CN106905423A - The Disease-causing gene of verticillium dahliae, albumen and its application - Google Patents
The Disease-causing gene of verticillium dahliae, albumen and its application Download PDFInfo
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Abstract
The present invention relates to a kind of pathogenic protein of verticillium dahliae, the albumen has causes the effect of cotton verticillium wilt, is following protein 1) or 2)：1) amino acid sequence such as SEQ ID NO：Protein shown in 2；2) by SEQ ID NO：2 amino acid sequence causes a disease the related protein as derived from 1) by the substitution of one or several amino acid residues and/or missing and/or addition and to verticillium dahliae.Further relate to the application of the gene and the gene and albumen of encoding said proteins.The Disease-causing gene, albumen and verticillium dahliae cause the mechanism of cotton verticillium wilt with being closely connected, and the pathogenic of verticillium dahliae can be reduced by knocking out the Disease-causing gene.
Description
Technical field
The present invention relates to biological technical field, and in particular to the Disease-causing gene of verticillium dahliae, albumen and
Its application.
Background technology
Cotton as caused by soil filamentous fungi verticillium dahliae (Verticillium dahliae Kleb.)
Verticillium wilt is a kind of vascular bundle diseases of soil-borne, and Variation Pathogenic Bacteria is very fast, with distribution is wide, harm
Weight, the time-to-live is long, chemical pesticide is difficult to the features such as preventing and treating, and is most destroyed in cotton growth process
One of disease of property, seriously threatens the production and development of cotton.Cotton verticillium wilt 1914 first appeared in
The Fei Jiniya states in the U.S., then successively find (Shen Qiyi, 1992) in other states and Ge Zhimian states of the world,
Nineteen thirty-five does not weigh with the U.S. incoming China of cotton variety of introduction, but harm.After to twentieth century fifties,
Verticillium wilt occurs successively in China north and south part cotton region, and diffusion rate of propagation is accelerated.It is the end of the eighties, yellow
Disease of withering is throughout national 478 Ge Zhimian counties (city).Since the nineties, Cotton verticillium wilt expands
Exhibition spreads rapidly, and especially continuous big generation in China in 1993,1995,1996,2002 years, damages
Lose serious.According to the report of the 11st international Verticillium dahliae conference in 2012, cotton 2005-2010 generation
23,520,000 tons of boundary's average annual productivity, the loss caused by Verticillium dahliae disease is up to 30% for producing per year, every 1%
The loss of yield is equivalent to 3.54 hundred million dollars of loss.China is Cotton Production big country, national cotton plantation
Nearly hundred million mu of area, ginnings accounts for a quarter of global cotton total output.The quality of Cotton Production is not only
Income and the life of cotton grower are directly affected, also has great influence to light textile, foreign trade and national defense construction.
But cotton verticillium wilt worldwide prevailing, seriously threaten the production and development of cotton.
The cotton major producing area Xinjiang of China, it is very tight because of the cotton underproduction problem that verticillium wilt causes every year
Weight, very big economic loss is caused to cotton grower and country.Cotton verticillium wilt has turned into Cotton Production can
One of major obstacle of sustainable development, is referred to as " cancer of cotton " (simple osmanthus is good to wait .2003), and turned into
The outstanding problem of restricting current Cotton in China production.
Verticillium dahliae (Verticillium dahliae Kleb.) belongs to Deuteromycotina, and clump stalk embraces mesh, light color
Spore section, Verticillium dahliae category.The host range of verticillium dahliae is very wide, be related to Cruciferae, the rose family,
Pulse family, Solanaceae, Labiatae, composite family etc., at present up to 660 various plants, and also year by year
Expand.Mechanism of causing a disease on cotton verticillium wilt has various explanations, wherein with catheter blockage and poisoning two
Plant based on viewpoint.The sixties, people were because thalline is default in conduit to the understanding of the germ mechanism of causing a disease
Grow, and amount reproduction, while stimulating neighbouring parenchyma cell to produce colloid substance and thylose and block
Conduit, hinders the operating of moisture, so as to cause here cotton plant withers (Garber, 1966).Ma Yuanli etc. (1990)
It is normal secondary wooden to thinking after the distribution situation research in the catheter of each position verticillium wilt pathogen of cotton
Considerably beyond the gross water requirement of plant, and blocked conduit number accounts for the potential conveyance power of water of portion's conduit
The ratio of whole vascular bundle less (maximum has 17.7%), therefore catheter blockage is not to cause here cotton withers
Main cause.Keen etc. (1972) thinks that the toxin that verticillium wilt pathogen is produced in metabolic process is have
The protein of poison, is a kind of complex of the lipopolysaccharides of acidic protein one.The compound is to susceptible cotton
The blade of kind has destruction with the cell membrane of root tissue, makes intracellular K+And Na+A large amount of seepages.
And the cell membrane of disease-resistant variety does not possess the acceptor site of detoxifying function without being destroyed by toxin.Wang etc.
(2004) be separated to from the mycelia of verticillium wilt opportunistic pathogen one it is new with there is the cause effect of withering to cotton leaf
Albumen VdNEP.The albumen can form necrotic plaque with evoking tobacco blade, it is also possible to produce arabidopsis
Raw disease resistance response, therefore the albumen may take part in interaction reaction when verticillium wilt pathogen infects to cotton.
But it is not immediately clear whether the albumen is same substance with the toxin protein that oneself was separate in the past.Now more
Show come more research, the toxin of verticillium wilt pathogen secretion is to cause the critical biochemical factor of verticillium wilt,
The occlusive effects Water Transportation of tissue tract may exacerbate the generation of illness simultaneously.
Verticillium dahliae is a kind of soil-borne fungus, and its dormancy knot can be produced in the case where rugged environment is dried
Structure Microsclerotia, so as to be survived for many years in soil.So the formation of Microsclerotia and its pathogenic breath manner of breathing
Close.2004, Dobinson KF etc. (Dobinson, K.F., et al., 2004) were using having transformed
EZ::TN transposon systems, the trypsase VTP1 to the verticillium dahliae from tomato successfully enters
Orientation of having gone is knocked out.The gene can promote the formation of Microsclerotia, but not influence it after knocking out
Pathogenic and growth characteristics.2005, Dobinson KF etc. were to from the big beautiful of lettuce and tomato
The mitogen activated protein kinase gene VMK1 of Verticillium dahliae is knocked out.After knocking out VMK1
Pathogenic slump of disastrous proportions of the bacterial strain to various hosts, illustrates the signal path of map kinase mediation in fungi
Played an important role on pathogenic.And the knockout of gene reduces the generation of spore and the shape of Microsclerotia
Into illustrating that the gene may participate in multiple cellular processes.
Due to the seriousness and the popularity of host of cotton verticillium wilt harm, sections of many countries in the world
Skilled worker author has made intensive studies to it.Plant is obtained during the long-term coevolution with pathogen
The defense mechanism for obtaining a series of complex protects oneself, and Resistant expression is that composing type resistance and induction type are anti-
Property, induction type resistance includes institutional framework resistance and Physiology and biochemistry resistance again.It is different to resistance to verticillium wilt
Cotton variety there is some difference in terms of institutional framework, oneself is much studied confirmation both at home and abroad.It is anti-
The space between cells of sick kind xylem is smaller, and cell membrane is thicker, and is penetrated containing more marrow in xylem
Line.In addition, the fiber core diameter of the catheter lumen of disease-resistant variety and xylem is less than susceptible variety, explanation
Cotton variety has solid xylem relevant the resistance of verticillium wilt with it.The Physiology and biochemistry of cotton resists
Oneself had more research to characteristic of disease aspect, and studying more disease-resistant correlation factor includes:Protective plant protecting agent, tannin,
Soluble sugar, amino acid and various enzymes.After germ invasion and attack are subjected to, inside produces various suppressions to cotton plant
Fungus matter, mainly including gossypol, protective plant protecting agent, tannin etc., in addition also with some enzymes, albumen and
Small-molecule substance such as H202.Their effect is non-specialization, and the basal resistance to plant is related.Cotton
The mechanism of flower resisting verticillium is an extremely complex problem, and the factor being related to is numerous, therefore constantly deep
Enter to study these rules of disease resistance response product on gene expression dose, for giving farther insight into
Disease resistance mechanisms and utilization genetic engineering means transformation cotton variety resistance are significant.
The acquisition of disease-resistant gene is the important foundation of breeding resistant variety, is learned to do by molecular biosciences at present
Oneself has more than 39 to section through the plant disease resistance genes of clone, and wherein disease-resistant fungal pathogen probably has more than 20
Individual, such as arabidopsis mildew-resistance gene RPW8, tomato anti-blight gene secretes Ve, and jowar is anti-common
Rust (Puccinia Sorghi) gene Rpl-D, and it is anti-that NBS-LRR classes have been cloned on sea island cotton
Ospc gene.On conventional breeding, domestic and international cotton breeding person payes attention to the screening and creation in anti-source always.
Lee 1983-1986 has carried out resisting verticillium identification into 161 pairs of 911 parts of upland cotton resources such as luxuriant growths, screens
Preferably a collection of anti-(resistance to) the disease kind of resistance.K.V.Srinvasin identifies 126 island cotton varieties
Resistance, as a result shows that performance is resistance to sick and disease-resistant and accounts for 85%.Anti- source is either found by conventional method,
Or disease-resistant gene is cloned by molecular biology method and all has been achieved for certain progress, but do not had also
There is the effective way for finding real preventing and treating cotton verticillium wilt.Basic reason is the crop genetics such as the cotton back of the body
Scape is complicated, it is difficult to molecular level carry out deeper into research, verticillium dahliae microspecies dissociation in addition
The reason such as different in nature strong is also for resistant heredity breeding brings numerous difficulties.Therefore, research cotton verticillium wilt disease
Former verticillium dahliae is most important with the molecule mechanism of host plant interaction.
The content of the invention
The invention provides a kind of Disease-causing gene of verticillium dahliae, albumen and its application, the pathogenic base
Cause, albumen and verticillium dahliae cause the mechanism of cotton verticillium wilt with being closely connected, should by knocking out
Disease-causing gene can reduce the pathogenic of verticillium dahliae.
A kind of one aspect of the present invention, there is provided pathogenic protein of verticillium dahliae, is named as CLP-1
(calpain clp-1), from verticillium dahliae (Verticillium dahliae Kleb.), the albumen tool
Play the role of to cause cotton verticillium wilt, be following protein 1) or 2)：
1) amino acid sequence such as SEQ ID NO：Protein shown in 2；
2) by SEQ ID NO：2 amino acid sequence by one or several amino acid residues substitution
And/or missing and/or addition and caused a disease the related protein as derived from 1) to verticillium dahliae.
A kind of another aspect of the present invention, there is provided Disease-causing gene of verticillium dahliae (being named as CLP-1),
The albumen of the gene code has causes the effect of cotton verticillium wilt, is following 1) to any institute in 4)
The gene stated：
1) nucleotide sequence such as SEQ ID NO：From 5 ' end 1-347,438-746 in 1
Position, 808-1311 and the gene shown in 1368-2901；
2) nucleotide sequence such as SEQ ID NO：Gene shown in 1；
3) under strict conditions with 1) or 2) described in the gene recombination and coding claim 1 for limiting
The gene of albumen；
1) or 2) 4) there is more than 90% homology and coding claim 1 with the gene for limiting
The gene of the albumen.
SEQ ID NO：1 is made up of 2901 deoxynucleotides, SEQ ID NO in sequence table：1
From 5 ' end 1-347；438-746；808-1311；1368-2901 is
ORF areas, coding SEQ ID NO：The protein of amino acid residue sequence described in 2, by SEQ ID
NO：Albumen shown in 2 is named as CLP-1, and the encoding gene of CLP-1 albumen is named as into CLP-1.
" stringent condition " is to be enough to make nucleotide sequence and SEQ ID NO：Gene sequence shown in 1
Arrange hybridization condition, these conditions be to those skilled in the art it is known, for example：Containing 0.1%SDS
0.1 × SSPE or the 0.1 × SSC solution containing 0.1%SDS in,
Hybridize at 65 DEG C, and film is washed with the solution.
Recombinant vector, expression cassette, transgenic cell line present invention also offers the above gene or
Recombinant bacterium.
" expression cassette " means that the nucleotide sequence for being adapted to specific nucleotide sequence expression in host cell can be instructed,
Comprising the controlling element being operably connected with purpose nucleotide sequence.The controlling element can be opened
The element that mover, enhancer, son of mourning in silence, terminator and/or other described nucleotide sequences of control are expressed,
Such as polyadenylation se-quence.
Another aspect of the invention, there is provided the purposes of the above gene, strikes in verticillium dahliae
Except above-described gene makes the pathogenic reduction of verticillium dahliae.
The pathogenic method of verticillium dahliae is reduced it is still another aspect of the present invention to provide a kind of, is knocked out
Gene described in claim 2, specifically includes following steps：
(1) SEQ ID NO are expanded respectively using two pairs of primers:3 and SEQ ID NO:Sequence shown in 4,
The fragment that will be obtained after amplification imports expression vector；
(2) SEQ ID NO are contained by what is obtained in step (1):3 and SEQ ID NO:Shown in 4
The expression vector conversion Agrobacterium of the fragment of sequence；
(3) the successful Agrobacterium of conversion is chosen；
(4) by the successful Agrobacterium infection verticillium dahliae of conversion described in step (3), choosing
Take resistant bacterial strain, the bacterial strain of as pathogenic reduction.
The process described above, wherein, two pairs of primer sequences are as follows described in step (1)：
Sense primer 1：5 ' → 3 ' directions：GGGTTTAAUGATGAATACTTCGCACCAC
G(SEQ ID NO:5)；
Sense primer 2：5 ' → 3 ' directions：GGACTTAAUGTCAGTGGTGCTGCCATCA
A(SEQ ID NO:6)；
Anti-sense primer 1：5 ' → 3 ' directions：GGCATTAAUACGCAAACCCAGGGCAAA
AC(SEQ ID NO:7)；
Anti-sense primer 2：5 ' → 3 ' directions：GGTCTTAAUAACTCACGCGGCGGGATAC
T(SEQ ID NO:8)。
The process described above, wherein, step also specifically includes following steps in (1)：
By the sense primer 1 and the sense primer 2 with verticillium dahliae genomic DNA as mould
Plate PCR amplification SEQ ID NO:The gene of sequence shown in 3, the anti-sense primer 1 and the downstream are drawn
Thing 2 with verticillium dahliae genomic DNA be template PCR amplifications SEQ ID NO:Sequence shown in 4
Gene, by amplification after two kinds of PCR primers be all connected to expression vector.
The process described above, wherein, step is contained by electroporated method in (2) by described
SEQ ID NO:3 and SEQ ID NO:The expression vector of the fragment of sequence shown in 4 imports Agrobacterium impression
State cell.
The process described above, wherein, coated containing anti-by by the Agrobacterium in step (3)
Cultivated on the flat board of raw element, the successful bacterial strain of screening conversion.
The process described above, wherein, the fungal bacterial strain in step (4) after by Agrobacterium-mediated Transformation
Coat and cultivated on the flat board containing antibiotic the screening successful bacterial strain of conversion.
The Disease-causing gene of present invention offer, albumen the experiment proved that and cause cotton yellow to wither with verticillium dahliae
The mechanism of disease has and is closely connected, can be with by knocking out in verticillium dahliae Disease-causing gene of the invention
Make the pathogenic reduction of verticillium dahliae, the verticillium dahliae mutation of Disease-causing gene of the present invention will have been knocked out
Body infects plant simultaneously with wild type verticillium dahliae, the cotton phase infected by verticillium dahliae mutant strain
More than 50%, disease index are reduced than the cotton verticillium wilt incidence of disease that wild type verticillium dahliae infects
Also there is larger reduction with sick series.Therefore the present invention is provided newly to study verticillium dahliae pathogenesis
Enlightenment, for treat and prevent cotton verticillium wilt provide new approach.
Brief description of the drawings
Fig. 1 is the contrast for detecting DNA level CLP-1 knockout mutations bodies in embodiment 2 by PCR
Electrophoretogram；Swimming lane 1 is marker, and swimming lane 2,3,4 is wild type verticillium dahliae V592；Swimming lane 5,6,7
It is CLP-1 knockout mutations bodies VdaΔclp-1-1；Swimming lane 8,9,10 is CLP-1 knockout mutations bodies VdaΔclp-1-2。
Wild type verticillium dahliae V592 expands not shaping band with F1-HptR and HptF-R1 these two pairs primer,
And Fg-Rg can be to amplify band；Conversely, CLP-1 knockout mutations bodies VdaΔclp-1- 1 and
VdaΔclp-1- 2 can amplify band with F1-HptR and HptF-R1 these two pairs primer, and Fg-Rg is
Band can not be amplified, illustrates that CLP-1 is knocked；
Fig. 2 is by Northern hybridization check rna level CLP-1 knockout mutations bodies in embodiment 2
The trace figure of middle CLP-1 expressions；It is CLP-1 in Northern detection knockout mutations bodies shown in figure
It is knocked, does not express.Swimming lane 1,2 can express CLP-1 for wild type verticillium dahliae V592；
Swimming lane 3,4 is knockout mutations body VdaΔclp-1- 1 and VdaΔclp-1- 2, it is impossible to express CLP-1.
Fig. 3 is the control cotton plants not infected in embodiment 3；
Fig. 4 is the cotton plants after being infected by verticillium dahliae V592 in embodiment 3；
Fig. 5 is by the knockout mutations body Vda of verticillium dahliae V592 in embodiment 3Δclp-1After infecting
Cotton plants；
Fig. 6 be in embodiment 3 cotton plants by wild type verticillium dahliae V592 and knockout mutations body
VdaΔclp-1Infect the incidence of disease comparison diagram after 20 days；
Fig. 7 be in embodiment 3 cotton plants by wild type verticillium dahliae V592 and knockout mutations body
VdaΔclp-1Infect the disease index comparison diagram after 20 days；
Fig. 8 be in embodiment 3 cotton plants by wild type verticillium dahliae V592 and knockout mutations body
VdaΔclp-1Infect the sick series comparison diagram after 20 days.
Specific embodiment
Below in conjunction with drawings and Examples, more detailed theory is carried out to specific embodiment of the invention
It is bright, so as to more fully understand the solution of the present invention and the advantage of its various aspects.However, with
The specific embodiment and embodiment of lower description are only descriptive purposes, rather than limitation of the present invention.
Experimental technique used in following embodiments is conventional method unless otherwise specified.
Material used, reagent etc. in following embodiments, unless otherwise specified, commercially
Obtain.
Agrobacterium tumefaciems EHA105 (Elizabeth E.Hood. in following embodiments
NewAgrobacteriumhelper plasmids for gene transfer to plants.Transgenic
Research, July 1993, Volume 2, Issue 4, pp 208-218) 20 years public can from the applying date
Obtained from Institute of Microorganism, Academia Sinica, the biomaterial is only attached most importance to again related experiment of the invention
It is used, can not be used as other purposes.
Verticillium dahliae V592 (Feng-Gao, Bang-JunZhou, A Glutamic in following embodiments
Acid-Rich Protein Identified in Verticillium dahliae from an Insertional
Mutagenesis Affects Microsclerotial Formation and Pathogenicity.PLoS ONE
5(12):E15319.) 20 years public can obtain from Institute of Microorganism, Academia Sinica from the applying date,
The biomaterial is only attached most importance to again used by related experiment of the invention, can not be used as other purposes.
" wild type " means not containing the organism of exogenous nucleic acid molecule in following embodiments, is non-turn
Organism change or non-transgenic.
The structure of embodiment 1, CLP-1 knockout carriers
Verticillium dahliae V592 genomic DNAs are extracted using CTAB methods, using following primer with the base
Because group DNA is template, utilizeCxHotstart archaeal dna polymerases (Agilent) enter
Performing PCR is expanded：Sense primer 1：5 ' → 3 ' directions：GGGTTTAAUGATGAATACTTCG
CACCACG(SEQ ID NO:5)；Sense primer 2：5 ' → 3 ' directions：GGACTTAAUGTC
AGTGGTGCTGCCATCAA(SEQ ID NO:6)；Anti-sense primer 1：5 ' → 3 ' directions：GG
CATTAAUACGCAAACCCAGGGCAAAAC(SEQ ID NO:7)；Anti-sense primer 2：5’
→ 3 ' directions：GGTCTTAAUAACTCACGCGGCGGGATACT(SEQ ID NO:8).
Two kinds of PCR primers are connected simultaneously with USER enzyme mix (New England Biolabs)
To pGKO-HPT carriers (Tian, L., Chen, J., Wang, J., Wang, J., and Dai, X.
2011,20 years public can obtain from Institute of Microorganism, Academia Sinica from the applying date, the biological material
Material is only attached most importance to again used by related experiment of the invention, can not be used as other purposes) on, sequencing knot
Fruit shows to contain SEQ ID No:3 and SEQ ID No:The recombinant vector of the DNA molecular shown in 4
It is defined as final knockout carrier.Wild type verticillium dahliae V592 is rotated into using Agrobacterium-medialed transformation
In obtain CLP-1 knockout mutations bodies.
The acquisition of embodiment 2, CLP-1 knockout mutations bodies
1) genetic transformation is carried out to fungi using agrobacterium-mediated transformation
Related culture medium：
A () looks into formula culture medium (30g/L sucrose, 3g/L NaNO3,0.5g/L MgSO4-7H2O,0.5
g/L KCl,100mg/L FeSO4-7H2O,1g/L K2HPO4,pH 7.2).LB fluid nutrient mediums：Egg
White peptone 10g, yeast extract 5g, water 1000ml, adjust pH to 7,121 DEG C, high steam with NaOH
Sterilizing 20min.
(b) LB fluid nutrient mediums：Peptone 10g, yeast extract 5g, NaCl5G, adds water to 1L,
121 DEG C, steam sterilizing 20min.Solid medium adds 1.5% agar.
(c) MM minimal mediums：10mL K-bufer(200g/L K2HPO4,145g/L KH2PO4,
H3PO3PH is to 7.0) for regulation, 20mL M-N buffer (30g/L MgSO4·7H2O, 15g/L NaCl),
The CaCl of 1mL 1%2·2H2The sucrose (w/v) of O (w/v), 10mL 20%, the FeSO of 1mL 0.1%4
(w/v), 0.5g NH4NO3, plus distilled water is to 1L.113 DEG C, steam sterilizing 20min.
(d) IM inducing cultures：10mL K-bufer (pH 7.0), 20mL M-N buffer, 1mL
1%CaC12·2H2O (w/v), the NH of 2.5mL 20%4NO3(w/v), 1mL 0.1%
FeSO4(w/v), 5mL glycerine, the sucrose of 5mL 2mol/L, 2mL 100mmol/L acetyl
Syringone, the MES (pH 5.3) of 40mL l mol/L, plus distilled water are to 1L.113 DEG C, steam goes out
Bacterium 20min.
E () CM co-cultures culture medium：1.5% agar powder is added in IM culture mediums, 113 DEG C, is steamed
Vapour sterilizing 20min.
Concrete operation step
(1) picking Agrobacterium single bacterium colony put into 5ml contain antibiotic (50ug/ml kanamycins,
50ug/ml rifampins) LB fluid nutrient mediums, be placed on 28 DEG C of shaking tables, 200rpm cultures are 24 small
When, at the same time it is put into the V592 bacteria cakes on 3 PDA solid mediums of toothpick picking and contains 80ml
Look into the triangular flask of formula culture medium, be placed on 26 DEG C of shaking tables, 200rpm cultures.
(2) take the Agrobacterium bacterium solution that 1ml cultivated 24 hours and be added to 20ml and contain antibiotic
In the MM fluid nutrient mediums of (50ug/ml kanamycins, 50ug/ml rifampins), it is placed in 28 DEG C and shakes
On bed, after 200rpm continues to cultivate 24 hours, 4000rpm centrifugations 10min, collects bacterium on centrifuge
Body, thalline is washed twice with IM culture mediums, then resuspended with IM culture mediums, adjusts OD600≈ 0.25, will train
48 hours wild type verticillium dahliae V592 afterwards, four layers of sterilized filtered through gauze have been supported, will
The bacterium solution for having filtered is placed in 4000rpm on centrifuge, and 10min is centrifuged, with the resuspended spores of IM+AS,
Blood counting chamber is counted, and it is 1.0 × 10 to adjust spore concentration6-7Individual spore/ml.
(3) Agrobacterium bacterium solution and fungal spore suspension are pressed 1：1 volume ratio mixes (respectively taking 1ml),
The glassine paper on CM culture medium flat plates is coated according to the μ l of every culture dish 200,26 DEG C of cultures 36 are small
When.
(4) coculture is rinsed with 3ml sterilized waters, is coated containing anti-according to 500ul/ plates
Raw element (hygromycin 50ug/ml, carbenicillin 200ug/ml, cephalosporin 200ug/ml, 20ug/ml five
Fluorouracil) PDA solid mediums on cultivate 5-7 days.
2) PCR identifies CLP-1 knockout mutationss body (Fig. 1)
A, CTAB method extract CLP-1 knockout mutations body genomic DNAs.
B, entered using three pairs of primers performing PCR amplification.First pair：In the upstream of genes of interest CLP-1
Fragment SEQ ID NO:3 one primers F 1 of upstream design:
5’-GGCTGACCTTGTCGGTGTCT-3’(SEQ ID NO:9), on HPT BOX draws
Thing HptR：5’-AAATTTTGTGCTCACCGCCTGGAC-3’(SEQ ID NO:10) carry out
Fragment upstream PCR is expanded.Second pair：In genes of interest segments downstream SEQ ID NO:4 downstream is again
One primer of design, is named as R1:5’-CCGTTGGTGGGTAGGTATCA-3’(SEQ ID
NO:11), another primer HptF on HPT BOX:5’-
TCTCCTTGCATGCACCATTCCTTG-3’(SEQ ID NO:12) expansion of segments downstream is carried out
Increase.If primer pair 1 and primer pair 2 can be amplified and the equal-sized fragment of expection, explanation
Restructuring is that occur in the position of genes of interest.3rd pair of primer：Fg：5’-
CGAAATCGATGGATCCCAGGATCAAAAGCCCTCTAC-3’(SEQ ID
NO:13), Rg：5’-AGGCTACGTAGGATCCTTACCACTGCGGTGCTTACA-3’
(SEQ ID NO:14) for expanding the genes of interest of knockout, it is impossible to amplify segment surfaces genes of interest true
It is knocked successfully in fact.
CLP-1 expressions in embodiment 3, Northern hybridization check CLP-1 knockout mutations bodies
A, Trizol reagent method extract fungal tissue RNA
(1) fungal material is used into liquid nitrogen grinding into powder in mortar, every 50~100mg tissues are added
1ml RNAVzol, are homogenized in centrifuge tube to cracking completely；Room temperature places 5min.
(2) 0.2 times of chloroform of volume is added in the centrifuge tube equipped with lysate, and (1ml RNAVzol add
Enter 0.2ml chloroforms), mixing 30 seconds is fully vibrated with oscillator, room temperature places 2~3min.
(3) 4 DEG C, 14000rpm is centrifuged 10min, in absorption upper strata aqueous phase to a new centrifuge tube,
Every milliliter of RNAVzol can draw about 0.5~0.55ml.
(4) 0.5ml isopropanols are added by every milliliter of initial RNAVzol, are overturned for several times, mixed,
Precipitation at room temperature 10min.
(5) 4 DEG C, Isosorbide-5-Nitrae 000rpm is centrifuged 10min, in the visible RNA precipitate of ttom of pipe.Abandon supernatant,
Every milliliter of initial RNAVzol adds the ethanol of 1ml 75%, gently overturns and mixes, to clean RNA
Precipitation.Liquid is discarded, RNA precipitate is not abandoned carefully.Room temperature is inverted and is dried (5~10min).
(6) add appropriate 50% deionized formamide dissolving RNA precipitate, deposit in -80 DEG C it is standby.
B, RNA electrophoresis
(1) related reagent
(175.3g NaCl, 88.2g trisodium citrates, pH is adjusted to 7.0 with HCl to 20 × SSC, fixed
Hold to 1L, autoclaving), 10 × MOPS (41.8g MOPS, 6.56g NaAc, 20ml 0.5M EDTA,
PH is adjusted to 7.0 with NaOH, is settled to 1L, in brown bottle, autoclaving or filtration sterilization.
Be presented faint yellow available after autoclaving, yellow is unavailable), 37% formaldehyde (Formaldehyde), go from
Sub- formamide (Formamide deionized), methylene blue staining liquid (0.03% methylene blue, 0.3M
NaAc, pH5.2).
(2) RNA electrophoresis
A () 1.2% agarose-formaldehyde is denatured the preparation of glue：
The treatment of RNA sample before (b) loading：
| Sample buffer mix | Volume:25.5μl |
| 10×MOPS | 5μl |
| 37% methyl alcohol | 8μl |
| Deionized formamide | 12.5μl |
Sample buffer mix are mixed in equal volume with RNA sample, 65 DEG C, be denatured 5min, put
In 5min on ice, sample-loading buffer is added, can loading after mixing.
(c) RNA electrophoresis.Buffer solution used is 1 × MOPS, 120V about 3h.
(3) transferring film (up capillary transfer)
A () is all higher than the offset plate of gel as platform with long and width, be placed in vinyl disc, pours into
20 × SSC, cut one it is wide with platform, length more than platform filter paper, first filter paper one end is soaked
In vinyl disc, be slowly placed on platform, until the other end is also soaked in 20 × SSC, drive out of filter paper and
Bubble between platform；
B () cuts a nylon membrane for being slightly larger than gel long and wide, cut off the upper left corner, soaks completely in nothing
In bacterium water, film is taken out, then immerse in 20 × SSC, at least 5 minutes；
C () electrophoresis terminates after, the nonuseable part of gel is cut away, and one jiao is cut in the upper left corner, as
Azimuth mark.Gel is placed in 20 × SSC and rinses a moment；
D () gel is placed upside down in the center of filter paper on platform, drive the bubble between glue and filter paper out of, is used
Parafilm should not encounter the sample on gel around gel；
E () soaks gel with 20 × SSC, the nylon membrane of moistening is placed on above gel, makes the two corner cut
Overlap, drive the bubble between nylon membrane and gel out of；
F () soaks 5 and an equal amount of filter paper of nylon membrane with 20 × SSC, be placed on above nylon membrane,
Drive filter paper and the intermembranous bubble of nylon out of；
G () cuts a folded 10cm thickness, be slightly less than the paper handkerchief of filter paper, is placed on above filter paper, and one is put on paper handkerchief
Block glass plate, compresses the weight of 500g, and transfer is overnight；
H () throws off paper handkerchief above gel, filter paper and Parafilm, overturn gel and nylon membrane, with solidifying
Glue one side is placed on a vinyl disc upper, marks the position of gel well on nylon membrane with pencil；
I () peels off gel from nylon membrane and abandons it, nylon membrane is immersed in into 5min in 20 × SSC, takes out
Nylon membrane, is placed on the filter paper of moistening, UV-crosslinked fixed RNA；
J () uses methylene blue staining, until seeing clearly RNA bands, distilled water flushing decolourizes,
Wrapped with preservative film, be placed on 4 DEG C of preservations stand-by.
(4) probe mark
(Amersham companies random primer labelling kit, RediprimeTMII Random Primer
Labelling System)。
A () takes DNA 25ng to be marked, plus sterilized water, make volume augmentation to 45 μ l.
B 98 DEG C of (), is incubated 5 minutes, denatured DNA.
C () is centrifuged, DNA is gathered in centrifuge tube ttom of pipe, is placed on ice.
D be added to the DNA of denaturation in mark mixture by (), gently mix, until pellet melts completely
Solution (can not be blown and beaten with rifle).
E () plus 1 μ l Klenow (prevent from marking the Klenow in mixture to inactivate), centrifugation.
F () plus 5 μ l α -32P-dCTP, uniform, centrifugation is gently blown and beaten with rifle.
G 37 DEG C of (), reacts 30 minutes；98 DEG C, 5 minutes, marked good DNA probe is denatured,
Centrifugation, is placed on ice.
H () takes appropriate probe for hybridizing, remaining probe is stored in 4 DEG C of refrigerators.
C, hybridization
The preparation of hybridization buffer
| Hybridization buffer:20.4ml | Required volume | Final concentration |
| 8.6ml | 43mM | |
| 20%SDS | 7ml | 7% |
| 5%BSA | 4ml | 1% |
| 0.5M EDTA | 0.8ml | 20mM |
(a) prehybridization：Hybridization buffer is poured into hybrid pipe and is put into crosslinking after 65 DEG C of preheating 15min
The fixed pre- miscellaneous 1~2h of 65 DEG C of film；
B () hybridizes：The probe that will have been marked is put into hybrid pipe, 65 DEG C of hybridized overnights；
C () washes film：With film 2 × SSC/0.2%SDS of buffer solution is washed, washed under the conditions of 65 DEG C/15min
Film 2~3 times；
(d) compressing tablet：Washed film is taken out from hybrid pipe, is transferred in two-layer plastic film, detection hybridization
Signal strength, film is put into phosphorus screen, according to signal strength pressure a few hours or overnight；
E () detects hybridization signal：Scanning phosphorus screen.
Result is as shown in Figure 2.
The pathogenic detection of the knockout mutations body of embodiment 3
Water planting cotton is infected by bacterial strain pathogenic to identify its.Select full cotton seed with 15% time
After sodium chlorate immersion 30min, aseptic water washing 2-3 times, then overnight put down afterwards with sterilized water Steeping and budding
Moisturizing in culture box is layered on, treats that bud is long to 3cm, planted in the box that germinates.The seedling transfer of cotyledon will be grown
To in the plastic casing (8-10cm high) for filling with clear water, in 25 DEG C, illumination 16h, dark 8h cultures.
Clear water is changed into 1/3 MS nutrient solutions when true leaf grows, a nutrient solution is changed weekly, 1 true
It is inoculated with when leaf is flattened.By -80 DEG C of verticillium dahliae V592 bacterial strains and CLP-1 knockout mutations bodies of preservation
VdaΔclp-13-4d is activated through PDA plate, Cha Shi nutrient solutions is put into from colony edge picking bacterium block,
25 DEG C, 220rpm shakes training 5d, and filtering, filtrate 5000rpm centrifugation 5min, clear water dilutes spore,
Blood counting chamber is counted, and concentration is adjusted into 1 × 107Individual spore/ml.The spore suspension of concentration will be mixed up
Add in empty plastic casing, cotton seedling soaks root 40min.Afterwards with 1/3 25 DEG C of illumination 16h of MS nutrient solutions,
Dark is lower to be continued to cultivate cotton seedling 8h.12 young plants are planted in per box, 3 repetitions of each kind compare cotton seedling
40min is soaked with clear water.Incidence is observed after 20.
By the above method, we demonstrate CLP-1 knockout mutationss body and the pathogenic of cotton are substantially subtracted
It is weak
Calculate the incidence of disease and disease index：
The incidence of disease=morbidity number/investigation sum × 100% (see Fig. 6)
Disease index=∑ [sick series × this grade disease leaf (fringe, strain) number]/(investigation sum × highest disease series)
× 100 (see Fig. 7)
Morbidity grade scale：
0 grade of plant health does not have symptom；
1 grade of blade of 0.1%-25% is wilted；
2 grades of blades of 25%-50% are wilted；
3 grades of blades of 50%-75% are wilted；
4 grades of blades of 75%-100% are wilted or dead；
The cotton that wild type verticillium dahliae V592 infects and knockout mutations body VdaΔclp-1The cotton infected
Morbidity grade scale statistical chart (see Fig. 8).
By the result figure of the above method, we can see that knockout mutations body VdaΔclp-1Infect cotton phase
It is all more obvious than infecting the cotton incidence of disease, disease index and morbidity classification in wild type verticillium dahliae V592
Reduce, illustrate that CLP-1 genes are pathogenic related to verticillium dahliae.
Finally it should be noted that：Obviously, above-described embodiment is only intended to clearly illustrate the present invention and is made
Citing, and not to the restriction of implementation method.For those of ordinary skill in the field,
Can also make other changes in different forms on the basis of the above description.Here need not
All of implementation method cannot be exhaustive.And the obvious change or change thus amplified out
Among moving still in protection scope of the present invention.
Claims (10)
1. a kind of pathogenic protein of verticillium dahliae, it is characterised in that the albumen has causes cotton
The effect of verticillium wilt, is following protein 1) or 2)：
1) amino acid sequence such as SEQ ID NO：Protein shown in 2；
2) by SEQ ID NO：2 amino acid sequence by one or several amino acid residues substitution
And/or missing and/or addition and caused a disease the related protein as derived from 1) to verticillium dahliae.
2. a kind of Disease-causing gene of verticillium dahliae, it is characterised in that the albumen of the gene code has
Cause the effect of cotton verticillium wilt, be following 1) to any described gene in 4)：
1) nucleotide sequence such as SEQ ID NO：From 5 ' end 1-347,438-746 in 1
Position, 808-1311 and the gene shown in 1368-2901；
2) nucleotide sequence such as SEQ ID NO：Gene shown in 1；
3) under strict conditions with 1) or 2) described in the gene recombination and coding claim 1 for limiting
The gene of albumen；
1) or 2) 4) there is more than 90% homology and coding claim 1 with the gene for limiting
The gene of the albumen.
3. recombinant vector, expression cassette, transgenic cell line or weight containing gene described in claim 2
Group bacterium.
4. the purposes of gene as claimed in claim 2, it is characterised in that knocked out in verticillium dahliae
Gene described in claim 2 makes the pathogenic reduction of verticillium dahliae.
5. it is a kind of to reduce the pathogenic method of verticillium dahliae, it is characterised in that to knock out claim 2
Described gene, specifically includes following steps：
(1) SEQ ID NO are expanded respectively using two pairs of primers:3 and SEQ ID NO:Sequence shown in 4,
The fragment that will be obtained after amplification imports expression vector；
(2) SEQ ID NO are contained by what is obtained in step (1):3 and SEQ ID NO:Shown in 4
The expression vector conversion Agrobacterium of the fragment of sequence；
(3) the successful Agrobacterium of conversion is chosen；
(4) by the successful Agrobacterium infection verticillium dahliae of conversion described in step (3), choosing
Take resistant bacterial strain, the bacterial strain of as pathogenic reduction.
6. method as claimed in claim 5, it is characterised in that two pairs are drawn described in step (1)
Thing sequence is as follows：
Sense primer 1：5 ' → 3 ' directions：GGGTTTAAUGATGAATACTTCGCACCAC
G；
Sense primer 2：5 ' → 3 ' directions：GGACTTAAUGTCAGTGGTGCTGCCATCA
A；
Anti-sense primer 1：5 ' → 3 ' directions：GGCATTAAUACGCAAACCCAGGGCAAA
AC；
Anti-sense primer 2：5 ' → 3 ' directions：GGTCTTAAUAACTCACGCGGCGGGATAC
T。
7. method as claimed in claim 6, it is characterised in that step is also specifically included in (1)
Following steps：
By the sense primer 1 and the sense primer 2 with verticillium dahliae genomic DNA as mould
Plate PCR amplification SEQ ID NO:The gene of sequence shown in 3, the anti-sense primer 1 and the downstream are drawn
Thing 2 with verticillium dahliae genomic DNA be template PCR amplifications SEQ ID NO:Sequence shown in 4
Gene, by amplification after two kinds of PCR primers be all connected to expression vector.
8. method as claimed in claim 5, it is characterised in that turned by electric shock in step (2)
The method of change described will contain SEQ ID NO:3 and SEQ ID NO:The expression of the fragment of sequence shown in 4
Vector introduction Agrobacterium competent cell.
9. method as claimed in claim 5, it is characterised in that by will be described in step (3)
Agrobacterium is coated and cultivated on the flat board containing antibiotic, the successful bacterial strain of screening conversion.
10. method as claimed in claim 5, it is characterised in that by by agriculture bar in step (4)
Fungal bacterial strain after bacterium conversion is coated and cultivated on the flat board containing antibiotic the screening successful bacterium of conversion
Strain.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108384799A (en) * | 2018-01-22 | 2018-08-10 | 浙江理工大学 | Regulate and control the method for verticillium dahliae gene expression using plant viral vector multiple target point |
| CN113604489A (en) * | 2021-09-09 | 2021-11-05 | 石河子大学 | Application of verticillium dahliae acetolactate synthase catalytic subunit gene VdIV 2A |
| CN113699170A (en) * | 2021-09-09 | 2021-11-26 | 石河子大学 | Application of VdIV 6 gene in growth and development, pathogenicity and branched chain amino acid synthesis of verticillium dahliae |
| CN113717956A (en) * | 2021-09-09 | 2021-11-30 | 石河子大学 | Application of verticillium dahliae acetolactate synthase catalytic subunit gene VdIV 2B |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108384799A (en) * | 2018-01-22 | 2018-08-10 | 浙江理工大学 | Regulate and control the method for verticillium dahliae gene expression using plant viral vector multiple target point |
| CN113604489A (en) * | 2021-09-09 | 2021-11-05 | 石河子大学 | Application of verticillium dahliae acetolactate synthase catalytic subunit gene VdIV 2A |
| CN113699170A (en) * | 2021-09-09 | 2021-11-26 | 石河子大学 | Application of VdIV 6 gene in growth and development, pathogenicity and branched chain amino acid synthesis of verticillium dahliae |
| CN113717956A (en) * | 2021-09-09 | 2021-11-30 | 石河子大学 | Application of verticillium dahliae acetolactate synthase catalytic subunit gene VdIV 2B |
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