CN104497115B - The clone of R genes TN2 of regulation and control plant powdery mildew resistance a kind of and application - Google Patents
The clone of R genes TN2 of regulation and control plant powdery mildew resistance a kind of and application Download PDFInfo
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- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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- C12N15/8282—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 fungal resistance
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Abstract
Clone and application the invention discloses a kind of R genes TN2 of regulation and control plant powdery mildew resistance.A kind of method the present invention provides regulation and control plant to powdery mildew resistance.The amino acid sequence of the TN2 albumen is sequence 2 in sequence table, wherein the change of single amino acids occurs for 7 sites and 2 site mutations cause translation to terminate in advance, can suppress the cell death that exo70B1 mutant induces the resistance and powdery mildew of powdery mildew.But the growth and development to plant is not produced and significantly affected.TN2 can be applied to gene engineering technology field, Resistant reaction of the regulation and control plant to powdery mildew.
Description
Technical field
The present invention relates to biological technical field, more particularly to a kind of clone of the R genes TN2 of regulation and control plant powdery mildew resistance
And application.
Background technology
In natural environment, plant is constantly infected be subject to various pathogens, including bacterium, fungi and virus etc..For
The invasion of pathogen is resisted, plant evolution goes out the system of defense of complexity, is divided into two levels：Pathogen associated molecular pattern
The immune response PTI and effector that (pathogen-associated molecular patterns, PAMPs) triggers
(effector) the immune response ETI triggered.First, by stomata, epidermis gap even injury enters plants pathogen
In object, pathogen associated molecular pattern, such as flagellin, elongation factors and chitin etc., by the mould on cell membrane
Formula identification receptor (pattern recognition receptor, PRR) identifies, triggers PTI immune responses；But pathogen
During long-term evolution, new mechanism is evolved to suppress PTI.If bacterium is by three type excretory systems, released into plant
Effector is put, suppresses the basal resistance of plant.Meanwhile plant evolves R albumen again, the effector of pathogen secretion can be identified,
Trigger the ETI immune responses of plant.
In plant, maximum a kind of R albumen is NBS-LRR classes, different according to the structure of N-terminal, and is divided into CC-NB-LRR
And TIR-NB-LRR.R albumen plays a very important role in plant, it can be directly mutual with the effector of pathogen
Effect, or trigger ETI by monitoring modification or the change of target protein to activate R albumen.Disease resistance response caused by ETI is to plant
Thing invades the hypersensitivity (Hypersensitive Response, HR) at position, causes plant to invade position in pathogen
The accumulation (oxidative burst) of active oxygen, deposition and apoptosis (PCD) of callose etc..PCD has cut off disease
The source of nutrition of opportunistic pathogen, while antibacterial material is discharged, so as to limit the growth and diffusion of pathogen.Research shows, in plant
Interior importing external source disease-resistant gene can improve resistance of the plant to pathogen.Such as：RPW8 is one cloned on arabidopsis
Atypical R genes, overexpression RPW8 genes can improve resistance of the tobacco to Powdery Mildew in tobacco.Xa21 is in rice
The R genes of one bacterial blight-resisting being cloned, height of the main cultivated rice cultivars performance to bacterial leaf-blight is transferred to by Xa21 genes
Resistance and resistance of wide spectrum.Therefore, more disease-resistant genes are found, the disease resistance of plant is improved by genetic engineering becomes very heavy
Will.
Powdery mildew is a kind of biotroph fungi, can infect the leaf of multiple plant species such as barley, wheat, grape, tomato
The positions such as piece, stem, flower and fruit.In worldwide, probably there are more than 500 kinds of powdery mildew microspecies to infect 10, kind more than 000
Plant, huge economic loss is brought to agricultural production.
In research plant in the disease resistance mechanisms of powdery mildew, it is found that the subunit member EXO70B1 of arabidopsis exocytosis complex
After mutation, to the resistance of powdery mildew performance enhancing, and compared with wild type, the expression enhancing of PR1 genes, salicylic content
Rise, the accumulation increase of callose and hydrogen peroxide.Described above, EXO70B1 is regulation and control plant to the important of powdery mildew resistance
The factor.
The content of the invention
It is an object of the present invention to provide a kind of method for suppressing plant excessive activation resistance.
Method provided by the invention, includes the following steps：TN2 protein coding genes in mutant plant, realize and suppress to plant
The excessive activation of thing resistance；
The amino acid sequence of the TN2 albumen is sequence 2 in sequence table.
In the above method, the excessive activation for suppressing plant resistance to environment stress is embodied in the bacterium for promoting plant leaf blade surface powdery mildew
Silk growth, the generation of increase plant leaf blade surface powdery mildew conidiophore number, suppress in plant the expression of PR1 genes and/or
Suppress the salicylic accumulation that dissociates in plant.
In the above method, the plant is monocotyledon or dicotyledon, and the plant is specially arabidopsis mutant
Body exo70B1；
TN2 protein coding genes in the exo70B1tn2 mutant plants are the TN2 genes of mutation.Utilize arabidopsis
Exo70B1 mutant hybridizes with arabidopsis tn2 mutant, obtains filial generation exo70B1tn2.
Another object of the present invention is to provide a kind of TN2 mutains.
TN2 mutains provided by the invention, it is following 1) -9)：
1) amino acid sequence of TN2 mutains is the sequence that 2 1-43 amino acids residue of sequence forms in sequence table；
2) amino acid sequence of TN2 mutains is that the 27th arginine of sequence 2 in sequence table is sported histidine, sequence
The constant obtained amino acid sequence of other amino acid residues of sequence 2 in list；
3) it by the 82nd alanine mutation of sequence 2 in sequence table is threonine that the amino acid sequence of TN2 mutains, which is, sequence
The constant obtained amino acid sequence of other amino acid residues of sequence 2 in list；
4) it by the 82nd alanine mutation of sequence 2 in sequence table is valine that the amino acid sequence of TN2 mutains, which is, sequence
The constant obtained amino acid sequence of other amino acid residues of sequence 2 in list；
5) amino acid sequence of TN2 mutains is the sequence for forming 2 1-218 amino acids residue of sequence in sequence table
Row；
6) it by the 222nd glycine mutation of sequence 2 in sequence table is arginine that the amino acid sequence of TN2 mutains, which is,
The constant obtained amino acid sequence of other amino acid residues of sequence 2 in sequence table；
7) it by the 310th glutamic acid mutation of sequence 2 in sequence table is lysine that the amino acid sequence of TN2 mutains, which is,
The constant obtained amino acid sequence of other amino acid residues of sequence 2 in sequence table；
8) it by the 318th glycine mutation of sequence 2 in sequence table is arginine that the amino acid sequence of TN2 mutains, which is,
The constant obtained amino acid sequence of other amino acid residues of sequence 2 in sequence table；
9) it by the 333rd alanine mutation of sequence 2 in sequence table is valine that the amino acid sequence of TN2 mutains, which is,
The constant obtained amino acid sequence of other amino acid residues of sequence 2 in sequence table.
The non-coding DNA molecules of above-mentioned albumen, for following 1) -9)：
1) non-coding DNA molecules 1) in above-mentioned albumen are 1-132 nucleotide of sequence A, and the sequence A is by sequence
Sequence 1 sports T from the 130th C in table, the nucleotide sequence that other invariant nucleotides of sequence 1 obtain in sequence table；
2) non-coding DNA molecules 2) in above-mentioned albumen are that the 80th G of sequence 1 in sequence table is sported A, in sequence table
The nucleotide sequence that other invariant nucleotides of sequence 1 obtain；
3) non-coding DNA molecules 3) in above-mentioned albumen are that the 244th G of sequence 1 in sequence table is sported A, sequence table
The nucleotide sequence that other invariant nucleotides of middle sequence 1 obtain；
4) non-coding DNA molecules 4) in above-mentioned albumen are that the 245th C of sequence 1 in sequence table is sported T, sequence table
The nucleotide sequence that other invariant nucleotides of middle sequence 1 obtain；
5) non-coding DNA molecules 5) in above-mentioned albumen are 1-657 nucleotide of sequence B, and the sequence B is by sequence
The 656th G of sequence 1 sports A in table, the nucleotide sequence that other invariant nucleotides of sequence 1 obtain in sequence table；
6) non-coding DNA molecules 6) in above-mentioned albumen are that the 664th G of sequence 1 in sequence table is sported A, sequence table
The nucleotide sequence that other invariant nucleotides of middle sequence 1 obtain；
7) non-coding DNA molecules 7) in above-mentioned albumen are that the 928th G of sequence 1 in sequence table is sported A, sequence table
The nucleotide sequence that other invariant nucleotides of middle sequence 1 obtain；
8) non-coding DNA molecules 8) in above-mentioned albumen are that the 952nd G of sequence 1 in sequence table is sported A, sequence table
The nucleotide sequence that other invariant nucleotides of middle sequence 1 obtain；
9) non-coding DNA molecules 9) in above-mentioned albumen are that the 998th C of sequence 1 in sequence table is sported T, sequence table
The nucleotide sequence that other invariant nucleotides of middle sequence 1 obtain.
Above-mentioned albumen or above-mentioned non-coding DNA molecules or recombinant vector containing its encoding gene are in regulation and control plant to white powder
Application in sick resistance is also the scope of protection of the invention；
The regulation and control plant is specially to suppress plant to powder mildew resistance to powder mildew resistance；
The plant is specially monocotyledon or dicotyledon, and the plant is specially further arabidopsis.
R albumen TN2 or its encoding gene or recombinant vector containing its encoding gene are in regulation and control plant to powder mildew resistance
In application be also the scope of protection of the invention；The amino acid sequence of the R albumen TN2 is sequence 2 in sequence table；
The regulation and control plant is specially to improve plant to powder mildew resistance to powder mildew resistance；
The plant is monocotyledon or dicotyledon, and the plant is specially arabidopsis.
Exo70B1 mutant plants depend on TN2 to the resistance of powdery mildew.The albumen of TN2 gene expressions is white in Genes For Plant Tolerance
Application in powder disease is also the scope of protection of the invention；The amino acid sequence of the TN2 albumen is sequence 2 in sequence table.
In above application, exo70B1 Biology of Plant-Powdery Mildew Interaction depends on TN2.Promote plant leaf blade surface white powder after TN2 missings
The generation of the mycelial growth, increase plant leaf blade surface powdery mildew conidiophore number of bacterium, the expression for suppressing PR1 genes in plant
And/or suppress the salicylic accumulation that dissociates in plant, show that TN2 plays a significant role in mildew-resistance.
In above application, the plant is monocotyledon or dicotyledon, and the dicotyledon is specially to intend south
Mustard, above-mentioned arabidopsis are specially Arabidopsis Mutants exo70B1.
The material that TN2 protein coding genes are expressed in above-mentioned plant is the TN2 albumen for the loss of function being mutated.
The experiment proves that being mutated TN2 encoding genes, resistance of the exo70B1 mutant to powdery mildew can be suppressed
And the cell death of powdery mildew induction, but the growth and development to plant is not produced and significantly affected.TN2 can be applied to gene
Field of engineering technology, disease resistance response of the regulation and control plant to powdery mildew.It is all that the mutation of TN2 not only inhibits exo70B1 to mediate
Disease-resistant phenotype, and by powdery mildew induced expression, imply important function of the R genes TN2 in Plant defense responses are regulated and controled.This is
In the future by technique for gene engineering, TN2 genes are applied in crop, the bad character of Crop Improvement, there is provided valuable gene
Resource.
Brief description of the drawings
Fig. 1 is the cell that the inhibition from mutation exo70B1 mutant of TN2 genes induces the resistance and powdery mildew of powdery mildew
It is dead
Fig. 2 be TN2 genes inhibition from mutation exo70B1 mutant in PR1 genes expression
Fig. 3 is the salicylic accumulation of dissociating in the inhibition from mutation exo70B1 mutant of TN2 genes
Fig. 4 is the clone of TN2 genes
Fig. 5 is TN2 genes by powdery mildew induced expression
Embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples, is commercially available unless otherwise specified.
Embodiment 1, TN2 inhibition from mutation exo70B1 mutant powdery mildew resistances
1st, the acquisition of mutant exo70B1tn2
By EMS mutagenesis, the mutant library of exo70B1 is obtained.In M2 generations, the white of exo70B1 mediations can be suppressed by screening
The mutant exo70B1tn2 of powder bacterium resistance.
Mutant exo70B1tn2 can also be prepared as follows：By mutant exo70B1 (N717829) and mutation
Body tn2 hybridizes, and obtains first familiar generation, can obtain the double-mutant of homozygosis in the F2 generations that F1 generation selfing obtains by screening
exo70B1tn2。
Wildtype Arabidopsis thaliana Col-0 (hereinafter abbreviation wildtype Arabidopsis thaliana), is recorded in the following literature：Catherine
A.Frye,Dingzhong Tang,and Roger W.Innes(2001).Negative regulation of defense
responses in plants by a conserved MAPKK kinase.PNAS 98(1):373-378；The public can be therefrom
The heredity of the academy of sciences of state is obtained with Developmental Biology research.
Mutant exo70B1 (the allelic variant body of N717829, EXO70B1, are T-DNA insertion mutations) is purchased from The
European Arabidopsis Stock Centre, NASC), compared with wildtype Arabidopsis thaliana Col-0, only EXO70B1 genes
(nucleotides sequence is classified as sequence 3, its encode protein amino acid sequence be sequence 4) have mutation, remaining nucleotide sequence with open country
Raw type arabidopsis is identical；
Mutant tn2, purchased from ABRC, SALK_204239；Compared with wildtype Arabidopsis thaliana Col-0, only tn2 genes (nucleosides
Acid sequence is sequence 1, its protein amino acid sequence encoded is sequence 2) there is mutation, remaining nucleotide sequence is intended with wild type
Southern mustard is identical.
Detect and be sequenced by genomics, double-mutant exo70B1tn2 is compared with wildtype Arabidopsis thaliana Col-0, only
EXO70B1 genes and TN2 genes have mutation, remaining nucleotide sequence is identical with wildtype Arabidopsis thaliana；Double-mutant
For exo70B1tn2 compared with mutant exo70B1, only TN2 genes have mutation, remaining nucleotide sequence with mutant exo70B1
It is identical.
2nd, TN2 inhibition from mutation exo70B1 mutant powdery mildew resistances
1) Phenotypic Observation
Arabidopsis WT lines Col-0 (WT), mutant exo70B1 and double-mutant exo70B1tn2 are planted in 22
DEG C, 9 it is small when illumination greenhouse in, it is a large amount of to be inoculated with Powdery Mildew Golovinomyces Cichoracearum after growth 4 weeks
(Yiping Wang,Marc T.Nishimura,Ting Zhao and Dingzhong Tang (2011)ATG2,an
autophagy-related protein,negatively affects powdery mildew resistance and
Mildew-induced cell death in Arabidopsis.The Plant 68,74-87,100 spores of Journal
Son/mm2).After connecing bacterium 7 days, disease-resistant phenotypic evaluation is carried out.Take a picture, and carry out to representational whole strain and individual blade
Trypan blue (sigma) dyes (Trypan Blue Staining).
As a result as shown in figs. 1A-1 c, 1A and 1B is the table of representational whole strain and blade of the inoculation Powdery Mildew after 7 days
Type picture, 1C are to carry out the picture that platform expects blue dyeing after being inoculated with Powdery Mildew.As can be seen that after powdery mildew connects bacterium 7 days, wild type
The blade surface of arabidopsis has substantial amounts of powdery mildew；The blade of exo70B1 mutant only has the growth of a small amount of powdery mildew, and goes out
Now obvious cell death, illustrates it compared with wildtype Arabidopsis thaliana, has mildew-resistance resistance；And the double mutation of exo70B1tn2
Body is similar to wild type, and blade surface has substantial amounts of mycelia and spore, does not produce cell death (1A, 1B), show its with
Exo70B1 mutant is compared, and powder mildew resistance reduces.Platform expects that orchid can dye the mycelial structure of Powdery Mildew and dead cell
Blueness, platform expect that the result (1C) of blue dyeing also demonstrates the plant powdery mildew resistance of TN2 inhibition from mutation exo70B1 mediations.
2) integration detection of hydrogen peroxide
For further reactions of the analysis exo70B1tn2 to powdery mildew, by arabidopsis WT lines Col-0 (WT), it is mutated
Body exo70B1 and double-mutant exo70B1tn2 is planted in 22 DEG C, 9 it is small when illumination greenhouse in, after growth 4 weeks, each strain
In a small amount inoculation Powdery Mildew Golovinomyces Cichoracearum (1 spore/mm2).After connecing bacterium 2 days, to blade into
Row DAB is dyed, and detects the accumulation of hydrogen peroxide.DAB stains can be combined with the hydrogen peroxide produced into the cell, formed it is insoluble,
Visually identifiable reddish-brown material.
As a result as shown in figure iD, it can be seen that mutant exo70B1 generates substantial amounts of hydrogen peroxide, exo70B1tn2
Double-mutant is similar to wild type, the accumulation of only a small amount of hydrogen peroxide.
5 days after bacterium is connect, microscopy after Trypan Blue is carried out to each strain individual blade, to monospore conidiophore
Counted, and statistical analysis is carried out to count results.
As a result as referring to figure 1E, exo70B1 mutant is substantially less than wild type, and exo70B1tn2 double-mutants return to open country
The level of raw type.
The above results show that suppressing the expression of tn2 genes in plant makes its mutation, causes the powder mildew resistance of the plant to drop
It is low.
(AY064023, on November 19th, 2001 carry PR1 genes in embodiment 2, TN2 inhibition from mutation exo70B1 mutant
Hand over) expression
In order to verify whether TN2 genes participate in the disease resistance response of plant, with the method for real-time quantitative PCR, detection Col-0,
The expression pattern of mutant exo70B1 and double-mutant exo70B1tn2 PR1 genes before and after powdery mildew is inoculated with.Instrument is
German Eppendorf Mastercyclereprealplex 4S, it is specific as follows：
Arabidopsis WT lines Col-0 (WT), mutant exo70B1 and the double-mutant of 4 weeks will be grown
After exo70B1tn2 is largely inoculated with powdery mildew 3 days, takes not connecing respectively and bacterium and connect the bacterium plant leaf of 3 days, be put into it is quick-frozen in liquid nitrogen,
Then in -70 DEG C of storages, it is spare to carry RNA.
With the total serum IgE of Trizol (Invitrogen) reagent extraction blade, polluted with DNase I (Promega) digestion
DNA half an hour, then with reverse transcriptase M-MLV (Invitrogen), invert 2 μ g total serum IgEs in 20 μ L reaction systems and obtain the first chain
cDNA；It is spare that 5 times of dilution gives over to pcr template.PCR reaction systems include：SYBR Green supermix reagent
(Takara)：10 μ L, cDNA templates：2 μ L, 10 μM of primers：1 μ L, are mended system to 20 μ L with redistilled water.PCR response procedures：95
DEG C pre-degeneration 2min, into after circulation, 95 DEG C of denaturation 15s, 55 DEG C of annealing 15s, 72 DEG C of extension 20s, run 40 circulations, each
Sample is repeated 3 times.Primer sequence：PR1F:5-CGGAGCTACGCAGAACAACT-3；R:5-CTCGCTAACCCACATGTTCA-
3；
ACT2 is as internal standard, ACT2 F：5---TCTCCCGCTATGTATGTCGCC---3, ACT2 R：5---
GTCACGTCCAGCAAGGTCAAGA----3。
The results are shown in Figure 2, be inoculated with powdery mildew before and after, in exo70B1 mutant the expression of PR1 genes be significantly higher than wild
Type, after TN2 gene mutations, in exo70B1tn2 double-mutants the expression of PR1 genes return to the level of wild type, show TN2
Gene take part in the Resistant reaction of plant pair Powdery Mildew.
Dissociate salicylic accumulation in embodiment 3, TN2 inhibition from mutation exo70B1 mutant
Salicylic acid is the central element for regulating and controlling Plant defense responses.Rely on salicylic signal transduction and part is established to plant
It is most important with systemic resistance.Col-0, mutant exo70B1 and double-mutant exo70B1tn2 be have detected in inoculation white powder
The content of free salicylic acid before and after bacterium.
The seedling of arabidopsis WT lines Col-0 (WT), mutant exo70B1 and double-mutant exo70B1tn2 is big
Amount inoculation powdery mildew, at 22 DEG C, 9 it is small when illumination/15 it is small when dark condition under normal growth after 4 weeks, take each material respectively
Blade, it is quick-frozen in liquid nitrogen, then in -70 DEG C of storages, it is spare to carry salicylic acid.
Salicylic content is measured with high performance liquid chromatography, bibliography the method Xin Li, Yuelin Zhang,
Joseph D Clarke,Yan Li,Xinnian Dong(1999)Identification and cloning of a
negative regulator of systemic acquired resistance,SNI1,through a screen for
suppressors of npr1-1.Cell 98:329-339。
The results are shown in Figure 3, is inoculated with before and after powdery mildew, and the content for salicylic acid of dissociating in exo70B1 mutant is significantly higher than
Wild type, the content for salicylic acid of dissociating after TN2 gene mutations, in exo70B1tn2 double-mutants drop to the level of wild type.
Show the Resistant reaction of TN2 gene regulations plant pair Powdery Mildew.
The acquisition of embodiment 4, TN2 genes
In order to clone TN2 genes, by exo70B1tn2 mutant and arabidopsis wild type Landsberg ecotypes plant into
Row hybridization, the F1 generation selfing of acquisition produce F2 generations.It is mutated containing exo70B1 for selection in plant from F2 but dashes forward with tn2exo70B1
40 similar single plants of variant phenotype, select 5 telltale marks for being evenly distributed on chromosome respectively from 5 chromosomes
(http://signal.salk.edu/genome/SSLP_info/SSLPsordered.html) carry out genotype identification.Slightly
TN2 is positioned at No. 1 chromosome by the result of positioning.The nucleotides sequence of TN2 genes is classified as sequence 1 in sequence table, its egg encoded
White amino acid sequence is sequence 2 in sequence table.
Then, using the mark of finely positioning, genotype identification is carried out to 2000 plants or so of F2 plant, TN2 is positioned
F1L3 is cloned to BAC.All genes in the region are sequenced, find significant mutator.It is meanwhile right
Exo70B1tn2 genomes carry out resurveying sequence, compared with wild type gene group, find the gene mutation in localization region.
Found through sequencing, the CDS in TN2 (AT1G17615) gene, weight occur for the mutational site for there are 9 allelic variant bodies
Sequencing result is same.TN2 genes include two domains of TIR and NBS (Fig. 4 A), are an atypical R genes.This 9
The allelic variant body of a TN2 genes, has 3 missense mutation occurs in TIR domains, 4 occur in NBS domains, have 2 to dash forward
Change causes translation to terminate (Fig. 4 B) in advance.
9 tn2 mutant are specific as follows：
Tn2 mutant 1 is the sequence that 2 1-43 amino acids residue of sequence forms in sequence table；Its encoding gene nucleosides
Acid sequence is 1-132 nucleotide of sequence A, and sequence A is that sequence in sequence table 1 is sported T from the 130th C, in sequence table
The nucleotide sequence that other invariant nucleotides of sequence 1 obtain；Compared with TN2, the 130th C of sequence 1 in sequence table is sported
T, terminates protein expression in advance；
2 amino acid sequence of tn2 mutant is that the 27th R (arginine) of sequence 2 in sequence table is sported H (histidine),
The constant obtained amino acid sequence of other amino acid residues of sequence 2 in sequence table；2 encoding gene nucleotides sequence of tn2 mutant
It is classified as and the 80th G of sequence 1 in sequence table is sported into A, the nucleotides sequence that other invariant nucleotides of sequence 1 obtain in sequence table
Row；
3 amino acid sequence of tn2 mutant is that the 82nd A (alanine) of sequence 2 in sequence table is sported T (threonine),
The constant obtained amino acid sequence of other amino acid residues of sequence 2 in sequence table；3 encoding gene nucleotides sequence of tn2 mutant
It is classified as and the 244th G of sequence 1 in sequence table is sported into A, the nucleotide that other invariant nucleotides of sequence 1 obtain in sequence table
Sequence；
4 amino acid sequence of tn2 mutant is that the 82nd A (alanine) of sequence 2 in sequence table is sported V (valine),
The constant obtained amino acid sequence of other amino acid residues of sequence 2 in sequence table；4 encoding gene nucleotides sequence of tn2 mutant
It is classified as and the 245th C of sequence 1 in sequence table is sported into T, the nucleotide that other invariant nucleotides of sequence 1 obtain in sequence table
Sequence；
5 amino acid sequence of tn2 mutant is the sequence for forming 2 1-218 amino acids residue of sequence in sequence table；
5 encoding gene nucleotides sequence of tn2 mutant is classified as 1-657 nucleotide of sequence B, and sequence B is by sequence in sequence table 1 the
656 G sport A, the nucleotide sequence that other invariant nucleotides of sequence 1 obtain in sequence table；
Tn2 mutant six amino acids sequence is that the 222nd G (glycine) of sequence 2 in sequence table is sported R (arginine),
The constant obtained amino acid sequence of other amino acid residues of sequence 2 in sequence table；6 encoding gene nucleotides sequence of tn2 mutant
It is classified as and the 664th G of sequence 1 in sequence table is sported into A, the nucleotide that other invariant nucleotides of sequence 1 obtain in sequence table
Sequence；
7 amino acid sequence of tn2 mutant is that the 310th E (glutamic acid) of sequence 2 in sequence table is sported K (lysine),
The constant obtained amino acid sequence of other amino acid residues of sequence 2 in sequence table；7 encoding gene nucleotides sequence of tn2 mutant
It is classified as and the 928th G of sequence 1 in sequence table is sported into A, the nucleotide that other invariant nucleotides of sequence 1 obtain in sequence table
Sequence；
Tn2 mutant eight amino acids sequence is that the 318th G (glycine) of sequence 2 in sequence table is sported R (arginine),
The constant obtained amino acid sequence of other amino acid residues of sequence 2 in sequence table；8 encoding gene nucleotides sequence of tn2 mutant
It is classified as and the 952nd G of sequence 1 in sequence table is sported into A, the nucleotide that other invariant nucleotides of sequence 1 obtain in sequence table
Sequence；
Tn2 mutant nine amino acids sequence is that the 333rd A (alanine) of sequence 2 in sequence table is sported V (valine),
The constant obtained amino acid sequence of other amino acid residues of sequence 2 in sequence table；9 encoding gene nucleotides sequence of tn2 mutant
It is classified as and the 998th C of sequence 1 in sequence table is sported into T, the nucleotide that other invariant nucleotides of sequence 1 obtain in sequence table
Sequence.
Embodiment 5, TN2 genes are by powdery mildew induced expression
As a plant R genes, the resistance of the powdery mildew of the inhibition from mutation exo70B1 mediations of TN2 and powdery mildew induction
Cell death.It is wild with the method for real-time quantitative PCR, detection in order to whether find out TN2 genes directly by powdery mildew induced expression
The expression of raw type Col-0 TN2 genes after inoculation powdery mildew 1 day, 3 days and 5 days.
The wild type Col-0 plant of 4 weeks will be grown and be largely inoculated with powdery mildew, respectively before bacterium is connect, connect bacterium 1 day, 3 days and 5
Clip plant leaf after it, be put into it is quick-frozen in liquid nitrogen, then -70 DEG C storage, it is spare to carry RNA.RNA extraction method, PCR reactions
Liquid and PCR response procedures are as previously described.Primer sequence：TN2 F：5---GGCTCATGAGTCAGAAAG---3；TN2 R：5---
GAAGATTCAGTCCCGGAT---3。
The results are shown in Figure 5, and for inoculation powdery mildew after 3 days and 5 days, the expression of TN2 genes does not connect 9 times and 25 of bacterium about
Times, show TN2 genes by powdery mildew induced expression.
The application of embodiment 6, arabidopsis powdery mildew resistance related gene TN2 in crop disease-resistant improvement
The Col-0 ecotypes share 21 TIR-NBS genoids in arabidopsis, it has been reported that some of them TN genes are in plant
Work in immune.AtTN10, AtTN11, AtTN2 and AtTN3 infection powdery mildew (Blumeriagraminis,
) and pseudomonas syringae (Pseudomonas syringae) a large amount of induced expressions afterwards Erysiphecichoracearum.
Some TN genes are instantaneously overexpressed in tobacco, hypersensitivity can occur, cause cell death.
TN2 genes, as one of TIR-NBS classes R genes, not only by powdery mildew induced expression, but also suppress exocytosis
The disease-resistant phenotype of all powdery mildews of complex subunit mutant exo70B1, illustrates it in regulation and control plant in powdery mildew resistance
Important function.Using technique for gene engineering, by being overexpressed TN2 in plant, being hopeful to obtain has excellent resistant character
Germplasm materials.
Claims (6)
1. a kind of method for suppressing plant excessive activation resistance, includes the following steps：In mutant A thaliana exo70B1 mutant
TN2 genes, realize the excessive activation for suppressing plant resistance to environment stress；
The nucleotides sequence of the TN2 genes is classified as sequence 1 in sequence table；
TN2 genes in the mutant plant are following 1) -9)：
1) the 130th C of sequence 1 in sequence table is sported into T, the nucleosides that other invariant nucleotides of sequence 1 obtain in sequence table
Acid sequence；
2) the 80th G of sequence 1 in sequence table is sported into A, the nucleotide that other invariant nucleotides of sequence 1 obtain in sequence table
Sequence；
3) the 244th G of sequence 1 in sequence table is sported into A, the nucleosides that other invariant nucleotides of sequence 1 obtain in sequence table
Acid sequence；
4) the 245th C of sequence 1 in sequence table is sported into T, the nucleosides that other invariant nucleotides of sequence 1 obtain in sequence table
Acid sequence；
5) the 656th G of sequence 1 in sequence table is sported into A, the nucleosides that other invariant nucleotides of sequence 1 obtain in sequence table
Acid sequence；
6) the 664th G of sequence 1 in sequence table is sported into A, the nucleosides that other invariant nucleotides of sequence 1 obtain in sequence table
Acid sequence；
7) the 928th G of sequence 1 in sequence table is sported into A, the nucleosides that other invariant nucleotides of sequence 1 obtain in sequence table
Acid sequence；
8) the 952nd G of sequence 1 in sequence table is sported into A, the nucleosides that other invariant nucleotides of sequence 1 obtain in sequence table
Acid sequence；
9) the 998th C of sequence 1 in sequence table is sported into T, the nucleosides that other invariant nucleotides of sequence 1 obtain in sequence table
Acid sequence.
2. according to the method described in claim 1, it is characterized in that：The suppression plant excessive activation resistance is embodied in promotion and plants
The generation of the mycelial growth, increase plant leaf blade surface powdery mildew conidiophore number of thing blade surface powdery mildew, suppress plant
Dissociate salicylic accumulation in the expression of middle PR1 genes and/or suppression plant.
3. a kind of TN2 mutains, it is following 1) -9)：
1) amino acid sequence of TN2 mutains is the sequence that 2 1-43 amino acids residue of sequence forms in sequence table；
2) amino acid sequence of TN2 mutains is that the 27th arginine of sequence 2 in sequence table is sported histidine, sequence table
The constant obtained amino acid sequence of other amino acid residues of middle sequence 2；
3) it by the 82nd alanine mutation of sequence 2 in sequence table is threonine that the amino acid sequence of TN2 mutains, which is, sequence table
The constant obtained amino acid sequence of other amino acid residues of middle sequence 2；
4) it by the 82nd alanine mutation of sequence 2 in sequence table is valine that the amino acid sequence of TN2 mutains, which is, sequence table
The constant obtained amino acid sequence of other amino acid residues of middle sequence 2；
5) amino acid sequence of TN2 mutains is the sequence for forming 2 1-218 amino acids residue of sequence in sequence table；
6) it by the 222nd glycine mutation of sequence 2 in sequence table is arginine that the amino acid sequence of TN2 mutains, which is, sequence
The constant obtained amino acid sequence of other amino acid residues of sequence 2 in table；
7) it by the 310th glutamic acid mutation of sequence 2 in sequence table is lysine that the amino acid sequence of TN2 mutains, which is, sequence
The constant obtained amino acid sequence of other amino acid residues of sequence 2 in table；
8) it by the 318th glycine mutation of sequence 2 in sequence table is arginine that the amino acid sequence of TN2 mutains, which is, sequence
The constant obtained amino acid sequence of other amino acid residues of sequence 2 in table；
9) it by the 333rd alanine mutation of sequence 2 in sequence table is valine that the amino acid sequence of TN2 mutains, which is, sequence
The constant obtained amino acid sequence of other amino acid residues of sequence 2 in table.
4. the non-coding DNA molecules of albumen described in claim 3, for following 1) -9)：
1) non-coding DNA molecules 1) in albumen described in claim 3 are 1-132 nucleotide of sequence A, and the sequence A is
The 130th C of sequence 1 in sequence table is sported into T, the nucleotide sequence that other invariant nucleotides of sequence 1 obtain in sequence table；
2) non-coding DNA molecules 2) in albumen described in claim 3 are that the 80th G of sequence 1 in sequence table is sported A, sequence
The nucleotide sequence that other invariant nucleotides of sequence 1 obtain in list；
3) non-coding DNA molecules 3) in albumen described in claim 3 are that the 244th G of sequence 1 in sequence table is sported A, sequence
The nucleotide sequence that other invariant nucleotides of sequence 1 obtain in list；
4) non-coding DNA molecules 4) in albumen described in claim 3 are that the 245th C of sequence 1 in sequence table is sported T, sequence
The nucleotide sequence that other invariant nucleotides of sequence 1 obtain in list；
5) non-coding DNA molecules 5) in albumen described in claim 3 are 1-657 nucleotide of sequence B, and the sequence B is
The 656th G of sequence 1 in sequence table is sported into A, the nucleotide sequence that other invariant nucleotides of sequence 1 obtain in sequence table；
6) non-coding DNA molecules 6) in albumen described in claim 3 are that the 664th G of sequence 1 in sequence table is sported A, sequence
The nucleotide sequence that other invariant nucleotides of sequence 1 obtain in list；
7) non-coding DNA molecules 7) in albumen described in claim 3 are that the 928th G of sequence 1 in sequence table is sported A, sequence
The nucleotide sequence that other invariant nucleotides of sequence 1 obtain in list；
8) non-coding DNA molecules 8) in albumen described in claim 3 are that the 952nd G of sequence 1 in sequence table is sported A, sequence
The nucleotide sequence that other invariant nucleotides of sequence 1 obtain in list；
9) non-coding DNA molecules 9) in albumen described in claim 3 are that the 998th C of sequence 1 in sequence table is sported T, sequence
The nucleotide sequence that other invariant nucleotides of sequence 1 obtain in list.
5. albumen described in claim 3 or the non-coding DNA molecules described in claim 4 or the recombinant vector containing its encoding gene
In regulation and control plant to the application in powder mildew resistance；
The regulation and control plant is specially to suppress the excessive activation of plant resistance to environment stress to powder mildew resistance；
The plant is specially monocotyledon or dicotyledon, and the plant is specially further arabidopsis exo70B1.
6. application according to claim 5, it is characterised in that:The resistance for suppressing plant excessive activation is embodied in promotion
The generation of the mycelial growth, increase plant leaf blade surface powdery mildew conidiophore number of plant leaf blade surface powdery mildew, suppress to plant
In thing PR1 genes expression and/or suppress plant in dissociate salicylic accumulation.
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| CN109796526B (en) * | 2017-11-16 | 2020-12-08 | 中国科学院遗传与发育生物学研究所 | Method for regulating transcription factor HvWRKY3 through phosphorylation of barley HvSnRK1 kinase and application of method |
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