CN106754966B - Plant disease resistance related gene LL G1 and application thereof - Google Patents

Plant disease resistance related gene LL G1 and application thereof Download PDF

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CN106754966B
CN106754966B CN201710040307.8A CN201710040307A CN106754966B CN 106754966 B CN106754966 B CN 106754966B CN 201710040307 A CN201710040307 A CN 201710040307A CN 106754966 B CN106754966 B CN 106754966B
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llg1
edr1
resistance
disease resistance
powdery mildew
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CN106754966A (en
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唐定中
沈秋静
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Fujian Agriculture and Forestry University
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    • C12N15/8279—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
    • 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

The invention provides a plant disease resistance related geneLLG1（LORELEI‑LIKE‑GPI‑ANCHORED PROTEIN 1) The invention relates to a method for screening inhibitor mutants and cloning map, and a resistance related geneLLG1The separation and the functional identification are carried out, and the first discovery and the proof are carried outLLG1The role of the gene in plant resistance to powdery mildew, pseudomonas bacteria and oomycetes. In agricultural production, the invention can be applied to the creation of transgenic crops for improving the resistance of crops and the like.

Description

Plant disease resistance related gene LL G1 and application thereof
Technical Field
The invention relates to a plant disease resistance related geneLLG1And applications thereof.LLG1Mutation of gene can inhibit arabidopsis mutantedr1Resistance to powdery mildew, pseudomonas and oomycetes, and participate in regulating resistance responses to pathogenic bacteria. The invention belongs to the field of genetic engineering.
Background
Powdery mildew is an important plant disease, belongs to parasitic fungi of ascomycetes, is widely distributed around the world, can infect nearly ten thousand plants including important crops such as wheat and barley, and causes great harm to agricultural production. After the powdery mildew infects the plants, the produced spores can cover the stems, leaves, inflorescences and fruit surfaces of the plants to form white cobweb or form a layer of white powder, so the powdery mildew is called powdery mildew. Under the condition of proper environmental conditions, the powdery mildew can be propagated in large quantity, which affects the growth and development of plants and even causes the withered leaves and death in severe morbidity. Similar to powdery mildew, pseudomonas bacteria and oomycetes can infect various plants, causing great harm to agricultural production.
In order to combat infestation by various pathogenic microorganisms, plants have evolved a number of strategies to defend. In general, plant defense responses are divided into two levels, namely basal resistance and resistance mediated by disease resistance genes. Basal resistance is the first level of defense response by the Recognition of pathogen-associated molecular patterns (PAMPs) by Pattern receptors (PRRs) located on the cell membrane, triggering PAMPs-induced immune responses, also known as PTIs. Pathogenic bacteria have developed a new mechanism to inhibit PTI during long-term evolution, such as bacteria passing through the type III secretory system and fungi releasing effector (effector) into plants through haustoria. Some effector factors, also known as avirulence proteins (Avr proteins), can be recognized by proteins encoded by Resistance (R) genes. This level of defense response is also known as effector-triggered immune response (ETI).
Early studies found Arabidopsis thalianaedr1The mutants exhibited enhanced erysiphe necator resistance and an erysiphe necator-induced cell death phenotype.EDR1The gene codes a Raf-like protein kinase and has kinase activity. The genetic analysis experiment shows thatedr1The phenotype of the mutant can bepad4，sid2，npr1The mutant inhibits, which shows thatedr1The phenotype of (a) requires the action of the salicylic acid signaling pathway. However, at present we are dealing withEDR1The molecular mechanisms mediating cell death and the disease resistance response of plants remain poorly understood. In order to find genes involved in regulating plant resistance, we carried outedr1And (4) screening inhibitor mutants. We obtained one of the mutants, and by map-based cloning we obtained the corresponding geneLORELEI-LIKE-GPI-ANCHORED PROTEIN 1I.e. byLLG1. Through the functional analysis of the gene, we find for the first time thatLLG1Has important effect on the disease resistance of plants. Our results show thatLLG1Is an excellent candidate gene for creating transgenic crops with enhanced disease resistance and crop molecular design, and has important theoretical value and wide application prospect.
Disclosure of Invention
The invention aims to provide a plant disease resistance related geneLLG1And applications thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
disease-resistant related gene of arabidopsis thalianaLLG1The CDS has a total length of 507 bp. The CDS full-length sequence is shown as SEQ ID NO. 1.
LL G1 gene codes protein sequence, codes 168 amino acids, size 18.46 Kd. and its amino acid sequence is shown in SEQ ID NO. 2.
LLG1Can inhibit after mutationedr1Resistance to powdery mildew.
LL G1 is applied to powdery mildew, pseudomonas and oomycetes resistance, LL G1 can be applied to the technical field of genetic engineering, and disease resistance of crops is improved.
The invention has the advantages that: in order to find genes involved in regulating plant resistance, we carried outedr1And (4) screening inhibitor mutants. We obtained one of the mutants, and by map-based cloning we obtained the corresponding geneLLG1. Through the functional analysis of the gene, we find for the first time thatLLG1Has important effect on the disease resistance of plants. Our results show thatLLG1Is an excellent candidate gene for creating transgenic crops with enhanced disease resistance and crop molecular design, and has important theoretical value and wide application prospect.
Drawings
FIG. 1 shows the wild type of Arabidopsis thaliana,edr1Mutants andedr1 llg1inoculating powdery mildew after 4-5 weeks of growthGolovinomyces cichoracearumUCSC1, phenotype after day 8 of infection (a), staining of infected leaves with Trypan blue (Trypan blue) (B) and counting of the quantity of conidiophores of the monospores (C).
FIG. 2 Arabidopsis thaliana wild type andedr1mutants andedr1 llg1inoculated with oomycetes 2 weeks after growth: (H.aNoco 2) results of spore quantification after 7 days (a), and inoculation with pseudomonas syringae (c.syringae) after 4 weeks of growthPtoDC 3000) results of the statistics of the number of colony clones after 3 days (B).
FIG. 3 cloning by map-based cloningLLG1Schematic representation of genes (a), andLLG1gene structure, protein sequence and mutation site (b),LLG1all genes can be complementaryllg1To pairedr1Inhibition of the mutants ((fig. 3c, d) where fig. 3c shows the powdery mildew growth phenotype, fig. 3d shows the results of quantitative analysis of powdery mildew in plants of fig. 3 c.
Detailed Description
Example 1llg1Mutation suppressionedr1Phenotypic analysis of mutant powdery mildew resistance
(1) Materials and methods
For Arabidopsis thalianaedr1EMS mutagenesis is carried out on the mutant, and the M2 generation is screened for inhibitor mutant, so that a mutant capable of inhibitingedr1Mutants resistant to powdery mildew, followed by map-based cloning of the mutated gene, we named the gene based on its encoded proteinLLG1。
The Arabidopsis thaliana wild type plant Col-0 and the mutant are planted in a greenhouse with the light of 9 hours at the temperature of 22 ℃, and inoculated with powdery mildew after 5 weeks of growth. Disease resistance phenotype was identified 8 days after inoculation, representative leaves were photographed and Trypan Blue Staining (Trypan Blue Staining) was performed on representative leaves to observe the growth of powdery mildew.
To quantitatively analyze the resistance of the plants, the above plants were grown under the same conditions for 5 weeks, inoculated with powdery mildew in small amounts, 5 days after inoculation, leaf stained with trypan blue, and subjected to single spore conidiophore counting under a microscope. The counting results of 20-30 monospores are subjected to statistical analysis.
(2) Results and analysis
After 8 days of inoculation of powdery mildew, a large amount of powdery mildew is generated on the surface of the leaf of the wild type Col-0, andedr1the leaves of the mutant supported only a small amount of powdery mildew growth and showed significant cell death. Whileedr1 llg1The mutant did not produce similarities on the leaf surface 8 days after Erysiphe cichoracearumedr1But, similar to Col-0, there was a large number of spore attachments (fig. 1A). In order to better observe the phenotype of the powdery mildew after inoculation, trypan blue staining was carried out (figure 1B), as can be seen,edr1after staining, significant cell death was observed and only a small number of spores were produced, whereas Col-0 andedr1 llg1cell death does not occur, but a large amount of hyphae and spores are produced. The quantity of conidia generated on the monospores on the leaves of various genotypes is quantitatively analyzed, and the result shows thatedr1The number of conidiophores is obviously lower than that of wild Col-0llg1Inhibitedr1The disease-resistant phenotype (FIG. 1C), these results showedr1For white powderResistance of the bacteria and powdery mildew-induced cell death require LL G1.
Example 2llg1Mutant inhibitionedr1Phenotypic analysis of mutant other pathogens
(1) Materials and methods
The wild plant Col-0 of arabidopsis thaliana,edr1the mutant is a mutant of a microorganism,edr1 llg1grown for 2 weeks in a greenhouse at 22 ℃ for 9 hours. Will be provided withpad4Oomycetes grown on the mutants for 7 days (H. a. Noco2) As a bacterial source, the oomycete spores are diluted to 5 × 10 by vortex shaking with water4And/ml. The spores were uniformly sprayed onto the surface of 2-week-sized plant leaves using a watering can. The seedling tray was covered with a transparent cover, sealed with an adhesive tape for the periphery to be moisturized, placed in an incubator (temperature 16 ℃, relative humidity 90%), and after 7 days, the disease-resistant phenotype was observed, and the spores were counted using a blood cell counting plate.
Pseudomonas syringae (Pto DC 3000) was streaked on KB solid medium containing the corresponding resistance for 12 h, using 10 mM MgCl2The bacteria grown on the medium were collected and diluted by gradient to OD600=5x10-4. Bacteria were injected into the back of 4-week-sized arabidopsis leaves and sampled 3 h after injection as a 0-day control. The blade was removed with a punch. 600 μ l of 10 mM MgCl was added2Thoroughly grinding the blade, and diluting the obtained blade homogenate to 10 deg.C-1And 10-2Two concentrations, spread on KB plates, and after 2-3 days of incubation at 28 ℃, colonies on the medium were counted. The leaves 3 days after infection were again sampled with the punch and counted as above.
(2) Results and analysis
For Pseudomonas syringae, the quantitative results showedr1To pairPtoDC3000 showed enhanced disease resistance, supporting significantly lower numbers of bacterial growth than wild type. In addition, theedr1 llg1It is more susceptible than the wild type. Show thatllg1Complete recoveryedr1For Pseudomonas syringaePtoDisease resistance of DC 3000.
The statistic result of oomycetes shows that the Arabidopsis thaliana wild type plant Col-0 supports the spore growth of a large amount of oomycetesedr1This number is significantly reduced. Show thatedr1Has disease resistance to oomycetes. In addition, theedr1 llg11In (1), the number of spores was even higher than that of the wild type, indicating thatllg1Can completely inhibitedr1Disease resistance to oomycetes.
In the resistance reaction to Pseudomonas syringae and Oomycetes,LLG1all genes can be complementaryllg1To pairedr1Inhibition of the mutant.
Example 3LLG1Map-based cloning of genes
(1) Materials and methods
To clone the suppressor gene, we will cloneedr1 llg1The mutant is crossed with Arabidopsis thaliana wild type L andsberg ecotype plant, the obtained F1 generation plant is self-crossed to generate F2 generation group, from F2 generation plant, the plant containingedr1Is mutated but is not reacted withedr1 llg140 individuals with similar mutant phenotypes were genotyped with a gross location marker (http:// signal. salk. edu/genome/SS L P _ info/SS L Psordered. html) evenly distributed on the Arabidopsis genomeLLG1Mapped to chromosome 5. Subsequently, we designed a finely-located marker and genotyped about 2000F 2 plantsLLG1Mapped to the BAC clone MDA 7. All genes in this region were sequenced to find mutant genes (FIG. 3).
(2) Results and analysis
We analyzed the sequencing results and found thatLLG1(AT5G56170) A base change from G to A in the gene results in a change in amino acid 114 (G114R) (FIGS. 3a, b) in the protein sequenceLLG1The genes can be complemented with each otherllg1To pairedr1Inhibition of the mutants ((fig. 3c, d).
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
SEQUENCE LISTING
<110> Fujian agriculture and forestry university
<120> cloning and application of plant disease resistance related gene LL G1
<130>2
<160>2
<170>PatentIn version 3.3
<210>1
<400>1
000
<210>2
<211>168
<212>PRT
<213> amino acid sequence
<400>2
Met Glu Leu Leu Ser Arg Ala Leu Phe Phe Phe Leu Leu Leu Ser Val
1 5 10 15
Leu Ser Ser Phe Ser Ser Ser Ser Phe Ile Ser Asp Gly Val Phe Glu
20 25 30
Ser Gln Ser Leu Val Leu Gly Arg Asn Leu Leu Gln Thr Lys Lys Thr
35 40 45
Cys Pro Val Asn Phe Glu Phe Met Asn Tyr Thr Ile Ile Thr Ser Lys
50 55 60
Cys Lys Gly Pro Lys Tyr Pro Pro Lys Glu Cys Cys Gly Ala Phe Lys
65 70 75 80
Asp Phe Ala Cys Pro Tyr Thr Asp Gln Leu Asn Asp Leu Ser Ser Asp
85 90 95
Cys Ala Thr Thr Met Phe Ser Tyr Ile Asn Leu Tyr Gly Lys Tyr Pro
100 105 110
Pro Gly Leu Phe Ala Asn Gln Cys Lys Glu Gly Lys Glu Gly Leu Glu
115 120 125
Cys Pro Ala Gly Ser Gln Leu Pro Pro Glu Thr Ser Ala Glu Val Asn
130 135 140
Ala Ala Thr Thr Ser Ser Ser Arg Leu Trp Leu Thr Val Ser Ala Ala
145 150 155 160
Leu Leu Val Phe Val Lys Leu Phe
165

Claims (1)

1. Plant disease resistance related geneLLG1The plant is arabidopsis thaliana, and can be used for resisting powdery mildew, pseudomonas bacteria and oomycetes.
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Non-Patent Citations (1)

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