CN103333231B - Plant disease-resistant protein Xa23 and its coding gene and use - Google Patents
Plant disease-resistant protein Xa23 and its coding gene and use Download PDFInfo
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Abstract
The invention discloses a paddy rice bacterial blight-resistant protein Xa23 and its coding gene. A nucleotide sequence of the coding gene is shown in the formula of SEQ ID NO: 1 and an amino acid sequence of the paddy rice bacterial blight-resistant protein Xa23 is shown in the formula of SEQ ID NO: 2. The invention also relates to a use of the coding gene in cultivation of disease-resistant crops and especially in cultivation of a paddy rice bacterial blight-resistant paddy rice variety.
Description
Technical field
The invention belongs to biological technical field, relate to paddy disease-resistant albumen and encoding gene thereof, be specifically related to a kind of Gene For Resistance To Rice Bacterial Blight and application thereof.
Background technology
Paddy rice is the most important food crop of China, and the population of about 60% take rice as staple food, and the Sustainable development of Rice Production is directly connected to the grain security of China.Bacterial blight of rice (Bacterial blight, BB) be one of Major Diseases in world's Rice Production, found in area, Japanese Fukuoka early than 1884, from the fifties, morbidity scope constantly expands, occurrence scope each paddy rice producing region all over the world (Mew TW. Current status and future prospects of research on bacterial blight of rice. Annu Rev Phytopathol of current bacterial leaf-blight, 1987,25:359-382; Ou SH. Rice disease, Commonwealth Agricultural Bereaux, England, 1985,380pp.).Bacterial blight of rice is especially serious in Asia, and in China, from south to north, except Xinjiang, there is distribution in Wai Gedao district.
Bacterial blight of rice be by Gram-negative bacteria Xanthomonas campestris rice varieties (
xanthomonas oryzaepv
. oryzae, Xoo) a kind of vascular bundle diseases of causing, susceptiblely generally can cause the paddy rice underproduction 20% ~ 30% afterwards, even have no harvest time serious.Produce harm because pathogenic bacteria invades vascular bundle by wound or water hole, with medicament prevention effect is not good, not only expensive, take a lot of work and contaminate environment.Facts have proved, plantation disease-resistant variety is water prevention bacterial blight of rice economy and effective means the most.So from the sixties in 20th century, excavate and utilize bacterial leaf spot resistant new gene to be one of focus of research both at home and abroad always.The Gene For Resistance To Rice Bacterial Blight reported at present reaches 38, and in these disease-resistant genes, great majority have been positioned on karyomit(e), wherein has 8 to be successively cloned, comprising:
xa1(Yoshimura S et al. Expression of Xa1, a bacterial blight-resistance gene in rice, is induced by bacterial inoculation. Proc Natl Acad Sci USA, 1998,95:1663-1668)
, Xa3/Xa26(same gene) (Sun XL, et al. Xa26, a gene conferring resistance to
xanthomonas oryzaepv
oryzaein rice, encodes an LRR receptor kinase-like protein. The Plant Journal, 2004,37:517-527; Xiang Y, et al. Xa3, conferring resistance for rice bacterial blight and encoding a receptor kinase-like protein, is the same as Xa26. Theoretical and Applied Genetics, 2006,113:1347 – 1355.)
, xa5(Iyer AS; McCouch SR. The rice bacterial blight resistance gene xa5 encodes a novel form of disease resistance. Molecular Plant Microbe Interactions; 2004,17 (12): 1348-1354; Jiang GH, et al. Testifying the rice bacterial blight resistance gene xa5 by genetic complementation and further analyzing xa5 (Xa5) in comparison with its homolog TFIIAg1. Molecular Genetics and Genomics, 2006,275:354 – 366.)
, xa13(Chu Z, et al. Promoter mutations of an essential gene for pollen development result in disease resistance in rice. Genes Dev, 2006,20:1250 – 1255)
, Xa21(Song WY, et al. A receptor kinase-like protein encoded by the rice disease resistance gene, Xa21. Science, 1995,270:1804-1806)
, xa25(Liu QS, et al. A paralog of the MtN3/saliva family recessively confers race-specific resistance to
xanthomonas oryzaein rice. Plant Cell and Environment, 2011,34:1958-1969) and
xa27(Gu KY, et al. R gene expression induced by a type-III effector triggers disease resistance in rice. Nature, 2005,435,23 June, 1122-1125).In these disease-resistant genes above-mentioned, their most of anti-spectrums are narrow, Resistance durability is poor or be recessive gene, not convenient use in production.What be used effectively at present only has
xa3, Xa4, Xa7with
xa21deng a few.Due to the continuous variation of Pathogenic, the long-term extensively utilization of minority Bacterial blight resistance gene always faces the risk of resistant lose.In order to the harm of sustainable control bacterial blight of rice, constantly must excavating new Resistance resource and therefrom find the new gene that there is breeding utilization and be worth, laying the foundation for cultivating excellent paddy disease-resistant new variety.
In the previous works such as the present inventor, from China's common wild-rice, qualification is excavated out one to differentiate that fungus strain all shows as high resistance, complete dominance, the time of infertility to domestic and international bacterial leaf-blight disease-resistant
xa23gene.Early-stage Study finds,
xa23gene is that broad spectrum antidisease gene (resists the representative bacterial leaf spot pathogenic bacteria strain from states such as China, Philippines, Japan, Korea S, Bangladesh, and do not find so far to overcome
xa23the bacterial leaf spot pathogenic bacteria Natural strains of gene resistance, Wang CL et al., 2009, Generation and characterization of Tn5-tagged
xanthomonasoryzaepv.
oryzaemutants that overcome
xa23-mediated resistance to bacterial blight of rice. European Journal of Plant Pathology, 123 (3): 343-351), and
xa23the resistance of gene mediated has complete dominance, the time of infertility disease-resistant feature, therefore to the genetic improvement of paddy rice particularly hybridisation rice, comprises molecular mark and transgenic engineering breeding all has very wide application prospect.
xa23the disease-resistant principle of gene has singularity:
xa23gene is not expressed under normal circumstances, but has a promoter trap strategy, contains when bacterial leaf spot pathogenic bacteria infects
xa23during trans-genetic hybrid rice, another patent of invention of the effector albumin A vrXa23(contriver of pathogenic bacteria, patent name: the avrXa23 albumen and the encoding gene thereof that excite rice to generate disease resistance response, the patent No. is: ZL 201010256332.8) specific activation
xa23the expression of gene, and its proteins encoded Xa23 just causes rice cell generating program death (PCD) or hypersensitive necrosis (HR), the breeding within plant tissue of restriction germ and diffusion, thus reach disease-resistant object.Therefore, clone
xa23gene has very important value.The present invention will early stage
xa23gene transformation is in susceptible Cultivar Buddha's warrior attendant 30, be bred as near isogenic line CBB23(Zhang Q, et al. Development of Near-Isogenic Line CBB23 with a New Resistance Gene to Bacterial Blight in Rice and Its Application. Chinese J Rice Sci, 2002,16 (3): 206-210).Subsequently will
xa23the RFLP be positioned on paddy rice the 11st chromosome long arm marks 69B and EST and marks between CP02662, thus will
xa23within the scope of 1.7cM, (Wang Chunlian etc. utilize genomic library to accelerate to gene frame
xa23the chromosome walking of the assignment of genes gene mapping. rice in China science, 2006,4(20): 355-360).After again Fine Mapping is carried out to it, find 4 with
xa23the closely linked molecule marker of gene, one of them label L j74(and the application have submitted patent application on the same day, and its denomination of invention is: paddy disease-resistant gene
xa23molecule marker and application) with
xa23be divided into from, this molecule marker be clone
xa23gene provides the foundation, and provides approach for molecular mark.The present invention completes just on the basis of above-mentioned work.
Summary of the invention
On the basis that phase is studied before this invention, in order to effective utilization
xa23gene, in Rice molecular breeding, builds and has screened the BAC library of rice material kind CBB23 further, then by a series of test that has complementary functions, has finally cloned
xa23gene, thus completes the present invention.
Therefore, the invention provides a kind of paddy disease-resistant albumen, it is characterized in that, its aminoacid sequence is as shown in SEQ ID NO:2.
Correspondingly, the present invention also provides the encoding gene of above-mentioned paddy disease-resistant albumen, comprising its genome sequence.Preferably, its nucleotide sequence is as shown in SEQ ID NO:1.
And then the present invention goes back the expression vector of providing package containing said gene.Be preferably plant expression vector, best described gene and inducible promoter are operatively connected.
In addition, the present invention further provides described gene and cultivate the application in disease-resistant crop varieties.Preferably, by described vector introduction plant, screening obtains resistant plant.Described plant is monocotyledons or dicotyledons, especially farm crop, and most preferably, described plant is paddy rice, describedly disease-resistantly refers to the water resistant bacterial blight of rice.
The present invention has following beneficial effect: successful clone of the present invention
xa23gene, this gene is broad spectrum antidisease gene.Meanwhile, prove (see embodiment 3):
xa23gene is expressed in the dicotyledonss such as tobacco, also can cause dicotyledonous plant cells generation hypersensitive necrosis (HR), if controlled with other inducible promoter
xa23the expression of gene, just can be used for the disease control of other plant, therefore has purposes widely.Control with other inducible promoter
xa23the expression of gene, just can be used for other diseases controlling paddy rice, such as bacterial stripe, rice blast etc.Such as, not only can adopt the promotor that bacterial blight of rice is induced, also the OsQ16p promotor (Wang Guang etc. that Pyricularia oryzae is induced can be adopted, the clone of rice blast fungus abduction delivering promotor OsQ16p and functional analysis, Acta Agronomica Sinica the 6th phase in 2012), when paddy rice is subject to will inducing in transgenic plant when rice blast infects
xa23the expression of gene, causes hypersensitive necrosis and produces resistance to rice blast.Again such as, (bacterial leaf streak of rice is caused containing Xanthomonas campestris other mutation
xanthomonas oryzaepv.
oryzicola, cause capsicum and tomato spotted disease
xanthomonas campestrispv.
vesicatoria, cause citrus bacterial canker disease
xanthomonas citripv. citri, causes cotton angular leaf spot
xanthonwnas campestrispv.m
alvacearumetc.) artificial induction's type promotor (Humme AW of effector protein recognition sequences, Doyle EL and Bogdanove AJ, Addition of transcription activator-like effector binding sites to a pathogen strain-specific rice bacterial blight resistance gene makes it effective against additional strains and against bacterial leaf streak. New Phytologist, 2012,195 (4): 883-93) drive
xa23gene, just can be used for controlling bacterial leaf streak of rice, capsicum or tomato spotted disease, citrus bacterial canker disease, cotton angular leaf spot etc.
Accompanying drawing explanation
Fig. 1 is that pulse electrophoresis detects DNA blob of viscose Quality Map.
Wherein: 1,2: detect from the high molecular genome dna electrophoresis of the etiolated seedling extracting of CBB23, M:50kb marker.
Fig. 2 is that pulse electrophoresis detects the BAC library external source Insert Fragment size and joint efficiency figure that build.
Wherein: the mono-clonal plasmid DNA in 1-28:CBB23 BAC library, cut with Not I enzyme respectively, M1:50kb marker M2:1kb marker.
Fig. 3 molecule marker Lj74 screens BAC clone.
Fig. 4 screens TAC expressing gene library, A:Lj74 label screening TAC expressing gene library B: restriction enzyme
bamh I enzyme cuts TAC clone.
Fig. 5 transfer-gen plant is to the Resistant reaction of PXO99.
Fig. 6 transfer-gen plant (T189-11) to the Resistant reaction of PXO99, MDJ8: transformation receptor plant.S: disease-resistant R: susceptible.
Fig. 7 Southern detects external source in T189 transfer-gen plant and inserts the copy number of segment.
In Fig. 8 CBB23
xa23gene by the abduction delivering of bacterial leaf spot pathogenic bacteria PXO99, and associates with disease resistance response, A: disease-resistant blade (CBB23) and infected leaves (JG30) symptom, B:q-RTPCR detection by quantitative
xa23the expression of gene
In Fig. 9 transfer-gen plant
xa23gene by the abduction delivering of bacterial leaf spot pathogenic bacteria PXO99, and associates with disease resistance response, A: disease-resistant transgenic plant (T114-151) and susceptible recipient plant (MDJ8), and B:q-RTPCR detects in transfer-gen plant
xa23the expression of gene
Figure 10 RNAi transfer-gen plant is to the Resistant reaction of PXO99.
Figure 11 builds
xa23gene plant expression vector mode chart.
Figure 12
xa23the expression of gene in tobacco and HR detect.A, after injection, 2d samples blade; B, decolouring rear blade.
Embodiment
Detailed description below by embodiment illustrates the present invention further, but is not limitation of the present invention, only does example explanation.
embodiment 1 is separated and contains
xa23candidate gene sequence is cloned
1 vegetable material
Contain
xa23the near isogenic line CBB23 of gene.
carrier
For building the CopyControl that the carrier in the BAC library of CBB23 is Epicentre company
tMpCC1BAC
tMtransformable artificial expression vector pYLTAC747(Agricultural University Of South China doctor Liu Yaoguang provides), RNAi interference expression vector pTCK303 (WANG Z et al. A Practical Vector for Efficient Knockdown of Gene Expression in Rice (
oryza satival.). Plant Molecular Biology Reporter 22:409-417)..
3 BAC library constructions
To carry
xa23the seed of the near isogenic line CBB23 of gene is cultivated in 28-30 DEG C of darkroom and is obtained etiolated seedling, after liquid nitrogen grinding powdered, stirs gently successively at 50 orders, 200 orders, 400 order nylon mesh screen suspension filters in the Extraction buffer of precooling.Finally nucleus is embedded in sepharose, obtains the high molecular genomic dna (Fig. 1) of CBB23.Select effective dna, use
hindiII enzyme carries out partially digested to the high molecular genomic dna be embedded in sepharose block, chooses the DNA fragmentation of 100-150Kb, with the method eluted dna of electroelution.
By 100-150Kb DNA fragmentation and carrier pCC1BAC
tMconnect under the effect of T4 ligase enzyme, connect product conversion to competent cell DH10B.On LB solid medium, cultivate 20-30 hour, grow mono-clonal, random picking mono-clonal for 37 DEG C, extract plasmid DNA, use
not digested plasmid DNA, pulse electrophoresis detects transformation efficiency and Insert Fragment size.Result shows, this library reliable in quality, Insert Fragment size distribution between 70-150Kb, average Insert Fragment 108Kb (Fig. 2).Picking 36096 clone forms library altogether, and calculate by rice genome 430Mb, this library overing number rice genome 9.06 times, can be used for library screening.
screening BAC clone
After in toothpick picking white single bacterium colony to 384 orifice plate substratum, then dipped in by toothpick in the 1.5ml centrifuge tube containing LB substratum, namely each 1.5ml centrifuge tube contains 384 different single bacterium colonies, sets up BAC and clones pond.Alkaline lysis method of extracting mixing plasmid DNA after recovery.Early stage screened with
xa23gene is divided into from label L j74(as SEQ ID NO:4) (have submitted patent application on the same day with the application, its denomination of invention is: paddy disease-resistant gene
xa23molecule marker and application), therefore the mixing plasmid DNA in pond is cloned for template with each BAC, with the amplification of Lj74 mark, and set the DNA of CBB23 as contrast, select amplified production and CBB23 amplified production clone pond of the same size, after agarose gel electrophoresis detects, having one to clone pond amplification banding pattern consistent with CBB23 is BAC6.Again 384 the mono-clonal bacterium liquid being kept at (BAC6) in 384 orifice plates are dipped again with toothpick, respectively with the amplification of Lj74 mark, and set the DNA of CBB23 as contrast, amplification and CBB23 is selected to increase mono-clonal of the same size, after agarose gel electrophoresis detects, there is a clonal expansion banding pattern consistent with CBB23, called after B6O8(Fig. 3).
the Shotgun order-checking of clone and bioinformatic analysis
Shotgun (Shotgun) complete sequence determination is carried out to B6O8 clone, obtains 72114bp continuous print rice genomic DNA sequence.By predictive genes, it comprises 12 open reading frame (ORFs), different albumen of encoding respectively, and according to the tetraploid rice of ORF coded product and the position of Lj74 equimolecular mark, finally locking 6 ORFs is candidate gene.
expression vector establishment and screening
Because B6O8 clone can not be directly used in Genetic Transformation in Higher Plants, and the oryza sativa genomic dna fragment that external source is inserted is longer.Therefore adopt the artificially colored body expression vector pYLTAC747 that can be used for Plant Transformation, structure contains
xa23the plant expression vector of gene.PYLTAC747 more than bearing capacity 50Kb, in view of small segment easily transforms, can comprise again the consideration of more than 2 candidate genes, therefore BAC6O8 is built into the TAC expression library of about 15-20Kb.Direct is template with each monoclonal bacterium liquid, and with the amplification of Lj74 mark, screened 282 clones altogether, obtained 43 positive colonies, part clone identification is as Fig. 4 A, and alkaline lysis method of extracting plasmid DNA, uses restriction enzyme
bamh I enzyme is cut, and part clone enzyme is cut as Fig. 4 B.Compare according to the position size of actual endonuclease bamhi and predictive genes endonuclease bamhi (according to acquired 72114bp restriction enzyme site), consider the scope that candidate gene is crossed over simultaneously, finally select 10 candidate gene TAC to clone to be used for transforming, comprise T189 clone.And end sequencing is carried out to the two ends of these clones, obtain accurate location and the length of each clone.
embodiment 2 is obtained by rice transformation and expression analysis
xa23gene
1 vegetable material
Acceptor No. 8, susceptible rice varieties Mudanjiang (MDJ8), in acceptor disease resisting rice kind, military No. 1 (contains
xa23gene).
rice leaf spot bacteria system
The wide pathogenic bacterium PXO99 of bacterial blight of rice for inoculated identification, draws from International Rice Research Institute (IRRI), vacuum Bao Cun Yu – 70 DEG C, with front rejuvenation on side of body Ben Zheshi substratum, be placed in 28 DEG C and cultivate 48h, with sterilized water preparation inoculation bacterium liquid, concentration is adjusted to OD=1.0.
agrobacterium tumefaciens-mediated Transformation
Above-mentioned T189 etc. 10 is contained
xa23the TAC cloned plasmids DNA of candidate gene is transformed in Agrobacterium EH105 competent cell respectively, is transformed into respectively in No. 8, acceptor susceptible variety Mudanjiang by these 10 clones by agrobacterium-mediated transformation.Through a series of resistance screening, break up, the process such as to take root obtains transfer-gen plant.By 1350 the transgenosis test-tube plantlets obtained (wherein T189 clone has 258 strains), all transplant in Rice Cropping pond.
the Field inoculation qualification of transfer-gen plant and Molecular Detection
Adopt artificial leaf-cutting inoculation method, wide pathogenic bacterium PXO99 is inoculated on transfer-gen plant, and set No. 8, donor susceptible variety Mudanjiang as contrast.Latter about 2 weeks of inoculation, investigates when No. 8, susceptible variety Mudanjiang state of an illness tends towards stability.Result shows, in the 258 strain transgenic seedlings in TAC cloning vector T189 transformation receptor Mudanjiang, has 23 strains performance disease-resistant (Fig. 5,6).Other has 2 candidate genes clone (T30, T180) also to obtain 11 and 8 transgenosis disease-resistant plant respectively.By the verity of Molecular Detection checking transgenosis disease-resistant plant, the partial sequence selecting hygromycin gene is probe, the disease-resistant strain of Southern hybridization portion and susceptible strain, result shows that disease-resistant transgenic plant is integrated the existing single copy of exogenous array and has again two copy (Fig. 7), also confirms that disease-resistant plant is caused by the insertion due to foreign gene really further.Result shows that T189 clones the DNA fragmentation contained and carries disease-resistant gene
xa23.
the Subclone Library of clone builds
The external source of T189 clone inserts segment total length 18Kb, containing 2 ORFs.Do actually so a gene works, or 2 genes all work? therefore subclone has been carried out to T189 clone, built the TAC Subclone Library that fragment length is about 7-9Kb.Therefrom select 2 end sequencings that 58 clones carry out Insert Fragment, according to sequencing result, select 4 candidate clones such as W17 to carry out genetic transformation.
xa23the functional verification of gene
4 TAC subclones such as W17 are transformed into (method is with embodiment 2.3) in No. 8, acceptor susceptible variety Mudanjiang respectively by agrobacterium-mediated transformation, and W17 transforms acquisition 98 strain transgenic seedling, has 13 strains performance disease-resistant (Fig. 9 A) with PXO99 inoculated identification.It is exactly target gene
Xa23 that result shows that W17 clones the gene contained.w17 clone only contains a complete ORF, this gene DNA sequence total length 342bp(SEQ ID NO:1), encode one and comprise 113 amino acid whose ill-resistant protein (SEQ ID NO:2).
7
expression analysis proves
xa23genetic transcription by the induction of bacterial leaf spot pathogenic bacteria, and associates with disease resistance response
inoculate CBB23 and JG30, CBB23 apparent altitude with bacterial leaf spot pathogenic bacteria PXO99 disease-resistant, and JG30 seriously susceptible (Fig. 8 A).Get the blade of inoculation 1d, 2d, 3d, extract RNA respectively, become cDNA with the Reverse Transcription box reverse transcription of TIANGEN Biotech (Beijing) Co., Ltd., carry out fluorescence real-time quantitative PCR (qRTPCR) analysis with the SYBR SELECT MASTER MIX test kit of Invitrogen company.
According to
xa23gene order design quantitative fluorescent PCR primer:
Xa23BDF3:5’- GTAGAACAGCATGACCGAGAGAC，
Xa23BDF3:5’- GTAGCCGGTATACACATGATCCTC。
Conserved sequence design internal reference primer according to rice genome ubiquitin protein (Ubiquitin):
UbiqF:5’- GCTCCGTGGCGGTATCAT
UbiqR:5’- CGGCAGTTGACAGCCCTAG。
Primer after synthesis is diluted to 5 μMs.Real-time fluorescence quantitative PCR reaction system is each 1.6 μ L, cDNA template 1 μ L of 20 μ L:2 × SYBR Mix 10 μ L, primers F and R primer, Nuclease-Free Water 7.4 μ L.Each sample arranges 3 repetitions.Response procedures, 95 DEG C of denaturation 10min; 95 DEG C of sex change 15s, 60 DEG C of annealing 1min, read fluorescent value, 40 circulations.Be interior mark reference with the Ubiquitin gene of paddy rice, relative expression quantity=2
-Δ Ct, Δ Ct=(Ct
target gene-Ct
actin), Ct is fluorescence threshold, 7500 of instrument selection ABi company.Result shows
xa23the transcriptional expression of gene is induced by PXO99, and expression amount is the highest to 3d, and JG30 can't detect
xa23the expression (Fig. 8 B) of gene.
Again to the positive transgenic plant T114-151(Fig. 9 A obtained), and qRTPCR analysis (method is the same) has been carried out in No. 8, susceptible acceptor Mudanjiang.In transfer-gen plant T114
xa23the expression amount of gene is very high (Fig. 9 B), and No. 8, acceptor Mudanjiang can't detect
xa23the expression (Fig. 9 B) of gene.Further illustrate
xa23gene by the abduction delivering of bacterial leaf spot pathogenic bacteria, and associates with disease resistance.
8
rNAi interference experiment is verified further
xa23the DNA sequence dna of gene and function thereof
Verify further by gene knockout (RNAi interference) method
xa23the DNA sequence dna of gene and disease-resistant function.Get
xa23the DNA fragmentation (SEQ ID NO:3) that gene and 5 ' non-coding sequence thereof are total to 379bp builds RNAi interference expression vector pTCK23, is transformed into is contained by agrobacterium-mediated transformation (method is with embodiment 2)
xa23in the disease-resistant japonica rice variety of gene in military No. 1 genome, obtain 105 strain transgenic seedlings, after inoculated identification, have 69 strains performance susceptible (Figure 10), to show in disease-resistant japonica rice variety military No. 1
xa23after genetic expression is suppressed, transfer susceptible phenotype to by disease-resistant, this demonstrates further
xa23the DNA sequence dna of gene and function thereof.
embodiment 3 transient expression proves that Xa23 albumen causes dicotyledonous plant cells to produce anaphylaxis
1 vegetable material
This bright cigarette (
nicotiana benthamiana) plant in phytotron, culture condition is: illumination 16h/d, light intensity 30 ~ 40 μm of ols
-1m
-2, temperature 25 ~ 20 DEG C, humidity 50 ~ 60%; This bright cigarette seed directly broadcasts sowing in Nutrition Soil, transplant seedlings after growing two panels cotyledon, move in new Nutrition Soil and (add isopyknic vermiculite, be beneficial to Soil ventilation), every basin moves 3 ~ 4 strains, and period notes preventing soil insect pest, water and nutritive medium interval is carried out.
carrier
Fast PCR Clone Kit (Beijing Bioche-Service Sci and Tech company), plant expression vector pCAMBIA1305.
xa23the structure of gene plant expression vector
Adopt the method (Fast PCR Clone) of restructuring to construct 35S promoter to drive
xa23the plant expression vector of genes encoding frame.As shown in figure 11, increase
xa23the primer of gene with
ncoi and
pmli restriction enzyme site, PCR primer is recombinated pCAMBIA1305 carrier
pmlon I restriction enzyme site, use
ncoi enzyme is cut above-mentioned recombinant plasmid and is removed
gusgene fragment, is built into
xa23expression vector, called after 35S::
xa23aTG
.similarly, we construct
xa23two expression vector 35S: of gene start codon single base mutation:
xa23aCG and 35S::
xa23aGG.
Amplification
xa23aTG,
xa23aCG and
xa23the upstream primer of AGG is respectively: CACCATCACCATCACCCATGGCAAATGTTGCATCATCTCAAGGAG, CACCATCACCATCACCCATGGCAAAGCTTGCATCATCTCAAGGAG, CACCATCACCATCACCCATGGCAAAGGTTGCATCATCTCAAGGAG
Downstream primer is identical: GTCACCAATTCACACTCTCAAAACTTTGAGTTATAACATATA.
4 bright cigarette transient expressions
xa23gene and anaphylaxis
By above-mentioned 35S::
xa23aTG, 35S::
xa23aCG, 35S::
xa23aGG and not containing
xa23the empty carrier pCAMBIA1305 plant expression vector of gene proceeds in Agrobacterium EHA105 competent cell, with MMA damping fluid (10mM MgCl after rejuvenation
2, 10mM MES pH 5.5,100 μMs of Syringylethanones) and resuspended, adjust OD
600to 0.5 ~ 1.0, for injecting tobacco leaf after 30 DEG C of standing 3h.
Injection tobacco and decolouring: use punctures leaf epidermis before injection, go the syringe of syringe needle to puncture from vacuum side of blade with 1mL during injection and be expelled in blade by bacterium liquid, inject 3 strains, two blades are injected in every strain, after injecting 36h, 35S::
xa23aTG produces anaphylaxis (HR).Get the blade of injection after 2 days and put into culture dish, add after dehydrated alcohol is placed in 37 DEG C of incubators decolouring, 3 ~ 5h, add 70% ethanol and be placed in 37 DEG C of incubators and continue decolouring to leaf chlorophyll and all slough, preserve, take a picture.
5
xa23the protein Xa23 of genes encoding causes vegetable cell to produce anaphylaxis
Utilize above-mentioned bright cigarette transient expression system result display, 35S::
xa23aTG carrier can be expressed
xa23gene, and make this bright Tobacco Leaves produce anaphylaxis (Figure 12) in injection zone
.and
xa23gene start codon single base mutation body expression vector 35S::
xa23aCG and 35S::
xa23aGG contrasts with empty carrier pCAMBIA1305() the same, this bright Tobacco Leaves can not be caused to produce anaphylaxis (Figure 12), show
xa23gene exercises by coded protein Xa23 the function causing vegetable cell to produce anaphylaxis.
<110> Institute of Crop Science, Chinese Academy of Agricultural Science
<120> plant disease-resistant albumin X a23 and encoding gene thereof and application
<160> 4
<210> 1
<211> 342
<212> DNA
<400> 1
ATGTTGCATC ATCTCAAGGA GCTGGCAGCC GTAGCCGGTA TACACATGAT 50
CCTCATCTAC CTCTGCCGCT TTCTCCTCCG CCGCAGCCGC AACGTATTAT 100
TCACCGTTTC CAACAGCCTC CGTTTTCGCC TCAAGGTATT AACTGTATTG 150
TTGTACATAT GTCTCTCGGT CATGCTGTTC TACCTGTTTG GCTCCATCAT 200
GCCGCTGCCG CCGTGGGGCC TCGTGGTCGG TTGGGTCATG GCCCTCATCG 250
CCGTCGAGCT CGCCTACGCC TTCATCTTTC CATATAGCTT TCGCTACATC 300
GCTGACAACG ACGACGACAA GATGGTTATT CTCCCTGTTT AA 342
<210> 2
<211> 113
<212> amino acid
<400> 2
Met Leu His His Leu Lys Glu Leu Ala Ala Val Ala Gly Ile His Met Ile Leu Ile Tyr 20
Leu Cys Arg Phe Leu Leu Arg Arg Ser Arg Asn Val Leu Phe Thr Val Ser Asn Ser Leu 40
Arg Phe Arg Leu Lys Val Leu Thr Val Leu Leu Tyr Ile Cys Leu Ser Val Met Leu Phe 60
Tyr Leu Phe Gly Ser Ile MET Pro Leu Pro Pro Trp Gly Leu Val Val Gly Trp Val Met 80
Ala Leu Ile Ala Val Glu Leu Ala Tyr Ala Phe Ile Phe Pro Tyr Ser Phe Arg Tyr Ile 100
Ala Asp Asn Asp Asp Asp Lys Met Val Ile Leu Pro Val 113
<210> 3
<211> 379
<212> DNA
<400> 3
CGAAACATCT TCCTCCCGCA TCACTAACAT CAGCTTCTAT AAAAGCCCTT 50
CCTTGTTGCA TCATCTCAAG GAGCTGCAAG CACTTCCTCT CTGGCAGCAC 100
TTCCTCATCT CAAGGAGTTG CAAATGTTGC ATCATCTCAA GGAGCTGGCA 150
GCCGTAGCCG GTATACACAT GATCCTCATC TACCTCTGCC GCTTTCTCCT 200
CCGCCGCAGC CGCAACGTAT TATTCACCGT TTCCAACAGC CTCCGTTTTC 250
GCCTCAAGGT ATTAACTGTA TTGTTGTACA TATGTCTCTC GGTCATGCTG 300
TTCTACCTGT TTGGCTCCAT CATGCCGCTG CCGCCGTGGG GCCTCGTGGT 350
CGGTTGGGTC ATGGCCCTCA TCGCCGTCG 379
<210> 4
<211> 983
<212> DNA
<400> 4
AAGCCATTTG ATGAGCAACC CTGTAATTTG GTTGAACAAG GTAAATTCAC 50
AAGATAATGA CAGCATGTAT ATTTGCGGAT CAGTTAGTGG AAACTTAAAA 100
AATAGAATAG AATAGACCAA AAAGCTGAGC AACCCTGTAA TTTGGTTGAA 150
CAAGGTAAAT TCACAAGATA ATGACAGCAT GTATATTTGC GGATCAGTTA 200
GTGGAAACTT AAAAAATAGA ATAGAATAGA CCAAAAAGCA TATTGATCTT 250
TTAAATGCTC TAATACTGAC ATAATTATTG CAATACGAGA AGTTAAATAT 300
AGCTGGTTAT GAGGTCTCCA AACAACAGAA GAAATAGGAG GTTTAATTTA 350
CCTTGCTCCA TTCTTCAATG ACACAAGTGA ATATTACATG AGAACAACCT 400
TTTCAAGTTCA TACTGGTGG TGCCAACTTT TCTGTTTGAA AGGCCCTGTA 450
AAGAAAAACA AGCTTAGACA AGCTATCAAT AACGCTGGAG CAATGGTAGA 500
CTTCAATTCA GATTTTAATC TCTTAGAGGA GAAGGCAGTG ACTCAGTTAT 550
TAGGACAACA TGAAACAACT AGTATTGAGA GAGCATGATA CAAGAACTAA 600
TATATGTGAT CTTAACAAAA ATACAGCTTG TTATCAAGAC AAAGGTCTTT 650
GTGACTACAG GGTAGCTCAA GCAGAGGTCC TGGTTGTGAT CCTCATCCAT 700
GCAGAGGTGA AAATGAAAAG CAAAGAAACC CTAGTTTTAA AAGCAAAATA 750
GAAAGGACAA TTTCTTTCTG CTGCTCTTGT AGATCAAAAT TATAGCTTCA 800
TCAAGATTTC ATTGACGTCC TCAACCGTTG ACACATACTC ATCGATCAAG 850
TTCTCAACAT GTACACCATT GCAAGCATTC TCTCTTATCT ATAATAATGG 900
AGTTGGAAAA GTTTACTGCA ATCCATTAGC AAACACCAAA TACAGGATTA 950
GAATATACCT AAAGGTTATG CTGAAATGGA TCC 983
Claims (9)
1. a paddy disease-resistant albumen, is characterized in that, its aminoacid sequence is as shown in SEQ ID NO:2.
2. the encoding gene of paddy disease-resistant albumen as claimed in claim 1.
3. gene as claimed in claim 2, it is characterized in that, its nucleotide sequence is as shown in SEQ ID NO:1.
4. comprise the expression vector of the gene described in Claims 2 or 3.
5. expression vector as claimed in claim 4, it is characterized in that, described expression vector is plant expression vector.
6. the expression vector as described in claim 4 or 5, is characterized in that, described gene and inducible promoter are operatively connected.
7. the gene described in Claims 2 or 3 is cultivating the application in disease-resistant crop varieties.
8. apply as claimed in claim 7, it is characterized in that: by vector introduction plant as claimed in claim 6, screening obtains resistant plant, and described plant is unifacial leaf or dicotyledons.
9. apply as claimed in claim 8, it is characterized in that: described plant is paddy rice, and described resistance refers to the water resistant bacterial blight of rice.
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| CN103805624B (en) * | 2014-01-23 | 2015-10-07 | 中国农业科学院作物科学研究所 | A kind of simplified method and plasmid building TAL effector and TALENs |
| CN103923197B (en) * | 2014-04-16 | 2015-10-28 | 中国农业科学院作物科学研究所 | Derive from the disease resistance associated protein TaVIP2 of wheat and relevant biological material thereof and application |
| CN105063036B (en) * | 2014-04-25 | 2017-11-14 | 山东农业大学 | The molecular labeling of Xanthomonas oryzae pv oryzae and its application |
| CN103952403B (en) * | 2014-04-28 | 2016-09-28 | 中国农业科学院作物科学研究所 | The closely linked molecular marker of rice bacterial blight resistance new gene Xa39 |
| CN106146634B (en) * | 2015-04-15 | 2019-08-16 | 中国农业科学院作物科学研究所 | Plant disease-resistant protein B jMYB9 and its encoding gene and application |
| CN110272915B (en) * | 2018-12-11 | 2020-10-30 | 中国农业科学院作物科学研究所 | Method for cultivating bacterial leaf blight resistant rice by gene editing technology |
| CN111662367B (en) * | 2019-03-08 | 2021-06-22 | 广东省农业科学院植物保护研究所 | Rice bacterial leaf blight-resistant protein and coding gene and application thereof |
| CN111269915B (en) * | 2020-03-10 | 2022-01-04 | 中国农业科学院作物科学研究所 | Bacterial blight resistance related gene Xa39(t), related biological material thereof and method for cultivating bacterial blight resistant rice |
| CN111423498B (en) * | 2020-04-10 | 2021-11-26 | 华中农业大学 | Wild citrus resource AbTFIIA gamma gene and application thereof in citrus canker resistance |
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Application publication date: 20131002 Assignee: Guizhou Zhunong High tech Seed Industry Co.,Ltd. Assignor: INSTITUTE OF CROP SCIENCES, CHINESE ACADEMY OF AGRICULTURAL SCIENCES Contract record no.: X2023990000891 Denomination of invention: Plant Disease Resistance Protein Xa23 and Its Encoding Genes and Applications Granted publication date: 20150624 License type: Common License Record date: 20231030 |
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