CN102586268B - Cloning and application of arabidopsis oidium disease resistance suppressor gene EDTS3 - Google Patents
Cloning and application of arabidopsis oidium disease resistance suppressor gene EDTS3 Download PDFInfo
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Abstract
The invention relates to separated cloning, functional verification and application of arabidopsis oidium disease resistance suppressor gene EDTS3, and further relates to a mutant gene of the EDTS3 gene, protein encoded by the EDTS3 gene and application of the EDTS3 gene.
Description
Technical field
The application relates to the plant gene engineering technology field, is specifically related in the model plant Arabidopis thaliana clone, functional verification and application to the positive regulatory factor of powdery mildew resistance.
Background technology
Powdery Mildew is the Plant diseases that the Erysiphaceae in fungi causes.When plant is infected morbidity, can produce the macroscopic white meal formed by mycelium, conidiophore and conidium in a large number and gain the name therefrom.Powdery Mildew is widely distributed in the whole world, and the harm dicotyledons is particularly general.It also infects multiple grass simultaneously, as wheat, barley, oat and multiple herbage.For example wheat powdery mildew is to be caused by specialization parasitical fungi standing grain dlumeria graminis, is one of Major Diseases affected Wheat Production.In China, wheat powdery mildew once was very popular continuously several times, had caused great financial loss.
Because there are various pathogenic bacterias in occurring in nature, plant has formed gradually multi-level defense system in long-term coevolution, and it mainly comprises the resistance of non-host-resistance, basic resistance and disease-resistant gene mediation.
Non-host-resistance is mainly by plant self structure and secondary metabolite defence, as PLANT CYTOSKELETON and de novo synthesis phytoalexin-antibiotic complex, so there are wide spectrum, characteristics that resistance is strong and lasting, be that plant can be resisted the major cause that most of latent pathogens infect.
The basis resistance has another name called the resistance (PAMP-Triggered Immunity, PTI) excited based on pathogen-associated molecular pattern (Pathogen-associated molecularpatterns, PAMPs).Pathogen-associated molecular pattern is that host self does not have, but extensively exists in pathogenic bacteria, and to its life history of the characterization of molecules of most important and high conservative again, as flagellin, the elongation factor of bacterium, the lipopolysaccharides in fungi and chitin etc.The physical efficiency that is subject on host cell surface is identified these molecules, and the cascade signal path of the MAPK in activation downstream, signal is transmitted into core the most at last, activate the protein induced course of disease (Pathogen-Responsive of WKRY, PR) expression of gene, the generation of active oxygen (Reactive OxygenSpecies, ROS) and dextran (callose) be in the accumulation (1) of p of E, thereby resist the invasion of pathogenic bacteria.The characteristics of basis resistance are that anti-spectrum is wide, but resistance level is lower usually.
The resistance of disease-resistant gene mediation has another name called the resistance (Effector TriggeredImmunity, ETI) that effector excites.After host plant is evolved out PTI, the infection mechanism that the part pathogenic bacteria develops and makes new advances.For example, bacterium utilizes III type excretory system that effector is directly imported to vegetable cell inside, suppresses the generation of PTI.In order to resist infecting of these pathogenic bacterias, plant evolution has gone out disease-resistant gene (Resistance gene, the R gene) come the recognition effect factor (at this moment just can be called Avr albumen) induced hypersensitivity reaction (Hypersensitive Response, HR) the growth that limits pathogenic bacteria and spread (Chisholm et al., 2006).The characteristics of the resistance of R gene mediated are high specificities, and anti-spectrum is narrow, and resistance is higher.
Adjusting and the control of three kinds of signaling molecules in the disease resistance response Chang Shouti of plant, these signaling molecules are respectively Whitfield's ointment SA, jasmonic JA and ethene ET.Wherein SA is the center regulatory factor of various Plant defense responses, is the basis of setting up (System Acquire Resistance, SAR).And be mutual antagonism by disease-resistant signal path and the SA path of JA/ET mediation.Comparatively typical example is exactly that false pseudomonas bacillus a kind of plant growth substance psendomonas syringae (coronatine) of producing similar JA/MeJA suppresses the resistance of SA-mediation, thereby reduces the resistance (2) of plant.
At present, the cloned resistance gene method mainly contains: subtractive hybridization, yeast two-hybrid system, map-based cloning, transposon tagging and homologous gene clone etc.For the model plant Arabidopis thaliana, the map based cloning isolated genes becomes one of topmost method, especially the genome sequencing plan complete the discovery with various molecule markers, and the simplification increasingly of DNA extraction method, significantly shorten the clone gene spended time.
Current disease-resistant research shows, Arabidopis thaliana is similar with the unifacial leaf grass with other dicotyledonss on disease-resistant molecule mechanism.With the R gene, compare, disease-resistant related gene can provide more wide spectrum and long-acting resistance for plant.Utilizing the purpose of model plant clone plant disease-resistant related gene, is the homologous gene that has similar functions in important plant in order to find, and by plant gene engineering technology, improves the resistance of wide spectrum of plant to pathogenic bacteria.
The disease-resistant gene of cloning from Arabidopis thaliana has potential using value in production.For example, RPW8 is a disease-resistant gene of cloning on Arabidopis thaliana, and NPR1 is the important regulating and controlling factor of the disease resistance response of cloning in Arabidopis thaliana.In tobacco, expressing the RPW8 gene can improve the resistance of tobacco to Powdery Mildew excessively; In paddy rice, expressing arabidopsis gene NPR1 can improve the resistance of paddy rice to bacterium excessively.
EDR1, EDR2 and EDR3 are the genes involveds of regulation and control powder mildew resistance in Arabidopis thaliana, the sudden change of these genes cause plant to strengthen the disease resistance of powdery mildew and the necrocytosis (Program Cell Death) of inducing generation (3-5).Wherein, the EDR1 protein kinase of encoding, have very high homology with the CTR1 in ethylene reaction; The EDR3 macromolecular GTPase that encodes.The edr2 mutant, with edr1, edr3 has similar phenotype, also has the old and feeble reaction of the powder mildew resistance of enhancing and ethene, and its disease resistance also needs the Whitfield's ointment signal path, does not need jasmonic or Ethylene Signal path.The EDR2 novel albumen of encoding, there are three structural domains, comprise a Pleckstrin Homology (PH) structural domain, STeroidogenic Acute Regulatoryprotein-related lipid-Transfer (START) structural domain and Unknown Function but the conservative carboxyl terminal structural domain DUF1336 of sequence.The function in the first two zone may be and contaminated with lipid that the carboxyl terminal structural domain may participate in the interaction of albumen and albumen.EDR2 has two lipid molecule lands, and the disease resistance of hint lipid metabolism and Powdery Mildew and the programmed death of vegetable cell are closely related.
Take edr2 as point of penetration, utilize the comparatively complete and system of the research of model plant Arabidopis thaliana, technology is advanced person and ripe advantage comparatively, excavate disease-resistant related gene, accelerate the understanding of enantiopathy gene function and the understanding of signal path and even network structure and expand, by very possible disease-resistant control and Molecular design breeding for farm crop, provide theoretical foundation and technical support.
Summary of the invention
The application is included in the model plant Arabidopis thaliana clone, functional verification and the application to the positive regulatory factor EDTS3 of powdery mildew resistance.The sudden change of EDTS3 gene can suppress the powdery mildew resistance of Arabidopsis Mutants edr2, and suppresses powdery mildew simultaneously and induce generation programmed cell death (Programmedcell death, PCD).
(1) a kind of Arabidopis thaliana mildew-resistance suppressor gene EDTS3 sequence, CDS full length sequence 264bp, intronless.Its nucleotide sequence is as shown in SEQ ID No.1.
(2) a kind of protein sequence of Arabidopis thaliana mildew-resistance genes involved EDTS3 coding, 87 amino acid of encoding, big or small 9.784kD.Its aminoacid sequence is as shown in SEQ ID No.2.
(3) after EDTS3 transgenation forfeiture self function, show the powdery mildew resistance that suppresses the edr2 mutant and suppress to be subject to powdery mildew to induce the generation necrocytosis.Its phenotype is shown in Fig. 1.
(4), according to the disease-resistant phenotype of the inhibition after the EDTS3 transgenation, can its expression amount be raise by genetic engineering technique, thereby improve the disease resistance of vegetables and crop.
Particular content is as follows:
1. an Arabidopis thaliana powdery mildew disease-resistant suppressor gene EDTS3, it one of is following nucleotide sequences:
1) nucleotide sequence of SEQ ID No.1;
2) there is the nucleotide sequence of 90% above homology and coding identical function protein with the nucleotide sequence of SEQ ID No.1.
2. a protein, it is by gene EDTS3 coding of above 1.
3. above 2 protein, it is:
1) by the albumen shown in the aminoacid sequence of SEQ ID No.2; Or
2) the derivative albumen by (1) through replacing, lack or adding one or several amino acid and there is the function identical with (1) by the aminoacid sequence of (1).
4. the mutator gene edts3 of above 1 gene EDTS3, the difference of the nucleotide sequence of its nucleotide sequence and SEQ ID No.1 is wherein to occur the A104G sudden change.
5. the protein that above 4 mutator gene edts3 encodes, the difference of the aminoacid sequence of its aminoacid sequence and SEQ ID No.2 is wherein to occur the R35K sudden change.
6. above 1 gene EDTS3 or above 2 or 3 protein induce for regulation and control (enhancer or inhibitor) Biology of Plant-Powdery Mildew Interaction resistance or regulation and control (promote or suppress) powdery mildew the application that produces programmed cell death, wherein said plant comprises Arabidopis thaliana, barley, wheat, is preferably Arabidopsis Mutants edr2.
7. above 6 application, wherein strengthen the Biology of Plant-Powdery Mildew Interaction resistance or promote powdery mildew to induce the application that produces programmed cell death by excessive in described plant or more than overexpression 1 gene EDTS3 or above 2 or 3 protein realize, suppress the Biology of Plant-Powdery Mildew Interaction resistance or suppress powdery mildew and induce the application that produces programmed cell death to realize by 1 gene EDTS3 more than suppressing in described plant or above 2 or 3 protein expression, wherein suppressing the gene EDTS3 of claim 1 or the protein expression of claim 2 or 3 can realize by the gene EDTS3 generation mistake function mutation that makes claim 1.
8. above 4 mutator gene edts3 is for suppressing the Biology of Plant-Powdery Mildew Interaction resistance and suppressing powdery mildew and induce the application that produces programmed cell death, and wherein said plant comprises Arabidopis thaliana, barley, wheat, is preferably Arabidopsis Mutants edr2.
The accompanying drawing explanation
Fig. 1. wild-type and mutant edr2, edts3/edr2 growth inoculate afterwards in 4-6 week the phenotype of Powdery Mildew (G.cichoracearum) after 8 days (on), Trypan blue coloration result (under).
Fig. 2. separate the sketch map of EDTS3 gene by the method for map based cloning.
Fig. 3. wild-type, mutant edr2, edts3/edr2 and complementary carrier transform mutant edts3/edr2 (T1) growth and inoculate afterwards the phenotype of Powdery Mildew (G.cichoracearum) after 8 days 4-6 week.
Embodiment
Embodiment mono-edts3 mutant suppresses disease-resistant phenotype analytical
1.edts3/edr2 mutant suppresses the powder mildew resistance of edr2 mutant and the necrocytosis that powdery mildew is induced
(1) materials and methods
Edts3/edr2 (wherein edts3 is enhanced disease resistance two suppressor 3) is by with 0.03%EMS (purchased from sigma company) mutagenesis mutant edr2 (4), filters out the mutant that can suppress the enhancing of edr2 Powdery Mildew resistance obtained from mutagenesis colony.An and then mutant called after edts3/edr2 that will obtain.
At first, mutant edr2, the seed of edts3/edr2 and wild-type col-0 (purchased from ArabidopsisBiological Resource Center (ABRC)) is broadcast MS substratum (being purchased the Laboratories from PhytoTechnology), after 4 ℃ of vernalization 2-3 days, transfer to the phytotron under 9h light/15hdark illumination condition.Approximately 7-10 days, move to seedling in soil.After growth of seedling 4-6 week, inoculation Powdery Mildew (G.cichoracearum) (6).This Powdery Mildew is preserved with the pad4 of the mutant to this Powdery Mildew susceptible (7) of Arabidopis thaliana.While connecing bacterium, growth is had the plant of the pad4 of a large amount of white powder spores to scrape lightly on the plant leaf of white powder waiting, the plant that then will connect bacterium first covers moisturizing 1 day with preservative film, after remove preservative film, the plant continued growth, after 7 days, is carried out disease-resistant phenotypic evaluation.The blade that will have typical phenotype is cut, and takes a picture and carries out trypan blue dyeing.
(2) results and analysis
After 8 days, on the blade of wild-type and double-mutant edts3/edr2, cover with white powder at the inoculation powdery mildew, and essentially no visible powdery mildew spore on the blade of mutant edr2, and show the necrocytosis that powdery mildew is induced.Dyeing has also proved the above results: the blade of wild-type and double-mutant edts3/edr2 has been covered with mycelia and conidiophore, and in mutant, dead mesophyll cell is dyed to blueness (Fig. 1).The above results shows that the edts3/edr2 mutant has suppressed edr2 powdery mildew resistance.
The acquisition of embodiment bis-EDTS3 genes
1. utilize map based cloning to isolate the EDTS3 gene
We utilize map based cloning to separate the EDTS3 gene.Specific practice is: mutant edts3/edr2 and environmental Lansberg (being purchased from ABRC) do hybridization, and the F1 of acquisition produces F2 generation for selfing.In F2 generation, the individual plant of the wild-type phenotype of selecting to contain the edr2 sudden change, then make its selfing produce F3 generation.Select the individuality of wild-type in for each strain at F3, be evenly distributed on chromosomal telltale mark on 5 karyomit(e)s (http://signal.salk.edu/genome/SSLP_info/SSLPsordered.html) by PCR method and utilization, the front end by the gene Primary Location at the disconnected arm of the 1st karyomit(e).Subsequently, utilize again about 3000 strain F3 for individual plant, by the zone of gene Fine Mapping 40kb between F9H16 and F26F24 (8) (Fig. 2).This zone comprises 12 genes.Find to contain a base mutation A104G in the CDS of Gene A t1g21327 by order-checking, and other genes are without any variation, thereby determine the candidate gene that this gene is the EDTS3 gene.
2.EDTS3 the structure of gene
EDTS3CDS total length 264bp, do not contain intron; EDTS3 albumen is a small protein that Serine is abundant, wherein a low complex degree structural domain, 23 amino acid, consists of.
The functional verification of embodiment tri-EDTS3 genes
For whether the resistance of checking edts3 sudden change to edr2 mutant powdery mildew suppresses because the At1g21327 base mutation causes, we have built the genetic transformation carrier with the At1g21327 of self promotor, utilize the method that flower infects to transform double-mutant edts3/edr2 plant, in T1 generation, transgenic line has recovered the phenotype of edr2 mutant.
We take the DNA of wild-type col-0 is template, with one couple of PCR primers F:5 '-AACTGCAGAAGTGGTGCTCACGTTGTTGTATTT-3 ' and R:5 '-AACTGCAGAAGTGGTGCTCACGTTGTTGTATTT-3 ', amplifies the At1g21327 gene fragment with the 5.7kb of KpnI and PstI.
The PCR reaction system
DNA： 2.0ul
10xbuffer： 5.0ul
dNTPs(2.0mM)： 5.0ul
MgSO4(25mM)： 2.0ul
Primers F (10uM): 1.6ul
Primer R (10uM): 1.6ul
KOD Taq(1U)： 1.0ul
H2O：31.8ul
The PCR response procedures: denaturation, 95 ℃ 3 minutes; Thermal cycling, in 95 ℃ of 30 second of sex change, anneal 55 ℃ of 30 second, extend 72 ℃ 5 minutes, 30 of cycle numbers.
Whole fragment comprises 3 of the coding region of promoter region, 0.26kb of 3.6Kb and 1.8Kb '-UTR.The fragment of amplification and pCAMBIA1300 (9) be double digestion (KpnI and PstI simultaneously, purchased from TaKaRa company, 37 ℃), connect (T4 ligase enzyme (purchased from NEB company), 16 ℃) and after the heat shock method transforms intestinal bacteria E.coli DH10B (purchased from full formula King Company), identify correct plasmid through order-checking (Hua Da genome company), transform Agrobacterium GV3101 (9) with freeze-thaw method subsequently.The method (10) of contaminating with flower transforms double-mutant edts3/edr2 plant, obtains T1 after 7 weeks for seed.To seed disinfection, cleaning, then be seeded in the MS substratum that adds Totomycin (80 μ g/ml).After 7-10 days, select T1 for the transgenic positive plant.
At 22 ℃, under 9h light/15h dark condition, when seedling grows to 4-6 week, we,, simultaneously wild-type, mutant and transgenosis T1 pickup kind powdery mildew, observe phenotype after 8 days.The double-mutant edts3/edr2 phenotype that proceeded to complete complementary of At1g21327 gene, show as edr2 phenotype (seeing Fig. 3) as a result.The disease-resistant phenotype that shows thus mutant is because the sudden change of At1g21327 gene causes.Therefore the EDTS3 gene is the At1g21327 gene.
The application of embodiment tetra-Arabidopis thaliana powdery mildew resistance related gene EDTS3 in the crop disease-resistant improvement
After EDTS3 transgenation forfeiture self function, show the powdery mildew resistance that suppresses the edr2 mutant and suppress to be subject to powdery mildew to induce the generation necrocytosis.Can make its excessive or overexpression by genetic engineering technique according to this characteristic, thereby improve the disease resistance of vegetables and crop, cultivate the crop varieties with disease-resistant proterties.
Reference
1.Chisholm ST，Coaker G，Day B，& Staskawicz BJ(2006)Host-microbeinteractions：shaping the evolution of the plant immune response.Cell124(4)：803-814.
2.Mudgett MB(2005)New insights to the function of phytopathogenic bacterialtype III effectors in plants.Annu Rev Plant Biol 56：509-531.
3.Frye CA，Tang D，& Innes RW(2001)Negative regulation of defense responsesin plants by a conserved MAPKK kinase.Proc Natl Acad Sci USA98(1)：373-378.
4.Tang D，Ade J，Frye CA，& Innes RW(2005)Regulation of plant defenseresponses in Arabidopsis by EDR2，a PH and START domain-containingprotein.Plant J 44(2)：245-257.
5.Tang D，Ade J，Frye CA，& Innes RW(2006)A mutation in the GTP hydrolysissite of Arabidopsis dynamin-related protein 1E confers enhanced cell death inresponse to powdery mildew infection.(Translated from eng)Plant J47(1)：75-84(in eng).
6.Adam L，et al.(1999)Comparison of Erysiphe cichoracearum and E.cruciferarum and a survey of 360 Arabidopsis thaliana accessions for resistanceto these two powdery mildew pathogens.Mol Plant Microbe Interact12(12)：1031-1043.
7.Jirage D，et al.(1999)Arabidopsis thaliana PAD4 encodes a lipase-like gene thatis important for salicylic acid signaling.(Translated from eng)Proc Natl AcadSci USA 96(23)：13583-13588(in eng).
8.Jander G，et al.(2002)Arabidopsis Map-Based Cloning in the Post-Genome Era.Plant Physiol.129(2)：440-450.
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Claims (3)
1. the mutator gene edts3 of an Arabidopis thaliana powdery mildew disease-resistant suppressor gene EDTS3, the difference of the nucleotide sequence of its nucleotide sequence and SEQ ID No.1 is wherein to occur the A104G sudden change.
2. the protein that the mutator gene edts3 of claim 1 encodes, the difference of the aminoacid sequence of its aminoacid sequence and SEQ ID No.2 is wherein to occur the R35K sudden change.
3. the mutator gene edts3 of claim 1 is for suppressing the Biology of Plant-Powdery Mildew Interaction resistance and suppressing powdery mildew and induce the application that produces programmed cell death, and wherein said plant is Arabidopsis Mutants edr2.
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