CN103361365B - Coniothyrium-minitans siderophore transporter (CmSit1) gene as well as preparation method and application thereof - Google Patents
Coniothyrium-minitans siderophore transporter (CmSit1) gene as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a coniothyrium-minitans siderophore transporter (CmSit1) gene as well as a preparation method and application thereof. A coniothyrium-minitans insertion mutant library containing a plurality of T-DNA (transferred deoxyribonucleic acid) markers is obtained by adopting a mature agrobacterium-mediated genetic transformation method of the coniothyrium minitans. Transformants and sclerotinia sclerotiorum Ep-1PNA367 are cultured at the same time in the PDA (potato dextrose agar) culture medium, and as the comparison of a strain ZS-1, a strain ZS-1N1812 with enhanced anti-fungal capacity is selected. The further study shows that the expression of the CmSit1 gene can be improved significantly when T-DNA in the strain ZS-1N1812 is inserted into a promoter area of the CmSit1 gene, and the capacity of resisting the sclerotinia sclerotiorum of a plant can be improved significantly if the CmSit1 gene is transferred into the nicotiana benthamiana. The CmSit1gene can be used for improving the capacity of resisting pathogenic fungi of various biocontrol bacteria and can be also used for the transgenic breeding for disease resistance of crops.
Description
Technical field
The invention belongs to biological technical field, more specifically relate to the mould siderophore transporter gene of a kind of shield shell, also relate to the preparation method of the mould siderophore transporter gene of a kind of shield shell simultaneously, also relate to this gene in the application of transforming biocontrol strain and cultivate in anti-sclerotium disease transfer-gen plant.
Background technology
Sclerotinite (Sclerotinia sclerotiorum) is a kind of very important plant pathogenic fungi, and host range is very extensive, can parasitic 64 section 540 various plants.The common important host of sclerotinite comprises the oil crops such as rape, Sunflower Receptacle, soybean and lettuce, Radix Dauci Sativae and multiple Cruciferae, leguminous vegetable, and the yield and quality of sclerotium disease on these crops caused by it has very important impact.For rape, all have generation by the microbial sclerotium disease of nuclear disk in each rapeseed cultivation region of China, wherein the morbidity of the vast planting area in the middle and lower reach of Yangtze River is especially serious, and long-term sickness rate is 15-30%, and the serious time is more than 80%.Germ infects rape senescence petal at the florescence, and petal of catching an illness afterwards drop to blade and limb, brings out contact and infects, often cause fall ill plant trunk and branch withered, cause Semen Brassicae campestris quality and thousand seed weight to decline, production loss between 10-70%, without receipts time serious.
The control of current sclerotium disease mainly relies on chemical pesticide, but the long-term use of chemical pesticide easily causes resistance to occur, make cost accounting increase, our preliminary monitored results also shows that the sclerotinite bacterial strain frequency of occurrences of China's resisting carbendazim increases, and long-term big area uses single agricultural chemicals to there is high risk.Meanwhile, chemical prevention also brings the problem such as food safety and environmental pollution.Based on this, greatly developing security, to substitute prevention and control measure imperative, wherein creates the research and practice main direction that disease-resistant variety and biological control are prevention and control sclerotium diseases, creates disease-resistant variety and seem and be even more important.And wait microorganism that to find disease-resistant related gene resource be one of important directions solving anti-sclerotium disease breeding from the biocontrol microorganisms of sclerotinite is as mould in shield shell.
First we establish the mould T-DNA insertional mutagenesis library of shield shell, therefrom find the mutant that a strain fungal resistance strengthens, empirical tests T-DNA is that unit point inserts, the flanking sequence of T-DNA that utilized the methods such as Tail-PCR to clone, find that T-DNA is inserted in the promoter region of an encoding gene further across bioinformatic analysis, utilize RACE technology to clone a new gene siderophore translocator CmSit1.This T-DNA inserts the expression that have activated CmSit1 in mutant to utilize RT-PCR technology to confirm, the expression of result prompting CmSit1 may strengthen relevant with the fungal resistance of mutant.Inoculate rape leaf after activating the mutant culturing filtrate process sclerotinite of expressing with CmSit1, significantly can suppress the expansion of scab on blade, the fungal resistance further illustrating the product of CmSit1 mould with shield shell is closely related.Simultaneously we construct the plant virus-based expression vector of CmSit1, and carrier is imported Ben Shi cigarette by the method for injection, found that virus vector import after CmSit1 can effective expression, and the resistance of plant to sclerotinite obviously strengthens.These results show shield shell mould in siderophore translocator CmSit1 gene not only can be used as the mould target of transformation shield shell, its fungal resistance is significantly strengthened, and also have boundless application prospect in breeding for disease resistance.
Through retrieve the domestic and foreign literature of prior art, so far there are no about shield shell mould in the research relevant to fungal resistance of siderophore translocator CmSit1 gene report.
Summary of the invention
The object of the invention is to there are provided the mould siderophore transporter gene of a kind of shield shell, this gene is after the mould middle overexpression of biocontrol fungi shield shell, biocontrol fungus Coniothyrium minitans Campbell can be made to produce antifungal substance, significantly improve the mould antagonistic effect to pathogenic fungi sclerotinite of shield shell.
Another object of the present invention is the preparation method that there are provided the mould siderophore transporter gene of a kind of shield shell, and the method is simple and quick, is conducive to oppositely finding goal gene according to desired phenotype, can carry out in a large number screening and analyzing.
Another object of the present invention there are provided a kind of shield shell mould siderophore transporter gene to strengthen the application in transfer-gen plant anti-sclerotium disease (can be used in the anti-sclerotium disease breeding of rape, soybean and Sunflower Receptacle).
The present invention is achieved through the following technical solutions:
Applicant is by gene clone technology, and be isolated and cloned into and antimycotic relevant gene and proteins encoded thereof from shield shell mould (Coniothyrium minitans), this unnamed gene is CmSit1 by applicant.
Described CmSit1 gene, nucleotide sequence is as shown in sequence table SEQ ID NO:1, and the aminoacid sequence of coding is as shown in sequence table SEQ ID NO:2.
Embodiments of the invention show, the CmSit1 of the present invention clone does not express in the mould wild strain of shield shell, activate after CmSit1 expresses and cause the mould fungal resistance of shield shell obviously to strengthen.Important use of the present invention is: utilize CmSit1 gene overexpression, the mechanism that the mould antifungal substance of research shield shell produces, and transforms out the shield shell trichoderma strain that fungal resistance strengthens, improves its biocontrol effect; Also can in other biocontrol strain overexpression CmSit1 gene, transform out fungal resistance strengthen other biocontrol strain, improve biocontrol effect; Simultaneously also can in the important crops such as rape overexpression CmSit1 gene, be applied to breeding for disease resistance.
A preparation method for the mould siderophore transporter gene of shield shell, the steps include:
The mould fungal resistance of shield shell strengthens the screening of mutant ZS-1N1812: according to the mould genetic transforming method of agriculture bacillus mediated shield shell of the maturation that this laboratory is set up, and obtains the mould insertional mutagenesis library of shield shell containing more than 45000 T-DNA marks.Transformant and sclerotinite Ep-1PNA367 are carried out opposite culture at PDA substratum, with ZS-1 bacterial strain for contrast.Observations after 7d, a kind of shield shell mould (Coniothyrium minitans) the bacterial strain ZS-1N1812 (China typical culture collection center, deposit number is CCTCC NO:M2012054) that screening acquisition one strain fungal resistance strengthens.This bacterial strain is compared with starting strain ZS-1, and poor growth, bacterium colony is easily fanned and become deformity, produces spore and reduces to some extent, but show that the fungal resistance of this bacterial strain obviously strengthens with the experimental result such as sclerotinite opposite culture and filtrate process.
Fungal resistance strengthens T-DNA insertion point number in bacterial strain ZS-1N1812 and analyzes: the genomic dna Kpn I extracting ZS-1N1812 and ZS-1 bacterial strain carries out complete degestion (about 12h), with recombinant plasmid pTFCM for positive control.Southern hybridization is carried out after electrophoresis transferring film.Results of hybridization display, the mould fungal resistance of shield shell strengthens bacterial strain ZS-1N1812 only a hybrid belt, and containing T-DNA mark in the genome of card ZS-1N1812, and this is labeled as unit point insertion.
To the clone of the relevant CmSit1 of the mould fungal resistance of shield shell: utilize T-DNA insertion point side fragment in TAIL-PCR technology clone strain ZS-1N1812, after obtaining respective segments sequence, the genome sequence that itself and our laboratory genome sequencing obtain is carried out Blast comparative analysis, found that T-DNA is inserted in the promoter region of CmSit1, we are through this gene of RACE and sequence verification subsequently, found that 2052 Nucleotide (SEQ ID NO:1) are contained in the coding region of CmSit1,673 amino acid (SEQID NO:2) of encoding.This gene is siderophore translocator, is the functional protein of assisting siderophore transdermal delivery and then metabolism.And siderophore is microorganism class low molecular weight substance for high specific chelating ferric ion under iron deficiency.There are some researches show that the function of this gene is relevant with resistance to growth, the function of this gene and the relation of antifungal property then have no report.We study and find that this gene overexpression can significantly improve the mould antagonistic properties to sclerotinite of shield shell in shield shell is mould, antagonism loop diameter is increased to about 1.0cm from 0.0cm, further analysis finds that this gene overexpression backing shell mould culturing filtrate process rape leaf significantly can suppress the Spot expansion of sclerotium disease, after shield shell mould ZS-1 filtrate treatment group inoculation 72h, lesion diameter is about 2cm, and has not yet to see obvious scab after CmSit1 overexpression shield shell mould filtrate treatment group inoculation 72h.
Advantage of the present invention is: 1) gene C mSit1 overexpression of the present invention can significantly improve the mould antagonistic ability to sclerotinite of shield shell, is conducive to transforming out efficient shield shell trichoderma strain; 2) CmSit1 of the present invention derives from fungi, can facilitate, effectively and the transformation being applied to other biocontrol fungis of safety, improve the fungal resistance of other biocontrol microorganisms; 2) gene C mSit1 of the present invention expresses and can significantly improve the resistance of plant to sclerotinite in tobacco, has the potentiality being applied to the anti-sclerotium disease breeding such as soybean and rape.
A kind of shield shell mould siderophore transporter gene is strengthening the application in transfer-gen plant anti-sclerotium disease (can be used in the anti-sclerotium disease breeding of rape, soybean and Sunflower Receptacle), the steps include: that the expression vector this gene being utilized retrovirus-mediated method imports the plant such as rape, soybean or Sunflower Receptacle, this system mainly comprises pTRV1, pTRV2 and pTRV2EX tri-carriers.Carrier (PSIT1V2EX) containing goal gene CmSit1 is proceeded in Agrobacterium GV3101, be cultured to two kinds of bacterium liquid balanced mix (PTRV1+PTRV2EX in this test under appropriate condition, PTRV1+PSIT1V2EX mixes), bacterium liquid is injected in the lower epidermis cell of rape, soybean or Sunflower Receptacle.The healthy leaves that before inoculating afterwards for 15 days, clip is injected blade and do not injected, extracts the expression that RNA, RT-PCR analyze CmSit1 gene.PDA flat board is inoculated sclerotinite Ep-1PNA367 mycelia block, after cultivating 3d, inoculates sclerotinite in plant, measure after inoculation 2d and record Lesion size, analyzing the resistance situation of transfer-gen plant.
The present invention has the illustration of practicality:
(1) CmSit1 gene provided by the present invention can be used for mould or other biocontrol strain of transformation shield shell, improves the ability of its anti-plant pathogenic fungi;
(2) CmSit1 gene provided by the present invention can be used for farm crop transgenosis breeding for disease resistance, improves crop to the resistance of pathogenic fungi;
(3) mutant library that the present invention can be utilized to introduce and research method thereof, other disease-resistant fungal pathogens gene of Screening and Identification, promotes the discovery of new effective gene.
Accompanying drawing explanation
Fig. 1 be mould ZS-1 and the ZS-1N1812 of a kind of shield shell on PDA and fungal resistance compare schematic diagram.
A, the colonial morphology figure of shield shell trichoderma strain ZS-1;
The colonial morphology figure of B, ZS-1N1812;
C, ZS-1 are to the antagonistic effect result of sclerotinite;
D, ZS-1N1812 are to the antagonistic effect result of sclerotinite.
Fig. 2 is the structural representation of a kind of vectors for transformation pTFCM.
Fig. 3 is that in a kind of ZS-1N1812, T-DNA insertion point number detects schematic diagram.
Swimming lane M, λ-HindIII DNA weight marker; Swimming lane 1, the mould ZS-1 bacterial strain of shield shell; Swimming lane 2, transform plastids pTFCM; Swimming lane 3, ZS-1N1812 bacterial strain.
Fig. 4 is the obvious elevated view of CmSit1 expression level in a kind of ZS-1N1812.
Fig. 5 is that the culturing filtrate process of a kind of ZS-1N1812 can significantly improve the resistance schematic diagram of rape to sclerotinite.
A, ddH
2sclerotinite inoculation test result after O process live body rape;
Sclerotinite inoculation test result after B, substratum PDB process live body rape;
Sclerotinite inoculation test result after the culturing filtrate process live body rape of C, the mould ZS-1 of shield shell;
Sclerotinite inoculation test result after the culturing filtrate process live body rape of D, ZS-1N1812;
E, ddH
2sclerotinite inoculation test result after the in vitro rape leaf of O process;
F, the sclerotinite inoculation test result after the in vitro rape leaf of substratum PDB process;
Sclerotinite inoculation test result after the in vitro rape leaf of culturing filtrate process of G, the mould ZS-1 of shield shell;
Sclerotinite inoculation test result after the in vitro rape leaf of culturing filtrate process of H, ZS-1N1812.
Fig. 6 be CmSit1 in a kind of overexpression transformant expression level and on PDA its fungal resistance and the mould ZS-1 bacterial strain of shield shell compare schematic diagram.
A, in overexpression transformant, the expression level of CmSit1 is obviously high than ZS-1, and P1, P2 and P3 represent three different overexpression transformants respectively;
B, ZS-1 are to the antagonistic effect result of sclerotinite;
C, CmSit1 overexpression transformant P1 is to the antagonistic effect result of sclerotinite.
Fig. 7 is the VIGS carrier through transformation and expression vector collection of illustrative plates schematic diagram that use in a kind of test.
Fig. 8 is that a kind of pSIT1V2EX1 of importing overexpression CmSit1 can strengthen the resistance of Ben Shi cigarette to sclerotinite.Schematic diagram.
A, the detection that after importing pSIT1V2EX1, in Ben Shi cigarette, CmSit1 expresses, 1 is Marker, and 2 and 3 is contrast, and 4 and 5 is transfer-gen plant;
B, process LAN CmSit1 make Ben Shi cigarette strengthen the resistance of sclerotinite, and after inoculation 6d, the expansion of observations display mycelia is obviously slowed down.
Embodiment
Below in conjunction with specific embodiment, further illustrate the present invention.It should be noted that following examples are only not used in for illustration of the present invention to limit the scope of the invention.
Embodiment 1:
A preparation method for the mould siderophore transporter gene of shield shell, the steps include:
The mould fungal resistance of A, shield shell strengthens the screening of bacterial strain ZS-1N1812:
The Agrobacterium (EHA105) of the maturation set up according to this laboratory mediates the mould genetic transforming method (LiMoxiao of shield shell, et al.Transformation of Coniothyrium minitans, a parasite of Sclerotiniasclerotiorum, with Agrobacterium tumefaciens.FEMS Microbiology Letters.2005,243:323-329.), transform this laboratory and be separated the mould ZS-1 bacterial strain of shield shell obtained from field, obtain the mould insertional mutagenesis library of shield shell containing more than 45000 T-DNA mark.With PDA substratum, (PDA adopts conventional making method: 200g peeled potatoes, after adding appropriate distilled water (500-800ml) boiling, add glucose 20g, agar powder 13g, supplement distilled water to 1000ml) activation culture shield shell mould ZS-1 bacterial strain and the mould transformant of shield shell under 20 DEG C of conditions, the mycelia block at the edge getting activation is beaten with punch tool (diameter is 5mm), transfer in culture dish (diameter the is 90mm) edge containing quantitative PDA (20ml/ ware), face down containing mycelia, at 20 DEG C, cultivate 4d.Activate the sclerotinite Ep-1PNAa bacterial strain that this laboratory is separated acquisition on the disease plant of field, and be connected to the culture dish edge relative with transformant, two bacterial strains are carried out opposite culture, with ZS-1 bacterial strain for contrast.The suppression situation that after 7d, observed and recorded mutant and ZS-1 bacterial strain grow sclerotinite.The mutant ZS-1N1812 (Fig. 1) that screening acquisition one strain fungal resistance strengthens, mould for this shield shell (Coniothyrium minitans) ZS-1N1812 bacterial strain have been delivered the China typical culture collection center (CCTCC) in the Wuhan University of Wuhan City, Hubei Province by applicant on March 5th, 2012, deposit number is No. NO:M2012054, CCTCC.
B, fungal resistance strengthen T-DNA insertion point number in bacterial strain ZS-1N1812 and analyze:
In order to verify the insertion point number of the T-DNA in mutant ZS-1N1812, genome Southern being carried out to it and has hybridized.Due to build transformant storehouse recombinant plasmid pTFCM (Fig. 2) used hygromycin gene in not containing restriction enzyme KpnI restriction enzyme site, so the genomic dna KpnI extracting ZS-1N1812 and ZS-1 bacterial strain carries out complete degestion (about 12h), be cut to positive control with the same enzyme of recombinant plasmid pTFCM.By digestion products in the sepharose of 1% (V/W) under 4 DEG C of conditions the slow electrophoresis 16h (1V/cm) of low voltage, transferring film is carried out by the following method: first gel is immersed 0.25mol/L HCL 10min after electrophoresis terminates, make DNA depurination, gel is washed twice with distillation, then gel is soaked 20min in 0.1mol/L NaOH solution, proceed in 0.1M Tris (pH8.0) and soak 20min, (1000ml water is containing 87.65g NaCl to use 10 × SSC again, 44.1 Trisodium Citrate, pH7.0) process 15min and can carry out transferring film.Using a synthetic glass slightly larger than gel as platform, put it in large pallet, pour 10 × SSC into and make liquid level a little less than platform surface, tile above Plexiglas platform Xinhua's filter paper, makes filter paper evenly moistening, and ensure that two ends are immersed among SSC; The filter paper that upper berth one deck is slightly larger than gel, make it evenly moistening, gel is faced down and is placed on filter paper moistening on platform, above gel, place the use 10 × SSC onesize with gel soak the nylon membrane (Hybond N+) of 5min, the filter paper that placement two is onesize with gel on film; Filter paper is put onesize thieving paper, thieving paper is put one piece of sheet glass, with the weight compacting of about 500g.After transferring film 20h with 10 × SSC cleaning filter membranes to remove the agarose fragment on filter membrane, place it in the middle of two filter paper after room temperature (20-25 DEG C) airing, vacuum oven 80 DEG C baking 2h.
Subsequently by filter membrane at 3 × SSC, 0.1% (V/W) Sodium dodecylbenzene sulfonate (SDS, purchased from sigma biotech firm) in prewet after put into hybrid pipe, make it just facing to the inside of pipe, add the 3 × SSC of appropriate (10-15ml), 0.1%SDS solution in 65 DEG C of process 15min.Abandon above-mentioned solution, add appropriate (10-15ml) prehybridization solution (1% bovine serum albumin, 1mM EDTA, 0.25M Na
2hPO
4-naH
2pO
4pH 7.2,7%SDS), 65 DEG C of prehybridization 5h.
The probe used in hybridization cuts recombinant plasmid pTFCM with Sac I and Xho I enzyme, recovery contains the fragment of hygromycin gene and trpC promotor as template, adopt random primering (this test kit purchased from precious biotechnology (Dalian) company limited, the method introduced according to the specification sheets of test kit operates) with (α-
32p) dCTP marks.
Recovery method containing hygromycin gene and trpC promoter fragment is as follows: the recombinant plasmid after Sac I and Xho I enzyme are cut through 1% agarose gel electrophoresis, separate targets fragment, then ethidium bromide (EB) dyes 20min.Ultraviolet transilluminator cuts the sepharose containing target DNA fragments with clean scalpel, test kit is reclaimed (purchased from AXYGEN company with AxyPrep DNA gel, Zhejiang Province, China province Hangzhou), reclaim object fragment according to the method for test kit specification sheets, be placed in-20 DEG C of preservations.
With Random Primer DNA Labeling Kit (this test kit is purchased from precious biotechnology (Dalian) company limited), carry out probe mark with reference to this test kit specification sheets, concrete steps are as follows: in centrifuge tube, add 25ng template DNA, 2 μ l random primers, add distilled water to 14 μ l.Be placed in rapidly ice after 95 DEG C of heating 3min and cool 5min.Add in above-mentioned reaction solution 10 × damping fluid 2.5 μ l, dNTP 2.5 μ l, Klenow 1 μ l, finally add α-
32dCTP (50 μ Ci, purchased from Beijing Fu Rui company) the 5 μ l of P mark, compressing mixing are placed on 37 DEG C of reaction 20min.Add 175 μ l distilled waters after reaction terminates, add 10NNaOH and make its final concentration be 0.1N, standing 5min makes probe sex change (or after 95 DEG C of heating 3min, putting rapidly quenching in ice).The probe marked is added at the bottom of the hybrid pipe after prehybridization and (be not directly added on film), 65 DEG C of hybridized overnight.
Carry out after having hybridized washing film.First use 2 × SSC, 0.1%SDS to wash film 15min in 65 DEG C after abandoning hybridization solution in hybrid heater, then use 1 × SSC, 0.1%SDS 65 DEG C washes film 15min, finally use 0.1 × SSC, 0.1%SDS 65 DEG C wash film 15min.To press phosphorus to shield (making it just facing to intensifying screen face) after preservative film coating, be placed in phosphorus screen box and place 3h, FUJIFILM phosphorus screen scanner scanning phosphorus screen in room temperature.
Genomic DNA hybridization result shows, the mould fungal resistance of shield shell strengthens bacterial strain ZS-1N1812 only a hybrid belt, plasmid pTFCM for transforming has a hybrid belt, there is not hybrid belt in ZS-1 bacterial strain, prove containing T-DNA mark in the genome of ZS-1N1812, and this is labeled as unit point insertion (Fig. 3).
C, clone to the relevant CmSit1 of the mould fungal resistance of shield shell:
Utilize T-DNA insertion point side fragment in TAIL-PCR technology clone strain ZS-1N1812, the T-DNA flanking fragment of acquisition is delivered to Beijing three and win the order-checking of biological company limited, this DNA fragmentation of sequencing result marks the genomic dna of insertion point from T-DNA.The genome sequence that this sequence and our laboratory genome sequencing obtain is carried out Blast comparative analysis, found that T-DNA is inserted in the promoter region of CmSit1, we are through this gene of RACE and sequence verification subsequently, found that 2052 Nucleotide (SEQ ID NO:1) are contained in the coding region of CmSit1,673 amino acid (SEQ ID NO:2) of encoding.
Embodiment 2:
The detection of CmSit1 expression level in ZS-1N1812
First ZS-1 and ZS-1N1812 mycelia block is connected on glassine paper, after cultivating 4d, collect mycelia to add 3ml sterilized water and mill in mortar, then draw 200 μ l with rifle head and coat and be covered with on the flat board of glassine paper, collect respectively and grow the fresh mycelia of 48h, 72h, 96h and 120h.Adopt the TRIzol test kit of Invitrogen company to extract RNA, carry out RT-PCR and detect target gene expression level (primer sequence SIT1-FP:CCACTT CCA ACC CGA CAC; SIT1-RP:CTT ACG CCT CCG ACA AAT), contrast (primer sequence ACTIN-FP:ACC GTG AGA AGA TGA CCC using ACTIN gene as internal reference; ACTIN-RP:AAG GAC AGA AGG CTG GAA G) concrete steps are see test kit specification sheets.Test-results finds in ZS-1N1812, and the expression level of 4 the time point CmSit1 detected all obviously rises, and the expression (Fig. 4) of CmSit1 all cannot be detected at each time point of the mould ZS-1 bacterial strain of shield shell.
Embodiment 3:
The restraining effect that the ZS-1N1812 culturing filtrate that CmSit1 activates expression causes a disease to sclerotinite
Virulence detection rape (general business rape variety), at greenhouse (15-20 DEG C) growth 30d, gets eugonic blade in the middle part of each rapeseed plants and tests.The mould mycelia block of shield shell is inoculated in be completed on the PDA flat board of glassine paper, collects mycelia after cultivating 6d; To collect mycelia add in mortar sterilized water grinding, add in PDB be placed in 20 DEG C of shaking tables shake again training 9d.PDA flat board is inoculated sclerotinite Ep-1PNA367 mycelia block, after cultivating 3d, beats the mycelia block getting colony edge, respectively at aseptic ddH
2soak 30min in O, PDB, ZS-1 filtrate, ZS-1N1812 filtrate, be then inoculated in vitro rape leaf or potted plant rape (5-6 leaf phase), cultivate at 20 DEG C, 5-6 repetition is established in each process, observes blade incidence after 48h.After found that the process of ZS-1N1812 filtrate, the pathogenecity of Sclerotinia sclerotiorum excised leaf and live body all obviously reduces (Fig. 5).
Embodiment 4:
CmSit1 gene overexpression is on the impact of the mould fungal resistance of biocontrol fungi shield shell
CmSit1 gene is connected to the carrier of band Ptrp C promotor, utilizes agrobacterium mediation converted technical system by mould for vector introduction shield shell ZS-1 bacterial strain.Carry out the expression level (specific experiment scheme is see embodiment 2) that RT-PCR detects CmSit1 after extracting RNA, found that the expression level of CmSit1 gene in transformant obviously rises (Fig. 6 A).At the transformant of the mould ZS-1 bacterial strain of PDA substratum activation culture shield shell and CmSit1 gene overexpression, get the mycelia block at the edge of activation with punch tool, transfer in PDA culture dish edge, face down containing mycelia, at 20 DEG C, cultivate 4d.Active nuclei cup fungi Ep-1PNAa bacterial strain, and be connected to the culture dish edge relative with transformant, two bacterial strains are carried out opposite culture, with ZS-1 bacterial strain for contrast.The suppression situation that after 7d, observed and recorded CmSit1 gene overexpression transformant and ZS-1 bacterial strain grow sclerotinite, finds that the mould resistance to sclerotinite of biocontrol fungi shield shell significantly strengthens (Fig. 6 B) after the mould middle overexpression CmSit1 gene of shield shell.
Embodiment 5:
CmSit1 transgenosis can strengthen the resistance of plant to pathogenic fungi sclerotinite
The plant such as transformation of tobacco utilize the silencing system of retrovirus-mediated method, this system mainly comprises pTRV1, pTRV2 and pTRV2EX tri-carriers, wherein the cDNA of pTRV1 and pTRV2 derives from the TRV-Ppk20 infecting Ben Shi cigarette (Nicotiana benthamiana), RNA1 in pTRV1 is complete, and pTRV2 eliminates nematode propagation associated protein, only remain CP.The genetic expression of RNA2 adopts subgenomic RNA strategy, CP sub-genomic promoter (selected sequence is according to report document) is introduced after the CP of pTRV2EX, introduce the ccdB gene containing XcmI simultaneously, carrying out Agrobacterium injection tobacco as GFP being cloned into pTRV2EX, can green fluorescence be detected.The sequence of RNA1 with RNA2, compared with TRV-Ppk20, transformed several base position, and infectivity is the same with wild-type, but the symptom caused not obvious (Fig. 7).Therefore after injecting tobacco, cause systemic infection but do not cause manifest symptom, can the next generation be passed to by seed simultaneously, and stable existence more than 2 years.
Systemic infection conversion process: in advance at 26 DEG C, cultivates Ben Shi cigarette one month under continuous light condition.Carrier (PSIT1V2EX) containing goal gene is proceeded in Agrobacterium GV3101, under 28 DEG C of conditions, shake training in containing the LB substratum of 50 μ g/ml kantlex and 50 μ g/ml Rifampins to spend the night, to get in the 10ml LB substratum that 1ml bacterium liquid goes to containing 50 μ g/ml kantlex, 50 μ g/ml Rifampins and 15 μMs of Syringylethanones enlarged culturing to bacterial growth logarithmic phase; Under room temperature condition, 4000g collected by centrifugation thalline, then with containing 10mM MgCl
2with the 10mM 2-N-morpholino ethanesulfonic acid buffer resuspension of 150 μMs of Syringylethanones, adjust OD
600=1.0, room temperature leaves standstill 2h.By two kinds of bacterium liquid balanced mix (in this test PTRV1+PTRV2EX, PTRV1+PSIT1V2EX mixing), bacterium liquid is injected in the lower epidermis cell of tobacco.At 26 DEG C, under continuous light condition, cultivate tobacco 15d.After injection, every day records tobacco growing change.
Genetic expression detects: the healthy leaves that before inoculation, clip tobacco is injected blade and do not inject, extract the expression (primer is SIT1SP:CCACTTCCAACCCGACAC and SIT1AP:CTTACGCCTCCGACAAAT) that RNA, RT-PCR analyze CmSit1 gene.Result shows that this transfection system effectively can realize the expression of goal gene CmSit1 in Ben Shi cigarette (Fig. 8 A).
Sclerotinite inoculates: on PDA flat board, inoculate sclerotinite EP-1PNA367 mycelia block, after cultivating 3d, beats the mycelia block getting colony edge, and inoculation sclerotinite is the 4th leaf in tobacco top.Measure and record Lesion size after inoculation 2d, afterwards every day time recording Spot expansion and the details such as infection rate.Found that after importing CmSit1, the resistance of Ben Shi cigarette to sclerotinite obviously strengthens (Fig. 8 B).
By the specific procedure of the farm crop such as this channel genes rape, soybean and Sunflower Receptacle and testing process the same.
SEQUENCE LISTING
<110> Hua Zhong Agriculture University
The mould siderophore transporter gene of <120> shield shell and preparation method and purposes
The mould siderophore transporter gene of <130> shield shell and preparation method and purposes
<160> 2
<170> PatentIn version 3.1
<210> 1
<211> 2052
<212> DNA
<213> shield shell is mould
<400> 1
atggcgggca acccccgccc ccaatcggca cccatgcttg acacatccgg cactgctgct 60
atcacgccgg cacaccttct gcccatgaat acggctgata gctacgaccc cgatgccatc 120
accatggtcg atccaaaacg cggctctttc agatccagcg agccactcga gcccggtgcg 180
aggcccacag acaaggacga cgtcgacaat gcagctgagt attgcgatgg ttgtagctcg 240
ctcgcaggcg ctgagtctca aggcatacct cgaaaacacg cggagccccc gactggcgcg 300
gatagcccag gctatcttcg catggctata acgagaaaga gcatccgatc ctcagagaag 360
acgtttgtac tcgtgtgcat cctgttgttg gcgactctac aaagcctgga caacttcatg 420
cgtgtcctgt accagtatga gattgccaca aactaccaaa agagtgggat gcttggcgcc 480
ctcaataccg tccctactct tgtcgctgcc gccatgaccc cattggtctc aaagagtgca 540
gatgtatatg gtcgtccgga gactctcatc acctctatca tgttctatat actaggcact 600
gtcttgcaag ccacggctac cacaatccaa atcttcttgg gcggcaccat catttgggct 660
atcggttttc tgggtgtcat ttccatgttc gaaatcatca ttgcagacct tacctcgatg 720
agactgaggg ttgtcgtgtt ctacttaccc gccctcccat atcttgtaac aacctggttc 780
agtgccgctt tgaggaatgc gcttgtcgaa acctgtcctt cagtgcactg gcgctttgct 840
tggtcggcaa ttatgtactt tgcatgctct gtacctttgg taataggcct ccttacaatc 900
gagcgcaacg cggagaaaag actgtcccct tctgagcttg agaacgcgtt gagaatacct 960
gtccgaggtc gcgtctctcg tcagctctta cgcctccgac aaatggacat actcggctgg 1020
atttgcttca tgggcatcgc gacatgtctc ttggcgcctt gggcatcgtt gccgctcgtg 1080
cttccacaca accagggcta tgggccaagc cccgctacca tctccatgac cattacgggc 1140
ttgctttgta ttgttatatt ctactatgtg gagaagtatt cgaagtatcc catgttccca 1200
attcagctgc ttggggagaa aagaatcgta acagccctca ctatgggatt cctctatcat 1260
atggcatact atgtgcaatc cacatacctt ctgatcgggc tgggaattcg atacaacaat 1320
gatgacgaca gctcagcaca catcgtgggc ctgtatacat tcacctccac actggttggc 1380
ctcttcatcg gggtcattat cagcataacc cgcgacctca ggtggtatct tcgatttggc 1440
gcaatattct atcttgcctc cttcgtgctc cagtacaatc gtgcgagtgg aaatgacaac 1500
gtcagccagc ttgctgtcac ttgttcacaa gtacttttgg gcatcgctgg cggattgttc 1560
ccatttccag caatggcttt cgttcaagga gcgcgtgatc atacgcagct gtctacactc 1620
cttggcgcct atatgacggc gtgtcgggtt ggaagtggcg tggggcagtc gcttgctggc 1680
gccatctgga cgaatgcact gctccccaag cttcaccaac gtctcgaaca gataattacg 1740
gaaagcgaaa tcgacatgct ttacgctatg cccacagctc atgaagacct ctacccttgg 1800
ggatcgcctc cacgagtgcc tatggtggaa gccttcgtcc agagccaccg ctatctatgc 1860
gccattggca ttctagcttc tacgctcttg atcatattgg cctttctggt ccgagatgcg 1920
tctttggatt cactaccgga agatcatgag atcgacctga agccactgcc agaagatttg 1980
cagcctaaaa aggcgtcgga gacaacggta ccatgcccgc ttcgtcccag ccgccatccg 2040
atcatcatgt aa 2052
<210> 2
<211> 683
<212> PRT
<213> shield shell is mould
<400> 2
Met Ala Gly Asn Pro Arg Pro Gln Ser Ala Pro Met Leu Asp Thr Ser
1 5 10 15
Gly Thr Ala Ala Ile Thr Pro Ala His Leu Leu Pro Met Asn Thr Ala
20 25 30
Asp Ser Tyr Asp Pro Asp Ala Ile Thr Met Val Asp Pro Lys Arg Gly
35 40 45
Ser Phe Arg Ser Ser Glu Pro Leu Glu Pro Gly Ala Arg Pro Thr Asp
50 55 60
Lys Asp Asp Val Asp Asn Ala Ala Glu Tyr Cys Asp Gly Cys Ser Ser
65 70 75 80
Leu Ala Gly Ala Glu Ser Gln Gly Ile Pro Arg Lys His Ala Glu Pro
85 90 95
Pro Thr Gly Ala Asp Ser Pro Gly Tyr Leu Arg Met Ala Ile Thr Arg
100 105 110
Lys Ser Ile Arg Ser Ser Glu Lys Thr Phe Val Leu Val Cys Ile Leu
115 120 125
Leu Leu Ala Thr Leu Gln Ser Leu Asp Asn Phe Met Arg Val Leu Tyr
130 135 140
Gln Tyr Glu Ile Ala Thr Asn Tyr Gln Lys Ser Gly Met Leu Gly Ala
145 150 155 160
Leu Asn Thr Val Pro Thr Leu Val Ala Ala Ala Met Thr Pro Leu Val
165 170 175
Ser Lys Ser Ala Asp Val Tyr Gly Arg Pro Glu Thr Leu Ile Thr Ser
180 185 190
Ile Met Phe Tyr Ile Leu Gly Thr Val Leu Gln Ala Thr Ala Thr Thr
195 200 205
Ile Gln Ile Phe Leu Gly Gly Thr Ile Ile Trp Ala Ile Gly Phe Leu
210 215 220
Gly Val Ile Ser Met Phe Glu Ile Ile Ile Ala Asp Leu Thr Ser Met
225 230 235 240
Arg Leu Arg Val Val Val Phe Tyr Leu Pro Ala Leu Pro Tyr Leu Val
245 250 255
Thr Thr Trp Phe Ser Ala Ala Leu Arg Asn Ala Leu Val Glu Thr Cys
260 265 270
Pro Ser Val His Trp Arg Phe Ala Trp Ser Ala Ile Met Tyr Phe Ala
275 280 285
Cys Ser Val Pro Leu Val Ile Gly Leu Leu Thr Ile Glu Arg Asn Ala
290 295 300
Glu Lys Arg Leu Ser Pro Ser Glu Leu Glu Asn Ala Leu Arg Ile Pro
305 310 315 320
Val Arg Gly Arg Val Ser Arg Gln Leu Leu Arg Leu Arg Gln Met Asp
325 330 335
Ile Leu Gly Trp Ile Cys Phe Met Gly Ile Ala Thr Cys Leu Leu Ala
340 345 350
Pro Trp Ala Ser Leu Pro Leu Val Leu Pro His Asn Gln Gly Tyr Gly
355 360 365
Pro Ser Pro Ala Thr Ile Ser Met Thr Ile Thr Gly Leu Leu Cys Ile
370 375 380
Val Ile Phe Tyr Tyr Val Glu Lys Tyr Ser Lys Tyr Pro Met Phe Pro
385 390 395 400
Ile Gln Leu Leu Gly Glu Lys Arg Ile Val Thr Ala Leu Thr Met Gly
405 410 415
Phe Leu Tyr His Met Ala Tyr Tyr Val Gln Ser Thr Tyr Leu Leu Ile
420 425 430
Gly Leu Gly Ile Arg Tyr Asn Asn Asp Asp Asp Ser Ser Ala His Ile
435 440 445
Val Gly Leu Tyr Thr Phe Thr Ser Thr Leu Val Gly Leu Phe Ile Gly
450 455 460
Val Ile Ile Ser Ile Thr Arg Asp Leu Arg Trp Tyr Leu Arg Phe Gly
465 470 475 480
Ala Ile Phe Tyr Leu Ala Ser Phe Val Leu Gln Tyr Asn Arg Ala Ser
485 490 495
Gly Asn Asp Asn Val Ser Gln Leu Ala Val Thr Cys Ser Gln Val Leu
500 505 510
Leu Gly Ile Ala Gly Gly Leu Phe Pro Phe Pro Ala Met Ala Phe Val
515 520 525
Gln Gly Ala Arg Asp His Thr Gln Leu Ser Thr Leu Leu Gly Ala Tyr
530 535 540
Met Thr Ala Cys Arg Val Gly Ser Gly Val Gly Gln Ser Leu Ala Gly
545 550 555 560
Ala Ile Trp Thr Asn Ala Leu Leu Pro Lys Leu His Gln Arg Leu Glu
565 570 575
Gln Ile Ile Thr Glu Ser Glu Ile Asp Met Leu Tyr Ala Met Pro Thr
580 585 590
Ala His Glu Asp Leu Tyr Pro Trp Gly Ser Pro Pro Arg Val Pro Met
595 600 605
Val Glu Ala Phe Val Gln Ser His Arg Tyr Leu Cys Ala Ile Gly Ile
610 615 620
Leu Ala Ser Thr Leu Leu Ile Ile Leu Ala Phe Leu Val Arg Asp Ala
625 630 635 640
Ser Leu Asp Ser Leu Pro Glu Asp His Glu Ile Asp Leu Lys Pro Leu
645 650 655
Pro Glu Asp Leu Gln Pro Lys Lys Ala Ser Glu Thr Thr Val Pro Cys
660 665 670
Pro Leu Arg Pro Ser Arg His Pro Ile Ile Met
675 680
Claims (7)
1. the mould siderophore transporter gene of shield shell be separated, its sequence is the nucleotide sequence shown in SEQ ID NO:1.
2. the mould siderophore translocator of shield shell be separated, its sequence is SEQ ID NO:2 aminoacid sequence.
3. a shield shell trichoderma strain, is characterized in that: this shield shell trichoderma strain (Coniothyrium minitans), ZS-1N1812, CCTCC NO:M2012054.
4. the application of shield shell mould siderophore transporter gene in the anti-sclerotinite transformation of biocontrol fungus Coniothyrium minitans Campbell of a kind of separation according to claim 1.
5. the application of shield shell mould siderophore transporter gene in the anti-sclerotinite breeding of rape transgenosis of a kind of separation according to claim 1.
6. the application of shield shell mould siderophore transporter gene in the anti-sclerotinite breeding of soybean transgene of a kind of separation according to claim 1.
7. the application of shield shell mould siderophore transporter gene in the anti-sclerotinite breeding of Sunflower Receptacle transgenosis of a kind of separation according to claim 1.
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| CN108672310B (en) * | 2018-05-14 | 2023-08-25 | 电子科技大学中山学院 | Automatic clamp detection machine |
| CN111254184A (en) * | 2019-12-03 | 2020-06-09 | 湖南农业大学 | Method for rapidly identifying pathogenicity of sclerotinia sclerotiorum mutant and method for rapidly identifying disease resistance of plant |
| CN116445531A (en) * | 2022-08-31 | 2023-07-18 | 四川省农业科学院植物保护研究所 | Application of coniothyrium minitans CmHMG1 gene in coniothyrium minitans for enhancing biocontrol capability |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101864449A (en) * | 2010-05-10 | 2010-10-20 | 华中农业大学 | Method for genetic transformation of agrobacterium tumefaciens mediated sclerotinia sclerotiorum |
| CN101906428A (en) * | 2010-05-17 | 2010-12-08 | 华中农业大学 | Biocontrol fungus coniothyrium minitans sporulation-related gene CmPex2 and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101864449A (en) * | 2010-05-10 | 2010-10-20 | 华中农业大学 | Method for genetic transformation of agrobacterium tumefaciens mediated sclerotinia sclerotiorum |
| CN101906428A (en) * | 2010-05-17 | 2010-12-08 | 华中农业大学 | Biocontrol fungus coniothyrium minitans sporulation-related gene CmPex2 and application thereof |
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