CN109652425A - The purposes of rice Os HIR3 gene and the method for obtaining disease resisting rice - Google Patents
The purposes of rice Os HIR3 gene and the method for obtaining disease resisting rice Download PDFInfo
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- CN109652425A CN109652425A CN201910016244.1A CN201910016244A CN109652425A CN 109652425 A CN109652425 A CN 109652425A CN 201910016244 A CN201910016244 A CN 201910016244A CN 109652425 A CN109652425 A CN 109652425A
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- C12N15/8281—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for bacterial resistance
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- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
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- C12N15/8283—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for virus resistance
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Abstract
本发明提供水稻OsHIR3基因在制备植物防御病毒或者细菌侵染危害上的用途以及制备方法,其中所述的方法包括：水稻OsHIR3基因构建到植物双元表达载体中，通过电击导入农杆菌菌株；通过水稻成熟胚诱导法获得过表达OsHIR3的转基因水稻。这样的植物具有基础抗病毒或者细菌病害的能力，主要是提高了植物体内SA的水平。
The present invention provides the use and preparation method of rice OsHIR3 gene in preparing plants to defend against virus or bacterial infection . Transgenic rice overexpressing OsHIR3 was obtained by induction of rice mature embryos. Such plants have basic ability to resist viral or bacterial diseases, mainly by increasing the level of SA in plants.
Description
Technical field
The present invention relates to gene engineering technology field and field of plant disease control more particularly to plant OsHIR3s genes
Application field in plant basal resistance.
Background technique
Rice stripe virus (Rice stripe virus, RSV) can cause stripe disease.Small brown rice planthopper
(Lapdelphax striatellus, SBPH) is the mediator that RSV is propagated, and rice stripe leaf caused by propagating through small brown rice planthopper is withered
Disease is virus disease important in China's Rice Production, causes great threat to agricultural production security.
Allergic reaction (HR) is that plant prevents pathogen from infecting a kind of defense mechanism of propagation, is mainly characterized by infecting week
The quick death for enclosing local cells closes injury site by the allergic reaction of induction part to resist the further of pathogen
It infects.Allergy induced reaction gene (HIR) family is considered as being closely related with HR, and it is anti-to the resistance of pathogen to participate in plant
Ying Zhong.Identify that many members of HIR family and common cigarette HR inducible protein NG1 are closely similar at present, activation of the NG1 as HR
Son can induce the necrotic plaque of class HR.35 times of HvHIR3 up-regulated expression on barley fast neutron mutant plants, blade are shown certainly
The HR of hair shows that HIR gene can induce HR.Then, the HIRs in leguminous plant, cucumber, rice and wheat be accredited as with
Microspore development is related to bacterial invasion reaction.
Research before is concentrated mainly on HIR1, studies other HIRs less.It is overexpressed the transgenosis of rice Os HIR1
Arabidopsis shows the resistance to pseudomonas syringae Pst.DC3000.It is not immediately clear HIR3s whether with other HIR families
Family member is similar, can induce HR, participates in plant in the defense reaction of pathogen.
Host plant is to adapt to external environment stimulation, biology and injury of the abiotic stress to itself is effectively reduced, in length
In phase evolutionary process, is expressed, formd a series of by perception, signal transduction and the various defensin genes of induction to adverse circumstance signal
Complicated and fine HR signal transduction mechanism.Ca2+The signal of ion, active oxygen, a variety of hormones and other signaling molecules in HR passes
Critical function is played during leading.Salicylic acid (Salicylic acid, SA), jasmonic (Jasmonic acid, JA), ethylene
(Ethylene, ETH) three parahormone participates in HR signal transduction process, and the important function in plant defense response has obtained extensively
Research and application.
For at present, the research about HIR is concentrated mainly on HIR1, resists bacterium and nosomycosis in plant to HIRs
The Effect study of pathogens is more.Functional study about other HIRs is not much, and does not also know whether HIRs participates in plant
The antiviral process infected.It is necessary to find or screen some other and disease-resistant related gene, to utilize these
Gene manufactures disease-resistant plant, to obtain disease-resistant plant, reduces the harm of chemical pesticide.
Summary of the invention
Natural host of the rice as rice stripe virus, OsHIR3 and experiment host's Ben Shi cigarette NbHIR3s sequence are high
Spend homologous (amino acid identity up to 80%).We have found that RSV is infected induction of rice Os HIR3 up-regulated expression.We have successful gram
The grand OsHIR3 gene to OryzasativaLcv.Nipponbare rice (Oryza sativa L.spp.japonica.cv.Nipponbare) constructs wink
When expression vector.Said gene is successfully overexpressed in rice body by Mature Embryos of Rice revulsion, turns OsHIR3 gene water
The germination of rice, plant shoots growth, plant height, solid situation no significant difference compared with wild type OryzasativaLcv.Nipponbare rice.
Using the malicious small brown rice planthopper of band to wild type feeding parallel with transgenic rice plant progress poison processing, discovery turns OsHIR3 base
Because rice plant is after RSV infects, symptom mitigates, and plant, which is downgraded, to be alleviated, and disease plant only shows striped phenotype, disease plant leaf
On piece RSV RNAs is substantially reduced.Thus, it is believed that OsHIR3 assigns rice to the tolerance to diseases of RSV.
In addition, turning the important bacterial pathogen Huang unit cell rice varieties of OsHIR3 trans-genetic hybrid rice plant Inoculated Rice
(Xanthomonas oryzae pv.oryzae, Xoo) turns the full scab length of OsHIR3 trans-genetic hybrid rice plant leaf and is significantly shorter than
Wild type illustrates to turn the enhancing of OsHIR3 trans-genetic hybrid rice plant to the resistance of Xoo.To sum up, OsHIR3 assigns rice to RSV and Xoo
Basal resistance.
Thus, the present invention will overexpress in rice Os HIR3 plant, and obtain resistance to RSV's and Xoo by Resistance Identification
Transgenic paddy rice with basal resistance, this is to be used to turn base using HIR3 gene as a kind of gene with basal resistance for the first time
Because resistance to viral rice is formulated.
The first purpose of the invention is to provide rice Os HIR3 genes.
A second object of the present invention is to provide the purposes of said gene.
In order to realize first above-mentioned purpose, the invention adopts the following technical scheme:
There are 6 HIR families in OryzasativaLcv.Nipponbare rice (Oryza sativa L.spp.japonica.cv.Nipponbare)
Gene, sequence analysis shows, in addition to Os06g0136000 belongs to HIR3 family, the nucleotides sequence of gene is classified as SEQ ID NO:1
Shown, other belong to HIR1 family.Said gene sequence is expanded using rice cDNA as template by regular-PCR using primer
Increase.
In order to realize second above-mentioned purpose, the invention adopts the following technical scheme:
Above-mentioned OsHIR3 is building up to plant binary expression vector pCV1300, is named as pCV:OsHIR3, electric shock imports agriculture
Bacillus strain EHA105.The transgenic paddy rice for being overexpressed OsHIR3 is obtained by Mature Embryos of Rice revulsion.
The malicious identification that connects that above-mentioned genetically modified plants are carried out with RSV, obtains the transgenic paddy rice of tolerance RSV.
Above-mentioned genetically modified plants are carried out with the inoculated identification of Xoo, obtains the transgenic paddy rice of tool basal resistance.
What the present invention obtained turns OsHIR3 trans-genetic hybrid rice, is mainly used in plant RSV and Xoo and infects, and mitigates viral and thin
Fungus diseases are caused harm.The present invention has important theory and practical significance to the genetically modified plants for cultivating tool basal resistance, to plant
Object disease control other field also has directive function.Compared with the resistance strain obtained by other viral resistant strategies, the present invention
Main advantage be OsHIR3 gene be gene existing for plant itself, relative to other viral sources resistant gene or
Segment has apparent safety；Turning OsHIR3 trans-genetic hybrid rice has basal resistance to virus and bacteriosis, and resistance is wider.
Detailed description of the invention
Fig. 1 .1: the expression vector map containing OsHIR3 gene
Fig. 1 .2: insertion plasmid pCV-eGFP-N1 Vector map.
Fig. 2: turn the molecular Biological Detection of OsHIR3 trans-genetic hybrid rice
Fig. 3: turn the development phenotype of OsHIR3 trans-genetic hybrid rice
Fig. 4: turn the anti-RSV analysis of OsHIR3 trans-genetic hybrid rice
Fig. 5: turn the anti-Xoo analysis of OsHIR3 trans-genetic hybrid rice
The Regulation Mechanism of Fig. 6: the OsHIR3 basal resistance mediated is probed into.
Fig. 7 is OsHIR3 genetic sequence map.
Specific embodiment
It should be noted that present embodiment only by way of example come illustrate we have found that gene new function
Energy.By demonstrating effectiveness of the invention in model plant Ben Shi cigarette, it is not to be construed as limiting the invention
The clone of embodiment 1:OsHIR3 gene
The turned plant of the present invention is OryzasativaLcv.Nipponbare rice.
1. the acquisition of recombinational agrobacterium
1) clone of OsHIR3 gene
Pass through regular-PCR using OryzasativaLcv.Nipponbare rice cDNA as template using primer OsHIR3-ORF-f and OsHIR3-ORF-r
Amplification.The nucleotides sequence of said gene is classified as shown in SEQ ID NO:1.
Cloning primer is as follows:
OsHIR3-ORF-f:5'-ATGGTGAGCGCCTTCTTCCTGCT-3'(SEQ ID NO:2)
OsHIR3-ORF-r:5'-TTACACGTTGCTGCAGGACGCTT-3'(SEQ ID NO:3)
Trizol method extracts plant total serum IgE: to avoid RNA from degrading, RNA extractive process need to wear mask and gloves.
1. taking appropriate amount of sample in import 2mL Eppendorf (EP) pipe containing steel ball, rapidly in stone roller after liquid nitrogen flash freezer
18rps (Revolutions Per Second, revolutions per second) concussion 30s-1min on instrument is ground, is added after being fully ground appropriate
Trizol (1mL/100mg sample, to ensure that sample sufficiently cracks), shakes vigorously and mix well, and places 5min on ice and (is conducive to nucleic acid egg
The dissociation of white matter complex).4 DEG C, 13,000rpm centrifugation 10min.
2. taking supernatant in new 2mL EP pipe, 1/5 volume of chloroform is added, acutely shakes 30s, mixes well, it is quiet on ice
Set 2~3min.4 DEG C, 13,000rpm centrifugation 30min.
3.EP pipe top layer is the colourless aqueous phase containing RNA, and middle layer white layer is albumen phase, and bottom is chloroform phase.Take upper layer
Water phase is transferred in new 2mL EP pipe (movement is as far as possible softly to avoid absorption middle protein phase), repeats step 2.
4. extracting upper strata aqueous phase in new 1.5mL EP pipe, the isopropanol that isometric (about 600 μ L) has been pre-chilled is added, on
Under be mixed by inversion, -70 DEG C of placement 1h.Taking-up is placed on ice after it sufficiently thaws, and 4 DEG C, 13,000rpm centrifugation 30min.
5. abandoning supernatant, 75% ethyl alcohol (preparation of RNase-free water) that 1mL has been pre-chilled is added, washing precipitating (fills precipitating
Divide and suspend, to ensure that washes clean is thorough).4 DEG C, 13,000rpm centrifugation 5min.
6. repeating step 5, residual salt is thoroughly cleaned.
7. abandoning supernatant, 4 DEG C of blank pipe, 13,000rpm centrifugation 2min carefully suck residual liquid with liquid-transfering gun, and room temperature is dried,
To be precipitated piece by white to transparent, add appropriate RNase-free water to dissolve.
8.RNA concentration can be measured by ultraviolet specrophotometer, RNA mass be also referred to RNA OD260/OD280,
OD260/OD230Ratio.RNA sample be placed in -80 DEG C it is spare.
CDNA is inverted system and condition is as follows by the total serum IgE reversion extracted by Trizol method:
Preceding four kinds of reagents are added first in the micro EP pipe of no RNA enzyme, mix, 70 DEG C of denaturation 5min；It is immediately placed in ice
On, place 2min.Three kinds of reagents next are sequentially added, are inverted in PCR instrument after mixing by the following conditions.
42 DEG C, 2h → 72 DEG C, 10min
PCR amplification system is as follows:
PCR cycle is carried out by the following conditions after mixing:
Examples of implementation 2: vector construction
It is and above-mentioned same using the OsHIR3 full length gene of above-mentioned acquisition as template using primer OsHIR3-f and OsHIR3-r
Amplification obtains the OsHIR3 sequence for containing corresponding restriction enzyme site under conditions of sample, by conventional digestion, is connected into double base table
Up to the multiple cloning sites of carrier pCV1300.The Vector map completed is constructed as shown in Fig. 1 .1, is inserted into plasmid vector such as Fig. 1 .2 institute
Show, specifically, exactly replaced in the position of carrier GFP (fluorescin) by target gene OsHIR3, is ultimately formed complete
Plasmid vector.Such plasmid vector is that those skilled in the art are readily appreciated that, any plasmid vector is all possible and this field
Common means.
Following (the underscore of primerTCTAGAWithGGATCCRespectively indicate Xba I and Bam HI restriction enzyme site):
OsHIR3-f:5'-tgcTCTAGAATGGTGAGCGCCTTCTTCCTGCT-3'(SEQ ID NO:4)
OsHIR3-r:5'-cgcGGATCCTTACACGTTGCTGCAGGACGCTT-3 (SEQ ID NO:5) '
Examples of implementation 3: the identification and preservation of Agrobacterium-mediated Transformation, positive colony
Positive plasmid is converted into Agrobacterium by electric shocking method, the specific steps are as follows:
1. 1 μ L, which is smoked pure plasmid DNA (examples of implementation 2 obtain), is added to the Agrobacterium competence EHA105 to have thawed
In (take out to be placed in advance and thaw on ice), gently piping and druming is mixed, and adds in electric shock cup；
2. above-mentioned electric shock cup is put into electric shock tank (electric shock instrument voltage is 2.2kV), shock button is pressed, hears drop sound i.e.
It can；
3. bacterium solution is extracted into EP pipe, 900 μ L non-resistant LB culture solutions of addition, 220rpm, 28 DEG C, shaken cultivation 1h.
4. extracting the 200 cultured bacterium solutions of μ L, LB plate is uniformly coated on (containing 50 μ g/mL kanamycins (Kan) and 50 μ
G/mL rifampin (Rif)), 28 DEG C of culture 2d.
The Agrobacterium single colonie of picking conversion is inoculated in containing 50 μ g/mL kanamycins (Kan) and 50 μ g/mL rifampins
(Rif) in LB liquid medium, 28 DEG C, 200rpm shakes training overnight, and 1 μ L bacterium solution is taken to carry out PCR detection.Detection primer is special
Property primer OsHIR3-detec-f and vector primer NOS-r (schematic diagram of primer bonding position is as shown in Figure 2 A).Detection is taken to tie
Fruit is positive bacterium solution, mixes 30% glycerol, sets in glycerol tube, save in -70 DEG C of ultra low temperature freezers.
Detection primer is as follows:
OsHIR3-detec-f:5'-AAGGTGATGGGAGATTATGGTTAC-3'(SEQ ID NO:6)
NOS-r:5 '-GATAATCATCGCAAGACCGG-3 ' (SEQ ID NO:7)
PCR detection architecture is as follows:
PCR cycle is carried out by the following conditions after mixing:
Examples of implementation 4: Mature Embryos of Rice callus induction method obtains transgenic plant
1) preparation of bacterium solution
Take the positive transformants bacterial strain (examples of implementation 3) of -70 DEG C of preservations, the LB of 50 μ g/mL Kan of Yu Han and 50 μ g/mL Rif
Flat lining out, 28 DEG C of cultures fall within the LB liquid containing 50 μ g/mL Kan, 100 μ g/mL Rif to single colonie, picking single bacterium is formed
It is cultivated in body culture medium, 28 DEG C, 220rpm shaken cultivation is stayed overnight；Bacterium solution is diluted by the fresh LB culture medium of 1:100, is continued
Shake culture is to OD600It is 1.0 or so.
2) transgenic plant is obtained by Mature Embryos of Rice callus induction method, the specific steps are as follows:
1. disinfection:
1. taking OryzasativaLcv.Nipponbare flowering two weeks or so (pustulation period) young fringes, full light is selected in artificial or mechanical threshing decladding
The seed of clean no bacterial plaque removes the empty grain floated with aseptic water washing seed；
2. seed is put into sterile glass tube, with aseptic water washing seed 2~3 times；
3. 70% alcohol disinfecting 1min is added, alcohol is removed, with aseptic water washing 2~3 times；
4. 30% sodium hypochlorite (NaClO, effective chlorine 5.2% contain few drops of Tween-20s) solution is added, it is standing and soaking
30min；Liquor natrii hypochloritis is outwelled, with aseptic water washing 2~3 times, finally seed is impregnated with sterile water, stands 30~45min.
2. Fiber differentiation:
Seed is laid on aseptic filter paper, excessive moisture is blotted, by 5~10 seeds of every ware, is placed in mature embryonal induction
In culture medium；With sealed membrane plastic packaging culture dish, it is placed in 28 DEG C of illumination boxs and cultivates about 20d.
3. squamous subculture:
When seed grows faint yellow, densification opens culture dish in superclean bench, uses tweezer in after spherical embryo callus subculture
The complete embryo callus that sub- picking divides naturally, is placed in subculture medium, in 28 DEG C of illumination boxs, squamous subculture 1
Week (if do not used immediately, can be moved to dark place, 22 DEG C are continued culture 1 week).
4. co-culturing:
1. picking Agrobacterium monoclonal shakes bacterium to bacterium solution OD600It is 1.0 or so；Thallus is collected, (contains 200 μ with AAM sense bacterium solution
M As) thallus is resuspended, adjust bacterial concentration OD600About 0.1；
2. the sizeable callus of picking is put into the above-mentioned agrobacterium suspension prepared, sufficiently immersion 5min；
Callus is taken out in 0.5~1h dry on aseptic filter paper；Callus is laid in and is co-cultured on base, 25 DEG C of dark cultures 2~
2.5d。
5. screening and culturing:
1. taking out callus, with sterile water wash, ceaselessly vibrate therebetween；It is sterile with the Cefradine containing 500mg/L
Water cleaning and dipping 30min is cleaned 3 times, callus is laid on aseptic filter paper and dries 2h；
It is carried out 2. the callus dried is transferred on Selective agar medium (Cefradine containing 500mg/L and 50mg/L hygromycin)
The first round screens, and cultivates 14d in 28 DEG C of illumination boxs；
3. the initial callus that picking newly bears kanamycin-resistant callus tissue be placed in new Selective agar medium (Cefradine containing 500mg/L and
50mg/L hygromycin) in carry out the second wheel selection, in 28 DEG C of illumination boxs, culture 10d or so, until growing graininess
Resistant calli.
6. differentiation culture:
The resistant calli of picking color cadmium yellow moves in the plastic jar containing differential medium (every bottle of placement
4-5), it is placed in constant incubator, seedling differentiation (15~30d).
7. strong plantlets and rootage and transplanting:
The seedling differentiated to callus is long to 2~3cm or so, takes out seedling, removes root callus, move to root media
In, it cultivates 1~2 week；Appropriate amounts of sterilized water (at the top of Miao Changzhi test tube, uncapping in time), hardening 3 are added into the seedling to grow fine
~7d；Root culture medium is washed away, seedling replanting is buried, the water surface is advisable with not flooding seedling, normal greenhouse-grown Environment cultivation.
Turn 14 plants of OsHIR3 trans-genetic hybrid rice by the acquisition of Mature Embryos of Rice callus induction method.
3) molecular Biological Detection of transgenic plant
Turn OsHIR3 trans-genetic hybrid rice DNA using the extraction of CTAB method is above-mentioned, the specific steps are as follows:
1. appropriate vegetable material is taken to be put into 2mL EP pipe, is quickly ground through liquid nitrogen completely, 500 μ 2 × CTAB of L is added,
Acutely concussion；
2. 65 DEG C of water-baths react 30min, every 10min, which turns upside down, to be mixed once；
3. 500mL chloroform is added, concussion is mixed, room temperature 12, and 000rpm is centrifuged 10min, takes supernatant into new EP pipe；
4. it is 3. primary to repeat step；
5. the NaAc (3M, pH 5.2) of isometric isopropanol and 1/10 volume is added, concussion is mixed, -20 DEG C of placements
15min；
6. room temperature 12,000rpm is centrifuged 10min；
7. abandoning supernatant, 75% ethanol washing, room temperature 12 are precipitated, 000rpm is centrifuged 5min；
8. it is 7. primary to repeat step；
9. abandoning supernatant, uncapping is placed at room temperature for 15min with drying precipitated, 40 μ L ddH of precipitating2O dissolution.
It is rice endogenous gene in view of OsHIR3, so selection specific primer (OsHIR3-detec-f:5'-
AAGGTGATGGGAGATTATGGTTAC-3'(SEQ ID NO:8)) and vector primer (NOS-r:5 '-
GATAATCATCGCAAGACCGG-3 ' (SEQ ID NO:9)) PCR detection is carried out to it, the results showed that, turn OsHIR3 gene water
12 plants of rice positive plant, 2 plants of negative plant, positive rate is 86% (Fig. 2 B).
In order to detect whether OsHIR3 is successfully integrated into genome and high expression, we are extracted positive strain T2 for plant
Leaves total protein and total serum IgE, it is carried out Western blot and qRT-PCR detection, the results showed that, in positive plant
OsHIR3 expression quantity difference, the OsHIR3 protein level of transfer OsHIR3 trans-genetic hybrid rice strain OE6, OE8, OE12 and
OsHIR3mRNA expression quantity is significantly higher than wild type (Fig. 2 C and Fig. 2 D).In Phenotypic Observation discovery, T2 generation, turn OsHIR3 trans-genetic hybrid rice
Germination, plant shoots growth, solid situation no significant difference (Fig. 3) compared with wild type OryzasativaLcv.Nipponbare rice, illustrate,
OsHIR3 up-regulated expression does not significantly affect the growth and development situation of rice plant.
Examples of implementation 5: turn the RSV inoculated identification of OsHIR3 trans-genetic hybrid rice
The present invention chooses T2 and turns OsHIR3 trans-genetic hybrid rice progress RSV inoculated identification for the positive.Concrete operations content is as follows:
1) purifying and identification of high band poison rate small brown rice planthopper
Small brown rice planthopper group 5~7 days (day, d) is raised with infection RSV rice plant, to guarantee that polypide can sufficiently obtain poison.
Then the female adult for individually capturing for 5 ages carries out single worm raising in test tube (plant 2~3 plants of rice seedlings in test tube and supply its feeding).
Single worm raises 2~3 weeks (growing to or so 2~3 ages to second generation larva), and every pipe captures 3 low instar larvaes and carries out single worm RT-PCR
Positive worm system, is then transferred in large beaker respectively and carries out expanding propagation by the malicious rate detection of band.Inspection is sampled to raising up seed
It surveys, raising will be summarized with malicious small brown rice planthopper after the malicious rate of band is stablized.
2) turn OsHIR3 trans-genetic hybrid rice inoculation RSV
It selecting to turn OsHIR3 trans-genetic hybrid rice T2 for strain as material to be tested, wild rice (OryzasativaLcv.Nipponbare) is used as control,
It sows simultaneously.Sterile water process, seed soaking and vernalization are carried out in 37 DEG C of constant incubators, and each strain chooses 20~40 health kind
Son.2 days or so, seed was sowed after showing money or valuables one carries unintentionally, and each strain is planted in respectively in nutritive cube (10cm × 10cm), greenhouse ring
It is cultivated in border.
To paddy growth to 3 leaf phases, move it to and connect in worm cage, according to 3~5 every plant effectively connect worm amount, will be above-mentioned
Malicious 2~4 instar larvae of (RSV) rate small brown rice planthopper of the high band purified, which is transferred to, connects worm cage, parallel simultaneously with transgenic plant to wild strain
It carries out raising 2~3d of poison.During connecing worm, it is ensured that the rice seedling of inoculation uniformly obtains poison, catches up with worm daily twice.After the completion of raising poison, institute is removed
After thering is small brown rice planthopper, rice seedling to alleviate 2~3d under greenhouse, by plantlet of transplant to crop field, disease survey and analysis are carried out.
3) turn the RSV inoculated identification of OsHIR3 trans-genetic hybrid rice
It is inoculated with 4~6d of RSV or so, rice seedling starts to crimp, and connects 8~12d of poison or so susceptible serious seedling and opens
Begin dead, connects serious newborn blade of falling ill after malicious 20d and obvious scab and curling occur, then, some serious plant of morbidity
Gradually dead, there is scab in the plant leaf with stronger resistance capacity, some can with plant strain growth and gradually hidden disease.
Rice plant whether is assigned to RSV resistance for analysis OsHIR3 overexpression, we, which choose, turns OsHIR3 trans-genetic hybrid rice
The T2 of three independent strain OE6, OE8, OE12 carry out anti-RSV identification for seedling, and each strain plants 30 plants, with wild type Japan
It is fine to be used as control, do same treatment.After the transplanting of the rice plant after the completion of poison crop field will be connect, continue to observe and count its death
Rate.After meeting malicious 20d, mortality statistics are substantially less than the results show that examining three independent strain OE6, OE8, OE12 death rates
Adjoining tree (Fig. 4 A).After meeting malicious 30d, transgenic rice plant growing state is significantly better than adjoining tree.It is more in adjoining tree
The susceptible serious plant dead of number；And examine in three transgenic lines, it is small and weak that susceptible serious plant only shows growing way.It deposits
Control disease plant growing way living is short and small, blade severe curl；And the susceptible strain of transgenic paddy rice survived only is presented in blade
Striped phenotype (Fig. 4 B) out.
It is analyzed by Northern blot and turns RSV RNAs accumulation in OsHIR3 trans-genetic hybrid rice and wild type disease plant
Amount, the results show that the RSV RNA3 accumulation for examining three transgenosis independence strain OE6, OE8, OE12 disease plant blades is equal
Lower than adjoining tree (Fig. 4 C).The above result shows that OsHIR3 is overexpressed the accumulation for effectively inhibiting RSV RNAs, turn OsHIR3 base
Because rice plant is shown to RSV tolerance to diseases.
Embodiment 6: turn the anti-Xoo analysis of OsHIR3 trans-genetic hybrid rice
1) Xoo is inoculated with
By bacterial leaf-blight bacteria strain P10 (Xoo)It transfers in association's Ben Zheshi fluid nutrient medium, 28 DEG C of shaking table 200r/min trainings
1d is supported, thallus is collected, uses ddH2O is configured to OD600 about 0.5.It selects to turn OsHIR3 trans-genetic hybrid rice T2 for strain as test material
Material, wild rice (OryzasativaLcv.Nipponbare) are sowed simultaneously as control, are planted in greenhouse.To (heading in paddy growth 2 months or so
Before), bacterial leaf-blight bacteria strain P10 is inoculated with to rice using artificial leaf-cutting inocalation method, after inoculation 1~2 week, observation rice leaf hair
State of an illness condition, measures the scab length of different rice material incidence of leaf, and statistical comparison evaluates rice resistance
2) full scab length measurment
It is inoculated with Xoo P10 microspecies after two weeks, the full scab length of measurement infection blade.Statistical result showed, with wild type
It compares, the scab length for turning OsHIR3 trans-genetic hybrid rice three independent strain OE6, OE8, OE12 is shorter (Fig. 5 A).Wild type Japan
Fine scab length is (10.5 ± 0.6) cm；And turn the scab of OsHIR3 trans-genetic hybrid rice three independent strain OE6, OE8, OE12
Length is respectively (5.2 ± 0.5) cm, (2.1 ± 0.3) cm, (6.1 ± 0.2) cm, statistical result showed, three independent strains
There is significant difference (Fig. 5 B) two-by-two compared with wild type in OE6, OE8, OE12.The above result shows that turning OsHIR3 trans-genetic hybrid rice
Plant enhances the tolerance to diseases to Xoo.To sum up, the basal resistance that OsHIR3 is mediated also targets other cause of diseases not only for RSV,
Such as Xoo.
Embodiment 7:NbHIR3s is by positive regulation SA approach to obtain basal resistance
The higher positive plant of OsHIR3 expression quantity is filtered out through above-mentioned detection, carries out SA assay and SA pathway key
Gene real time fluorescent quantitative qRT-PCR analysis.Referring to qRT-PCR specification, concrete operations are as follows:
Mentioned reagent is successively added in RNase-free EP pipe, is mixed well, it is quantitative by 10 μ L/ hole point samples to 384 holes
In plate, it is covered with film, brief centrifugation is placed in qRT-PCR instrument and is reacted: 95 DEG C of 5min；40 are carried out to recycle: 95 DEG C of 20s →
58℃20s→72℃20s；72℃10min.
It is as follows that real-time fluorescence quantitative PCR analyzes specific quantification primer used:
Compared with wild type, turn in three independent strain OE6, OE8, OE12 blade cells of OsHIR3 trans-genetic hybrid rice, SA
Content dramatically increases (Fig. 6 A), SA pathway key gene PBZ1 (also known as NPR1), the significant up-regulated expression of PR1 and PR5 gene (figure
6B).This absolutely proves that OsHIR3 gene is by positive regulation SA approach to obtain basal resistance.
This is because system resistant causes not infecting position distally and generate to resist pathogen after plants by phytopathogenic bacterium is infected
Property, this induction of resistance are referred to as systemic acquired resistance (Systemic Acquired Resistance, SAR), the phenomenon
It is confirmed in many plants and pathogen interaction.The typical performance of SAR is limitation growth of pathogenic bacteria and inhibits its disease
Shape development.In plant, there are many report, Main Viewpoints think for effect of the SA in SAR, SA be during SAR one it is important
Signaling molecule, the accumulation of SA can excite SAR to react.Pathogenesis-related proteins (pathogenesis related protein, PR
Albumen) great expression be SAR reaction important symbol, a variety of common coordinative roles of PR albumen rather than a certain specific PR albumen list
It solely acts on and then SAR is caused to react.In the tobacco body handled by SA or aspirin, PR albumen is largely accumulated, and to tobacco
Mosaic virus (Tobacco mosaic virus, TMW) infects generation resistance；TMV infects can the endogenous SA content urgency of evoking tobacco
Increase severely and add, and the SA content of resistant variety is apparently higher than susceptible variety；Sid1 and sid2 mutant plants, internal SA can not be accumulated
It is tired, SAR can not be started, sensibility is shown to pseudomonas syringae, further prove that SA is the signal point of key during SAR
Son.
Modulin NPR1 is a key component in the signal transduction pathway that SA is mediated, and NPR1 can induce PR-1 etc.
The expression of resistant gene, to enhance disease resistance of plant.When pathogen infects, the impacted mutant of NPR1 gene expression
Nim1, SA are horizontal normal, but can not induce SAR, show that NPR1 acts on the downstream SA, are one in SAR signal transduction pathway
Key regulator.Despres etc. is the study found that NPR1 can be rich in the TGA of leucine (bZIP) transcription factor with arabidopsis
Interaction occurs for family member, and npr1 mutant then loses the interaction with TGA2, illustrates that the TGA2 that NPR1 is mediated is combined to anti-
The activation of imperial gene is very crucial.When inducing SAR, NPR1 is swashed by the interaction of the transcription factor with the gene promoter area PR
PR-1 gene living, shows that NPR1 activity and PR gene expression regulation are closely related.The study found that four point mutation bodies of NPR1 hinder
Broken SA signal, and loses the interaction with TGA2 and TGA3, and TGA2 and TGA3 can be rung in conjunction with the SA of arabidopsis PR-1 promoter
Element is answered, research is connected NPR1 and SA induction PR-1 gene expression by TGA transcription factor.
Sequence table
<110>University Of Ningbo
<120>purposes of rice Os HIR3 gene and the method for obtaining disease resisting rice
<130> 19-100070-00011864
<141> 2019-01-08
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<170> SIPOSequenceListing 1.0
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<213>artificial sequence (Artificial Sequence)
<400> 1
atggtgagcg ccttcttcct gctgtgcggg tgcgtggacc aggcgagcgt cgcggtggtg 60
gagaagtggg gccgcttcct ccgcctcgcc gagccgggcc tccacttctt caacccgttc 120
gccggcgagt tcgtcgccgg gacgctctcc acccgcgtcc agtcgctcga cgtccgcgtc 180
gagaccaaga ccaaggataa tgtctttgtt cagcttatct gcacaatcca atatcgggtt 240
gttaaggaac atgctgatga tgcattctat gagttgcaga atccccaaca gcaaattcag 300
gcctacgtct ttgatgtggt ccgagctata gttccgagaa tgaatcttga tgatcttttt 360
gagcaaaaga atgatgtggc gaaagctgta cttcaggagc tagaaaaggt gatgggagat 420
tatggttaca gcattgagca cattctcatg gttgacatca tccctgatgc tgctgtacgc 480
agagcaatga atgaaataaa tgcagcacaa aggcttcagc ttgcaagtgt ctacaaagga 540
gaggcggaga agattcttct ggtgaagaaa gcagaagcag aggcagaggc aaaacacctt 600
tccggtgtcg gcattgctag acagcggcag gcgataactg atggcctgag agagaacatc 660
ctgaacttct cgcactcggt ttcaggcacc tcagcaaaag aagtcatgga tctcatcatg 720
gtcacgcagt acttcgacac catcaaagaa cttggggatg gctcgaagaa caccacggtg 780
ttcatacctc atggcccagg ccatgtcagg gatatcagcg agcaaatccg gaatggtatg 840
atggaagcgt cctgcagcaa cgtgtaa 867
<210> 2
<211> 23
<212> DNA/RNA
<213>artificial sequence (Artificial Sequence)
<400> 2
atggtgagcg ccttcttcct gct 23
<210> 3
<211> 23
<212> DNA/RNA
<213>artificial sequence (Artificial Sequence)
<400> 3
ttacacgttg ctgcaggacg ctt 23
<210> 4
<211> 32
<212> DNA/RNA
<213>artificial sequence (Artificial Sequence)
<400> 4
tgctctagaa tggtgagcgc cttcttcctg ct 32
<210> 5
<211> 32
<212> DNA/RNA
<213>artificial sequence (Artificial Sequence)
<400> 5
cgcggatcct tacacgttgc tgcaggacgc tt 32
<210> 6
<211> 24
<212> DNA/RNA
<213>artificial sequence (Artificial Sequence)
<400> 6
aaggtgatgg gagattatgg ttac 24
<210> 7
<211> 20
<212> DNA/RNA
<213>artificial sequence (Artificial Sequence)
<400> 7
gataatcatc gcaagaccgg 20
<210> 8
<211> 24
<212> DNA/RNA
<213>artificial sequence (Artificial Sequence)
<400> 8
aaggtgatgg gagattatgg ttac 24
<210> 9
<211> 20
<212> DNA/RNA
<213>artificial sequence (Artificial Sequence)
<400> 9
gataatcatc gcaagaccgg 20
<210> 10
<211> 25
<212> DNA/RNA
<213>artificial sequence (Artificial Sequence)
<400> 10
ggtatccatg agactacata caact 25
<210> 11
<211> 23
<212> DNA/RNA
<213>artificial sequence (Artificial Sequence)
<400> 11
tactcagcct tggcaatcca cat 23
<210> 12
<211> 20
<212> DNA/RNA
<213>artificial sequence (Artificial Sequence)
<400> 12
cacactcgac ggagacgaag 20
<210> 13
<211> 21
<212> DNA/RNA
<213>artificial sequence (Artificial Sequence)
<400> 13
gccatagtag ccatccacga t 21
<210> 14
<211> 20
<212> DNA/RNA
<213>artificial sequence (Artificial Sequence)
<400> 14
tatccaagct ggccattgct 20
<210> 15
<211> 20
<212> DNA/RNA
<213>artificial sequence (Artificial Sequence)
<400> 15
ttctctggct ggcgtagttc 20
<210> 16
<211> 20
<212> DNA/RNA
<213>artificial sequence (Artificial Sequence)
<400> 16
cgcaacaact gcacctacac 20
<210> 17
<211> 20
<212> DNA/RNA
<213>artificial sequence (Artificial Sequence)
<400> 17
ggctaggaac gagacgttgg 20
Claims (10)
1. riceOsHIR3Gene is preparing the purposes in plant defense virus or bacterial invasion harm.
2. purposes according to claim 1, describedOsHIR3Gene is sequence shown in SEQ ID NO:1.
3. purposes according to claim 1, the virus is RSV;Bacterium isXoo。
4. purposes according to claim 1, the riceOsHIR3Gene comes from OryzasativaLcv.Nipponbare rice.
5. a kind of method for obtaining rice transgenics, this method comprises: riceOsHIR3It is gene constructed to arrive plant binary table
Up in carrier, agrobacterium strains are imported by electric shock；It is overexpressed by Mature Embryos of Rice revulsionOsHIR3Transgenosis
Rice.
6. described according to the method described in claim 5, whereinOsHIR3Gene is sequence shown in SEQ ID NO:1.
7. according to the method described in claim 5, wherein, binary expression vector comprises the following structure: LB-35s PolyA-
HPTII-35s promoter-Nos- target gene -35s promoter-RB.
8. according to the method described in claim 5, the plant of the acquisition has the function of defending virus infection or bacterium.
9. according to the method described in claim 9, the virus is RSV;Bacterium isXoo。
10. purposes according to claim 2, wherein describedOsHIR3Gene passes through up-regulation PBZ1(also known as NPR1),
PR1 and PR5 and the up-regulation for causing SA, thus the ability with basal resistance.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910016244.1A CN109652425B (en) | 2019-01-08 | 2019-01-08 | Application of rice OsHIR3 gene and method for obtaining disease-resistant rice |
| US16/523,703 US20200216855A1 (en) | 2019-01-08 | 2019-07-26 | Disease Resistant Plants Containing HIR3 Gene and Method for making the plants thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910016244.1A CN109652425B (en) | 2019-01-08 | 2019-01-08 | Application of rice OsHIR3 gene and method for obtaining disease-resistant rice |
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| CN109652425A true CN109652425A (en) | 2019-04-19 |
| CN109652425B CN109652425B (en) | 2022-10-04 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116121297A (en) * | 2023-01-17 | 2023-05-16 | 宁波大学 | Rice OsNPR1 gene and application thereof in resisting rice stripe disease |
| WO2025246496A1 (en) * | 2024-05-28 | 2025-12-04 | 华中农业大学 | Use of athir4 gene and/or protein coded thereby in regulation of plant pathogen resistance |
| CN121204082A (en) * | 2025-11-27 | 2025-12-26 | 华南农业大学 | Application of OsHIR1 gene in improving rice planthopper resistance |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20100107295A (en) * | 2009-03-25 | 2010-10-05 | 대한민국(관리부서:농촌진흥청장) | Rice stripe virus resistant rice transformants and method of producing thereof |
| US20120060234A1 (en) * | 2009-03-02 | 2012-03-08 | Evogene Ltd. | Isolated polynucleotides and polypeptides, and methods of using same for increasing plant yield and/or agricultural characteristics |
| CN104877993A (en) * | 2015-04-24 | 2015-09-02 | 浙江省农业科学院 | Two plant eIF4A genes and application thereof in preparation of transgenic rice stripe virus resistant plant body |
-
2019
- 2019-01-08 CN CN201910016244.1A patent/CN109652425B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120060234A1 (en) * | 2009-03-02 | 2012-03-08 | Evogene Ltd. | Isolated polynucleotides and polypeptides, and methods of using same for increasing plant yield and/or agricultural characteristics |
| KR20100107295A (en) * | 2009-03-25 | 2010-10-05 | 대한민국(관리부서:농촌진흥청장) | Rice stripe virus resistant rice transformants and method of producing thereof |
| CN104877993A (en) * | 2015-04-24 | 2015-09-02 | 浙江省农业科学院 | Two plant eIF4A genes and application thereof in preparation of transgenic rice stripe virus resistant plant body |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116121297A (en) * | 2023-01-17 | 2023-05-16 | 宁波大学 | Rice OsNPR1 gene and application thereof in resisting rice stripe disease |
| CN116121297B (en) * | 2023-01-17 | 2025-10-17 | 宁波大学 | Rice OsNPR1 gene and application thereof in resisting rice stripe disease |
| WO2025246496A1 (en) * | 2024-05-28 | 2025-12-04 | 华中农业大学 | Use of athir4 gene and/or protein coded thereby in regulation of plant pathogen resistance |
| CN121204082A (en) * | 2025-11-27 | 2025-12-26 | 华南农业大学 | Application of OsHIR1 gene in improving rice planthopper resistance |
| CN121204082B (en) * | 2025-11-27 | 2026-02-17 | 华南农业大学 | Application of OsHIR1 gene in improving rice planthopper resistance |
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| CN109652425B (en) | 2022-10-04 |
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