CN106701783A

CN106701783A - Rice gene OsDF1 and application of disease control functions

Info

Publication number: CN106701783A
Application number: CN201710050197.3A
Authority: CN
Inventors: 蔡新忠; 徐幼平; 郭伟毅
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2017-01-23
Filing date: 2017-01-23
Publication date: 2017-05-24
Anticipated expiration: 2037-01-23
Also published as: CN106701783B

Abstract

The invention provides a rice disease control gene named as OsDF1. An open reading frame of the gene is 1512bp long, and the encoded protein is composed of 503 amino acids. The protein does not comprise any structural domain of known functions. By constructing overexpression transgenic rice of the gene and disease resistance analysis thereof, a broad spectrum control effect of the gene on the bacterial disease resistance of the rice is illustrated first. The overexpression transgenic rice plants widely reduce the resistance of bacterial leaf streak and bacterial leaf blight of rice, and the broad spectrum negative control function of the gene on the bacterial disease resistance of the rice is disclosed. The invention first discloses biological functions of the gene, and provides application of acquiring rice materials with general decrease of bacterial disease resistance by creating OsDF1 overexpression rice and application of acquiring broad spectrum bacterial disease resistant rice materials by creating OsDF1-RNAi rice. The gene is a novel gene resource applied to creating and breeding novel materials and novel varieties of rice with broad spectrum bacterial disease resistance.

Description

The application of paddy gene OsDF1 and disease-resistant adjusting function

Technical field

The invention belongs to biological technical field, it is related to the application of a paddy gene OsDF1 and its disease-resistant adjusting function.

Background technology

1st, Gene Clone in Plant

Methods of plant gene cloning has many kinds, as the plant species that genome sequence determination is completed increase, 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 transcriptase-PCR (RT-PCR), PCR primer With the connection of carrier, the Bacterial Transformation of connection product, plasmid extraction, digestion 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 Analyze phenotype (Phenotype) or Function situation in the case of unconventional expression and the normal expression of gene judge with it is bright Really.The unconventional expression of gene includes the expression higher than normal level and the major class of expression two less than normal level.Higher than normal The expression of level is mainly overexpression/overexpression (Over-expression), is mainly driven by connecting a strong promoter The mode of destination gene expression reaches.Less than normal level expression mainly by RNA disturb (RNA interfering, RNAi), the mode such as gene knockout (Knock-out) reaches.RNAi contains same sequence fragment by building and converting one Rightabout inserts the hairpin structure of an introne or other sequences do not expressed formation to realize, result is genes of interest table The reduction for reaching.Gene knockout (Knock-out) is then by inserting a non-plant long in the genes of interest in Plant Genome Thing sequence or excision genes of interest etc. mode reach, and result is that the nearly fully complete of destination gene expression suppresses.Structure Build gene overexpression plant and/or RNAi plant, knock out mutants body, compare the phenotype and proterties of these plant with it is wild The difference of type/normal plant, the regulatory function of the gene pairs proterties that can just have a definite purpose.

3rd, plant transgenic technology

For foreign gene to be imported into purpose plant, so as to obtain the genetically modified plants 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 genetic transformation and the detection and analysis of expression.

4th, disease resistance of plant detection and analysis technology

The phytopathy original includes fungi, oomycetes, bacterium, bacterium substance, virus and polytype etc. nematode.Different type cause of disease The inoculation method of thing is different.For fungi, can produce it is conidial, generally with conidial suspension spray inoculation plant Thing.Do not produce it is conidial, then with inoculated plants such as mycelia blocks.For oomycetes, typically first culture produces a large amount of sporangiums, and Low- temperature culture is allowed to discharge zoospore afterwards, finally with zoospore suspension inoculated plant.For bacterium, often suspended with thalline The method inoculated plant such as liquid leaf-cutting, acupuncture, injection, spraying.For virus, often produced with the virion for purifying or artificial in-vitro transcription Thing is rubbed, injected or by the communication media inoculated plant such as insect.For bacterium substance, it is inoculated with or transfers usually through vector Inoculation.For nematode, often with inoculated plant in a number of second instar larvae contact plant root basal part of stem or rhizosphere soil. In most cases, need to keep relative humidity higher, suitable temperature and illumination condition after inoculation.Different time is pressed after inoculation There is symptom, the statistics incidence of disease and morbidity severity in point Continuous Observation disease, calculate disease index, and cause of disease is detected using RT-PCR Thing biomass.By the comparative analysis 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 the major class of genetic engineering breeding for disease resistance two.Traditional breeding for disease resistance because Having its natural genetic isolation phenomenon is significantly limited Resistant germplasm usable range, can only apply the nearer disease-resistant money of genetic affinity Source, and repeatedly hybridization and backcrossing etc. are needed, therefore the seed selection cycle is long, and need a large amount of manpower and materials.And genetic engineering breeding Method is it is obtained originally by by the disease-resistant controlling gene of external source The disease resistance not having.Therefore, genetic engineering breeding method has broken the limitation of its natural genetic isolation phenomenon, has widened disease-resistant money Source usable range, and with operate relatively easy convenience, cultivation period it is short, without largely artificial material resources the characteristics of.In addition, can By importing a broad-spectrum disease resistance controlling gene, or multiple different disease-resistant spectrums gene, 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 to paddy bacterial Disease Resistance it is an object of the invention to provide a paddy rice (Oryza sativa) gene Regulating and controlling effect and thereby determine that name OsDF1 to the gene.The nucleotide sequence of the OsDF1 genes of present invention clone is such as SEQ ID：Shown in 1, ORFs (ORF) 1512bp long of the gene, the albumen of coding is made up of 503 amino acid, its sequence Row such as SEQ ID：Shown in 2.Domain of the gene encoding production not comprising any known function.The present invention clone sequence with The LOC_Os07g01060.3 of the genome annotation of rice varieties Nipponbare has similitude very high, only 5 differences of base： The G148 of LOC_Os07g01060.3 sequences replaces with A；T407 replaces with G；T427 replaces with C；A1171 replaces with C；G1309 Replace with T.These point mutation cause amino acid to produce corresponding change：The V50 of LOC_Os07g01060.3 sequences replaces with I； V136 replaces with G；C143 replaces with R；T391 replaces with P；V437 replaces with F.Before making the present invention, the gene does not have any name Claim, also any open report not on its function.First passage of the present invention builds the overexpression transgenic paddy rice of the gene And its disease-resistant analysis, illustrate the regulating and controlling effect of the gene pairs paddy bacterial Disease Resistance.Result shows, overexpresses transgenosis Rice plant is reduced to bacterial leaf streak of rice (Xanthomonas oryzae pv.oryzicola, Xoc) and water extensively The resistance of rice bacterial leaf spot pathogenic bacteria (Xanthomonas oryzae pv.oryzae, Xoo).Present invention is disclosed the gene pairs paddy rice The wide spectrum negative regulation function (being specifically shown in the explanation in embodiment 1) of bacteriosis resistance.Therefore, the unnamed gene is by the present invention Oryza sativa defense-related 1 (abbreviation OsDF1).

Obtain anti-by formulating transgenic paddy rice it is a further object to provide described rice Os DF1 genes Application in the rice material that characteristic of disease is changed, including (1) is in the transgenic paddy rice homozygous line by formulating overexpression OsDF1 To obtain the application (being specifically shown in the explanation in embodiment 1) in rice material of the reduction to paddy bacterial Disease Resistance；And (2) increase to paddy bacterial disease resistance of wide spectrum being obtained by formulating the transgenic paddy rice homozygous line of OsDF1-RNAi Application (being specifically shown in the explanation in embodiment 2) in rice material.

1. rice Os DF1 genes are obtaining reduction to water by formulating the transgenic paddy rice homozygous line of overexpression OsDF1 Application in the rice material of rice bacterial disease resistance.Realized by following steps：

(1) structure of OsDF1 genes overexpression structure and acquisition

OsDF1 gene opens reading frame (ORF) are cloned into a plant expression vector, it is ordered about by strong promoter Expression.

(2) acquisition of the Agrobacterium of conversion OsDF1 genes overexpression structure

The OsDF1 genes overexpression structure that will be built is converted by methods such as electric shocks has strong infection ability to paddy rice Agrobacterium strains.

(3) the transgenic paddy rice initiative and acquisition of overexpression OsDF1

OsDF1 genes are overexpressed by structure Introduced into Rice by agrobacterium-mediated transformation, acquisition turns OsDF1 genes overexpression knot The paddy rice of structure.

(4) acquisition of the transgenic paddy rice homozygous line of overexpression OsDF1

Respectively with antibiotic resistance and OsDF1 gene expressions as Testing index, the proterties point of transfer-gen plant offspring is detected From situation, the transgenic paddy rice homozygous line of overexpression OsDF1 that characters of progenies no longer separate and that heredity can be stablized is obtained.

(5) the transgenic paddy rice homozygous line Screening and Identification of the overexpression OsDF1 declined to bacteriosis resistance and acquisition

It is material with the transgenic paddy rice homozygous line for overexpressing OsDF1, detection and analysis resists to all kinds of paddy rice bacteriosises Property, obtain the decline of anti-bacterial disease ability turns OsDF1 trans-genetic hybrid rice.

2. rice Os DF1 genes increase to paddy rice by formulating the transgenic paddy rice homozygous line of OsDF1-RNAi to obtain Application in the rice material of bacterial disease resistance of wide spectrum.Realized by following steps：

(1) structure of OsDF1 gene RNAis structure and acquisition

One specific sequence fragment of OsDF1 genes is cloned into medial expression vector pENTR, then by recombination structure PENTR-OsDF1 carries out LR enzyme reactions with RNAi carrier (pANDA carriers), obtains RNAi structures pANDA-OsDF1.

(2) acquisition of the Agrobacterium of conversion OsDF1 gene RNAi structures

OsDF1 gene RNAis structure (pANDA-OsDF1) are converted to have paddy rice by methods such as electric shocks and infects energy by force The agrobacterium strains of power.

(3) initiative and acquisition of OsDF1 gene RNAi structure paddy rice are turned

By agrobacterium-mediated transformation by OsDF1 gene RNAi structure Introduced into Rice, acquisition turns OsDF1 gene RNAi structures Paddy rice.

(4) acquisition of OsDF1 gene RNAi structure paddy rice homozygous lines is turned

Respectively with antibiotic resistance and OsDF1 gene expressions as Testing index, the proterties point of transfer-gen plant offspring is detected From situation, the paddy rice homozygous line for turning OsDF1 gene RNAi structures that characters of progenies no longer separate and that heredity can be stablized is obtained.

(5) Screening and Identification for turning OsDF1 gene RNAi structure paddy rice homozygous lines of anti-bacterial disease and acquisition

It is material with the paddy rice homozygous line for turning OsDF1 gene RNAis structure (pANDA-OsDF1), detection and analysis is to all kinds of thin The resistance of disease caused by bacterium pathogen, obtains the OsDF1 gene RNAi paddy rice of anti-bacterial disease.

Advantages of the present invention：(1) the OsDF1 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 disease-resistant spectrum is wide.Paddy rice is to Xanthomonas campestris PV.oryzicola (Xoc) and the white leaf of paddy rice The resistance mechanism of rot bacterium (Xoo) has notable difference, therefore, while the genetic resources of simultaneous anti-Xoc and Xoo is little.In addition, paddy rice Disease-resistant gene (Resistance gene) to the resistance of Xoc also without dominant main effect is found and identifies, still lacks high at present Anti- resistant variety and resource.Function is totally unknown before this for the OsDF1 genes that the present invention is provided, and present invention research finds the gene Have concurrently to the effect of the negative regulation of Xoc and Xoo, can be formulated by building OsDF1-RNAi paddy rice and acquisition have concurrently it is wide to Xoc and Xoo Compose the new material of resistance.Therefore, OsDF1 genes are a kind of Rice New Materials suitable for formulating with seed selection broad spectrum antibiotic disease With the brand-new genetic resources of new varieties.(2) disease-resistant material periodicities are obtained short.The method for obtaining disease-resistant plants material and kind is main There is conventional traditional breeding way and the genetic engineering breeding method using disease-resistant controlling gene.Traditional breeding way has available Resistant germplasm scope is limited by its natural genetic isolation, and the seed selection cycle is long, it is necessary to the shortcomings of a large amount of artificial material resources.And genetic engineering is educated Kind of method then has that available Resistant germplasm scope is wide, operate relatively easy convenience, cultivation period it is short, without largely artificial material resources, The advantages of particularly suitable cultivation wide spectrum, lasting, resistance high.The present invention utilizes disease-resistant controlling gene OsDF1, using gene The rice material of strong anti-bacterial disease is cultivated in engineering method, initiative, with cycle is short, the features such as seed selection is quick.

Brief description of the drawings

Fig. 1 is after OsDF1 overexpresses paddy rice homozygous line and its parent's rice leaf Inoculated Rice Xanthomonas campestris PV.oryzicola Symptom and bacterium amount detection and analysis results contrast figure.OsDF1 overexpresses paddy rice homozygous line 69-1 and its parent's rice leaf uses pin Cylinder infusion method Inoculated Rice Xanthomonas campestris PV.oryzicola (Xoc) bacterial strain oxy04 and ROS105.It is left：Disease of the inoculation Xoc bacterial strains after 10 days Shape；It is right：Xoc bacterium amount testing results in inoculation rear blade.Result shows, compared with parent, OsDF1 overexpression paddy rice homozygous lines 69-1 forms longer scab, and bacterium amount is also dramatically increased.Show that the overexpression of OsDF1 genes is reduced to paddy bacterial streak The resistance of disease, discloses OsDF1 and plays negative regulation to bacterial leaf streak of rice resistance.

Fig. 2 is the symptom after OsDF1 overexpresses paddy rice homozygous line and its parent's rice leaf Inoculated Rice leaf spot bacteria Results contrast figure is tested and analyzed with bacterium amount.OsDF1 overexpresses paddy rice homozygous line 69-1 and its parent's rice leaf uses leaf-cutting method Inoculated Rice leaf spot bacteria (Xoo) bacterial strain PXO99.On：Symptom of the inoculation Xoo bacterial strains after 14 days；Under：In inoculation rear blade Xoo bacterium amount testing results.Result shows, compared with parent, OsDF1 overexpressions paddy rice homozygous line 69-1 forms longer withered disease Spot, bacterium amount is also dramatically increased.Show that the overexpression of OsDF1 genes reduces the resistance to bacterial blight of rice, disclose OsDF1 pairs Bacterial Blight Resistance in Rice plays negative regulation.

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, and first passage builds overexpression transgenic paddy rice, illustrates the gene Broad spectrum antibiotic adjusting function, disclosing the gene pairs paddy rice bacteriosis resistance has wide spectrum negative regulation function.Therefore, this hair Bright is Oryza sativa defense-related 1 (abbreviation OsDF1) by the unnamed gene.Because the super table of OsDF1 genes Up to causing paddy rice to be remarkably decreased bacteriosis resistance, therefore, it can the overexpression paddy rice homozygosis by building OsDF1 genes It is the Rice New Material to create acquisition to the decline of paddy rice bacteriosis resistance, for purposes such as gene function analysis.OsDF1 bases The clone of cause and functional analysis, the Rice New Material initiative declined to paddy rice bacteriosis resistance and the key step bag for obtaining Include：

1) clone of rice Os DF1 genes and preservation

The rice Os DF1 genes that the present invention is provided are cloned by following steps and obtained.First according to rice genome database Middle OsDF1 sequences Designs primer OsDF1-F (5 '-caggcgcgcc atg tgc att atc ttt gct ggt-3 ', tiltedly Body portion is the restriction enzyme sites of Asc I) (sequence such as SEQ ID：Shown in 3), and OsDF1-R2 (5 '-caggtacc cat gct Ctg aaa atg ctg ctg-3 ', italicized item is the restriction enzyme sites of Kpn I) (sequence such as SEQ ID：Shown in 4).Using TRIZOL Reagent extracts rice leaf total serum IgE, OsDF1cDNA is obtained using RT-PCR method amplification, by after 1% agarose gel electrophoresis PCR primer is reclaimed in rubber tapping purifying, connects pMD19-mT carriers, and thermal shock conversion bacillus coli DH 5 alpha shakes bacterium training in LB culture mediums Support overnight, extract plasmid, use the digestion methods of I/Kpn of Asc I and the PCR method with OsDF1-F/OsDF1-R2 as primer pair Whether the plasmid that inspection institute extracts respectively includes OsDF1 genes, company's sequence verification is finally sent, so that successful clone and acquisition OsDF1 gene cDNA full length sequences.OsDF1 nucleotide sequences such as SEQ ID：Shown in 1, the ORFs (ORF) of the gene is long 1512bp, the albumen of coding is made up of 503 amino acid, its sequence such as SEQ ID：Shown in 2.The gene encoding production does not include The domain of any known function.Found by BLASTn analyses, the OsDF1 trans-genetic hybrid rice kind Japan that present invention clone obtains The LOC_Os07g01060.3 of fine genome annotation has similitude very high, only 5 differences of base：LOC_ The G148 of Os07g01060.3 sequences replaces with A；T407 replaces with G；T427 replaces with C；A1171 replaces with C；G1309 is replaced It is T.These point mutation cause amino acid to produce corresponding change：The V50 of LOC_Os07g01060.3 sequences replaces with I；V136 Replace with G；C143 replaces with R；T391 replaces with P；V437 replaces with F.Before making the present invention, the gene function does not have any public affairs Open report.

The Escherichia coli of the carrier for carrying OsDF1 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 digestion, OsDF1 genes are subcloned into purpose carrier, be used for Transgenosis etc. is studied.

2) structure of OsDF1 genes overexpression structure and acquisition

Kpn I are first carried out respectively to above-mentioned 1) the middle pMD19-OsDF1 plasmids for obtaining and plant overexpression vector pCZD Digestion, Asc I digestions then are carried out, the genes of interest after digestion is attached with the pCZD carriers after same digestion, is connected Product carries out the steps such as Escherichia coli conversion, ammonia benzyl Antibiotic medium plate screening, digestion and PCR identification and obtains OsDF1 bases Because of overexpression structure pCZD-OsDF1.Overexpression vector pCZD drives the expression of genes of interest with corn Ubq promoters, while taking Band HA labels, are easy to Molecular Identification and gene functional research.

3) acquisition of the Agrobacterium of conversion OsDF1 genes overexpression structure pCZD-OsDF1

OsDF1 genes are overexpressed structure pCZD-OsDF1 and converted by methods such as electric shocks has strong infecting potential to paddy rice Agrobacterium strains, such as EHAl05, screen transformant on the YEP culture mediums containing streptomysin and kanamycins, then by Asc I+ Kpn I double digestions and PCR are identified, are obtained and carry the Agrobacterium that OsDF1 genes overexpress structure pCZD-OsDF1.For next step The genetic transformation of paddy rice.

4) initiative and acquisition that OsDF1 genes overexpress structure pCZD-OsDF1 paddy rice are turned

OsDF1 genes are overexpressed by structure pCZD-OsDF1 Introduced into Rice by agrobacterium-mediated transformation.It is divided into rice callus The induction of tissue, the Agrobacterium infection Rice Callus, the regeneration and identification of hygromycin resistance plant that carry pANDA-OsDF1 Etc. several big steps, the paddy rice T for turning pCZD-OsDF1 of regeneration is finally obtained₀Generation.Concrete operation step is as follows：

The induction of (i) Rice Callus

Plant the sterilization and cleaning of embryo surface：Ripe intact Nipponbare rice paddy seed is taken, kind of a skin is peelled off, complete kind is left Embryo.With 70% alcohol solution dipping embryo 1min, embryo is soaked with 50% liquor natrii hypochloritis after outwelling ethanol, be placed on shaking table 28 DEG C, 180rpm sterilizations 40min.Liquor natrii hypochloritis is outwelled, with the ddH of sterilizing₂O washings embryo at least 10 times, until liquid It is clean it is transparent untill.After air-drying embryo with the filter paper of sterilizing, carefully embryo is put on NB1 solid mediums with tweezers, each About 30 seeds of culture dish.Sealed with preservative film afterwards, be placed in 28 DEG C of light cultures in incubator.

The induction of callus：Embryo is placed on after being cultivated 15 days on NB1, the callus that will be differentiated is transferred to fresh NB2 culture mediums on, be further cultured under 28 DEG C of dark conditions 10 days.The bright orange callus of health is transferred on NB3 and is cultivated About 10 days.Note, in order to provide sufficient nutrition, a NB2 plate changes into 2 NB3 plates and cultivated.

The squamous subculture of callus：The callus of health is shifted on NB4 from NB3, is trained under 28 DEG C of dark conditions Support 9 days, callus now is best suitable for Agrobacterium infection.

(ii) the Agrobacterium infection of Rice Callus

The culture of Agrobacterium：The Agrobacterium tumefaciems EHA105 bacterium of OsDF1 gene RNAi structures pANDA-OsDF1 will be carried Strain is taken out from -80 DEG C, in the flat lining outs of LB containing streptomysin and kanamycins, is placed in 28 DEG C of insulating boxs and is cultivated (about 2d), picking single bacterium colony after bacterium colony is grown, is inoculated in respectively in the 50mL YEP nutrient solutions containing corresponding antibiotic, 27 DEG C of constant temperature shake Bed (180rpm) culture about 48h to OD₆₀₀It is 0.5-0.8, is transferred in sterile centrifugation tube, 5000 × g centrifugations under room temperature condition 10min, removes supernatant, and thalline 25mL AAM-AS nutrient solutions suspend after cleaning again, then one is centrifuged at identical conditions It is secondary, finally with about 50mL AAM-AS nutrient solutions again suspension thalline to OD₆₀₀It is 0.05-0.1 (about 10⁹Individual cell/mL), it is placed in Can be used for the conversion of paddy rice on ice.

Agrobacterium is infected：The callus of growth selection health care belt glassy yellow, is carefully blown into aseptic small with spoon In culture dish, then resuspended good agrobacterium liquid is poured into culture dish, bacterium solution is submerged all callus as far as possible, keep leaching Bubble about 30min, period jiggles every now and then.Bacterium solution is suctioned out as much as possible with the glue head dropper of sterilizing afterwards.

Co-culture：The callus that will be infected is put into 22 DEG C of dark culturing 3d on NB-AS culture mediums.

Carry disease germs cleaning of the callus before resistance screening：After co-culturing 3d, the callus for carrying disease germs is concentrated and is put into In 500mL aseptic bottles, callus is cleaned 8~10 times 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, and 30min is shaken under 180rpm, 27 DEG C of constant temperature, Then eluate is suctioned out with the glue head dropper of sterilizing, the callus that will finally clean up is transferred to NB-CHA culture mediums, at 28 DEG C It is used for resistance screening after the lower culture 14d of dark.

(iii) regeneration of hygromycin resistance plant

Resistance screening culture：It is to be transferred to containing antibiotic (Cef, 300mg/mL by callus point；Hyg, 50mg/mL) On NB-CH1 screening and culturing mediums, cultivated under 28 DEG C of dark, carry out antibiotic resistance screening；

Squamous subculture：The Hygromycin resistant calli that will be regenerated through resistance screening on NB-CH1, is transferred in the way of being Containing antibiotic (Cef, 300mg/mL；Hyg, 50mg/mL) NB-CH2 screening and culturing mediums on, the squamous subculture one under 28 DEG C of dark It is secondary, incubation time about 1 week.

The pre- differentiation culture of resistant calli：Callus with hygromycin is transferred to containing antibiosis in the way of being On element (Hyg, 50mg/mL) pre- differential medium Pre-H, 15d is cultivated under 28 DEG C of dark.

The dedifferentiation culture of resistant calli：Sufficient kanamycin-resistant callus tissue will in advance be broken up to be transferred to containing antibiotic in the way of being On (Hyg, 50mg/mL) redifferential medium R-50, cultivated under 27 DEG C of optical conditions about 3 weeks.

Culture of rootage：Treat that Hygromycin resistant calli differentiates rootlet and green budlet, and rootlet grows to 1cm with enterprising Row culture of rootage.The resistant budses of greening are chosen from differential medium with tweezers, is transferred to and is contained antibiotic (Hyg, 50mg/ ML culture of rootage is carried out on 1/2MS root medias).

Soil is planted：Deng hygromycin resistance seedling grow to root system it is complete after, by T₀Uncapped 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 carries out Routine Management in greenhouse, finally harvested and obtain T₀Seed.

5) Screening and Identification and the acquisition of the paddy rice homozygous line of OsDF1 genes overexpression structure pCZD-OsDF1 are turned

Respectively with hygromycin resistance and OsDF1 gene expressions as Testing index, the proterties point of transfer-gen plant offspring is detected From situation.Hygromycin resistance is screened to carry out for rice paddy seed on the flat board containing hygromycin and carried out, and can observation in hygromycin Normal growth is into healthy seedling in resistant panel.Gene expression is detected using methods such as real-time fluorescence quantitative PCRs.After acquisition The paddy rice homozygous line for turning OsDF1 genes overexpression structure pCZD-OsDF1 no longer separate for proterties and that heredity can be stablized.This A little paddy rice homozygous lines on hygromycin resistance flat board all can normal growth show into healthy seedling and OsDF1 gene expression doses Write higher than the control for turning empty carrier.

6) the disease resistance detection and analysis of OsDF1 genes overexpression paddy rice homozygous line

It is material with 5) the middle OsDF1 genes overexpression paddy rice homozygous line for obtaining, tests and analyzes it to paddy rice bacteria pathogeny The resistance of disease caused by thing, so that the regulating and controlling effect of clear and definite OsDF1 gene pairs paddy rice bacteriosis resistance, is to be created using the gene Anti-bacterial disease paddy rice is built and obtains to lay the foundation.

The pathogen of detection and analysis includes：Xanthomonas oryzae pv. oryzicola (Xanthomonas oryzae Pv.oryzicola, Xoc) bacterial strain oxy04 and ROS105, rice leaf spot bacteria (Xanthomonas oryzae Pv.oryzae, Xoo) bacterial strain PXO99.For Xoc, it is inoculated with using infusion method.First shake bacterium and be made concentration for OD₆₀₀=1.0 Xoc Bacterial suspension, then rice leaf is infiltrated with aseptic one-shot injector.For Xoo, using leaf-cutting method inoculation.First shake bacterium be made it is dense It is OD to spend₆₀₀=1.0 Xoo bacterial suspensions, then bacterium solution is dipped with sterile scissors, in the leaf-cutting away from blade tip 3cm, it is placed in big modeling Overlay film moisturizing 48h in charging basket.

Inoculation result shows that parent control rice leaf divides 10 days after infiltration inoculation Xoc bacterial strains oxy04 and ROS105 Not Xing Cheng the water stain shape scab that is extended along vascular bundle up to 10.1mm and 6.7mm of length, bacterial ooze is also formed above.OsDF1 genes After infiltration is inoculated with the two Xoc bacterial strains, the water stain shape scab of formation is averagely up to overexpression paddy rice homozygous line 69-1 blades 28.8mm and 9.7mm, is 2.9 times and 1.5 times of control.And the bacterial ooze of the 69-1 blades formation of inoculation oxy04 bacterial strains is notable More than control blade (Fig. 1 is left).Bacterium amount detection and analysis result show, parent control rice leaf inoculation Xoc bacterial strains oxy04 with The diseased region bacterium amount of ROS105 is respectively 9.42 × 10⁸Cfu/ leaves and 2.80 × 10⁸Cfu/ leaves.And OsDF1 overexpression paddy rice is pure The bacterium amount mean height of syzygy 69-1 blades is up to 2.95 × 10⁹Cfu/ leaves and 5.13 × 10⁸Cfu/ leaves, respectively parent control 3.13 times and 1.83 times (Fig. 1 is right).Similar analysis result to rice leaf spot bacteria Xoo bacterial strains PXO99 shows, parent control Rice leaf forms length up to the withered scab of 16.2cm 14 days after leaf-cutting inoculation, is mainly extended along blade both sides.And OsDF1 genes overexpression paddy rice homozygous line 69-1 blades then form the average withered scab up to 23.6cm, and length is control 1.5 times, and withered scab is that whole leaf extends forward (on Fig. 2).Bacterium amount tests and analyzes result and shows, parent control Rice Leaf The diseased region bacterium amount of piece is respectively 1.24 × 10⁹Cfu/ leaves.And the bacterium amount of OsDF1 overexpression paddy rice homozygous line 69-1 blades is put down Up to 2.65 × 10⁹Cfu/ leaves, are 2.14 times (under Fig. 2) of parent control.

These results indicate that OsDF1 overexpresses paddy rice homozygous line 69-1 to Xoc bacterial strains oxy04 and ROS105, Xoo bacterial strain The resistance of PXO99 is substantially less than parent control.The overexpression of OsDF1 genes cause paddy rice to these bacterial strain resistances it is notable under Drop, therefore, OsDF1 plays negative regulation to paddy rice bacterial leaf streak and bacterial leaf-blight.Can be by building OsDF1- RNAi paddy rice homozygous line obtains the Rice New Material and new varieties of anti-bacterial leaf streak of rice high to create.

Embodiment 2

The antibacterium negative regulation function of the paddy gene OsDF1 illustrated according to the present invention as described above, establishes a set of Using build the gene RNAi transgenic paddy rices, using technique for gene engineering formulate and obtain wide spectrum water resistant rice bacterial disease The technical system of Rice New Material.Key step includes：

(1) structure of OsDF1 gene RNAis structure and acquisition

According to OsDF1 primers OsDF1-F2 (the 5 '-ttggatcc gaa tct ttg gtt of present invention clone Gag aga agc-3 ', italicized item is Bam HI restriction enzyme sites) (sequence such as SEQ ID：Shown in 5), and OsDF1-R3 (5 '- Ttctcgag cat gct ctg aaa atg ctg ctg-3 ', italicized item is XhoI restriction enzyme sites) (sequence such as SEQ ID： Shown in 6).With 1) the middle pMD19-OsDF1 plasmids for obtaining in embodiment 1 as template, OsDF1-F2/OsDF1-R3 is primer It is right, the OsDF1 fragments for obtaining a length of 300bp are expanded by PCR, then by Bam HI/XhoI double digestions, connection, conversion, card The middle table that the step acquisitions such as the screening of that mycin culture medium flat plate, digestion and PCR identifications have been subcloned 300bp OsDF1 fragments reaches Carrier pENTR (pENTR-OsDF1).Again by recombination structure pENTR-OsDF1 and RNAi carrier (with paddy rice ubi promoters, The pANDA carriers of GUS Linker sequences and T-NOS terminators) carry out LR enzyme reactions overnight, plus 37 DEG C of reactions after protein kinase K 10min uses the Screening of Media positive colony containing kanamycins, so with abundant terminating reaction after product conversion Escherichia coli Identify whether positive and negative two-way inserted at the GUS Linker two ends of pANDA carriers with SacI+KpnI double digestions and PCR afterwards The OsDF1 fragments of 300bp, so as to obtain RNAi structures pANDA-OsDF1.

(2) acquisition of the Agrobacterium of conversion OsDF1 gene RNAi structures

OsDF1 gene RNAis structure (pANDA-OsDF1) are converted to have paddy rice by methods such as electric shocks and infects energy by force The agrobacterium strains of power, such as EHAl05, screen transformant, then pass through on the YEP culture mediums containing streptomysin and kanamycins SacI+KpnI double digestions and PCR identify that acquisition carries the Agrobacterium of OsDF1 gene RNAi structures pANDA-OsDF1.For under The genetic transformation of one step paddy rice.

By agrobacterium-mediated transformation by OsDF1 gene RNAi structure pANDA-OsDF1 Introduced into Rice.Key step includes water The induction of rice callus, Agrobacterium infection Rice Callus, the hygromycin resistance plant of carrying pANDA-OsASR2 are again Raw and identification etc., finally obtains the paddy rice T for turning pANDA-OsDF1 of regeneration₀Generation.Concrete operation step with embodiment 1 4) In it is as described above.

Respectively with antibiotic resistance and OsDF1 gene expressions as Testing index, the proterties point of transfer-gen plant offspring is detected From situation.Hygromycin resistance is screened to carry out for rice paddy seed on the flat board containing hygromycin and carried out, and can observation in hygromycin Normal growth is into healthy seedling in resistant panel.Gene expression is detected using methods such as real-time fluorescence quantitative PCRs.After acquisition The paddy rice homozygous line for turning OsDF1 gene RNAi structures pANDA-OsDF1 no longer separate for proterties and that heredity can be stablized.This A little paddy rice homozygous lines on hygromycin resistance flat board all can normal growth show into healthy seedling and OsDF1 gene expression doses Write less than the control for turning empty carrier.

(5) Screening and Identification for turning OsDF1 gene RNAi structure paddy rice homozygous lines of broad spectrum antibiotic disease and acquisition

It is material with the paddy rice homozygous line for turning OsDF1 gene RNAis structure (pANDA-OsDF1), detection and analysis is to all kinds of thin The resistance of disease caused by bacterium pathogen, the pathogen of detection and analysis includes：Xanthomonas oryzae pv. oryzicola (Xanthomonas Oryzae pv.oryzicola, Xoc), rice leaf spot bacteria (Xanthomonas oryzae pv.oryzae, Xoo) etc.. Inoculation method and disease resistance evaluation are with embodiment 1) in 6) described in.

Analyzed by above-mentioned inoculation and disease-resistant evaluation, screening and acquisition show resistance to many bacterial strains of Xoc and Xoo , the OsDF1 gene RNAi paddy rice of broad spectrum antibiotic disease.

The present invention combines the result of Fig. 1 and Fig. 2, OsDF1 is disclosed first and plays wide spectrum to paddy bacterial Disease Resistance Negative regulation is acted on.

<110>Zhejiang University

<120>The application of paddy gene OsDF1 and disease-resistant adjusting function

<160> 6

<210> 1

<211> 1512

<212> DNA

<213>Paddy rice（Oryza sativa）

<220>

<221> CDS

<222> (1)…(1512)

<400> 1

atgtgcatta tctttgctgg tgatgatggt cattcggagc aactagctct cctgaacaat 60

gaccatgaag tttcagaagt ttgtgttgaa gaaatttcag ctgataatac aggacgatct 120

ttcctcatca ggatttcaga atctaaaatt ttttactatt ggtgtgctga gaagtcaaag 180

aaacacggga tggaccttct tgcaaagatg aaaaatctac tgcagggaag gccaacactc 240

tctgatctca caggcatctc agattcacga ctggatgctt ttgccactca tctacacgct 300

tatcttgttg catcaagtat tggagatgtt aaatcgctgg gatcacttaa tgacttcctg 360

ggcgcttcaa gcccacaaga tcaatattta cagccctcat cagttggttc caagtcttca 420

cgcttccgca cctctgcagc taatgcagca aaagcaagtt ctgtctacca aactagtctg 480

agtcccagat gtggtgcttt taaagatgga gtgccaagga tgtcgtgtgc aaagattgct 540

ggaagagaca agctgaagcg acgtggggac tggctgagct catcaactgg tcctgatgac 600

gcaaatcttt tgacaccgaa gattgttagc tctgattctg ccagtgagaa gtgtggtggg 660

gactgttctg aaaacagtgc taattcacct cctctagact tgcctctttc atttcccttg 720

ttgccatctc tgtttcctct tgcgactcaa tatccacttc ctaaggattc tacagagcaa 780

ccatttaagc cttactattg ctggtgccca ccatgcccat cttctctgca gtacagtgta 840

acccctctgc atatgccagt tacatctgta gaaccactgc ccttgccgcc tttgagttct 900

ctgctatcaa atgatcagcc gccgacttca acagtttctg caaagatgga tacaactgat 960

ctcccgtcac ttaatcttcc atcgatactg cgtgatcctt tacttcacct gccgcttcct 1020

acttcaccac ttgtatcctt gcatggttca caagttccaa cattcacacc attgatgtct 1080

gaccccattg tgcatgtccc ggtcattgat gtgtgttcct ctggccaggc ttacctggtg 1140

agttgcgggc cttctatgtc tgcgactgtt cccctgctcc ctagcttgaa gcccctgatt 1200

ccagaaacag aatctttggt tgagagaagc gctagggaaa cactaatgag gcttatagcc 1260

tcaacaccat ctgcaagtaa tccccagctg gtgaacattc tccctgcatt ccttactgac 1320

gtgcctgaaa tgaatgtgag gaaacatctt ggtgtccatc caggcgacag gctaagctca 1380

tcttgtagcg tcgatgtgat tggacctggg tttgccgtca cagaggatga tgcatctgtt 1440

ggagatgggg ctcacgcaac ttttgcggaa tatgatgata tcggcgatca gcagcatttt 1500

cagagcatgt ag 1512

<210> 2

<211> 503

<212> PRT

<213>Paddy rice（Oryza sativa）

<400> 2

Met Cys Ile Ile Phe Ala Gly Asp Asp Gly His Ser Glu Gln Leu Ala

1 5 10 15

Leu Leu Asn Asn Asp His Glu Val Ser Glu Val Cys Val Glu Glu Ile

20 25 30

Ser Ala Asp Asn Thr Gly Arg Ser Phe Leu Ile Arg Ile Ser Glu Ser

35 40 45

Lys Ile Phe Tyr Tyr Trp Cys Ala Glu Lys Ser Lys Lys His Gly Met

50 55 60

Asp Leu Leu Ala Lys Met Lys Asn Leu Leu Gln Gly Arg Pro Thr Leu

65 70 75 80

Ser Asp Leu Thr Gly Ile Ser Asp Ser Arg Leu Asp Ala Phe Ala Thr

85 90 95

His Leu His Ala Tyr Leu Val Ala Ser Ser Ile Gly Asp Val Lys Ser

100 105 110

Leu Gly Ser Leu Asn Asp Phe Leu Gly Ala Ser Ser Pro Gln Asp Gln

115 120 125

Tyr Leu Gln Pro Ser Ser Val Gly Ser Lys Ser Ser Arg Phe Arg Thr

130 135 140

Ser Ala Ala Asn Ala Ala Lys Ala Ser Ser Val Tyr Gln Thr Ser Leu

145 150 155 160

Ser Pro Arg Cys Gly Ala Phe Lys Asp Gly Val Pro Arg Met Ser Cys

165 170 175

Ala Lys Ile Ala Gly Arg Asp Lys Leu Lys Arg Arg Gly Asp Trp Leu

180 185 190

Ser Ser Ser Thr Gly Pro Asp Asp Ala Asn Leu Leu Thr Pro Lys Ile

195 200 205

Val Ser Ser Asp Ser Ala Ser Glu Lys Cys Gly Gly Asp Cys Ser Glu

210 215 220

Asn Ser Ala Asn Ser Pro Pro Leu Asp Leu Pro Leu Ser Phe Pro Leu

225 230 235 240

Leu Pro Ser Leu Phe Pro Leu Ala Thr Gln Tyr Pro Leu Pro Lys Asp

245 250 255

Ser Thr Glu Gln Pro Phe Lys Pro Tyr Tyr Cys Trp Cys Pro Pro Cys

260 265 270

Pro Ser Ser Leu Gln Tyr Ser Val Thr Pro Leu His Met Pro Val Thr

275 280 285

Ser Val Glu Pro Leu Pro Leu Pro Pro Leu Ser Ser Leu Leu Ser Asn

290 295 300

Asp Gln Pro Pro Thr Ser Thr Val Ser Ala Lys Met Asp Thr Thr Asp

305 310 315 320

Leu Pro Ser Leu Asn Leu Pro Ser Ile Leu Arg Asp Pro Leu Leu His

325 330 335

Leu Pro Leu Pro Thr Ser Pro Leu Val Ser Leu His Gly Ser Gln Val

340 345 350

Pro Thr Phe Thr Pro Leu Met Ser Asp Pro Ile Val His Val Pro Val

355 360 365

Ile Asp Val Cys Ser Ser Gly Gln Ala Tyr Leu Val Ser Cys Gly Pro

370 375 380

Ser Met Ser Ala Thr Val Pro Leu Leu Pro Ser Leu Lys Pro Leu Ile

385 390 395 400

Pro Glu Thr Glu Ser Leu Val Glu Arg Ser Ala Arg Glu Thr Leu Met

405 410 415

Arg Leu Ile Ala Ser Thr Pro Ser Ala Ser Asn Pro Gln Leu Val Asn

420 425 430

Ile Leu Pro Ala Phe Leu Thr Asp Val Pro Glu Met Asn Val Arg Lys

435 440 445

His Leu Gly Val His Pro Gly Asp Arg Leu Ser Ser Ser Cys Ser Val

450 455 460

Asp Val Ile Gly Pro Gly Phe Ala Val Thr Glu Asp Asp Ala Ser Val

465 470 475 480

Gly Asp Gly Ala His Ala Thr Phe Ala Glu Tyr Asp Asp Ile Gly Asp

485 490 495

Gln Gln His Phe Gln Ser Met

500

<210> 3

<211> 31

<212> DNA

<213>Artificial sequence

<220>

<223>The rice genome sequence provided according to rice genome database is designed and artificial synthesized, as primer

<400> 3

caggcgcgcc atg tgc att atc ttt gct ggt

<210> 4

<211> 29

<212> DNA

<213>Artificial sequence

<220>

<400> 4

caggtacc cat gct ctg aaa atg ctg ctg

<210> 5

<211> 29

<212> DNA

<213>Artificial sequence

<220>

<223>Rice genome sequence according to clone is designed and artificial synthesized, as primer

<400> 5

ttggatcc gaa tct ttg gtt gag aga agc

<210> 6

<211> 29

<212> DNA

<213>Artificial sequence

<220>

<400> 6

ttctcgag cat gct ctg aaa atg ctg ctg

Claims

1. a paddy disease-resistant controlling gene, is named as Oryza sativa defense-related 1, abbreviation OsDF1, its Nucleotide sequence such as SEQ ID：Shown in 1, the ORFs 1512bp long of the gene, the albumen of coding is by 503 amino acid groups Into its sequence such as SEQ ID：Shown in 2, domain of the gene encoding production not comprising any known function.

2. a kind of paddy disease-resistant controlling gene OsDF1 according to claim 1 is obtained by formulating transgenic paddy rice Application in the rice material that disease resistance is changed, it is characterised in that in the transgenic paddy rice by formulating overexpression OsDF1 Homozygous line obtains the application reduced in rice material to paddy bacterial Disease Resistance；And by formulating OsDF1- The transgenic paddy rice homozygous line of RNAi obtains the application in the rice material for increasing to paddy bacterial disease resistance of wide spectrum.

3. application according to claim 2, it is characterised in that realizing obtaining by following steps reduces to paddy bacterial Application in the rice material of Disease Resistance：

(1) structure of OsDF1 genes overexpression structure and acquisition

OsDF1 genes ORF is cloned into a plant expression vector, it is ordered about expression by strong promoter；

The OsDF1 genes overexpression structure that will be built is converted by electric-shocking method has the Agrobacterium of strong infection ability to paddy rice Bacterial strain；

OsDF1 genes are overexpressed by structure Introduced into Rice by agrobacterium-mediated transformation, acquisition turns OsDF1 genes overexpression structure Paddy rice；

Respectively with antibiotic resistance and OsDF1 gene expressions as Testing index, the trait segregation feelings of transfer-gen plant offspring are detected Condition, obtains the transgenic paddy rice homozygous line of overexpression OsDF1 that is that characters of progenies no longer separate and can stablizing heredity；

It is material with the transgenic paddy rice homozygous line for overexpressing OsDF1, detection and analysis is obtained to the resistance of all kinds of paddy rice bacteriosises Take the decline of anti-bacterial disease ability turns OsDF1 trans-genetic hybrid rice.

4. application according to claim 2, it is characterised in that realize that acquisition is increased to paddy bacterial by following steps Application in the rice material of disease resistance of wide spectrum：

(1) structure of OsDF1 gene RNAis structure and acquisition

One specific sequence fragment of OsDF1 genes is cloned into medial expression vector pENTR, then by recombination structure pENTR- OsDF1 carries out LR enzyme reactions with RNAi carrier (pANDA carriers), obtains RNAi structures pANDA-OsDF1；

(2) acquisition of the Agrobacterium of conversion OsDF1 gene RNAi structures

OsDF1 gene RNAis structure (pANDA-OsDF1) are converted by electric-shocking method has the agriculture of strong infection ability to paddy rice Bacillus strain；

By agrobacterium-mediated transformation by OsDF1 gene RNAi structure Introduced into Rice, acquisition turns the water of OsDF1 gene RNAi structures Rice；

Respectively with antibiotic resistance and OsDF1 gene expressions as Testing index, the trait segregation feelings of transfer-gen plant offspring are detected Condition, obtains the paddy rice homozygous line for turning OsDF1 gene RNAi structures that is that characters of progenies no longer separate and can stablizing heredity；

(5) Screening and Identification for turning OsDF1 gene RNAi structure paddy rice homozygous lines enhanced to bacteriosis resistance and acquisition

It is material with the paddy rice homozygous line for turning OsDF1 gene RNAis structure (pANDA-OsDF1), tests and analyzes to all kinds of bacterial diseases The resistance of disease caused by the original, obtains the enhanced OsDF1 gene RNAis paddy rice of anti-bacterial disease ability.