CN107880098A

CN107880098A - Barley ill-resistant protein MLA10 mutant is improving plant to the application in powdery mildew resistance

Info

Publication number: CN107880098A
Application number: CN201711093399.2A
Authority: CN
Inventors: 沈前华; 张春雷
Original assignee: Institute of Genetics and Developmental Biology of CAS
Current assignee: Institute of Genetics and Developmental Biology of CAS
Priority date: 2017-11-08
Filing date: 2017-11-08
Publication date: 2018-04-06
Anticipated expiration: 2037-11-08
Also published as: CN107880098B

Abstract

The present invention relates to barley ill-resistant protein MLA10 mutant to improve plant to the application in powdery mildew resistance, and the mutant is MLA10 two self-activation mutant MLA10 (F99E) and MLA10 (D502V).Present invention firstly discovers that drive the arabidopsis of barley MLA10 (F99E) and MLA10 (D502V) expression to strengthen resistance of the arabidopsis to arabidopsis powdery mildew using PR1 promoters, and the influence of growing on plant in itself is little.Transgenic arabidopsis is not only restricted to MLA10 race specific resistance, and antimicrobial spectrum is expanded to other powdery mildews or pathogen by big wheat powdery mildew, has important directive function for agricultural production.

Description

Barley ill-resistant protein MLA10 mutant is improving plant in powdery mildew resistance Using

Technical field

The present invention relates to agricultural biological technical field, specifically, is related to barley ill-resistant protein MLA10 mutant and is improving Plant is to the application in powdery mildew resistance.

Background technology

The invasion and attack of plant reply pathogenic microorganism rely primarily on innate immune mechanisms defence pathogenic microorganism invasion and expanded Increase (Ausubel, 2005；Chisholm et al.,2006；Dangl and Jones,2001).The immunologic mechanism of plant is main It is made up of two-layer defense.First level system of defense is the pattern recognition receptors (pattern by plant cell membrane surface Recognition receptors, PRRs) the conservative model molecule (microbial-or of identification pathogenic microorganism Pathogen-associated molecular patterns, MAMPs or PAMPs) such as the flagellin of bacterium, fungi Chitin etc., and the disease resistance response triggered, referred to as PTI (PAMPs-triggered immunity).The defence of second level System is by the ill-resistant protein (disease resistance protein, R protein) in plant cell, mainly NLR The direct or indirect identification pathogenic microorganism of the ill-resistant protein of type is secreted into the effector in plant cell, the effect of initiation More strong immune response, referred to as ETI (effector-triggered immunity) (Dangl et al., 2013； Dodds and Rathjen,2010；Jones and Dangl, 2006), resistance protein mediated R is often also referred to as microspecies Specialization resistance.ETI is usually associated with plant and is produced substantial amounts of peroxide by the cell for infecting place, and cell death occurs, This phenomenon is also known as the hypersensitivity (hypersensitive response, HR) (Dangl et al., 2013) of plant.

NLRs immunity receptors in plant cell, nucleic-acid binding domains (NB-ARC) and C-terminal comprising centre are rich in The domain (LRR) of leucine, and CC the or TIR domains of N-terminal, according to the difference of R albumen n end domains, Ke Yifen For two types, i.e., the CC-NB-LRR (CNL) and contain TIR that N-terminal contains Coiled-coil (coiled-coil, CC) domain TIR-NB-LRR (TNL) two types (Sukarta et al., 2016) of domain.

MLA is distinctive a kind of ill-resistant protein in barley, mediates race specific resistance of the barley to big wheat powdery mildew, greatly Barley microspecies of the race specific resistance of wheat such as containing MLA10 genes, which can only be directed to, contains respective effects albumin A vrMLA10's Big wheat powdery mildew microspecies produce resistance, and can not produce resistance to other powdery mildew biological strains.

R albumen is triggering plant ETI simultaneously, is usually associated with ROS accumulation, cell death, grows suppressed phenomenon.Such as Barley ill-resistant protein MLA10 and its self-activation mutant MLA10 (F99E) and MLA10 (D502V) is instantaneously overexpressed in tobacco Tobacco cell can be triggered dead (Bai et al., 2012).Disease-resistant wheat Protein S R33 and SR50 can also draw in tobacco Send out cell death (Cesari et al., 2016).R albumen is overexpressed the growth that can influence plant in arabidopsis, and plant is often Show short and small phenotype.Such as arabidopsis TNL type R Gene As t2g32140 is overexpressed the table for causing plant short and small in arabidopsis Type (Kato et al., 2014).The acquired function mutation of R albumen can also influence the life of plant while strengthening disease-resistant It is long, cause the phenotype that plant is short and small.For example, SNC1 is the ill-resistant protein of TNL types in arabidopsis, its gain-of-function mutant Snc1 shows as that plant strain growth is short and small, leaf rolling isophenous (Zhang and Gassmann, 2003).Utilize mutant Snc1EMS mutagenesis builds mutant library, obtains a series of gene of regulation and control SNC1 functions and expression, so as to disclose the anti-of SNC1 Interpretation of the cause, onset and process of an illness system (Huang et al., 2016；Johnson et al.,2017；Wu et al.,2017).How to obtain both to have and resist The growing plants that sick function does not influence plant again is always the target for cultivating new varieties.Recently studies have found that, TBF1 genes The open reading frame element in upstream is uORFs_TBF1(upstream open reading frames of the TBF1gene) Can respond pathogenic bacterium inducing, TBF1 expression quantity can be regulated and controled in translation skill.Using TBF1 promoters and its uORFs_TBF1Sequence driving expression snc1 transgenic arabidopsis had both kept stronger disease resistance, and did not influenceed the growth of plant (Xu et al.,2017)。

Arabidopsis pen2pad4sag101 (pps) Trimutant loses the non―technological factors (Lipka to big wheat powdery mildew et al.,2005).Ill-resistant protein MLA1 in barley is expressed in arabidopsis Trimutant pps, can still play MLA1 To the race specific resistance of big wheat powdery mildew, and trigger the hypersensitivity (Maekawa et al., 2012) of plant cell.

The content of the invention

The purpose of the present invention is to break away from limits of the barley ill-resistant protein MLA10 to this race specific resistance of big wheat powdery mildew System, widens its antibacterial spectral limit, other powdery mildews or other pathogens can be obtained with the plant of resistance, so as to instruct Agricultural production.

Present inventive concept is as follows：MLA is the ill-resistant protein of a kind of CNL types of specifically expressing in barley, mediates barley pair The race specific resistance of big wheat powdery mildew.The MLA disease-resistant function of Race specificity is relatively conservative in barley and arabidopsis, is intending Overexpression MLA1 transgenic arabidopsis shows the race specific resistance to big wheat powdery mildew in southern mustard Trimutant pps (Maekawa et al.,2012).Compared with background resistance, the immune response that the race specific resistance of MLA mediations triggers is stronger It is strong.But the race specific resistance of MLA mediations can only be directed to a certain or several barley white powder containing corresponding nontoxic protein Bacterium biological strain plays immunization, causes the limitation of its application.Therefore, the present invention constructs overexpression and induced expression MLA10 self-activation mutant MLA (F99E), MLA (D502V) transgenic arabidopsis, find MLA (F99E), MLA (D502V) Expression can trigger the short and small phenotype of Arabidopsis plant, simultaneous pathogenesis related gene PR1, PR2 up-regulation in arabidopsis Expression, trigger plant superoxide accumulation, blade cell death.

In order to realize the object of the invention, the present invention provides barley ill-resistant protein MLA10 mutant and is improving plant to white powder Application in bacterium resistance, the mutant are MLA10 two self-activation mutant MLA10 (F99E) and MLA10 (D502V), Specifically such as SEQ ID NO:The 99th phenylalanine of barley ill-resistant protein MLA10 shown in 1 sports glutamic acid and formed Mutant MLA10 (F99E), or the mutant MLA10 that the 502nd Aspartic acid mutations are formed by valine (D502V).Barley ill-resistant protein MLA10 GenBank accession number is AAQ55541.

Mutant MLA10 (F99E) is that the 99th phenylalanine of CC domains of MLA10 albumen sports glutamic acid, mutation Body MLA10 (D502V) is that the 502nd Aspartic acid mutations of NB-ARC domains of MLA10 albumen are valine.

Plant of the present invention includes but is not limited to arabidopsis, wheat.

Powdery mildew of the present invention includes but is not limited to arabidopsis powdery mildew (Golovinomyces orontii), cloves Pseudomonas alba (Pseudomonas syringae), oomycetes (Hyaloperonospora arabidopsidis).

The present invention also provides a kind of barley ill-resistant protein MLA10 mutant expression cassettes, and it is started by pathogenic bacterium inducing type The encoding gene of son and the barley ill-resistant protein MLA10 mutant being driven by it composition.

Preferably, the pathogenic bacterium inducing type promoter is arabidopsis PR1 gene promoters, its nucleotide sequence such as SEQ ID NO:Shown in 2.

The present invention also provides the expression vector containing above-mentioned expression cassette.

The present invention is also provided containing above-mentioned expression cassette, the engineering bacteria and transgenic cell line of expression vector.

The present invention also provides a kind of method of raising arabidopsis to arabidopsis powdery mildew resistance, including：

1) arabidopsis is made to contain above-mentioned barley ill-resistant protein MLA10 mutant MLA10 (F99E) and/or MLA10 (D502V) Expression cassette；Or

2) arabidopsis is made to express barley ill-resistant protein MLA10 mutant MLA10 (F99E) and/or MLA10 (D502V).

In the specific embodiment of the present invention, it the described method comprises the following steps：

S1, structure barley ill-resistant protein MLA10 mutant MLA10 (F99E) and/or MLA10 (D502V) expression cassette；

On S2, the expression vector cTAPi (Fig. 4) for transforming the insertion of above-mentioned expression cassette, recombinant plasmid is obtained；

S3, by above-mentioned recombinant plasmid import arabidopsis in, screen positive transgenic plant.

The expression vector cTAPi transformed described in step S2 refers to the 35S promoter for removing initial carrier cTAPi, then will Herbicide BASTA resistances change kalamycin resistance into, and obtained behind target gene plus 3HA labels.The expression of transformation Carrier cTAPi structure is as shown in Figure 4.The structure of recombinant plasmid containing MLA10 (F99E) expression cassette is as shown in figure 5, contain The structure of the recombinant plasmid of MLA10 (D502V) expression cassette is as shown in Figure 6.

Compared with prior art, the present invention has advantages below：

Arabidopsis using PR1 promoters driving barley MLA10 (F99E) and MLA10 (D502V) expression can strengthen plan Southern mustard is to the resistance of arabidopsis powdery mildew, and the influence of growing on plant in itself is little.Transgenic arabidopsis MLA10 (F99E) and MLA10 (D502V) is not only restricted to MLA10 race specific resistance, and antimicrobial spectrum is expanded to it by big wheat powdery mildew Its powdery mildew or pathogen, there is important directive function for agricultural production.

Brief description of the drawings

Fig. 1 is to be overexpressed MLA10 (D502V) in the embodiment of the present invention 1 to trigger that Arabidopsis plant is short and small and PR gene expressions Amount rises；Wherein, A：Transfer-gen plant phenotypic analysis.35S:MLA10(D502V)-3HA#17-3、35S:MLA10(D502V)- 3HA#19-1 and wild type Col-0 grows 3 weeks phenotypes, scale 1cm；B：Semiquantitive PCR detection figure (A) transfer gene M LA10 (D502V) expression quantity；C：MLA10 (D502V), PR1 and PR2 genes expression in Real-time PCR detection figures (A).

Fig. 2 is that expression MLA10 (F99E), MLA10 (D502V) cause arabidopsis in arabidopsis in the embodiment of the present invention 2 Short and small phenotype, trigger the expression of PR gene upregulations, cause peroxide accumulation and cell death；Wherein, A：pER8:MLA10 (F99E)-HA and pER8:MLA10 (D502V)-HA transfer-gen plant phenotypic analyses.Respectively in 10 μM of 1/2MS culture mediums and addition The wild type and transfer-gen plant phenotype of 7 days, scale 1cm are grown in the 1/2MS culture mediums of estradiol；B：Western MLA10 (F99E) and MLA10 (D502V) protein level in Blotting detection figures A；C：In Real-time PCR detection figures A Add in the 1/2MS culture mediums of 10 μM of estradiol PR1 and PR2 gene expression amounts in transfer-gen plant and WT lines；D：DAB With the lower pER8 of Trypan Blue detection estradiol induction:MLA10(F99E)-HA、pER8:MLA10 (D502V)-HA and wild type The accumulation of peroxide and cell death situation, scale 1mm in Col-0 cotyledons.

Fig. 3 is that can to strengthen arabidopsis white to arabidopsis by MLA10 (F99E) and MLA10 (D502V) in the embodiment of the present invention 3 The resistance of powder bacterium；Wherein, A：Transgenic arabidopsis connects bacterium phenotypic analysis.pps、PR1:MLA10 (F99E)-HA (pps) and PR1: MLA10 (D502V)-HA (pps) grows 28 days under the conditions of short-day, inoculation G.orontii phenotype, scale 1cm after 8 days； B：Figure A meets the blade powdery mildew growing state after bacterium, scale 1cm；C：Arabidopsis is through DAB and Kao Ma in micro- sem observation figure B This light blue dyes the upgrowth situation and peroxide accumulation of rear blade mycelia, and scale is 100 μm.

Fig. 4 is the plasmid map for the expression vector cTAPi-native promoter-GW-3HA that the present invention transforms.

Fig. 5 is recombinant expression carrier PR1 in the embodiment of the present invention 3:MLA10 (F99E)-HA plasmid map.

Fig. 6 is recombinant expression carrier PR1 in the embodiment of the present invention 3:MLA10 (D502V)-HA plasmid map.

Fig. 7 is initial carrier in the embodiment of the present invention 1:CTAPi-3HA plasmid map.

Fig. 8 is the plasmid map of recombinant expression carrier CTAPi-MLA10 (F99E) -3HA in the embodiment of the present invention 1.

Fig. 9 is the plasmid map of recombinant expression carrier cTAPi-MLA10 (D502V) -3HA in the embodiment of the present invention 1.

Embodiment

Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.Unless otherwise specified, embodiment According to conventional laboratory conditions, as Sambrook equimoleculars Cloning: A Laboratory Manual (Sambrook J＆Russell DW, Molecular Cloning:A Laboratory Manual, 2001), or the condition according to manufacturer's specification suggestion.

Primer sequence information in embodiment 1-3 used in construction recombination plasmid is as shown in table 1.

Embodiment 1 builds the transgenic arabidopsis that 35S promoter driving is overexpressed MLA10 (F99E) and MLA10 (D502V) Strain

Drive overexpression MLA10's (F99E) and MLA10 (D502V) respectively by building 35S promoter in the present embodiment Transgenic arabidopsis strain, 17 and 29 transgenic lines are obtained respectively.

Specifically, using GATEWAY carrier construction method, by artificial synthesized MLA10 (F99E) and MLA10 (D502V) gene is cloned and is connected to purpose support C TAPi-GW-3HA (plasmid map is as shown in fig. 7, i.e. CTAPi- respectively 3xHA), recombinant expression carrier CTAPi-MLA10 (F99E) -3HA (Fig. 8) and CTAPi-MLA10 (D502V) -3HA (figures are obtained 9)。

Then Agrobacterium competence GV3101 is converted respectively, using flower-dipping method, is infected arabidopsis, is utilized herbicide BASTA Screened to obtain positive arabidopsis transfer-gen plant.

35S:MLA10 (F99E) -3HA and 35S:MLA10 (D502V) -3HA is overexpressed plant 50% compared with wild type T2 short and small phenotype is shown for plant.However, this short and small phenotype fades away during passage, wherein, 35S: All strain phenotypes of MLA10 (F99E) -3HA are all lost in T3 generations, are only obtained 2 T3 at present and are represented the more stable 35S of type: MLA10 (D502V) -3HA transgenic arabidopsis strains, are 35S respectively:MLA10 (D502V) -3HA#17-3 and 35S:MLA10 (D502V) -3HA#19-1, they show as short and small phenotype (Figure 1A).Transfer-gen plant is examined by semiquantitive PCR Survey, find compared with compareing Col-0,35S:MLA10 (D502V) -3HA#17-3 and 35S:MLA10 (D502V) -3HA#19-1 plants MLA10 genes in strain have obvious accumulation (Figure 1B) in rna level.Real-time PCR results show, 35S:MLA10 (D502V) -3HA#17-3 and 35S:Pathogenesis related gene PR1 and PR2 expression quantity also have in MLA10 (D502V) -3HA#19-1 Obvious up-regulation (Fig. 1 C).

Test result indicates that, MLA10 (D502V) is overexpressed and can result in the short and small phenotype of arabidopsis, still, its phenotype above It is unstable, easily weaken or lose in follow-on transfer-gen plant.MLA10 (D502V), which is overexpressed, can trigger course of disease phase Correlation gene PR1 and PR2 up-regulated expression.

Embodiment 2 builds estradiol inducible promoter and drives expression MLA10's (F99E) and MLA10 (D502V) to turn base Because of arabidopsis strain

Because 35S promoter drives overexpression MLA10 (F99E) and MLA10 (D502V) transgenic arabidopsis strain respectively It is that phenotype is unstable, and phenotype evanescence in succeeding generations.In order to further determine that MLA10 (F99E) and MLA10 (D502V) Expression can trigger short and small phenotype in arabidopsis, and we further utilize estradiol inducible promoter, drive MLA10 (F99E) expressed with MLA10 (D502V) in arabidopsis, structure inducible expression MLA10 (F99E) and MLA10 (D502V) turn Gene arabidopsis strain pER8:MLA10 (F99E)-HA and pER8:MLA10 (D502V)-HA, 47 and 53 are obtained respectively and is turned Gene arabidopsis strain.

Specifically, the carrier construction method connected using digestion, pER8 carriers are utilized into Xhol I and the double digestions of Spe I, enzyme The carrier segments reclaimed after cutting are connected with MLA10 (F99E)-HA and MLA10 (D502V)-HA PCR fragments respectively, are recombinated Expression vector pER8:MLA10 (F99E)-HA and pER8:MLA10 (D502V)-HA, Agrobacterium competence is then converted respectively GV3101, using flower-dipping method, arabidopsis is infected, screened to obtain positive arabidopsis transgenosis using hygromycin Hygromycin Plant.

Wherein there are 11 pER8:- HA and 17 pER8 of MLA10 (F99E):MLA10 (D502V)-HA transgenic lines, which tie up to, to be contained Have and short and small phenotype is shown on the 1/2MS culture mediums of estradiol.With 35S:MLA10 (F99E) -3HA and 35S:MLA10 (D502V) -3HA transgenic arabidopsis is compared, and the phenotype of this two classes transfer-gen plant is relatively stable.The pER8 of T3 generation homozygosis: MLA10 (F99E)-HA#8 and pER8:MLA10 (D502V)-HA#1 have obvious short and small on the culture medium of addition estradiol Phenotype (Fig. 2A).On normal 1/2MS culture mediums, transgenic arabidopsis phenotype no significant difference compared with wild type, but female In glycol inducing culture, transgenic Arabidopsis plants show as blade and diminish, crispatura, and petiole shortens isophenous.Western Blotting testing results show the pER8 on the 1/2MS culture mediums of addition estradiol:MLA10 (F99E)-HA#8 and pER8: MLA10 (D502V)-HA#1 has obvious MLA10 (F99E) and MLA10 (D502V) protein expression, and in 1/ without estradiol When on 2MS culture mediums, MLA10 (F99E) and MLA10 (D502V) expression (Fig. 2 B) can't detect.Above experimental result is further Indicate MLA10 (F99E) and MLA10 (D502V) and expressed in arabidopsis and can result in the short and small phenotype of Arabidopsis plant.

Real-time PCR detect hair to the PR gene expression amounts in transgenic arabidopsis on estradiol inducing culture It is existing, pER8:MLA10 (F99E)-HA#8 and pER8:Pathogenesis related gene PR1 and PR2 expression in MLA10 (D502V)-HA#1 Amount has obvious rising (Fig. 2 C) compared with wild type.pER8:MLA10 (F99E)-HA#8 and pER8:MLA10(D502V)-HA# 1 grows 7 days on normal incubation medium, and phenotype is normal, compared with wild type, without significant difference, but transfers them to containing female Continued growth 7 days on the culture medium of glycol, find plant in addition to grow retardation, its blade particularly cotyledon occur flavescence, Withered isophenous, we are dyed using DAB dyeing and trypan blue to its cotyledon, find pER8:MLA10(F99E)-HA#1 And pER8:There are obvious peroxide accumulation and Cell death (Fig. 2 D) in MLA10 (D502V)-HA#8.

It is above-mentioned test result indicates that, MLA10 (F99E) and MLA10 (D502V) are expressed in arabidopsis, can not only be influenceed The normal growth of arabidopsis, cause to produce short and small phenotype, while can also induce the up-regulated expression of arabidopsis PR genes, trigger plant Produce the reactions such as peroxide accumulation, cell death.These reactions and the HR that plant is triggered when resisting extraneous pathogen are anti- Should be quite similar.MLA10 is as an ill-resistant protein, and mediation barley is to the barley white powder containing nontoxic effector AvrMLA10 The race specific resistance of bacterium biological strain.So, MLA10 (F99E) and MLA10 (D502V) obtains as MLA10 two functions Obtain property mutant, if the disease resistance response to arabidopsis powdery mildew can be mediated in arabidopsis.Next, we are to growth 28 It pER8:MLA10 (F99E)-HA#8 and pER8:MLA10 (D502V)-HA#1 external sources spray estradiol induction MLA10 (F99E) expressed with MLA10 (D502V), and be inoculated with arabidopsis powdery mildew Golovinomyces orontii, observe disease-resistant table Type.But find that the external estradiol that sprays induces MLA10 (F99E) and MLA10 through Protein Detection (Wstern Blotting methods) (D502V) expression effect unobvious.

Embodiment 3 builds PR1 promoters driving expression MLA10 (F99E) and MLA10 (D502V) transgenic arabidopsis strain System

Many reports show PR1 under the induction of arabidopsis powdery mildew, expression quantity significantly rise (Wu et al., 2015； Zhao et al.,2015).In order to determine whether MLA10 (F99E) and MLA10 (D502V) mediate arabidopsis pair in arabidopsis The resistance of powdery mildew, the transgenosis that we construct PR1 promoter driving MLA10 (F99E) and MLA10 (D502V) expression are intended Southern mustard strain PR1:MLA10 (F99E)-HA and PR1:MLA10 (D502V)-HA, and it is inoculated with arabidopsis powdery mildew Golovinomyces orontii, observe its disease-resistant situation.

We have cloned arabidopsis PR1 upstream region of gene 2112bp promoter sequences (SEQ ID NO:2), respectively with MLA10 (F99E) merged with MLA10 (D502V) gene order, height sense arabidopsis powdery mildew Trimutant (pen2pad4sag101, Pps) under background, PR1 promoters driving expression MLA10 (F99E) and MLA10 (D502V) transgenic arabidopsis strain are constructed System, i.e. PR1:MLA10 (F99E)-HA (pps) and PR1:MLA10(D502V)-HA(pps).

Specifically, the carrier construction method connected using digestion, by cTAPi-native promoter-GW-3HA carriers (the expression vector cTAPi transformed, being named as CTAPi-native promoter-3xHA kan, Fig. 4 certainly) using Xbal I with The double digestions of Spe I, the carrier segments reclaimed after digestion respectively with PR1-MLA10 (F99E) and PR1-MLA10 (D502V) PCR fragment Using enzyme connection is seamlessly connected, recombinant expression carrier PR1 is obtained:MLA10 (F99E)-HA (i.e. CTAPi-P PR1：MLA10 (F99E) -3xHA kan, Fig. 5) and PR1:MLA10 (D502V)-HA (i.e. CTAPi-P PR1：MLA10(D502V)-3xHA Kan, Fig. 6), then respectively convert Agrobacterium competence GV3101, using flower-dipping method, infect arabidopsis Trimutant pps, containing Have and screened on the 1/2MS culture mediums of 100 μM of kanamycins, obtain transgenic arabidopsis positive plant..

Wherein, PR1-MLA10 (F99E) and PR1-MLA10 (D502V) PCR fragment design primer when need to be by PR1 promoters The ends of reverse primer PR1-R 5 ' connection MLA10 forward direction sequences, MLA10 positive sequence 5 ' end connection PR1-R sequence, using taking The method built, designs two-wheeled PCR, and amplification obtains PR1-MLA10 (F99E) and PR1-MLA10 (D502V) PCR fragment.

The transgenic arabidopsis phenotype in observation 28 days T2 generations of growth, find there is 10% or so plant compared with pps, show For similar to 35S:MLA10 (D502V) -3HA short and small phenotype, other transfer-gen plant sizes are compared with the control without obvious area Not.The transfer-gen plant of normal size is inoculated with arabidopsis powdery mildew Golovinomyces orontii by us, and discovery connects bacterium 3 After it, PR1:MLA10 (F99E)-HA (pps) and PR1:MLA10 (D502V)-HA (pps) blade shows dewatering symptom, 8 days After observe its phenotype and find transgenic arabidopsis PR1:MLA10(F99E)-HA(pps)、PR1:MLA10(D502V)-HA(pps) It is dead withered to show blade, white powder bacteria growing is suppressed, and shows as disease-resistant phenotype (Fig. 3 A, B).

Dock the product that the Arabidopsis leaf after bacterium carries out peroxide in DAB dyeing and coomassie brilliant blue staining observation blade Involve the growing state of mycelia.Experiment finds that control pps blade surface has a large amount of powdery mildew mycelia and its spore to gather, almost Accumulation without peroxide.And the growth of the mycelia of transgenic arabidopsis MLA10 (F99E), MLA10 (D502V) powdery mildew by It is obvious to suppress, only a small amount of mycelia and spore, and there is accumulating for peroxide (to scheme in the blade cell infected by mycelia 3C).When can be seen that transgenic Arabidopsis plants normal in size from above experimental result, and being inoculated with arabidopsis powdery mildew, turn Gene arabidopsis can start the disease resistance response to arabidopsis powdery mildew rapidly, trigger the reactions such as ROS accumulation and cell death, suppression Powdery mildew growth and breeding processed, strengthens the resistance to arabidopsis powdery mildew.

Although above the present invention is described in detail with a general description of the specific embodiments, On the basis of the present invention, it can be modified or improved, this will be apparent to those skilled in the art.Cause This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.

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Sequence table

<110>Inst. of Genetics and Development Biology, CAS

<120>Barley ill-resistant protein MLA10 mutant is improving plant to the application in powdery mildew resistance

<130> KHP171117219.0

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 951

<212> PRT

<213>Barley (Hordeum vulgare L.)

<400> 1

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

1 5 10 15

Glu Leu Leu Thr Glu Glu Phe Lys Leu His Lys Gly Val Lys Lys Asn

20 25 30

Ile Glu Asp Leu Gly Lys Glu Leu Glu Ser Met Asn Ala Ala Leu Ile

35 40 45

Lys Ile Gly Glu Val Pro Arg Glu Gln Leu Asp Ser Gln Asp Lys Leu

50 55 60

Trp Ala Asp Glu Val Arg Glu Leu Ser Tyr Val Ile Glu Asp Val Val

65 70 75 80

Asp Lys Phe Leu Val Gln Val Asp Gly Ile Lys Ser Asp Asp Asn Asn

85 90 95

Asn Lys Phe Lys Gly Leu Met Lys Arg Thr Thr Glu Leu Leu Lys Lys

100 105 110

Val Lys His Lys His Gly Ile Ala His Ala Ile Lys Asp Ile Gln Glu

115 120 125

Gln Leu Gln Lys Val Ala Asp Arg Arg Asp Arg Asn Lys Val Phe Val

130 135 140

Pro His Pro Thr Arg Thr Ile Ala Ile Asp Pro Cys Leu Arg Ala Leu

145 150 155 160

Tyr Ala Glu Ala Thr Glu Leu Val Gly Ile Tyr Gly Lys Arg Asp Gln

165 170 175

Gly Leu Met Arg Leu Leu Ser Met Glu Gly Asp Asp Ala Ser Asn Lys

180 185 190

Arg Leu Lys Lys Val Ser Ile Val Gly Phe Gly Gly Leu Gly Lys Thr

195 200 205

Thr Leu Ala Arg Ala Val Tyr Glu Lys Ile Lys Gly Asp Phe Asp Cys

210 215 220

Arg Ala Phe Val Pro Val Gly Gln Asn Pro Asp Met Lys Lys Val Leu

225 230 235 240

Arg Asp Ile Leu Ile Asp Leu Gly Asn Pro His Ser Asp Leu Ala Met

245 250 255

Leu Asp Ala Asn Gln Leu Ile Lys Lys Leu His Glu Phe Leu Glu Asn

260 265 270

Lys Arg Tyr Leu Val Ile Ile Asp Asp Ile Trp Asp Glu Lys Leu Trp

275 280 285

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

290 295 300

Leu Ile Thr Thr Thr Arg Ile Val Ser Val Ser Asn Ser Cys Cys Ser

305 310 315 320

Ser Asp Gly Asp Ser Val Tyr Gln Met Glu Pro Leu Ser Val Asp Asp

325 330 335

Ser Arg Met Leu Phe Tyr Lys Arg Ile Phe Pro Asp Glu Asn Ala Cys

340 345 350

Ile Asn Glu Phe Glu Gln Val Ser Arg Asp Ile Leu Lys Lys Cys Gly

355 360 365

Gly Val Pro Leu Ala Ile Ile Thr Ile Ala Ser Ala Leu Ala Gly Asp

370 375 380

Gln Lys Met Lys Pro Lys Cys Glu Trp Asp Ile Leu Leu Arg Ser Leu

385 390 395 400

Gly Ser Gly Leu Thr Glu Asp Asn Ser Leu Glu Glu Met Arg Arg Ile

405 410 415

Leu Ser Phe Ser Tyr Ser Asn Leu Pro Ser Asn Leu Lys Thr Cys Leu

420 425 430

Leu Tyr Leu Cys Val Tyr Pro Glu Asp Ser Met Ile Ser Arg Asp Lys

435 440 445

Leu Ile Trp Lys Trp Val Ala Glu Gly Phe Val His His Glu Asn Gln

450 455 460

Gly Asn Ser Leu Tyr Leu Leu Gly Leu Asn Tyr Phe Asn Gln Leu Ile

465 470 475 480

Asn Arg Ser Met Ile Gln Pro Ile Tyr Asn Tyr Ser Gly Glu Ala Tyr

485 490 495

Ala Cys Arg Val His Asp Met Val Leu Asp Leu Ile Cys Asn Leu Ser

500 505 510

Asn Glu Ala Lys Phe Val Asn Leu Leu Asp Gly Thr Gly Asn Ser Met

515 520 525

Ser Ser Gln Ser Asn Cys Arg Arg Leu Ser Leu Gln Lys Arg Asn Glu

530 535 540

Asp His Gln Ala Arg Pro Phe Thr Asp Ile Lys Ser Met Ser Arg Val

545 550 555 560

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

565 570 575

Ser Arg Phe Asp Val Leu Arg Val Leu Asp Leu Ser Arg Cys Asn Leu

580 585 590

Gly Glu Asn Ser Ser Met Gln Leu Asn Leu Lys Gly Val Gly His Leu

595 600 605

Thr His Leu Arg Tyr Leu Gly Leu Glu Gly Thr Asn Ile Ser Lys Leu

610 615 620

Pro Ala Glu Ile Gly Lys Leu Gln Phe Leu Glu Val Leu Asp Leu Glu

625 630 635 640

Asn Asn His Asn Leu Lys Glu Leu Pro Ser Thr Val Cys Asn Phe Arg

645 650 655

Arg Leu Ile Tyr Leu Asn Leu Val Gly Cys Gln Val Val Pro Pro Val

660 665 670

Gly Val Leu Gln Asn Leu Thr Ser Ile Glu Val Leu Ser Gly Ile Leu

675 680 685

Val Ser Leu Asn Ile Ile Ala Gln Glu Leu Gly Asn Leu Lys Arg Leu

690 695 700

Arg Glu Leu Asn Ile Leu Phe Asn Asp Gly Ser Leu Asp Phe Tyr Glu

705 710 715 720

Gly Phe Val Lys Ser Leu Cys Asn Leu His His Ile Glu Ser Leu Ile

725 730 735

Phe Asp Cys Lys Ser Ile Glu Thr Ser Ser Phe Glu Leu Met Asp Leu

740 745 750

Leu Gly Glu Arg Trp Ile Pro Pro Val His Leu Arg Glu Phe Lys Ser

755 760 765

Phe Met Pro Ser Gln Leu Ser Ala Leu Arg Gly Trp Ile Gln Arg Asp

770 775 780

Pro Ser His Leu Ser Asn Leu Ser Glu Leu Thr Leu Thr Ser Val Lys

785 790 795 800

Glu Val Gln Gln Asp Asp Val Val Ile Ile Gly Ala Leu Ser Ser Leu

805 810 815

Arg Arg Leu Cys Ile Arg Ser Thr His Gln Thr Gln Arg Leu Leu Val

820 825 830

Ile His Ala Asp Gly Phe Arg Cys Ile Val Tyr Phe Gln Leu Asp Cys

835 840 845

Gly Ser Ala Thr Gln Ile Leu Phe Glu Pro Gly Ala Leu Pro Arg Ala

850 855 860

Glu Val Val Ala Phe Ser Leu Ala Val Arg Val Ala Lys Glu Asp Gly

865 870 875 880

Asn Cys Gly Phe Asp Leu Gly Leu Gln Gly Asn Leu Phe Ser Leu Arg

885 890 895

Gln Phe Val Ser Val Ile Ile Tyr Cys Gly Gly Ala Arg Val Gly Glu

900 905 910

Ala Lys Glu Ala Glu Ala Ala Val Arg Arg Ala Leu Asp Ala His Pro

915 920 925

Asn His Pro Gln Ile Ala Ile Phe Met His Pro Pro Ile Ala Glu Gly

930 935 940

Ala Gln Asp Asp Asp Leu Met

945 950

<210> 2

<211> 2112

<212> DNA

<213>Arabidopsis (Arabidopsis thaliana)

<400> 2

taggcagcaa gtcatttaca aagtaaaaaa tttctccatg catgtaacct tcatttatca 60

ttcattttag tttgtaactt tttattagat tttgatcaag ttaaccgcta aaatctcatt 120

ttatccgttc gcattaaagt taaatagatt gctgacatat tttaaatcta atagaaaatg 180

ccatctggca aataaacaac ggacacgatt ttaaactaaa ttttaccaaa aagaaaaaac 240

ttatacgact tttcttgctt agaagtcttt gcattgttaa tagattgttg aaaaggttta 300

ttcattactt tcatgcagag agataacata tcatcgcgtg gggatttatt caatccaaag 360

aaaagcttcc aaaaactgac tttgcttcat gaaacactca ctctaatttg cttcatcaat 420

cttaggactg acttttccaa atcaatatgc gaactatctt ctaatttaca ttggtttcgt 480

gttttttcga aaggagacaa ctatcttttt aaaagctttt ctatagtgtg atgacaaaaa 540

aaaaatgtaa ttgttagttg caaaagaaaa gtacaatagt cttttctagt tttgagagtt 600

taaggtttat gatcggaact tagagtttaa atttaaacta ttttgttaat ttttggactg 660

ataacagttt ttttttgaaa atattgaaac gttgtttacc taatgtaaca tgttattcta 720

cttaaattac tttatatttt aataacatat aatattgaat aggatatcat aggatattat 780

tacgtaataa tatcctatgg tgtcatttta taagttagca caagcttgtt ttaacttata 840

aaatgattct ccctccatat aaaaaagttt gattttatag aatgtttata ccgattaaaa 900

aaataataat gcttagttat aaattactat ttattcatgc taaactattt ctcgtaacta 960

ttaaccaata gtaattcatc aaattttaaa attctcaatt aattgattct tgaaattcat 1020

aaccttttaa tattgattga taaaaatata cataaactca atctttttaa tacaaaaaaa 1080

ctttaaaaaa tcaatttttc tgattcggag ggagtatatg ttattgctta gaatcacaga 1140

ttcatatcag gattggaaaa ttttaaagcc agtgcatatc agtagtcaaa attggtaaat 1200

gatatacgaa ggcggtacaa aattaggtat actgaagata gaagaacaca aaagtagatc 1260

ggtcacctag agtttttcaa tttaaactgc gtattagtgt ttggaaaaaa aaaacaaagt 1320

gtatacaatg tcaatcggtg atcttttttt tttttttttt tttttttttt ctttttggat 1380

aaatctcaat gggtgatcta ttgactgttt ctctacgtca ctattttact tacgtcatag 1440

atgtggcggc atatattctt caggactttt cagccatagg caagagtgat agagatactc 1500

atatgcatga aacactaaga aacaaataat tcttgacttt ttttctttta tttgaaaatt 1560

gactgtagat ataaactttt attttttctg actgtaaata taatcttaat tgccaaactg 1620

tccgatacga tttttctgta ttatttacag gaagatatct tcaaaacatt ttgaatgaag 1680

taatatatga aattcaaatt tgaaatagaa gacttaaatt agaatcatga agaaaaaaaa 1740

aacacaaaac aactgaatga catgaaacaa ctatatacaa tgtttcttaa taaacttcat 1800

ttagggtata cttacatata tactaaaaaa atatatcaac aatggcaaag ctaccgatac 1860

gaaacaatat taggaaaaat gtgtgtaagg acaagattga caaaaaaata gttacgaaaa 1920

caacttctat tcatttggac aattgcaatg aatattacta aaatactcac acatggacca 1980

tgtatttaca aaaacgtgag atctatagtt aacaaaaaaa aaaagaaaaa aatagttttc 2040

aaatctctat ataagcgatg tttacgaacc ccaaaatcat aacacaacaa taaccattat 2100

caacttagaa aa 2112

Claims

1. barley ill-resistant protein MLA10 mutant is improving plant to the application in powdery mildew resistance, it is characterised in that described prominent Variant is such as SEQ ID NO:The 99th phenylalanine of barley ill-resistant protein MLA10 shown in 1 sports glutamic acid formation Mutant, or the mutant that the 502nd Aspartic acid mutations are formed by valine.

2. application according to claim 1, it is characterised in that the plant includes arabidopsis, wheat.

3. application according to claim 1 or 2, it is characterised in that the powdery mildew includes arabidopsis powdery mildew (Golovinomyces orontii), pseudomonas syringae (Pseudomonas syringae), oomycetes (Hyaloperonospora arabidopsidis)。

4. barley ill-resistant protein MLA10 mutant expression cassettes, it is characterised in that by pathogenic bacterium inducing type promoter and be driven by it Barley ill-resistant protein MLA10 mutant encoding gene composition；Wherein, the definition of the mutant is the same as described in claim 1.

5. expression cassette according to claim 4, it is characterised in that the pathogenic bacterium inducing type promoter is arabidopsis PR1 Gene promoter, its nucleotide sequence such as SEQ ID NO:Shown in 2.

6. the expression vector containing the expression cassette of claim 4 or 5.

7. the engineering bacteria containing expression vector described in the expression cassette of claim 4 or 5 or claim 6.

8. a kind of improve method of the arabidopsis to arabidopsis powdery mildew resistance, it is characterised in that including：

1) arabidopsis is made to contain the expression cassette of claim 4 or 5；Or

2) arabidopsis is made to express barley ill-resistant protein MLA10 mutant；

Wherein, the definition of the mutant is the same as described in claim 1.

9. according to the method for claim 8, it is characterised in that comprise the following steps：

S1, the structure expression cassette of claim 4 or 5；

On S2, the expression vector cTAPi for transforming the insertion of above-mentioned expression cassette, recombinant plasmid is obtained；

10. according to the method for claim 9, it is characterised in that the expression vector cTAPi transformed described in step S2 refers to Fall initial carrier cTAPi 35S promoter, then change herbicide BASTA resistances into kalamycin resistance, and in target gene Obtained below plus 3HA labels.