CN107955064A - A kind of cause a disease with Rhizoctonia solani Kuhn relevant effector R1YBXS8 and its encoding gene - Google Patents
A kind of cause a disease with Rhizoctonia solani Kuhn relevant effector R1YBXS8 and its encoding gene Download PDFInfo
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- CN107955064A CN107955064A CN201711376407.4A CN201711376407A CN107955064A CN 107955064 A CN107955064 A CN 107955064A CN 201711376407 A CN201711376407 A CN 201711376407A CN 107955064 A CN107955064 A CN 107955064A
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- C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- 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 R1YBXS8 and its encoding gene, the function and sequence of cause a disease the present invention relates to Rhizoctonia solani Kuhn relevant effector R1YBXS8 and its encoding gene；Show that R1YBXS8 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 R1YBXS8 and its encoding gene, belong to raw
Thing technical field.
Background technology
Fungal pathogens infect plant by various modes, and it is the hundreds and thousands of kinds of effects secreted by fungal pathogens to infect
Protein regulation；Fungi effect albumen can be between hypha,hyphae and host interaction zone be directly transferred in plant
And work；These effect proteins can suppress plant defense response, and regulation and control plant physiology mechanism can adapt to fungal infection, and
Nutriment is provided for fungi；The research of rhizoctonia effect protein is less, only one found at present in Rhizoctonia solani Kuhn
With the Avr-Pita1 genes for coming from Pyricularia oryzae, Rhiz-Pita genes are referred to as.
The heterologous gene expression system that the research of effect protein uses usually all is the prokaryotic expression using Escherichia coli；But
It is due to that destination protein is often expressed with inclusion bodies, product purification is difficult, and prokaryotic expression system post translational processing modified body
It is imperfection, the bioactivity of expression product is relatively low；So, complicated and success rate longer using the time used in prokaryotic expression
It is relatively low；And carry out gene transfer using the Ti-plasmids in Agrobacterium (Agrobacterium) as carrier and have the characteristics of easy operation,
All the time, but this transfering system is only applicable to dicotyledon, and the successful example of monocotyledon is less, with science
Development, has more and more reports to show to achieve very big success on monocotyledon recent years.
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 R1YBXS8 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 R1YBXS8, 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 (R1YBXS8) with the relevant effector R1YBXS8 that causes a disease, the core of shown gene in Rhizoctonia solani Kuhn
Nucleotide 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 R1YBXS8 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 R1YBXS8 imports plant tissue or cell, the symptom of observation of plant.
It is described cause a disease with rhizoctonia relevant effector gene R1YBXS8 can by containing described with relevant effect of causing a disease
The plant expression vector of the gene R1YBXS8 of Ying Zi imports explant：For building the carrier that sets out of the plant expression vector
Can be any binary vector, such as pBI101, pCAMBIA, pBIN19, pBI121 etc..
Using pBI121 as the carrier that sets out, structure causes a disease correlation effect subbase because R1YBXS8's containing described with rhizoctonia
Plant expression vector is pBI121-R1YBXS8.
The plant expression vector for carrying R1YBXS8 of the present invention can be agriculture bacillus mediated to wait standard biologic by using Ti-plasmids
Method converts plant cell or tissue, observes the symptoms；The plant host that is converted either monocotyledon, as rice,
Corn, 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 R1YBXS8 and
Its encoding gene, shows that R1YBXS8 is one of polygenes that Rhizoctonia solani Kuhn is caused a disease by transgenosis transient expression；The gene
It was found that the Study on Molecular Mechanism caused a disease to understanding rhizoctonia has certain contribution.
Brief description of the drawings
The PCR verifications that Fig. 1 is pBI121-R1YBXS8 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 lettuce blade, and B is control.
Fig. 4 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 similarly 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；4DNAligase 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 lettuce and rice leaf surface 1.0~1.2, with syringe, observes the symptoms.
As shown in Figure 3 and Figure 4, for the results show gene injection on lettuce and rice leaf, all there is disease in relatively control
Spot, so determining that 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
MNTATLLLSLSLLSAVRAQQVGTQKAEVHPTLTWQKCTKSGGCQTQSQGKIVLDANWRWVHNTGGYTNC
YTGQAWDSSLCPDPVKCAQGCAVDGADYSGTYGISANGNSLTMKFVTKTENTNVGSRVYLLADDSHYQMFKLKNQEF
TFDVDVSNLPCGLNGALYFSQMDADGGVAKYPNNKAGAKYGTGYCDAQCPKDIKFINGEANVLDWKGASNDPNSGTG
RYGTCCNEMDIWEGNAISTAYTPHPCTVQGQTRCSGSECTSFCDQPGCDFNSYRMGDKSFYGKGLTLDTSKKMTVVT
QFITADGTATGALKEIRRLYVQNGKVIQNSKTNIAGMTAYDSITEAFCSAQKTAFSDTNVFAQKGGYTNMGKAFDSG
MVLVLSIWDDHTANMLWLDSNYPTDRPATQPGTARGSCATTSGVPKDVEANSPNSSVTYSNIKFGDIGSTYSA
Sequence table 2
ccaacgtctactgtctgctcagcatgaataccgctactcttttgctttcactttcactcctctctgctgttcgtgct
cagcaagtcggcactcaaaaagccgaggtccaccccactctcacctggcagaaatgcaccaagtcaggcggttgcca
gacccagtctcagggaaagatcgtactcgacgccaactggcgctgggttcacaacactggcggctacaccaactgct
acaccggccaggcatgggactcttcgctttgccccgacccagtaaagtgtgctcaaggatgcgcggtcgatggcgcc
gactacagcggaacttatggtatctccgccaacggcaactcgttgaccatgaaattcgtcactaagaccgagaacac
caacgtcggctcccgcgtgtacttgctggccgacgactcccactaccagatgttcaagctcaagaatcaagagttta
cctttgacgtcgacgtctccaacctcccatgtggccttaacggcgcactctacttctctcagatggacgccgacggt
ggtgtggccaagtacccgaacaacaaggctggtgccaagtacggaactgggtactgcgatgctcaatgccccaagga
tatcaagttcatcaacggcgaggccaatgtcctcgactggaaaggtgcgagcaatgatcccaactcgggtaccggtc
gctacggcacctgctgcaacgagatggacatctgggagggcaacgccatctctaccgcatacaccccacacccttgc
actgttcaaggacagacccgctgctccggttccgagtgcacgagcttctgcgaccagcccggttgtgatttcaactc
gtatcgcatgggcgacaagagcttctacggcaagggcttgaccctcgataccagcaagaagatgaccgttgtcactc
agttcatcaccgctgacggtactgccactggtgctctcaaggaaattcgtcgcctctatgtccagaacggcaaggtc
atccagaactccaagaccaacattgccggcatgaccgcctatgactcgatcactgaggcattctgcagtgcccagaa
gaccgcgttcagcgataccaatgtcttcgcgcagaagggcggctacacgaacatgggcaaggctttcgattccggca
tggttctcgttctcagtatctgggatgaccatacggccaacatgctctggctcgacagcaactaccccaccgatagg
cccgccactcagcccggtactgctcgtggcagttgtgctactacctctggtgtccccaaggacgttgaagccaacag
ccccaactcgtctgtcacatactccaacatcaagtttggtgatatcggctcgacctactctgcctaaatggtcagtt
Claims (10)
- Relevant effector R1YBXS8 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 R1YBXS8 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 R1YBXS8 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 R1YBXS8 and its encoding gene with Rhizoctonia solani Kuhn according to Claims 2 or 3, It is characterized in that：Expression vector containing the gene.
- 5. causing a disease relevant effector R1YBXS8 and its encoding gene with Rhizoctonia solani Kuhn according to Claims 2 or 3, It is characterized in that：Transgenic cell line containing the gene.
- 6. causing a disease relevant effector R1YBXS8 and its encoding gene with Rhizoctonia solani Kuhn according to Claims 2 or 3, 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 R1YBXS8 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 R1YBXS8 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-R1YBXS8.
- Relevant effector R1YBXS8 and its encoding gene 10. according to claim 7 and Rhizoctonia solani Kuhn is caused a disease, its It is characterized in that：Described its plant tissue of application or cell come from lettuce and 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 CN201711376407.4A patent/CN107955064A/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 |
|---|
| CUBETA,M.等: "putative 1,4-beta-D-glucan cellobiohydrolase B [Rhizoctonia solani 123E]，GenBank: KEP49443.1", 《GENBANK》 * |
| 张红: "立枯丝核菌胞壁降解酶及其在致病中的作用", 《中国优秀博硕士学位论文全文数据库（硕士）农业科技辑》 * |
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