.For antibacterial, often suspended with thalline
The methods such as liquid leaf-cutting, acupuncture, injection, spraying are inoculated with plant.For virus, often produced with the virion or artificial in vitro transcription that purify
Thing friction, injection, or plant is inoculated with by the communication media such as insecticide.For bacterium substance, it is inoculated with or transfers usually through vector
Inoculation.For nematicide, often contacted in plant root basal part of stem or rhizosphere soil with a number of second instar larvae and be inoculated with plant.
In most cases, need to keep after inoculation higher relative humidity, suitable temperature and illumination condition.Different time is pressed after inoculation
There is symptom, statistics sickness rate and morbidity severity in point Continuous Observation disease, calculate disease index, detect cause of disease using RT-PCR
Thing Biomass.By the relative analyses with susceptible check plant incidence, the disease resistance of hard objectives plant.
5th, Resistant breeding technology
Can be largely classified into traditional breeding for disease resistance and by two big class of genetic engineering breeding for disease resistance.Traditional breeding for disease resistance because
There is its natural genetic isolation phenomenon to be significantly limited Resistant germplasm usable range, the disease-resistant money that genetic affinity can only be applied nearer
Source, and repeatedly hybridization and backcrossing etc. are needed, therefore the selection-breeding cycle is long, and need a large amount of manpower and materials.And genetic engineering breeding
Method be by by the disease-resistant controlling gene of external source by the technological sourcing plant such as agriculture bacillus mediated method so as to obtain original
The disease resistance not having.Therefore, genetic engineering breeding method has broken the restriction of its natural genetic isolation phenomenon, has widened disease-resistant money
Source usable range, and have the relatively easy convenience of operation, cultivation period it is short, without the need for artificial material resources in a large number the characteristics of.In addition, can
By importing a broad-spectrum disease resistance controlling gene, or the gene of multiple different disease-resistant spectrums, kind of the initiative with broad-spectrum disease resistance.
Therefore there is particularly suitable cultivation wide spectrum, permanent disease-resistant kind.
The content of the invention
It is an object of the invention to provide Oryza sativa L. (Oryza sativa) gene OsUEP3, anti-to paddy bacterial disease
Property has regulating and controlling effect.The nucleotide sequence such as SEQ ID of the paddy gene OsUEP3 of present invention clone：Shown in 1, the gene
The long 390bp of open reading frame (ORF), encodes a kind of ubiquitin extension protein (Ubiquitin extension protein), by
129 aminoacid compositions, its sequence such as SEQ ID：Shown in 2.The albumen n end is the ubiquitin molecule of 76 aminoacid, and C-terminal is then
The 60S ribosomal protein L 40e of 53 aminoacid.The sequence of the present invention clone genome annotation fine with rice varieties Japan
LOC_Os03g13170.1 has very high similarity, the only difference of 2 bases：The A6 of LOC_Os03g13170.1 sequences is replaced
For G；G15 replaces with C.Change of these point mutation without result in aminoacid.Before making the present invention, the gene function is not any
Open report.First passage of the present invention builds the overexpression transgenic paddy rice of the gene and its disease-resistant analysis, illustrates the gene
Regulating and controlling effect to paddy bacterial Disease Resistance.As a result show, overexpression transgenic rice plant is significantly reduced to Oryza sativa L.
The resistance of leaf spot bacteria (Xanthomonas oryzae pv.oryzae, Xoo) bacterial strain PXO99 and PXO112, so as to disclose
The positive adjusting function of the gene pairss Bacterial Blight Resistance in Rice (being specifically shown in the explanation in embodiment).
It is a further object to provide described rice Os UEP3 gene by formulate gene overexpression Oryza sativa L. come
The application of bacteria resistance disease rice material is obtained, is obtaining in the transgenic paddy rice homozygous line by formulating overexpression OsUEP3
The application that must be directed in the broad-spectrum disease resistance material of rice leaf spot bacteria different strains, is realized by following steps：
(1) structure of OsUEP3 genes overexpression structure and acquisition
OsUEP3 gene ORF are cloned into into a plant expression vector so as to expression is ordered about by strong promoter.
(2) convert the acquisition of the Agrobacterium of OsUEP3 gene overexpression structures
The OsUEP3 gene overexpression structures for building had into strong infection ability by the conversion of the methods such as electric shock to Oryza sativa L.
Agrobacterium strains.
(3) the transgenic paddy rice initiative and acquisition of overexpression OsUEP3
By agrobacterium-mediated transformation by OsUEP3 gene overexpression structure Introduced into Rice, acquisition turns OsUEP3 gene overexpressions
The Oryza sativa L. of structure.
(4) acquisition of the transgenic paddy rice homozygous line of overexpression OsUEP3
Respectively with antibiotic resistance and OsUEP3 gene expressions as Testing index, the character point of transfer-gen plant offspring is detected
From situation, obtain characters of progenies it is no longer detached, and can stable hereditary overexpression OsUEP3 transgenic paddy rice homozygous line.
(5) the transgenic paddy rice homozygous line Screening and Identification of the overexpression OsUEP3 of anti-bacterial disease and acquisition
As material, detection and analysis resists transgenic paddy rice homozygous line with overexpression OsUEP3 to all kinds of Oryza sativa L. bacteriosises
Property, obtain anti-bacterial disease turns OsUEP3 trans-genetic hybrid rice.
Advantages of the present invention：(1) the OsUEP3 genes that the present invention is provided are high-quality anti-bacterial disease controlling gene resources, profit
The disease-resistant material obtained with the gene has the advantages that resistance is strong, disease-resistant spectrum is wide.Rice leaf spot bacteria (Xoo) has significantly
Differentiation of Pathogenicity, can be divided into many microspecies.There were significant differences for resistance mechanism of the Oryza sativa L. to different Xoo microspecies, therefore, obtain
While the paddy gene resource of simultaneous anti-different Xoo microspecies has important value for the prevention and control of bacterial blight of rice.The present invention is carried
For OsUEP3 genes function is totally unknown before this, the present invention research find the gene have concurrently to Xoo bacterial strain PXO99 and PXO112
Positive regulating and controlling effect, can be had concurrently to Xoo different strains resistance of wide spectrum by building the initiative of OsUEP3- overexpressions Oryza sativa L. and obtaining
New material.Therefore, OsUEP3 genes are a kind of suitable for formulating the water with selection-breeding wide spectrum bacterial blight-resisting and other bacteriosises
The brand-new genetic resourceses of rice new material and new varieties.(2) disease-resistant material periodicities are obtained short.Obtain disease-resistant plants material and kind
Method mainly has conventional traditional breeding way and the genetic engineering breeding method using disease-resistant controlling gene.Traditional breeding way
Limited by its natural genetic isolation with available Resistant germplasm scope, the selection-breeding cycle is long, the shortcomings of need a large amount of artificial material resources.And base
Then have that available Resistant germplasm scope is wide because of Engineering Breeding method, operate relatively easy convenience, cultivation period it is short, without the need for a large amount of people
The advantages of work material resources, particularly suitable cultivation wide spectrum, lasting, high resistance.The present invention utilizes disease-resistant controlling gene OsUEP3,
Using gene engineering method, the rice material of strong anti-bacterial disease is cultivated in initiative, with cycle is short, the features such as selection-breeding is quick.
Description of the drawings
Fig. 1 is OsUEP3 overexpression Oryza sativa L. homozygous lines and its parent's rice leaf Inoculated Rice leaf spot bacteria different strains
Symptom afterwards and bacterium amount detection and analysis results contrast figure.OsUEP3 overexpressions Oryza sativa L. homozygous line 169-6 and its parent's rice leaf
Using leaf-cutting method inoculation rice leaf spot bacteria (Xoo) bacterial strain PXO99 and PXO112.On：Inoculation disease of the Xoo bacterial strains after 14 days
Shape；Under：Xoo bacterium amount testing results in inoculation rear blade.As a result show, compared with parent, OsUEP3 overexpression Oryza sativa L. homozygosis
It is that the withered scab that 169-6 is formed is considerably shorter, bacterium amount is significantly reduced.Show that the overexpression of OsUEP3 genes is significantly enhanced
Resistance to bacterial blight of rice different strains, discloses OsUEP3 and plays the positive regulating and controlling effect of wide spectrum to Bacterial Blight Resistance in Rice.
Specific embodiment
The present invention is further described in conjunction with the accompanying drawings and embodiments.
Embodiment 1
The present invention has cloned a paddy gene OsUEP3, and first passage builds overexpression transgenic paddy rice, illustrates this
The adjusting function of the wide spectrum bacterial blight-resisting different strains of gene.Meanwhile, the overexpression transgenic paddy rice that the present invention builds has
Enhanced disease resistance, therefore, the invention provides a set of overexpression transgenic paddy rice by building the gene, using gene work
Journey technology is formulated and obtains the technical system of wide spectrum water resistant rice bacteriosises Rice New Material.Key step includes：
1) clone of rice Os UEP3 gene and preservation
The rice Os UEP3 gene that the present invention is provided is obtained by following steps clone.First according to rice genome data base
Middle OsUEP3 sequential designs primer OsUEP3-F3 (5 '-caggcgcgcc atg cag atc ttc gtc aag acc-3 ',
Italicized item is Asc I restriction enzyme sites) (sequence such as SEQ ID：Shown in 3), and OsUEP3-R2 (5 '-caggtacc gtt
Ctt gat ctt ctt ctt ggg-3 ', italicized item are Kpn I restriction enzyme sites) (sequence such as SEQ ID：Shown in 4).Using
TRIZOL reagents extract rice leaf total serum IgE, obtain OsUEP3cDNA using RT-PCR method amplification, solidifying by 1% agarose
Purification of tapping rubber after gel electrophoresis reclaims PCR primer, connects pMD19-mT carriers, thermal shock conversion bacillus coli DH 5 alpha, in LB culture medium
In shake bacterium overnight incubation, extract plasmid, adopt AscI/KpnI digestion methods and with OsUEP3-F3/OsUEP3-R2 as primer
To the plasmid that extracts of PCR method difference inspection institute whether include OsUEP3 genes, finally send company's sequence verification, so as to success
Clone and acquisition OsUEP3 gene cDNA full length sequences.
The OsUEP3 nucleotide sequences such as SEQ ID that clone obtains：Shown in 1, the open reading frame (ORF) of the gene is long
390bp, encodes a kind of ubiquitin extension protein (Ubiquitin extension protein), is made up of 129 aminoacid, its
Sequence such as SEQ ID：Shown in 2.The albumen n end is the ubiquitin molecule of 76 aminoacid, and C-terminal is then the 60S ribose of 53 aminoacid
Body protein L40e.The LOC_Os03g13170.1 of the sequence of the present invention clone genome annotation fine with rice varieties Japan has very
High similarity, the only difference of 2 bases：The A6 of LOC_Os03g13170.1 sequences replaces with G；G15 replaces with C.These
Change of the point mutation without result in aminoacid.Before making the present invention, the gene function does not have any open report.
The escherichia coli of the carrier for carrying OsUEP3 gene orders have been converted, -80 DEG C of refrigerators have been stored in.Therefore, can be with
When by activated strains, extract plasmid, expanded by PCR and enzyme action, by OsUEP3 genes sub-clone to purpose carrier, be used for
Transgenic etc. is studied.
2) structure of OsUEP3 genes overexpression structure and acquisition
Kpn I are first carried out respectively to above-mentioned 1) the middle pMD19-OsUEP3 plasmids for obtaining and plant overexpression vector pCZD
Enzyme action, Asc I enzyme action (because the buffer differences of two kinds of enzymes are big, therefore double digestion can not be carried out simultaneously) is subsequently carried out, by enzyme
PCZD carriers after genes of interest after cutting and same enzyme action are attached, connection product carries out escherichia coli conversion, ammonia benzyl resists
The steps such as raw element culture medium flat plate screening, enzyme action and PCR identifications obtain OsUEP3 gene overexpression structures pCZD-OsUEP3.It is super
Expression vector pCZD is easy to Molecular Identification and base with the expression of Semen Maydiss Ubq promoters driven genes of interest while carrying HA labels
Because of functional study.
3) convert the acquisition of the Agrobacterium of OsUEP3 gene overexpression structures pCZD-OsUEP3
OsUEP3 gene overexpression structures pCZD-OsUEP3 had into strong infecting potential by the conversion of the methods such as electric shock to Oryza sativa L.
Agrobacterium strains, such as EHAl05 screens transformant in the YEP culture medium containing streptomycin and kanamycin, then by Asc I
+ Kpn I enzyme action and PCR identifications, obtain the Agrobacterium for carrying OsUEP3 gene overexpression structures pCZD-OsUEP3.For next
The genetic transformation of step Oryza sativa L..
4) initiative and acquisition of OsUEP3 gene overexpression structure pCZD-OsUEP3 Oryza sativa L. are turned
By agrobacterium-mediated transformation by OsUEP3 gene overexpression structure pCZD-OsUEP3 Introduced into Rice.It is divided into Oryza sativa L. to heal
The induction of injured tissue, the agroinfection Rice Callus for carrying pANDA-OsUEP3, the regeneration of hygromycin resistance plant and
Several big steps such as identification, finally obtain the Oryza sativa L. T for turning pCZD-OsUEP3 of regeneration0Generation.Concrete operation step is as follows：
The induction of (i) Rice Callus
Plant the sterilization and cleaning of embryo surface：Ripe intact Japanese fine rice paddy seed is taken, kind of a skin is peelled off, is left complete kind
Embryo.With 70% alcohol solution dipping embryo 1min, embryo is soaked with 50% liquor natrii hypochloritises after outwelling ethanol, be placed on shaking table
28 DEG C, 180rpm sterilization 40min.Liquor natrii hypochloritises are outwelled, with the ddH of sterilizing2O washings embryo at least 10 times, until liquid
It is clean it is transparent till.Air-dried after embryo with the filter paper of sterilizing, carefully embryo is put on NB1 solid mediums with tweezers, each
About 30 seeds of culture dish.Fresh-keeping film phonograph seal is used afterwards, is placed in 28 DEG C of light cultures in incubator.
The induction of calluss：Embryo is placed on after cultivating 15 days on NB1, the calluss for differentiating is transferred to fresh
NB2 culture medium on, 10 days are further cultured under 28 DEG C of dark conditions.Health bright orange calluss are transferred on NB3 and are cultivated
About 10 days.Note, in order to provide sufficient nutrition, a NB2 plate changes into 2 NB3 plates and cultivated.
The successive transfer culture of calluss：The calluss of health are shifted on NB4 from NB3, is trained under 28 DEG C of dark conditions
Support 9 days, calluss now are best suitable for agroinfection.
(ii) agroinfection of Rice Callus
The culture of Agrobacterium：The Agrobacterium tumefaciems of OsUEP3 gene overexpression structures pANDA-OsUEP3 will be carried
EHA105 bacterial strains are taken out from -80 DEG C, in the flat lining out containing the LB of streptomycin and kanamycin, are placed in training in 28 DEG C of calorstats
Support (about 2d), grow picking single bacterium colony after bacterium colony, be inoculated in the 50mL YEP culture fluid containing corresponding antibiotic respectively, 27 DEG C permanent
Warm shaking table (180rpm) cultivates about 48h to OD600For 0.5-0.8, proceed in sterile centrifugation tube, 5000 × g centrifugations under room temperature condition
10min, removes supernatant, and thalline 25mL AAM-AS culture fluid is suspended after cleaning again, then is centrifuged one at identical conditions
It is secondary, finally with about 50mL AAM-AS culture fluid again suspension thalline to OD600For 0.05-0.1 (about 109Individual cell/mL), it is placed in
Can be used for the conversion of Oryza sativa L. on ice.
Agrobacterium is infected：The jonquilleous calluss of growth selection health care belt, are carefully blown into spoon aseptic little
In culture dish, then resuspended good agrobacterium liquid is poured in culture dish, make all calluss of bacterium solution submergence as far as possible, keep leaching
Bubble about 30min, period are jiggled every now and then.Bacterium solution is suctioned out as much as possible with the glue head dropper of sterilizing afterwards.
Co-culture：The calluss for infecting are put into into 22 DEG C of dark culturing 3d in NB-AS culture medium.
Carry disease germs cleaning of the calluss before resistance screening：After co-culturing 3d, the calluss for carrying disease germs are concentrated and is put into
In 500mL aseptic bottles, calluss 8~10 times are cleaned until liquid-transparent with the distilled water of sterilizing, be subsequently adding 200mL and contain
The ddH of Amp (200mg/mL) and Cef (300mg/mL)2O, puts it to shaking table, shakes 30min under 180rpm, 27 DEG C of constant temperature,
Then eluate is suctioned out with the glue head dropper of sterilizing, the wound healing for cleaning up is proceeded to into NB-CHA culture medium finally, at 28 DEG C
It is used for resistance screening after dark lower culture 14d.
(iii) regeneration of hygromycin resistance plant
Resistance screening culture：It is to proceed to containing antibiotic (Cef, 300mg/mL by calluss point；Hyg, 50mg/mL)
In NB-CH1 screening culture medium, it is dark at 28 DEG C under cultivate, carry out antibiotic resistance screening；
Successive transfer culture：By the Hygromycin resistant calli of Jing resistance screenings regeneration on NB-CH1, proceeded in the way of being
Containing antibiotic (Cef, 300mg/mL；Hyg, 50mg/mL) NB-CH2 screening culture medium on, it is dark at 28 DEG C under successive transfer culture one
It is secondary, incubation time about 1 week.
The pre- differentiation culture of resistant calli：Calluss with hygromycin are proceeded to containing antibiosis in the way of being
On plain (Hyg, 50mg/mL) pre- division culture medium Pre-H, it is dark at 28 DEG C under cultivate 15d.
The dedifferentiation culture of resistant calli：Broken up sufficient kanamycin-resistant callus tissue in advance to proceed to containing antibiotic in the way of being
On (Hyg, 50mg/mL) redifferential medium R-50, about 3 weeks are cultivated under 27 DEG C of optical conditions.
Root culture：Treat that Hygromycin resistant calli differentiates rootlet and green budlet, and rootlet grows to 1cm with enterprising
Row root culture.The resistant budses of greening are chosen from division culture medium with tweezers, be transferred to containing antibiotic (Hyg, 50mg/
ML root culture is carried out on 1/2MS root medias).
Soil is planted：Deng hygromycin resistance Seedling grow to root system it is complete after, by T0Uncap at 27 DEG C culture for aseptic seedling
2d is tempered, is then transplanted in the mellow soil that paddy field is fetched, being placed in greenhouse carries out Routine Management, finally harvested and obtain T0Seed.
5) Screening and Identification and the acquisition of the Oryza sativa L. homozygous line of OsUEP3 gene overexpression structures pCZD-OsUEP3 are turned
Respectively with hygromycin resistance and OsUEP3 gene expressions as Testing index, the character point of transfer-gen plant offspring is detected
From situation.Hygromycin resistance screening is carried out for rice paddy seed on the flat board containing hygromycin to be carried out, and can observation in hygromycin
In resistant panel, normal growth is into healthy seedling.Gene expression is detected using methods such as real-time fluorescence quantitative PCRs.After acquisition
For character it is no longer detached, and can stable heredity the Oryza sativa L. homozygous line for turning OsUEP3 gene overexpression structures pCZD-OsUEP3.
These Oryza sativa L. homozygous lines on hygromycin resistance flat board all can normal growth into healthy seedling and OsUEP3 gene expression doses
It is significantly higher than the control for turning empty carrier.
6) the disease resistance detection and analysis of OsUEP3 genes overexpression Oryza sativa L. homozygous line
Which, as material, is tested and analyzed to bacterial blight of rice with 5) the middle OsUEP3 gene overexpression Oryza sativa L. homozygous lines for obtaining
The resistance of bacterium (Xanthomonas oryzae pv.oryzae, Xoo) bacterial strain PXO99 and PXO112.Using leaf-cutting method inoculation.First
Shake bacterium concentration is made for OD600=1.0 Xoo bacterial suspensions, then bacterium solution is dipped with sterile scissors, cutting away from blade tip 3cm
Leaf, is placed in overlay film moisturizing 48h in big plastic crate.
Inoculation result shows that, 14 days after leaf-cutting inoculation Xoo bacterial strain PXO99, parent control rice leaf forms length and reaches
The withered scab of 16.4cm, and OsUEP3 gene overexpression Oryza sativa L. homozygous line 169-6 blades then only form average a length of 9.3cm's
Withered scab, 56.7% (Fig. 1 upper lefts) that length is only compareed.Bacterium amount tests and analyzes result and shows, parent control rice leaf
Bacterium amount up to 1.25 × 109Cfu/ leaves, and the bacterium amount of OsUEP3 overexpression Oryza sativa L. homozygous line 169-6 blades averagely only 6.50 ×
10852.0% (Fig. 1 lower-lefts) of cfu/ leaves, only parent control.14 days after leaf-cutting inoculation Xoo another bacterial strain PXO112, parent
Withered scab of this control rice leaf formation length up to 15.4cm, and OsUEP3 gene overexpression Oryza sativa L. homozygous line 169-6 leaves
Piece then only forms the withered scab of average a length of 8.4cm, 54.5% (Fig. 1 upper rights) that length is only compareed.Bacterium amount is tested and analyzed
As a result show, the bacterium amount of parent control rice leaf is up to 3.10 × 108Cfu/ leaves, and OsUEP3 overexpression Oryza sativa L. homozygous line 169-
The bacterium amount of 6 blades averagely only 9.96 × 10732.1% (Fig. 1 bottom rights) of cfu/ leaves, only parent control.
These results indicate that OsUEP3 overexpressions Oryza sativa L. homozygous line 169-6 is to two Xoo bacterial strain PXO99 and PXO112
Resistance is all remarkably higher than parent control.The overexpression of OsUEP3 genes causes Oryza sativa L. significantly increasing to these bacterial strain resistances, because
This, OsUEP3 plays positive regulating and controlling effect to rice leaf spot bacteria different strains.Can be by building OsUEP3- overexpression Oryza sativa L.
Homozygous line obtains the Rice New Material and new varieties of wide spectrum water resistant rice bacterial leaf spot pathogenic bacteria different strains to create.
7) Screening and Identification of the overexpression OsUEP3 trans-genetic hybrid rice homozygous lines of broad spectrum antibiotic disease and acquisition
Oryza sativa L. homozygous line with overexpression OsUEP3 genes is tested and analyzed to disease caused by all kinds of bacterial diseases originals as material
Resistance, such as xanthomonas oryzae pv. oryzicola (Xanthomonas oryzae pv.oryzicola, Xoc) different strains, water
Other bacterial strains of rice bacterial leaf spot pathogenic bacteria (Xanthomonas oryzae pv.oryzae, Xoo) etc..Analyzed and disease-resistant by being inoculated with
Evaluate, screening and acquisition show resistance, broad spectrum antibiotic disease OsUEP3 genes and surpass to many bacterial strains of Xoc and Xoo
Expression Oryza sativa L..
<110>Zhejiang University
<120>The application of paddy gene OsUEP3 and disease-resistant adjusting function
<160> 4
<210> 1
<211> 390
<212> DNA
<213>Oryza sativa L.（Oryza sativa）
<220>
<221> CDS
<222> (1)…(390)
<400> 1
atgcagatct tcgtcaagac cctgactggg aagaccatca ccctcgaggt ggagagcagc 60
gacaccatcg acaatgtcaa ggctaagatc caggacaagg agggaatccc gccggaccag 120
cagcggctga tcttcgccgg gaagcagctg gaggacggac gcaccctggc tgactacaac 180
atccagaagg agtccaccct ccacctcgtc ctcaggctcc gtggcggtat catcgagccg 240
tcgcttcagg cgcttgcccg caagtacaac caggacaaga tgatctgccg caaatgctat 300
gcgcgcctgc accctagggc tgtcaactgc cgcaagaaga agtgtggtca cagcaaccag 360
ctgaggccca agaagaagat caagaactag 390
<210> 2
<211> 129
<212> PRT
<213>Oryza sativa L.（Oryza sativa）
<400> 2
Met Gln Ile Phe Val Lys Thr Leu Thr Gly Lys Thr Ile Thr Leu Glu
1 5 10 15
Val Glu Ser Ser Asp Thr Ile Asp Asn Val Lys Ala Lys Ile Gln Asp
20 25 30
Lys Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile Phe Ala Gly Lys
35 40 45
Gln Leu Glu Asp Gly Arg Thr Leu Ala Asp Tyr Asn Ile Gln Lys Glu
50 55 60
Ser Thr Leu His Leu Val Leu Arg Leu Arg Gly Gly Ile Ile Glu Pro
65 70 75 80
Ser Leu Gln Ala Leu Ala Arg Lys Tyr Asn Gln Asp Lys Met Ile Cys
85 90 95
Arg Lys Cys Tyr Ala Arg Leu His Pro Arg Ala Val Asn Cys Arg Lys
100 105 110
Lys Lys Cys Gly His Ser Asn Gln Leu Arg Pro Lys Lys Lys Ile Lys
115 120 125
Asn
<210> 3
<211> 31
<212> DNA
<213>Artificial sequence
<220>
<223>The rice genome sequence design provided according to rice genome data base and synthetic, as primer
<400> 3
caggcgcgcc atg cag atc ttc gtc aag acc
<210> 4
<211> 29
<212> DNA
<213>Artificial sequence
<220>
<223>The rice genome sequence design provided according to rice genome data base and synthetic, as primer
<400> 4
caggtacc gtt ctt gat ctt ctt ctt ggg
Claims (3)
1. a paddy disease-resistant controlling gene, is named as OsUEP3, its nucleotide sequence such as SEQ ID：Shown in 1, the gene is opened
The long 390bp of reading frame is put, a kind of ubiquitin extension protein is encoded, is made up of 129 aminoacid, its sequence such as SEQ ID：Shown in 2,
The albumen n end is the ubiquitin molecule of 76 aminoacid, and C-terminal is then the 60S ribosomal protein L 40e of 53 aminoacid.
2. a kind of paddy disease-resistant controlling gene according to claim 1 is in the transgenic water by formulating overexpression OsUEP3
Rice is obtaining the application in broad-spectrum disease resistance material, it is characterised in that pure in the transgenic paddy rice by formulating overexpression OsUEP3
Syzygy is obtaining the application in the broad-spectrum disease resistance material for rice leaf spot bacteria different strains.
3. application according to claim 2, it is characterised in that realized by following steps：
(1) structure of OsUEP3 genes overexpression structure and acquisition
OsUEP3 gene ORF are cloned into into a plant expression vector so as to expression is ordered about by strong promoter；
(2) convert the acquisition of the Agrobacterium of OsUEP3 gene overexpression structures
The OsUEP3 gene overexpression structures for building had into the agriculture of strong infection ability by the conversion of the methods such as electric shock to Oryza sativa L.
Bacillus strain；
(3) the transgenic paddy rice initiative and acquisition of overexpression OsUEP3
By agrobacterium-mediated transformation by OsUEP3 gene overexpression structure Introduced into Rice, acquisition turns OsUEP3 gene overexpression structures
Oryza sativa L.；
(4) acquisition of the transgenic paddy rice homozygous line of overexpression OsUEP3
Respectively with antibiotic resistance and OsUEP3 gene expressions as Testing index, the trait segregation feelings of transfer-gen plant offspring are detected
Condition, obtain characters of progenies it is no longer detached, and can be stable hereditary overexpression OsUEP3 transgenic paddy rice homozygous line；
(5) the transgenic paddy rice homozygous line Screening and Identification of the overexpression OsUEP3 of anti-bacterial disease and acquisition
Transgenic paddy rice homozygous line with overexpression OsUEP3 tests and analyzes the resistance to all kinds of Oryza sativa L. bacteriosises as material,
Obtain anti-bacterial disease turns OsUEP3 trans-genetic hybrid rice.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116064648A (en) * | 2022-11-04 | 2023-05-05 | 东北师范大学 | Application of OsGA1 protein or biological material related to OsGA1 protein in regulation of plant rice blast resistance |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110167514A1 (en) * | 2007-07-05 | 2011-07-07 | Ceres, Inc. | Nucleotide sequences and corresponding polypeptides conferring modulated plant characteristics |
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2017
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110167514A1 (en) * | 2007-07-05 | 2011-07-07 | Ceres, Inc. | Nucleotide sequences and corresponding polypeptides conferring modulated plant characteristics |
Non-Patent Citations (2)
| Title |
|---|
| XM_015774309.2: "PREDICTED: Oryza sativa Japonica Group ubiquitin-60S ribosomal protein L40-2-like (LOC4332169), mRNA", 《GENBANK数据库》 * |
| 付坚等: "云南疣粒野生稻抗白叶枯病相关基因分析", 《2015年中国作物学会学术年会论文摘要集》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116064648A (en) * | 2022-11-04 | 2023-05-05 | 东北师范大学 | Application of OsGA1 protein or biological material related to OsGA1 protein in regulation of plant rice blast resistance |
| CN116064648B (en) * | 2022-11-04 | 2024-02-02 | 东北师范大学 | Application of OsGA1 protein or biological material related to OsGA1 protein in regulation of plant rice blast resistance |
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