CN107955065A - A kind of cause a disease with Rhizoctonia solani Kuhn relevant effector R1YBX6 and its encoding gene - Google Patents
A kind of cause a disease with Rhizoctonia solani Kuhn relevant effector R1YBX6 and its encoding gene Download PDFInfo
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- CN107955065A CN107955065A CN201711376409.3A CN201711376409A CN107955065A CN 107955065 A CN107955065 A CN 107955065A CN 201711376409 A CN201711376409 A CN 201711376409A CN 107955065 A CN107955065 A CN 107955065A
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- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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- C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
- C12N15/8205—Agrobacterium mediated transformation
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Abstract
The invention discloses a kind of cause a disease with Rhizoctonia solani Kuhn relevant effector R1YBX6 and its encoding gene, the function and sequence of cause a disease the present invention relates to Rhizoctonia solani Kuhn relevant effector R1YBX6 and its encoding gene；Show that R1YBX6 is one of polygenes that Rhizoctonia solani Kuhn is caused a disease by transgenosis transient expression；The discovery of the gene has certain contribution to understanding the Study on Molecular Mechanism that rhizoctonia causes a disease.
Description
Technical field
The present invention relates to a kind of cause a disease with Rhizoctonia solani Kuhn relevant effector R1YBX6 and its encoding gene, belong to biology
Technical field.
Background technology
Rice sheath blight disease is caused by Rhizoctonia solani Kuhn (Rhzioctonia soalni), and Rhizoctonia solani Kuhn is to be distributed widely in
The soil inhabitant in each rice producing region in the world；It not only infects that scope is wide, often causes the disease of various plants, and in the soil
Saprophytic competitiveness it is strong, the time-to-live length；It is taken as that it, which is most destructive soil, passes one of phytopathy original；In China
Rice region, rice sheath blight disease become the hot and humid regional important disease in south China and the Yangtze river basin；To current, both at home and abroad to the disease
Harmful disease-resistant relevant molecule mechanism has done some researchs, but due to the related money of the pathogenic molecular mechanism for lacking Rhizoctonia solani Kuhn
Material, so breeding for disease resistance does not make a breakthrough；It is less for the research of rice pathogenic bacteria effect protein, mainly to other
The research of fungi；It is Pyricularia oryzae to be studied most ripe for effect protein at present；And both at home and abroad for Rhizoctonia solani Kuhn effect
The research of albumen is seldom；A same Avr-Pita1 gene for coming from Pyricularia oryzae is found in Rhizoctonia solani Kuhn in the prior art,
It is referred to as Rhiz-Pita genes；The gene illustrates the feature of multiple effect proteins, so Rhiz-Pita genes may be compiled
The effect protein of one good will of code, its expression formula are relevant to infecting for fungi inoculation early stage.
Carrying out gene transfer as carrier using the Ti-plasmids in Agrobacterium (Agrobacterium) has spy easily to operate
Point, but all the time, this transfering system is only applicable to dicotyledon, and the successful example of monocotyledon is less, existing skill
Mature Embryos of Rice being converted by Ti-plasmids in art, T-DNA has been integrated into cell chromosome and has been expressed, but
Normal plant is not obtained from transforming tissue；It is existing by optimizing the various factors, obtained in long-grained nonglutinous rice, japonica rice and naked body rice
High Transient Expression frequency；In addition, accounted for rice variety Australia green grass or young crops, early silk accounts for, the rataria that seven mountains account for and young fringe are explant, exploration
Using transfer of the Agrobacterium as the gus gene of mediation in long-grained nonglutinous rice.
The content of the invention
To solve the above problems, the present invention propose it is a kind of cause a disease with Rhizoctonia solani Kuhn relevant effector R1YBX6 and its
Encoding gene, there is provided one and the relevant effector that causes a disease in Rhizoctonia solani Kuhn.
The present invention provides one and the relevant effector that causes a disease, entitled R1YBX6, from silk core in Rhizoctonia solani Kuhn
Pseudomonas, be it is following 1) or 2) shown in protein：
1) protein being made of the amino acid sequence shown in sequence table 1；
2) amino acid sequence of the protein limited by sequence table 1 passes through the substitution of one or several amino acid residues
And/or missing and/or addition, and the protein that the regulation and control rhizoctonia with transcriptional activation function causes a disease.
Encode the gene (R1YBX6) with the relevant effector R1YBX6 that causes a disease, the nucleosides of shown gene in Rhizoctonia solani Kuhn
Acid sequence is following 1), 2) or 3) shown：
1) DNA sequence dna in sequence table 2；
2) DNA sequence dna under strict conditions with 1) limiting hybridizes and encodes the DNA sequences of protein described in claim 1
Row；
1) or 2) 3) DNA sequence dna with limiting has more than 90% homology and encodes protein described in claim 1
DNA sequence dna.
Expression vector, transgenic cell line and host strain containing gene of the present invention belong to protection scope of the present invention.
The primer pair of any fragment in R1YBX6 is expanded also within protection scope of the present invention.
It is a further object to provide pathogenic in a kind of pathophyte.
The present invention provides a kind of pathogenic method in pathophyte, is with the relevant effector that causes a disease by described
Gene R1YBX6 imports plant tissue or cell, the symptom of observation of plant.
It is described cause a disease with rhizoctonia relevant effector gene R1YBX6 can by containing described with relevant effect of causing a disease
The plant expression vector of the gene R1YBX6 of Ying Zi imports explant：The carrier that sets out for building the plant expression vector can
For any binary vector, such as pBI101, pCAMBIA, pBIN19, pBI121 etc..
Using pBI121 as the carrier that sets out, plant of the correlation effect subbase because of R1YBX6 of causing a disease containing described in rhizoctonia of structure
Thing expression vector is pBI121-R1YBX6.
The plant expression vector for carrying R1YBX6 of the present invention can be agriculture bacillus mediated to wait conventional biology by using Ti-plasmids
Method converts plant cell or tissue, observes the symptoms；The plant host that is converted is either monocotyledon, such as rice, jade
Rice, wheat etc. or dicotyledon, such as lettuce, Chinese cabbage, capsicum, celery.
The present invention compared with prior art, it is of the invention cause a disease with Rhizoctonia solani Kuhn relevant effector R1YBX6 and its
Encoding gene, shows that R1YBX6 is one of polygenes that Rhizoctonia solani Kuhn is caused a disease by transgenosis transient expression；The hair of the gene
The Study on Molecular Mechanism now caused a disease to understanding rhizoctonia has certain contribution.
Brief description of the drawings
The PCR verifications that Fig. 1 is pBI121-R1YBX6 after gene is connected with carrier.
Fig. 2 is the LB medium culture characters after connection carrier is transferred in Agrobacterium tumefaciems.
Fig. 3 is symptom of the Agrobacterium inoculation containing target gene after rice leaf, and B is control.
Embodiment
Embodiment 1, secreted protein gene import binary vector pBI121：
Used carrier is pBI121, insertion point XbaI/BamHI；
(1) purpose fragment expands：According to primers, the purpose product of sufficient amount is amplified by the method for PCR；
First round PCR is reacted using pfu polymerases；Reaction system 50ul；DNA 1ul；Primer MIX 10ul；Upper and lower primer is each
1ul；dNTP 1ul；10X pfu Buffer 5ul；Pfu 0.4ul, are settled to 50ul；
PCR response procedures：The first step, reaction temperature are 95 DEG C, reaction time 3min；Second step, reaction temperature 94
DEG C, reaction time 30s；3rd step, reaction temperature are 55 DEG C, reaction time 30s；4th step, reaction temperature are 72 DEG C, instead
It is 60s between seasonable；5th step, reaction temperature are 72 DEG C, reaction time 6min；Second step circulates 22 times to the 4th step；
1) using first round PCR product as template, carry out second and expand, it is therefore an objective to a large amount of amplifications；
2) reaction system is identical with reactions steps with first time；
3) after PCR terminates, electrophoresis recycling purpose fragment is carried out；
(2) PCR purified products and carrier digestion；
Digestion target gene system is 50ul；PCR purified products 20ul；Each 1ul of BamHI and XbaI；FD Buffer
5ul, is finally settled to 50ul；
Digestion carrier system is 50ul；pBI121 1.5ug；Each 1ul of BamHI and XbaI；FD Buffer 10ul, finally
It is settled to 50ul；
Above-mentioned digestion target gene system and digestion carrier system are respectively put into 37 DEG C of thermostat water baths and react 3h, electricity
Swimming recycling purpose band；
(3) purpose fragment is connected with carrier：Linked system 20ul；Digestion purpose fragment 6ul；Digestion carrier 5ul；10X
T4DNAligase Buffer 2ul；T4DNAligase 1ul, are settled to 20ul；
Above-mentioned connection mixed liquor is placed in 16 DEG C of water-bath 2h；
(4) convert, screening and cloning：Above-mentioned connection liquid is transferred in DH5a, detection filters out positive colony and is sequenced；Such as
Shown in Fig. 1, verification successful connection is expanded by carrier universal primer：Positive sequencing primer：GCAAGTGGATTGATGTGAT；Instead
To sequencing primer：GCTTTCCCACCAACGCTGAT.
Embodiment 2, the culture conversion of Agrobacterium tumefaciems：
Cacl2 freeze-thaw methods difference Pignus pignoris grain enters EHA105 Agrobacterium competent cells；
0.5~1 μ g Plasmid DNA is taken to add in the centrifuge tube of the EHA105 Agrobacterium competent cells containing 200 μ L, gently
Mix；After being placed in ice bath 30 minutes, then it is transferred in liquid nitrogen and freezes 5 minutes；It is immediately placed in 37 DEG C of water-baths 5 minutes；
The LB fluid nutrient mediums of 600 μ L are added, it is 200r/min to control rotating speed, and temperature is 28 DEG C of shaken cultivation 4h；
The Agrobacterium of activation is coated on the LB solid mediums of 50mg/L kanamycins and 20mg/L rifampins, 28 DEG C
It is inverted culture 2~3 days；
Monoclonal is selected in the LB fluid nutrient mediums of 500 μ L kanamycins containing 50mg/L and 20mg/L rifampins, is controlled
Temperature is 28 DEG C, rotating speed 200r/min, shaken cultivation 10h；
Agrobacterium nutrient solution 10ul is drawn in 500ul cards containing 0.5mg/L Na mycin and the LB liquid of the rifampin of 20mg/L
In culture medium, controlled at 28 DEG C, rotating speed 200r/min, shaken cultivation 10h, as shown in Fig. 2, using UV detector
Survey OD600；
OD600 is injected at rice leaf surface 1.0~1.2, with syringe, observes the symptoms.
As shown in figure 3, the results show gene injection, on rice leaf, all there is scab in relatively control, so determining
The effector and its encoding gene work in rhizoctonia pathogenic course.
Wherein, the preparation of solution：
[1] LB culture mediums：Tryptone 10g, dusty yeast 5g, NaCl 10g, agar 15g, supplement distilled water to 1000mL,
Adjust 7.0~7.2,121 DEG C of high pressure steam sterilization 30min of pH；
[2] 200 times of kanamycins solution：10g kanamycins is added in 1L solution；200 times of rifampin；In 1L solution
Add 4g rifampins；
[3] the LB fluid nutrient mediums of the rifampin of 500ml cards containing 0.5mg/L Na mycin and 20mg/L are：500ml culture mediums
In be separately added into 200 times of kanamycins solution and rifampin solution 2.5ml.
Above-described embodiment, is only the better embodiment of the present invention, therefore all structures described according to present patent application scope
Make, the equivalent change or modification that feature and principle are done, be included in the range of present patent application.
Sequence table 1
MNKLFLYTIDFLVLCGLSFASALHKQNEASFDYIVVGSGPAGIVVADRLSEAGKKVLLIERWPQYCRNR
RDRHAPVLTRFDIPGAFESFYDITAGIPYFWCRDLPALGGCIIGGGAAINSMLYWYPQDEDFATSNNWPVGFQNIAP
YVSKLHNRLPSTDVPSPDGKRYLTQVYDVFKGVLDAQGYQSITINNQRNKKDKIYGYSSQRGTRTGPMGTYLQTALA
RPNFKLLAYTKALAVARNGSTITGVHTNNTEGMRYTDEVWLKGSCSALCSCIIWSGIGPADSLANVEATPESAPYLP
PRSTYLDLPVGYNLQDNPSVSLVFSHPSVNAYQNFRPVWFNPNLTDAKQYVESQSGVLAQTSPRINWWKKYVGSDSK
VRWMQGNVVSWSRYSFFWKSINNQTRVTLTTTPWLTDPIDREVLITATKDVISTYQKVPGLELRYPNLNSTTIEDLV
STTVGGSNHWIGSTRAGTNSSTSVVDANLKVWDNLFVVDAGVFPGMPVGNPTASIMVMAEMAAAKLLKVIVN
Sequence table 2
tcagtttacgatcactttcaagagcttagcagcagccatctcagccatcaccatgatagaggcagttgggtttccca
cgggcatccctgggaatactcccgcatcgacgacaaacaaattatcccaaactttcaagttggcatcgaccacggac
gtggatgaattagtacccgcacgtgtactgccaatccaatggttcgaaccacccacggtcgtcgagacaagatcctc
gatggtagtactgttcaagttcgggtagcgcaactccaaacccggcactttttggtaagtggaaatgacgtctttag
tcgctgtgatcagtacttcccgatcgatcggatccgtaagccatggggttgtagtcagagtcactcgcgtttggtta
tttatactcttccagaagaagctatacctcgaccaagatacaacgttaccctgcatccaccttactttcgagtccga
acccacgtattttttccaccaattgattcgaggactcgtttgtgcaagtacaccgctttggctttcaacgtattgct
tggcgtcggtcagattcggattaaaccagacagggcggaaattctggtacgcattgactgatgggtgagagaatacg
agtgagacagatgggttatcctggaggttatagccaactggaaggtcgaggtatgtgcttctaggcggtaggtacgg
agccgactcaggtgttgcctctacatttgccaggctatcggcgggaccgatacctgaccatataatgcatgagcaca
gcgcagagcacgacccctttaaccacacctcatcagtgtaacgcataccctctgtgttgttagtatgaaccccggtt
atagtgctaccgttccgagcaactgctaaagcctttgtatatgccaatagtttgaagttaggtcgcgcaagggcggt
ctggagatatgtacccatagggcctgtgcgagtgcctcgttgactgctatatccataaattttgtccttcttgttcc
tttgattgttaatcgtgatcgattggtacccctgtgcgtctaaaacgcccttgaaaacgtcatagacctgcgtcaaa
taccttttcccatctggagaaggaacatccgttgacggaagacgattgtgtaacttggagacgtatggtgcaatatt
ctgaaatccaacaggccagttattgctagtcgcgaaatcctcatcctgaggataccagtaaagcatggaattaatcg
ctgctcctccaccaataatgcatccccctaaagctggtaagtctctgcaccaaaaatagggaattccagccgtgata
tcgtaaaatgactcgaaagccccaggtatatcaaaacgagtcaacacgggggcgtgtcggtccctccggttgcggca
atactggggccacctctcaattaaaagcactttcttcccagcttccgatagtcgatcggctaccacgattcctgcag
gaccagagccgacgacgatatagtcgaagctggcctcattttgtttatgaagggcagaggcaaagcttaagccacag
agcaccaggaaatcaatcgtatacagaaacagtttattcat
Claims (10)
- Relevant effector R1YBX6 and its encoding gene 1. a kind of and Rhizoctonia solani Kuhn is caused a disease, it is characterised in that：Including as follows Or 2) 1) protein shown in：1) protein being made of the amino acid sequence shown in sequence table 1；2) amino acid sequence of the protein limited by sequence table 1 by one or several amino acid residues substitution and/or Missing and/or addition, and the protein that the regulation and control rhizoctonia with transcriptional activation function causes a disease.
- Relevant effector R1YBX6 and its encoding gene 2. according to claim 1 and Rhizoctonia solani Kuhn is caused a disease, it is special Sign is：The gene is by encoding above-mentioned protein.
- Relevant effector R1YBX6 and its encoding gene 3. according to claim 2 and Rhizoctonia solani Kuhn is caused a disease, it is special Sign is：Its nucleotide sequence of the gene is following 1), 2) or 3) shown：1) DNA sequence dna in sequence table 2；2) under strict conditions with the DNA sequence dna hybridization 1) limited and code for said proteins its DNA sequence dna；1) or 2) 3) DNA sequence dna with limiting has more than 90% homology and code for said proteins its DNA sequence dna.
- 4. causing a disease relevant effector R1YBX6 and its encoding gene with Rhizoctonia solani Kuhn according to Claims 2 or 3, its It is characterized in that：Expression vector containing the gene.
- 5. causing a disease relevant effector R1YBX6 and its encoding gene with Rhizoctonia solani Kuhn according to Claims 2 or 3, its It is characterized in that：Transgenic cell line containing the gene.
- 6. causing a disease relevant effector R1YBX6 and its encoding gene with Rhizoctonia solani Kuhn according to Claims 2 or 3, its It is characterized in that：Host strain containing the gene.
- 7. the protein described in claim 1, or the gene described in Claims 2 or 3, or in claim 4~6 it is any described Expression vector, transgenic cell line or host strain it is following it is any in application：Import plant tissue or cell, inducing plant Morbidity.
- Relevant effector R1YBX6 and its encoding gene 8. according to claim 7 and Rhizoctonia solani Kuhn is caused a disease, it is special Sign is：The application is that the carrier that sets out for building its expression vector of plant is pBI121.
- Relevant effector R1YBX6 and its encoding gene 9. according to claim 7 and Rhizoctonia solani Kuhn is caused a disease, it is special Sign is：Described its expression vector of its plant of application is pBI121-R1YBX6.
- Relevant effector R1YBX6 and its encoding gene 10. according to claim 7 and Rhizoctonia solani Kuhn is caused a disease, it is special Sign is：Described its plant tissue of application or cell come from rice.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104447965A (en) * | 2013-09-17 | 2015-03-25 | 中国科学院微生物研究所 | Verticillium dahliae Kleb. pathogenic associated protein VdSENP1 and encoding gene thereof |
| CN105367665A (en) * | 2015-11-12 | 2016-03-02 | 山东农业大学 | Fungus pathogenesis-related protein PcCAT1 and its coding gene and use |
-
2017
- 2017-12-19 CN CN201711376409.3A patent/CN107955065A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104447965A (en) * | 2013-09-17 | 2015-03-25 | 中国科学院微生物研究所 | Verticillium dahliae Kleb. pathogenic associated protein VdSENP1 and encoding gene thereof |
| CN105367665A (en) * | 2015-11-12 | 2016-03-02 | 山东农业大学 | Fungus pathogenesis-related protein PcCAT1 and its coding gene and use |
Non-Patent Citations (2)
| Title |
|---|
| AIPING ZHENG 等: "The evolution and pathogenic mechanisms of the rice sheath blight pathogen", 《NATURE COMMUNICATIONS》 * |
| ZHENG,A.等: "cellobiose dehydrogenase [Rhizoctonia solani AG-1 IA]，GenBank: ELU43981.1", 《GENBANK》 * |
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Application publication date: 20180424 |