CN105481958A - Application of protein OsOSM1 to regulation and control over plant disease resistance - Google Patents
Application of protein OsOSM1 to regulation and control over plant disease resistance Download PDFInfo
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Abstract
The invention discloses application of protein OsOSM1 to regulation and control over plant disease resistance. The protein OsOSM1 provided by the invention is a1), a2) or a3): a1) is protein, of which, the amino acid sequence is shown as the sequence 2 in a sequence table; a2) is fused protein obtained by connecting the N-terminal or/and C-terminal of protein shown as sequence 2 in the sequence table with labels; a3) is protein obtained by replacing the amino acid sequence shown as sequence 2 in the sequence table with one or more amino acid residues, deleting one or more amino acid residues from the amino acid sequence shown as sequence 2 in the sequence table and/or adding one or more amino acid residues into the amino acid sequence shown as sequence 2 in the sequence table. Experimental results show that the resistance of rice to sheath blight can be improved through OsOSM1 gene expression in rice, the sheath blight grade is lower than that of wild rice and an important effect on breeding of sheath-blight-resistant new materials is realized.
Description
Technical field
The present invention relates to biological technical field, be specifically related to the application of protein OsOSM1 in regulating plant disease resistance.
Background technology
Banded sclerotial blight is one of most important disease of global paddy rice, and along with breeding wheat for semidwarfness and employing that is high fertile, planting culture technology, banded sclerotial blight harm increases the weight of gradually, and in many places in south China rice district, banded sclerotial blight has become the first disease of paddy rice.Rice sheath blight disease pathogenic bacterium are dry thread Pyrenomycetes (Rhizoctoniasolani), belong to wide host range, strong S fungi, its main harm rice leaf sheath, blade also can be fallen ill, cause leaves water loss dead, finally cause the significantly underproduction and quality to reduce.
Paddy rice is typical amounts proterties to the resistance of banded sclerotial blight, easily affected by environment, can only utilize Quantitative gene in breeding for disease resistance, causes the breeding of Rice Resistance banded sclerotial blight to be in progress slower always.Have so far more than 50 anti-banded sclerotial blight QTLs (quantitativetraitloci) by Primary Location, but the resistance effect of each QTL is all less, the report that so far there are no clones, affects the process of being cultivated anti-banded sclerotial blight breeding novel material by marker assisted selection technology.Therefore, in the urgent need to adopting molecular breeding means, by the introducing of foreign gene or the genetic manipulation to inherent gene, orientation adjustment plant, to the resistance of banded sclerotial blight, accelerates the seed selection process of anti-banded sclerotial blight novel material.
Summary of the invention
Technical problem to be solved by this invention how to improve the disease resistance of plant.
For solving the problems of the technologies described above, the present invention provide firstly the application of protein OsOSM1 in regulating plant disease resistance; Described protein OsOSM1 is a1) or a2) or a3):
A1) aminoacid sequence is the protein shown in sequence 2 in sequence table;
A2) in sequence table, the N of the protein shown in sequence 2 holds or/and C end connects the fused protein that label obtains;
A3) by the protein relevant to disease resistance that the aminoacid sequence shown in sequence in sequence table 2 obtains through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation.
Wherein, in sequence table, sequence 2 can be made up of 233 amino-acid residues.
In order to make a1) in protein be convenient to purifying, in sequence table, the N-terminal of the protein shown in sequence 2 or C-terminal can connect label as shown in table 1.
The sequence of table 1, label
Above-mentioned a3) in protein OsOSM1, the replacement of one or several amino-acid residue described and/or disappearance and/or be added to the replacement and/or disappearance and/or interpolation that are no more than 10 amino-acid residues.
Above-mentioned a3) in protein OsOSM1 can synthetic, also can first synthesize its encoding gene, then carry out biological expression and obtain.
Above-mentioned a3) in the encoding gene of protein OsOSM1 by the codon by lacking one or several amino-acid residue in the DNA sequence dna shown in sequence in sequence table 1, and/or carry out the missense mutation of one or several base pair, and/or the encoding sequence connecting the label shown in table 1 is held to obtain at its 5 ' end and/or 3 '.
The application of nucleic acid molecule in regulating plant disease resistance of code for said proteins OsOSM1 also belongs to protection scope of the present invention.
The nucleic acid molecule of described code for said proteins OsOSM1 can be following b1) or b2) or b3) shown in DNA molecular:
B1) nucleotide sequence is the DNA molecular shown in sequence 1 in sequence table;
B2) nucleotide sequence and b1) limited has more than 75% or 75% identity, and the DNA molecular of code for said proteins OsOSM1;
B3) nucleotide sequence hybridization limited with (b1) or (b2) under strict conditions, and the DNA molecular of code for said proteins OsOSM1.
Wherein, described nucleic acid molecule can be DNA, as cDNA, genomic dna or recombinant DNA; Described nucleic acid molecule can be also RNA, as mRNA or hnRNA etc.
Wherein, in sequence table, sequence 1 is made up of 702 Nucleotide, the aminoacid sequence shown in sequence 2 in polynucleotide.
Those of ordinary skill in the art can adopt known method easily, the method for such as orthogenesis and point mutation, suddenly change to the nucleotide sequence of protein OsOSM1 of the present invention.Those are through manually modified, have and be separated the nucleotide sequence 75% of the protein OsOSM1 obtained or the Nucleotide of higher identity with the present invention, as long as coded protein OsOSM1 and there is protein OsOSM1 function, be all be derived from nucleotide sequence of the present invention and be equal to sequence of the present invention.
Term used herein " identity " refers to the sequence similarity with native sequence nucleic acid.The nucleotide sequence that " identity " comprises the protein formed with the aminoacid sequence shown in sequence 2 in polynucleotide of the present invention has 75% or higher, or 85% or higher, or 90% or higher, or the nucleotide sequence of 95% or higher identity.Identity can with the naked eye or computer software evaluate.Use computer software, the identity between two or more sequence can represent with per-cent (%), and it can be used for evaluating the identity between correlated series.
In above-mentioned application, described plant can be following c1) to c5) in any one:
C1) dicotyledons;
C2) monocotyledons;
C3) grass;
C4) paddy rice;
C5) rice varieties Xu rice No. 3.
In above-mentioned application, described disease resistance can be the microbial disease of anti-miliary damping-off.Described dry thread Pyrenomycetes specifically can be rice sheath blight disease bacterial strain YN-7.
In above-mentioned application, described disease resistance can be anti-banded sclerotial blight.Described banded sclerotial blight can be the microbial disease of miliary damping-off.Described dry thread Pyrenomycetes specifically can be rice sheath blight disease bacterial strain YN-7.
For solving the problem, present invention also offers a kind of method of cultivating disease resistant transgenic plants.
The method of cultivation disease resistant transgenic plants provided by the present invention, comprises the steps: the encoding gene importing described protein OsOSM1 in recipient plant, obtains the transgenic plant that disease resistance is better than described recipient plant.
In aforesaid method, the encoding gene of described protein OsOSM1 can be following b1) or b2) or b3) shown in DNA molecular:
B1) nucleotide sequence is the DNA molecular shown in sequence 1 in sequence table;
B2) nucleotide sequence and b1) limited has more than 75% or 75% identity, and the DNA molecular of code for said proteins OsOSM1;
B3) nucleotide sequence hybridization limited with (b1) or (b2) under strict conditions, and the DNA molecular of code for said proteins OsOSM1.
In aforesaid method, described recipient plant can be following c1) to c5) in any one:
C1) dicotyledons;
C2) monocotyledons;
C3) grass;
C4) paddy rice;
C5) rice varieties Xu rice No. 3.
In aforesaid method, described " in recipient plant, importing the encoding gene of described protein OsOSM1 " realizes by importing recombinant vectors in recipient plant; Described recombinant vectors can be and the encoding gene of described protein OsOSM1 is inserted the recombinant plasmid that plasmid obtains that sets out.
Described recombinant vectors specifically can be recombinant plasmid pCAMBIA1301/Ubi-OsOSM1.Fragment (being limited property of plant expression vector pCAMBIA1301/Ubi endonuclease BamHI and KpnI is cut into a large fragment and a small segment, and this DNA is this small segment) between described recombinant plasmid pCAMBIA1301/Ubi-OsOSM1 specifically can be BamHI and the KpnI recognition sequence of plant expression vector pCAMBIA1301/Ubi replaces with the DNA molecular shown in sequence 1 in sequence table.
In aforesaid method, described disease resistance can be the microbial disease of anti-miliary damping-off.Described dry thread Pyrenomycetes specifically can be rice sheath blight disease bacterial strain YN-7.
In aforesaid method, described disease resistance can be anti-banded sclerotial blight.Described banded sclerotial blight can be the microbial disease of miliary damping-off.Described dry thread Pyrenomycetes specifically can be rice sheath blight disease bacterial strain YN-7.
Experiment proves, protein OsOSM1 provided by the present invention can improve the disease resistance of plant: T
2be experiment material for transgenic paddy rice strain L1, strain L2, strain L3, strain L4, strain L5, strain L6, strain L7, strain L8, strain L9 and wild rice, adopt at rice tillering latter stage to the jointing initial stage and embed inoculation method inoculating strain YN-7, at experiment material heading latter 35 days record experiment material field banded sclerotial blight incidences, result shows, the sick level of banded sclerotial blight of strain L1, strain L2, strain L3, strain L4, strain L5, strain L6, strain L7, strain L8 and strain L9 is all lower than the sick level of banded sclerotial blight of wild rice.Show, protein OsOSM1 can be utilized to improve paddy rice to the resistance of banded sclerotial blight, and the seed selection of antagonism banded sclerotial blight novel material has vital role.
Accompanying drawing explanation
Fig. 1 is the expression pattern analysis of OsOSM1 gene.
Fig. 2 is T
2for expression analysis and the sick level of banded sclerotial blight of OsOSM1 gene in transgenic paddy rice strain.
Fig. 3 is wild rice and T after inoculating strain YN-735 days
2field banded sclerotial blight morbidity for transgenic paddy rice strain is compared.
Fig. 4 is T
2for the transgenic paddy rice strain heading plant type of latter 30 days and the heading fringe portion phenotype of latter 60 days.
Fig. 5 is the Subcellular Localization result of OsOSM1 gene.
Embodiment
Below in conjunction with embodiment, the present invention is further described in detail, the embodiment provided only in order to illustrate the present invention, instead of in order to limit the scope of the invention.
Experimental technique in following embodiment, if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
In following embodiment, rice sheath blight disease bacterial strain YN-7, rice varieties YSBR1, rice varieties Lemont are all documented in as in Publication about Document: a left side shows quick etc., and Different resistance levels rice varieties is to the defense response of sheath blight fungus toxin and differences of Physiological.Rice in China science, 2014,28 (5): 551-558., the public can obtain from Yangzhou University (i.e. applicant), to repeat this experiment.Rice sheath blight disease bacterial strain YN-7 is referred to as bacterial strain YN-7; Rice varieties YSBR1 is disease-resistant variety, hereinafter referred to as YSBR1; Rice varieties Lemont is susceptible variety, hereinafter referred to as Lemont.
Carrier pGDG is documented in as in Publication about Document: GoodinMM, DietzgenRG, SchichnesD, RuzinS, JacksonAO.pGDvectors:versatiletoolsfortheexpressionofgre enandredfluorescentproteinfusionsinagroinfiltratedplantl eaves.PlantJ.2002Aug; 31 (3): the 375-83. public can obtain from Yangzhou University (i.e. applicant), to repeat this experiment.
Rice varieties Japan is fine to be documented in as in Publication about Document: Li Lei, Xue's fragrance, etc. the suitableeest fine Leaf rolling index research of rice varieties Japan. Yangzhou University's journal: hereinafter, rice varieties Japan is fine fine referred to as Japan for agricultural and life science version .2003 in June.
Xu rice No. 3 is documented in as in Publication about Document: Liu Chao, Wang Jiankang, Deng. round-grained rice new variety Xu's rice No. 3 features and cultivation technique in good quality and high output. Jiangsu's agriculture science .2003 (6): 42-43. hereinafter, and Xu rice No. 3 is referred to as XD3 or wild rice (WT).
This life of tobacco bred cigarette is documented in as in Publication about Document: Cui Haitao, Li Chunxia, etc. the foundation of this life cigarette tissue culture and genetic conversion system. Shandong science .2006 (19): this life of 23-27. tobacco bred cigarette is called for short tobacco hereinafter.
Agrobacterium tumefaciens AGL1 and agrobacterium tumefaciens EHA105 is all documented in as in Publication about Document: easily rely on oneself, Cao Shouyun, etc. improve the research of Agrobacterium-mediated Transformation paddy rice frequency. Acta Genetica Sinica .2001,28 (4): 352-358.
Reversed transcriptive enzyme is Roche Products.
Plant expression vector pCAMBIA1301/Ubi is documented in as in Publication about Document: Li Lei, Xue's fragrance, etc. a kind of novel method transforming vector multiple cloning site. biotechnology .2013 23 (4).
Embedding inoculation method in following embodiment is documented in as in Publication about Document: a left side shows quick etc., the establishment of field water sheath and culm blight of rice Resistance Identification system and perfect. Yangzhou University's journal: agricultural and life science version .2007 (27): 57-61.)
The acquisition of the transgenic paddy rice that embodiment 1, anti-banded sclerotial blight strengthen and qualification
One, the structure of recombinant vectors and recombinational agrobacterium
The present invention finds the genes involved by rice sheath blight disease bacterial strain YN-7 abduction delivering in YSBR1 by gene chip, and wherein 1 gene is positioned on the 12nd karyomit(e), by its called after OsOSM1 gene.
Recombinant vectors pCAMBIA1301/Ubi-OsOSM1 is built according to following step:
1, the acquisition of template: adopt Trizol method to extract the total serum IgE of the fine young leaflet tablet of rice varieties Japan, this total serum IgE reversed transcriptive enzyme reverse transcription is gone out the first chain cDNA.
2, synthetic primer CDS-F:5 '-CG
gGATCCaTGGCGAACAAGCTGCAGCTC-3 ' (underscore is restriction enzyme BamHI recognition sequence) and CDS-R:5 '-GG
gGTACCcTAGTTGTCGGCGACGTCGAC-3 ' (underscore is restriction enzyme KpnI recognition sequence).
3, after completing steps 1 and 2, the cDNA obtained with step 1 is for template, carry out pcr amplification with CDS-F and CDS-R of the 2-in-1 one-tenth of step for primer, the N end obtaining about 718bp holds the double chain DNA molecule containing restriction enzyme KpnI containing restriction enzyme BamHI and C.
4, the double chain DNA molecule containing restriction enzyme KpnII containing restriction enzyme BamHI and C end is held by the N obtained in step 3 to be connected to pMD
tM19 (simple) carrier T (Takara Products, catalog number is 3271), obtains recombinant plasmid pMD19-OsOSM1.
5, after completing steps 4, with restriction enzyme BamHI and KpnI double digestion recombinant plasmid pMD19-OsOSM1, the fragment of about 718bp is reclaimed.
6, with restriction enzyme BamHI and KpnI double digestion plant expression vector pCAMBIA1301/Ubi, the carrier framework of about 14088bp is reclaimed.
7, the carrier framework that fragment step 5 obtained and step 6 obtain is connected, and obtains recombinant plasmid pCAMBIA1301/Ubi-OsOSM1 (A in Fig. 2 is shown in by part-structure schematic diagram).
According to sequencing result, structrual description carries out to recombinant plasmid pCAMBIA1301/Ubi-OsOSM1 as follows: to plant expression vector pCAMBIA1301/Ubi BamHI and KpnI restriction enzyme site between to insert nucleotide sequence be the DNA molecular shown in sequence 1 in sequence table.OsOSM1 albumen shown in sequence 2 in recombinant plasmid pCAMBIA1301/Ubi-OsOSM1 expressed sequence table.
Recombinant plasmid pCAMBIA1301/Ubi-OsOSM1 is imported agrobacterium tumefaciens AGL1, obtains recombinational agrobacterium, called after AGL1/pCAMBIA1301/Ubi-OsOSM1.
Empty carrier plasmid pCAMBIA1301/Ubi is imported agrobacterium tumefaciens AGL1, obtains the recombinational agrobacterium containing plasmid pCAMBIA1301/Ubi, called after AGL1/pCAMBIA1301/Ubi.
Two, the regeneration of Transgenic Rice Plants
By wild rice seed shelling sterilizing, then adopt paddy rice such as transformed wild type such as AGL1/pCAMBIA1301/Ubi-OsOSM1 such as the method (Hiei, Y.etal.PlantJ, 1994,6 (2): 271-282) of Hiei etc., obtain T
0for transgenic rice plant.T
0after transgenic rice plant sowing, sow after hygromycin selection, obtain T
1for transgenic paddy rice, T
1after transgenic paddy rice sowing, again sow after hygromycin selection, obtain T
2for transgenic paddy rice, get 9 T respectively
2for the T of transgenic paddy rice strain (strain L1, strain L2, strain L3, strain L4, strain L5, strain L6, strain L7, strain L8 and strain L9)
2the experiment of subsequent step four is carried out for plant.
According to the method described above, AGL1/pCAMBIA1301/Ubi-OsOSM1 is replaced with AGL1/pCAMBIA1301/Ubi, other steps are all identical, obtain T
2for the plant turning empty carrier paddy rice, hereinafter referred to as turning empty carrier paddy rice.
Three, the expression pattern analysis of OsOSM1 gene
1, Real_time quantitative detection is in the relative expression quantity of OsOSM1 gene in the paddy rice sample of different growing
Carry out three revision tests, the step of each revision test is all as follows:
(1) sample is obtained
Get the blade of the wild rice being in seedling stage, put into Liquid nitrogen storage, obtain sample 1.
Then according to the method described above, will replace with tillering phase, boot stage, heading stage and filling stage respectively seedling stage, other step is all constant, obtains sample 2, sample 3, sample 4 and sample 5.
(2) expression pattern analysis of OsOSM1 gene
Adopt the total serum IgE of sample 1 in Trizol method extraction step (1), this total serum IgE reversed transcriptive enzyme reverse transcription is gone out the first chain cDNA, then with this cDNA for template, the relative expression quantity (using Actin gene as reference gene) of OsOSM1 gene in Real_time quantitative detection sample 1.In sample 1, the expression amount of Actin gene is as 1, and in sample 1, the relative expression quantity of OsOSM1 gene is shown in A in Fig. 1.
Identify that the primer of OsOSM1 gene is 5 '-CCGACAACTAGCTAGCCTA-3 ' and 5 '-CGCACGTACAAACATAAGG-3 '.Internal reference primer is Actin-F:5 '-CTTCATAGGAATGGAAGCTGCGGGTA-3 ' and Actin-R:5 '-CGACCACCTTGATCTTCATGCTGCTA-3 '.
According to above-mentioned steps, sample 1 is replaced with respectively sample 2, sample 3, sample 4 and sample 5, other step is all identical, obtains the relative expression quantity (in Fig. 1 A) of OsOSM1 gene in sample 2, sample 3, sample 4 and sample 5.
Experimental result shows, OsOSM1 gene is the highest at the relative expression quantity of boot stage (sample 3).
The relative expression quantity of OsOSM1 gene in the paddy rice sample of 2, Real_time quantitative detection Different Organs in boot stage
Carry out three revision tests, the step of each revision test is all as follows:
(1) sample is obtained
Get the root of the wild rice being in boot stage, put into Liquid nitrogen storage, obtain sample 6.
Then according to the method described above, root is replaced with stem, leaf, leaf sheath and fringe respectively, other step is all constant, obtains sample 7, sample 8, sample 9 and sample 10.
(2) expression pattern analysis of OsOSM1 gene
Adopt the total serum IgE of sample 6 in Trizol method extraction step (1), this total serum IgE reversed transcriptive enzyme reverse transcription is gone out the first chain cDNA, then with this cDNA for template, the relative expression quantity (using Actin gene as reference gene) of OsOSM1 gene in Real_time quantitative detection sample 6.In sample 6, the expression amount of Actin gene is as 1, and in sample 6, the relative expression quantity of OsOSM1 gene is shown in B in Fig. 1.
Identify that the primer of OsOSM1 gene is 5 '-CCGACAACTAGCTAGCCTA-3 ' and 5 '-CGCACGTACAAACATAAGG-3 '.Internal reference primer is Actin-F:5 '-CTTCATAGGAATGGAAGCTGCGGGTA-3 ' and Actin-R:5 '-CGACCACCTTGATCTTCATGCTGCTA-3 '.
According to above-mentioned steps, sample 6 is replaced with respectively sample 7, sample 8, sample 9 and sample 10, other step is all identical, obtains the relative expression quantity (in Fig. 1 B) of OsOSM1 gene in sample 7, sample 8, sample 9 and sample 10.
Experimental result shows, the relative expression quantity of OsOSM1 gene in leaf sheath (sample 9) is the highest.
The experimental result of step 1 and step 2 mainly seriously to occur and the feature of main harm leaf sheath fits like a glove in boot stage with rice sheath blight disease.
3, the relative expression quantity of OsOSM1 gene in the YSBR1 before and after Real_time quantitative detection inoculating strain YN-7
Carry out three revision tests, the step of each revision test is all as follows:
(1) sample is obtained
A, the seedling of the growth YSBR1 of 4 weeks adopted embed inoculation method inoculating strain YN-7, get the blade of 72h after the blade of 48h after the blade of 24h after the blade of 12h after the blade of 0h before inoculation, inoculation, inoculation, inoculation and inoculation respectively, put into Liquid nitrogen storage, by sample called after sample 11, sample 12, sample 13, sample 14 and the sample 15 successively obtained.
According to the method described above, the seedling of the growth YSBR1 of 4 weeks is not carried out any process, get blade at same time point, put into Liquid nitrogen storage, by sample called after sample 21, sample 22, sample 23, sample 24 and the sample 25 successively obtained.
B, the seedling of the growth Lemont of 4 weeks adopted embed inoculation method inoculating strain YN-7, get the blade of 72h after the blade of 48h after the blade of 24h after the blade of 12h after the blade of 0h before inoculation, inoculation, inoculation, inoculation and inoculation respectively, put into Liquid nitrogen storage, by sample called after sample 31, sample 32, sample 33, sample 34 and the sample 35 successively obtained.
According to the method described above, the seedling of the growth Lemont of 4 weeks is not carried out any process, get blade at same time point, put into Liquid nitrogen storage, by sample called after sample 41, sample 42, sample 43, sample 44 and the sample 45 successively obtained.
C, the seedling of the growth wild rice of 4 weeks adopted embed inoculation method inoculating strain YN-7, get the blade of 72h after the blade of 48h after the blade of 24h after the blade of 12h after the blade of 0h before inoculation, inoculation, inoculation, inoculation and inoculation respectively, put into Liquid nitrogen storage, by sample called after sample 51, sample 52, sample 53, sample 54 and the sample 55 successively obtained.
According to the method described above, the seedling of the growth wild rice of 4 weeks is not carried out any process, get blade at same time point, put into Liquid nitrogen storage, by sample called after sample 61, sample 62, sample 63, sample 64 and the sample 65 successively obtained.
(2) expression pattern analysis of OsOSM1 gene
Adopt the total serum IgE of sample 11 or sample 21 in Trizol method extraction step (1), this total serum IgE reversed transcriptive enzyme reverse transcription gone out the first chain cDNA, then with this cDNA for template, the relative expression quantity of OsOSM1 gene in Real_time quantitative detection sample 11.In sample 21, the expression amount of OsOSM1 gene is as 1, and in sample 11, the relative expression quantity of OsOSM1 gene is shown in C in Fig. 1.Identify that the primer of OsOSM1 gene is 5 '-CCGACAACTAGCTAGCCTA-3 ' and 5 '-CGCACGTACAAACATAAGG-3 '.
According to above-mentioned steps, sample 11 is replaced with sample 12, sample 21 replaces with sample 22, and other step is all constant, obtains the relative expression quantity of OsOSM1 gene in sample 12; Sample 11 is replaced with sample 13, and sample 21 replaces with sample 23, and other step is all constant, obtains the relative expression quantity of OsOSM1 gene in sample 13; Sample 11 is replaced with sample 14, and sample 21 replaces with sample 24, and other step is all constant, obtains the relative expression quantity of OsOSM1 gene in sample 14; Sample 11 is replaced with sample 15, and sample 21 replaces with sample 25, and other step is all constant, obtains the relative expression quantity of OsOSM1 gene in sample 15; Sample 11 is replaced with sample 31, and sample 21 replaces with sample 41, and other step is all constant, obtains the relative expression quantity of OsOSM1 gene in sample 31; Sample 11 is replaced with sample 32, and sample 21 replaces with sample 42, and other step is all constant, obtains the relative expression quantity of OsOSM1 gene in sample 32; Sample 11 is replaced with sample 33, and sample 21 replaces with sample 43, and other step is all constant, obtains the relative expression quantity of OsOSM1 gene in sample 33; Sample 11 is replaced with sample 34, and sample 21 replaces with sample 44, and other step is all constant, obtains the relative expression quantity of OsOSM1 gene in sample 34; Sample 11 is replaced with sample 35, and sample 21 replaces with sample 45, and other step is all constant, obtains the relative expression quantity of OsOSM1 gene in sample 35; Sample 11 is replaced with sample 51, and sample 21 replaces with sample 61, and other step is all constant, obtains the relative expression quantity of OsOSM1 gene in sample 51; Sample 11 is replaced with sample 52, and sample 21 replaces with sample 62, and other step is all constant, obtains the relative expression quantity of OsOSM1 gene in sample 52; Sample 11 is replaced with sample 53, and sample 21 replaces with sample 63, and other step is all constant, obtains the relative expression quantity of OsOSM1 gene in sample 53; Sample 11 is replaced with sample 54, and sample 21 replaces with sample 64, and other step is all constant, obtains the relative expression quantity of OsOSM1 gene in sample 54; Sample 11 is replaced with sample 55, and sample 21 replaces with sample 65, and other step is all constant, obtains the relative expression quantity of OsOSM1 gene in sample 55.
Experimental result is shown in C in Fig. 1, result shows, in YSBR1, the expression amount of OsOSM1 gene acutely raises because of infecting of bacterial strain YN-7, in Lemont and Xu rice No. 3, the expression amount of OsOSM1 gene also has certain rise because of infecting of bacterial strain YN-7, and visible OsOSM1 gene infects in the process of paddy rice at suppression bacterial strain YN-7 and has important effect.
Four, the sharp eyespot resistance qualification of transgenic paddy rice
1, the qualification of transgenic paddy rice
Adopt Trizol method to extract the RNA being in the leaf sheath tissue of the strain L1 in boot stage, obtain the RNA of strain L1, this total serum IgE reversed transcriptive enzyme reverse transcription is gone out the first chain cDNA, referred to as the cDNA of strain L1.
According to the method described above, strain L1 is replaced with strain L2, strain L3, strain L4, strain L5, strain L6, strain L7, strain L8 and strain L9 respectively, other step is all constant, obtains the cDNA of strain L2, the cDNA of strain L3, the cDNA of strain L4, the cDNA of strain L5, cDNA, the cDNA of strain L7 of strain L6, the cDNA of the cDNA of strain L8 and strain L9.
According to the method described above, strain L1 is replaced with wild rice, obtain contrasting cDNA.
According to the method described above, strain L1 is replaced with and turns empty carrier paddy rice, obtain unloaded contrast cDNA.
With above-mentioned cDNA for template, the relative expression quantity (with Actin gene for internal reference) of Real_time quantitative detection OsOSM1 gene.
The primer of qualification OsOSM1 gene and internal reference are with 1 in step 3.
Using the expression amount of OsOSM1 gene in wild rice as 1, in strain L1-strain L9, the relative expression quantity of OsOSM1 gene is shown in B in Fig. 2.Result shows, in strain L1, strain L2, strain L3, strain L4, strain L5, strain L6, strain L7, strain L8 and strain L9, the expression amount of OsOSM1 gene is respectively 4.63 times, 1.58 times, 3.49 times, 3.53 times, 4.17 times, 8.69 times, 13.82 times, 18.98 times and 11.07 times of the expression amount of OsOSM1 gene in wild rice.Wild rice and turn the expression amount of OsOSM1 gene in empty carrier paddy rice without significant difference.
2, the sharp eyespot resistance qualification of transgenic paddy rice
With wild rice, turn empty carrier paddy rice, strain L1, strain L2, strain L3, strain L4, strain L5, strain L6, strain L7, strain L8 and strain L9 for experiment material, measure the sharp eyespot resistance of paddy rice.
In triplicate, each strain plants 10 strains at every turn in experiment, adopts embed inoculation method inoculating strain YN-7 at rice tillering latter stage to the jointing initial stage.Every strain paddy rice inoculates 3 stem stalks, and inoculum embeds from top to bottom inside the 3rd leaf sheath with tweezers by each stem stalk.
Experiment material heading latter 35 days record experiment material field banded sclerotial blight incidences, and carry out the investigation of disease level according to the banded sclerotial blight disease scale standard be documented in document (left side shows quick etc., Yangzhou University's journal: agricultural and life science version .2007 (27): 57-61).Result shows (in Fig. 2 C and Fig. 3), the sick level of banded sclerotial blight of strain L1, strain L2, strain L3, strain L4, strain L5, strain L6, strain L7, strain L8 and strain L9 is all lower than the sick level of banded sclerotial blight of wild rice, and wild rice and the sick level of the banded sclerotial blight turning empty carrier paddy rice are without significant difference.Visible, in paddy rice, process LAN OsOSM1 gene can improve the resistance of paddy rice to banded sclerotial blight.
Five, the agronomy correlated character of transgenic rice plant and the observation of Correlated Yield Characters and statistics
With wild rice, strain L1, strain L2, strain L3, strain L4, strain L5, strain L6, strain L7, strain L8 and strain L9 for experiment material, carry out observation and the statistics of agronomy correlated character and Correlated Yield Characters.
Experiment in triplicate, each strain plants 10 strains at every turn, treat that experiment material is in the ripening stage, observe and add up following economical character: breeding time, angle of tillering, plant height, sword-like leave is high, sword-like leave is long, sword-like leave is wide, spike length, grain length, grain are wide, panicle number per hill, number of grain per ear, setting percentage, thousand seed weight, single plant yield.Experimental result is in table 2, table 3 and Fig. 4 (A is the heading plant type phenotype of latter 30 days, and B is the heading fringe portion phenotype of latter 60 days, and C is the heading seed phenotype of latter 60 days, and wherein left figure is wild rice, and right figure is strain L3).Result shows, the agronomy correlated character of strain L1, strain L2, strain L3, strain L4, strain L5, strain L6, strain L7, strain L8, strain L9 and wild rice and Correlated Yield Characters do not have difference substantially.
Table 2. agronomy correlated character statistics
Note: HD is heading stage, and TA is angle of tillering, and PH is plant height, and FLL is that sword-like leave is long, and FLW is that sword-like leave is wide.
Small English alphabet shows the significant difference of 5% level, and same letter shows do not have significant difference.
Table 3. Correlated Yield Characters statistics
Note: PL is spike length, and GL is grain length, and GW is that grain is wide, and PNP is panicle number per hill, and SNP is number of grain per ear, and RFG is setting percentage, and 1000GW is thousand seed weight, and PY is single plant yield.Small English alphabet shows the significant difference of 5% level, and same letter shows do not have significant difference.
Six, OsOSM1 gene Subcellular Localization
A, build recombinant vectors pGDG/OsOSM1-GFP according to following step:
1, the acquisition of template: adopt Trizol method to extract the total serum IgE of the fine young leaflet tablet of rice varieties Japan, this total serum IgE reversed transcriptive enzyme reverse transcription is gone out the first chain cDNA.
2, synthetic primer F:5 '-CCG
cTCGAGaTGGCGAACAAGCTGCAGCTC-3 ' (underscore is restriction enzyme XhoI recognition sequence) and R:5 '-CG
gGATCCcTAGTTGTCGGCGACGTCGAC-3 ' (underscore is restriction enzyme BamHI recognition sequence).
3, after completing steps 1 and 2, the cDNA obtained with step 1, for template, carries out pcr amplification with F and R of the 2-in-1 one-tenth of step for primer, and the N end obtaining about 719bp holds the double chain DNA molecule containing restriction enzyme BamHI containing restriction enzyme XhoI and C.
4, held by the N obtained in step 3 double chain DNA molecule containing restriction enzyme BamHI containing restriction enzyme XhoI and C end to be connected to carrier pGDG, obtain recombinant plasmid 35S::GFP-OsOSM1 (A in Fig. 5 is shown in by part-structure schematic diagram).
According to sequencing result, structrual description carries out to recombinant plasmid 35S::GFP-OsOSM1 as follows: to carrier pGDG XhoI and BamHI restriction enzyme site between to insert nucleotide sequence be the DNA molecular shown in sequence 1 in sequence table.
B, recombinant plasmid 35S::GFP-OsOSM1 steps A built import agrobacterium tumefaciens EHA105, obtain recombinational agrobacterium, called after EHA105/35S::GFP-OsOSM1.Then according to document (Guo Li, the clone of Dunaliella salina MAPK gene and the conversion to tobacco, Gansu Agriculture University master Diplomarbeit in 2006) EHA105/35S::GFP-OsOSM1 transforms the tobacco leaf in 4 week age by the method recorded, transform after 2 ~ 3 days, under Laser Scanning Confocal Microscope, observe the transient expression result of Tobacco Epidermis.
According to the method described above, recombinant plasmid 35S::GFP-OsOSM1 is replaced with plasmid pGDG, other step is all constant, observes the transient expression result of Tobacco Epidermis, in contrast under Laser Scanning Confocal Microscope.
Experimental result see B in Fig. 5 (left is fluorescence, in be light field, the right side is the integration of fluorescence and light field; 35S::GFP is contrast).Result shows, the OsOSM1 assignment of genes gene mapping is on cytoplasmic membrane.
Claims (10)
1. the application of protein OsOSM1 in regulating plant disease resistance; Described protein OsOSM1 is a1) or a2) or a3):
A1) aminoacid sequence is the protein shown in sequence 2 in sequence table;
A2) in sequence table, the N of the protein shown in sequence 2 holds or/and C end connects the fused protein that label obtains;
A3) by the protein relevant to disease resistance that the aminoacid sequence shown in sequence in sequence table 2 obtains through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation.
2. the application of nucleic acid molecule in regulating plant disease resistance of protein OsOSM1 described in coding claim 1.
3. apply as claimed in claim 2, it is characterized in that: described in described coding claim 1, the nucleic acid molecule of protein OsOSM1 is following b1) or b2) or b3) shown in DNA molecular:
B1) nucleotide sequence is the DNA molecular shown in sequence 1 in sequence table;
B2) nucleotide sequence and b1) limited has more than 75% or 75% identity, and the DNA molecular of protein OsOSM1 described in coding claim 1;
B3) nucleotide sequence hybridization limited with (b1) or (b2) under strict conditions, and the DNA molecular of protein OsOSM1 described in coding claim 1.
4. the application as described in as arbitrary in claims 1 to 3, is characterized in that: described plant is following c1) to c5) in any one:
C1) dicotyledons;
C2) monocotyledons;
C3) grass;
C4) paddy rice;
C5) rice varieties Xu rice No. 3.
5. the application as described in as arbitrary in Claims 1-4, is characterized in that: described disease resistance is anti-banded sclerotial blight.
6. cultivate a method for disease resistant transgenic plants, comprise the steps: the encoding gene importing protein OsOSM1 described in claim 1 in recipient plant, obtain the transgenic plant that disease resistance is better than described recipient plant.
7. method as claimed in claim 6, is characterized in that: the encoding gene of protein OsOSM1 described in described claim 1 is following b1) or b2) or b3) shown in DNA molecular:
B1) nucleotide sequence is the DNA molecular shown in sequence 1 in sequence table;
B2) nucleotide sequence and b1) limited has more than 75% or 75% identity, and the DNA molecular of protein OsOSM1 described in coding claim 1;
B3) nucleotide sequence hybridization limited with (b1) or (b2) under strict conditions, and the DNA molecular of protein OsOSM1 described in coding claim 1.
8. method as claimed in claims 6 or 7, is characterized in that: described recipient plant is following c1) to c5) in any one:
C1) dicotyledons;
C2) monocotyledons;
C3) grass;
C4) paddy rice;
C5) rice varieties Xu rice No. 3.
9. the method as described in as arbitrary in claim 6 to 8, is characterized in that: described disease resistance is anti-banded sclerotial blight.
10. the application as described in as arbitrary in Claims 1-4 or claim 6 to 8 arbitrary as described in method, it is characterized in that: described disease resistance is the microbial disease of anti-miliary damping-off.
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| CN106135239A (en) * | 2016-08-08 | 2016-11-23 | 扬州大学 | Basic element of cell division application in the adjusting and controlling rice resistance to banded sclerotial blight |
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| CN109705198A (en) * | 2019-01-25 | 2019-05-03 | 扬州大学 | The application of OsCKX7 protein and its encoding gene in regulation plant sharp eyespot resistance |
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