CN104211792B - Powdery mildew resistance associated protein, encoding gene thereof and application of both - Google Patents
Powdery mildew resistance associated protein, encoding gene thereof and application of both Download PDFInfo
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Abstract
The invention discloses powdery mildew resistance associated protein AT5G05190, encoding gene thereof and application of the both. The protein AT5G05190 is protein possessing one of the following amino acid residue sequences: 1) an amino acid residue sequence shown as SEQ ID No. 2 in sequences; and 2) protein which is derived from the amino acid residue sequence 1), is associated with the plant powdery mildew resistance and is obtained by substituting and/or deleting and/or adding one or more amino acid residues on the basis of the amino acid residue sequence shown as SEQ ID No. 2 in sequences. By reducing expression of the protein or the encoding gene, improvement on pathogenic bacteria resistance of plants and improvement on variety are realized, and important theoretical and practical significance is provided for agricultural production.
Description
Technical field
The invention belongs to biological technical field, is related to a kind of powder mildew resistance associated protein and its encoding gene and application.
Background technology
Plant growing is threatened in nature by various plant diseases.Identification and resist pathogen infect for
The survival of multicellular organism it is critical that (Aderem and Ulevitch, 2000).Receptor in animal such as Toll-like is received
Body, the identification to pathogen part can just induce innate immunity to react.Plant is different from animal, moveable due to lacking
Guard cell's (such as macrophage and granulocyte) and adaptive immune system (bone-marrow-derived lymphocyte as produced antibody), so only
The non-infected tissue's opposing of whole plant that the innate immune system and local disease resistance response initiation of each cell itself can be relied on is wide
Spectrum pathogen it is subsequent infect (Cao et al., 1997).
Plant has gradually formed the multi-level defense system similar to animal in order to adapt to environment, also, defines oneself
Innate immune system (innate immunity).It mainly includes basal resistance (basal immunity) and R gene mediateds
Resistance (R gene-mediated immunity) (Chisholm et al., 2006).
Basal resistance also known as the resistance (Pathogen-associated excited based on pathogen-associated molecular pattern
Molecular pattern Triggered Immunity, PTI).The pattern recognition mainly used on host cell membrane is received
Body (PRRs, Pattern recognition receptors) recognizes the PAMPs (Pathogen-associated of pathogen
Molecular patterns)/MAMPs (Microbe-associated molecular patterns), and then activate downstream
Blocking effect of mitogen activated protein kinases (MAPKs, Mitogen-activated protein kinases) cascade signal path or
Person is calcium-dependent protein kinase (CDPKs, Calcium depenedent protein kianses), most signal transmission at last
Enter core, activating transcription factor albumen is so as to inducing expression, active oxygen (the Reactive Oxygen of pathogenesis related gene PR genes
Species, ROS) generation and glucosan (callose) point of entry accumulation, cause plant disease resistance response resist it is various
Pathogen (Tena et al., 2011).
After host evolves PTI, pathogen also generates accordingly new mechanism of causing a disease.For example, some pathogen
Made using itself type III excretory system (Type III Secretion System, TTSS) or haustorium (haustoria)
Effector enters host cell and suppresses PTI, so as to effectively infect plant, promotes the growth and procreation of itself, that is, imitate
Answer the factor trigger susceptibility (ETS, Effector-Triggered Susceptibility) (Jones and Dangl,
2006)。
In order to defend infecting for pathogen, plant evolution to go out R genes (Resistance gene), the product of R genes can
Specifically recognize that (Flor, 1971), and activates the immunoreation in downstream, and induction produces anaphylaxiss for the effect protein of pathogen
(Hypersensitive Response, HR) limiting the growth of pathogen and spread, i.e., second system of defense R gene is situated between
The resistance led, or it is called the immunoreation (Effector Triggered Immunity, ETI) that effector is excited
(Chisholm et al., 2006).
Although it have been found that many albumen play an important role in plant disease-resistant.But, in plant disease-resistant network very
Polyprotein component is also unknown by the people.By the method for hereditism, biochemistry, cytobiology, genomics etc., can be effective
Find new protein component in disease resistance response.And genetic method is proved to very effective, many disease-resistant correlations
The encoding gene of albumen is obtained by genetic method separating clone.
The host that powdery mildew is distributed widely in all over the world and infects is numerous, including Semen Tritici aestivi, Fructus Hordei Vulgaris, Fructus Vitis viniferae and Chinese rose etc. are more
Plant cereal crops, industrial crops and flower plant.The growth and development of plants of powdery mildew infection can be subject to strong influence, cause to plant
The underproduction of strain is even withered.Therefore the research to powder mildew resistance not only has important theory value, and developing agricultural is produced
It is significant.
The content of the invention
It is an object of the invention to provide a kind of powder mildew resistance associated protein AT5G05190 and its encoding gene and application.
AT5G05190 albumen sources are in arabidopsiss (Arabidopsis thaliana).
Albumen provided by the present invention, is following albumen 1) or 2)：
1) protein of the aminoacid sequence composition shown in the SEQ ID № .2 in sequence table；
2) by amino acid residue sequence the taking through one or several amino acid residues of the SEQ ID № .2 in sequence table
Generation and/or disappearance and/or addition and by 1) derived from the protein related to plant powdery mildew resistance.
Encoding gene provided by the present invention has one of following nucleotide sequence：
1) SEQ ID № in sequence table：Nucleotide sequence shown in 1 1498-3520 positions；
2) SEQ ID № in polynucleotide：The polynucleotide sequence of 2 protein sequences；
3) can be with SEQ ID № in sequence table under high high stringency conditions：The nucleotide sequence of the 1 DNA sequence hybridization for limiting；
1) or 2) or 3) 4) there is more than 90% homology with the DNA sequence for limiting.
Above-mentioned high high stringency conditions can be, with 6 × SSC, the solution of 0.5%SDS, to hybridize at 65 DEG C, then with 2 × SSC,
0.1%SDS and 1 × SSC, 0.1%SDS respectively wash film once.
A further object is for the present invention provides the expression cassette comprising the encoding gene.The expression cassette has SEQ ID
№：Nucleotide sequence shown in 1 10-4741 positions.
A further object of the present invention is to provide the albumen, the encoding gene or the expression cassette and makes the white of plant
Application in powder disease resistance enhancing.
It is also another object of the present invention to provide the albumen, the encoding gene or the expression cassette are cultivating powdery mildew
Resistance strengthens the application in plant.
Specifically, the plant is dicotyledon or monocotyledon；The dicotyledon is specially arabidopsiss.
The present invention isolated gene related to powder mildew resistance in arabidopsiss by the method for map based cloning.
A total of 1848 nucleotide composition in the gene coding region, two exons.The 1498- of its sequence such as SEQ ID No.1
Shown in 1618 and 1794-3520 positions nucleotide.The albumen of the gene code has 615 aminoacid compositions.Its sequence
Row are as shown in SEQ ID No.2.
The method that above-mentioned cultivation powder mildew resistance strengthens plant (such as arabidopsiss), specifically loses the function of gene, makes
Plant (such as arabidopsiss) strengthens the resistance of powdery mildew.
The expression of the endogenous gene can be reduced by T-DNA (or being similar to such as, TALEN and RNA interference) technology.
Knockout technology or TALEN technologies are disturbed or pinpointed using RNA reduces the expression of gene of the present invention in crop, is capable of achieving
Crop is to the raising of pathogen resistance and the improvement of kind, so gene of the present invention and albumen have important meaning to agricultural production
Justice.
Description of the drawings
Fig. 1 is the phenotype of the wild type and mutant plants inoculation powdery mildew for growing 4 weeks.
Fig. 2 is the functional verification figure of AT5G05190 genes.
Specific embodiment
Experimental technique used in following embodiments if no special instructions, is conventional method.
Material used, reagent etc. in following embodiments, if no special instructions, commercially obtain.
The preparation of embodiment 1, powder mildew resistance related gene AT5G05190
(1), powder mildew resistance strengthens the screening of mutant arabidopsiss
0.03% ethylmethane sulfonate (EMS) mutation wildtype Arabidopsis thaliana Col-0, screens powdery mildew and resists from mutation colony
The enhanced mutant of property.Specifically screening technique is：It is in short photoperiod (9 hours illumination/15 hour dark) intensity of illumination
8000lux, relative humidity is under 70% growing environment, with powdery mildew (G.cichoracearum UCSC1) (Adam et
Al., the wild type and mutant 1999) to growing surrounding carries out inoculation experiments, observation experiment result (Fig. 1).Experimental result shows
Show：After connecing bacterium 7 days, different from producing the conidial wild type of a large amount of powdery mildews, mycelia and conidiophore can not be in mutation
The blade surface growth of body, and there is large-area death (Fig. 1 a) in the blade cell of mutant；Trypan blue (Trypan-
Blue) dyeing also demonstrate that the above results (Fig. 1 b).After connecing bacterium 7 days, the quantitative experiment of conidiophore shows, single in mutant
The conidiophore number that individual spore is formed is considerably less than wild type (Fig. 1 c).Fresh blade after being inoculated with 48 hours to powdery mildew
The double dyes of Trypan Blue-DAB are carried out, it is found that a large amount of hydrogen peroxide accumulation occurs at powdery mildew spores invasion point in mutant, and
Wild-type plant only occurs accumulating (Fig. 1 d) on a small quantity.Show in Aniline Blue dye tests, the product of callose in mutant
It is tired to improve about 7 times (Fig. 1 e) compared with wild type
(2), gene amplification
Using wildtype Arabidopsis thaliana Col-0 genomic DNAs template, PCR is expanded containing promoter and terminator sequence
AT5G05190 genetic fragments, primer sequence is：
Forward primer：5 '-the GCTCTAGAGGACTTGAGATAGCCCTACAAAACTT-3 ' (T/ of XbaI containing restriction enzyme site
CTAGA)
Downstream primer：5 '-the GCGTCGACGTGCCTACTTACCTCTGACTCATCTA-3 ' (G/ of Sal1 containing restriction enzyme site
TCGAC)
PCR reaction systems are：
DNA： 2.0μl
10xbuffer： 5.0μl
dNTPs(2.0mM)： 5.0μl
MgSO4(25mM)： 2.0μl
Primers F (10 μM)： 1.6μl
Primer R (10 μM)： 1.6μl
KOD Taq(1U)： 1.0μl
H2O： 31.8μl
PCR response procedures：Denaturation, 96 DEG C 3 minutes；Thermal cycle, degeneration 96 DEG C 30 seconds, annealing 55 DEG C 30 seconds, prolong
Stretch 68 DEG C 8 minutes, period 30.
Pcr amplification product is sequenced.Sequencing result shows, the common 4750bp of above-mentioned PCR primer, with sequence table
SEQ ID №：Nucleotide sequence shown in 1.Wherein SEQ ID №：1 10-4741 positions nucleotide is expression cassette sequence, is wrapped
Containing promoter sequence, exon sequence, intron sequences and terminator sequence；SEQ ID №：1 1498-3520 positions nucleoside
The sour gene order for AT5G05190, SEQ ID №：1 1498-1618 positions and 1794-3520 positions nucleotide are outer aobvious
Son, common 1848bp nucleotide sequences, SEQ ID № in polynucleotide：Aminoacid sequence shown in 2, totally 615 aminoacid are residual
Base.
The functional verification of embodiment 2, powder mildew resistance related gene AT5G05190
(1), back mutation experiment
With the gained pcr amplification product of Xba1 and Sal1 enzyme action embodiment 1；With Xba1 and Sal1 enzyme action pCAMBIAL1300,
Collect carrier large fragment；With T4 ligases in 16 DEG C of connections, DH10b competent cells are converted, Jing sequencings find to be dashed forward without base
Plasmid is extracted after change, and (Sambrook, 1989), the exogenous gene sequence inserted in plasmid is shown in SEQ ID No.1, to freeze afterwards
Melt method conversion Agrobacterium competence GV3101, then (Clough and Bent, 1998) turn with the method for Agrobacterium flower drop leaching
Change the mutant that embodiment 1 is filtered out, with hygromycin selection transfer-gen plant 25 positive plants are obtained.This 25 positive plants
Inoculation powdery mildew, has 22 transgenic plants to be returned to wild type phenotype (Fig. 2 b).
(2), AT5G05190 genes insertion mutation experiment
3 T-DNA insertion mutation body SALK_ of insertion AT5G05190 gene internals are ordered and identified from ABRC
009370th, SALK_048465 and SALK_064750.AT5G05190 genes in these three mutants are because T-DNA is inserted
It is silenced, but other sequences are consistent with wild type on genome.
The RNA of three mutant plants is extracted respectively with Trizol reagents and reverse transcription produces cDNA, with the cDNA as mould
Plate, expands AT5G05190 gene C DS sequences, and primer sequence is：
Forward primer：5’-ATGGCGAGCCAGACGGGTC-3’
Downstream primer：5’-CTATATAGAAGGAGGACGCTGTGAAA-3’
As a result show, in this 3 T-DNA insertion mutation bodies the gene (Fig. 2 a) cannot be all amplified.So as to show T-
The insertion of DNA causes the expression of gene to lose, and loses itself function.
It is to this 3 insertion mutation body inoculation powdery mildew concrete grammars subsequently：In short photoperiod, (illumination/15 hour are black within 9 hours
Secretly) intensity of illumination is 8000lux, and relative humidity is under 70% growing environment, with powdery mildew (G.cichoracearum
UCSC1) wild type and mutant to growing surrounding carries out inoculation experiments, observation experiment result after 7 days.As a result show, with open country
Raw type is compared, and three T-DNA insertion mutations bodies all occur in that plant leaf blade cell death and conidiophore grow less table
Type, it is similar with the mutant that embodiment 1 is filtered out, show the enhanced phenotype (Fig. 2 b) of powder mildew resistance.Because embodiment 1 is sieved
The mutant selected and three T-DNA insertion mutations bodies are all the allelic variant bodies of AT5G05190, and are inoculated with table after powdery mildew
Type is similar to, and it is because AT5G05190 gene expressions shift to an earlier date further to prove that mutant shows the enhanced phenotype of powder mildew resistance
Termination in turn results in gene lacks functionality generation.
Claims (4)
1. the aminoacid sequence shown in the SEQ ID No.2 in sequence table is constituted protein or the encoding gene of the protein
Application in the powder mildew resistance for making arabidopsiss strengthens；It is by losing sequence that the powder mildew resistance for making arabidopsiss strengthens
The encoding gene functional realiey of the protein of the aminoacid sequence composition shown in the SEQ ID No.2 in table.
2. application according to claim 1, it is characterised in that：In the nucleotide sequence of the encoding gene such as sequence table
Shown in the 1498-3520 positions of SEQ ID No.1.
3. the aminoacid sequence shown in the SEQ ID No.2 in sequence table is constituted protein or the encoding gene of the protein
Application in the enhanced arabidopsiss of powder mildew resistance are cultivated；The enhanced arabidopsiss of powder mildew resistance of cultivating are by losing
The encoding gene functional realiey of the protein of the aminoacid sequence composition shown in the SEQ ID No.2 in sequence table.
4. application according to claim 3, it is characterised in that：In the nucleotide sequence of the encoding gene such as sequence table
Shown in the 1498-3520 positions of SEQ ID No.1.
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| CN111197035B (en) * | 2020-01-08 | 2022-09-13 | 西北农林科技大学 | Powdery mildew-resistant grape calcium-dependent protein kinase gene VpCDPK13 and application thereof |
| CN113215188B (en) * | 2021-04-29 | 2023-05-16 | 上海师范大学 | Method for improving powdery mildew resistance and pathogen infection of China rose |
| CN113832124B (en) * | 2021-10-19 | 2023-04-21 | 武夷学院 | Application of related biological material of protein in enhancing bacterial leaf blight resistance of rice |
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| WO2008142036A2 (en) * | 2007-05-22 | 2008-11-27 | Basf Plant Science Gmbh | Plant cells and plants with increased tolerance and/or resistance to environmental stress and increased biomass production-ko |
| CN102586268A (en) * | 2011-01-13 | 2012-07-18 | 中国科学院遗传与发育生物学研究所 | Cloning and application of arabidopsis oidium disease resistance suppressor gene EDTS3 |
| CN102586266A (en) * | 2011-01-13 | 2012-07-18 | 中国科学院遗传与发育生物学研究所 | Cloning and application of arabidopsis powdery mildew resistance related gene EDR6 |
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| WO2008142036A2 (en) * | 2007-05-22 | 2008-11-27 | Basf Plant Science Gmbh | Plant cells and plants with increased tolerance and/or resistance to environmental stress and increased biomass production-ko |
| CN102586268A (en) * | 2011-01-13 | 2012-07-18 | 中国科学院遗传与发育生物学研究所 | Cloning and application of arabidopsis oidium disease resistance suppressor gene EDTS3 |
| CN102586266A (en) * | 2011-01-13 | 2012-07-18 | 中国科学院遗传与发育生物学研究所 | Cloning and application of arabidopsis powdery mildew resistance related gene EDR6 |
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