CN1951957A - Gene MgPTH12 for controlling mature and pathogenicity of fungi appressorium derived from Magnaporthe grisea and its uses - Google Patents
Gene MgPTH12 for controlling mature and pathogenicity of fungi appressorium derived from Magnaporthe grisea and its uses Download PDFInfo
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Abstract
The invention discloses a new necessary MgPTH12 gene and utility to make pear spore bacterium attach to mature and lead to disease, which is characterized by the following: the gene and its cDNA and coding product possess the sequence in the SEQ ID No.:1, No.:2 and No.:3; the protein of coded gene possesses homeo structural region, which is not similar to known functional protein; the gene removes abnormal and matured attaching cell, which reduces forming frequency of infecting tin and extending infecting hypha without pathogenic ability for rice; the homologous gene of gene lies in the plant pathogenic bacteria widely, which can be applied to design and sieve new drug to prevent fungus.
Description
Technical field
The invention belongs to plant pathology, Pesticide Science and microbiological genetic engineering field.The present invention has proved a MgPth12 gene that comes from the control appressorium maturation and the virulence of Magnaporthe grisea by inserting sudden change, gene complementation and gene knockout.This gene transcription factor of may encoding, its disappearance causes Magnaporthe grisea that the virulence of paddy rice is significantly reduced.Therefore, the expression of the functional protein of this gene and coding thereof can be used as the screening and design that target site is used for antifungal medicine.
Background technology
Magnaporthe grisea (Magnaporthe grisea) is the fungi of Ascomycotina, can infect paddy rice, wheat, barley, grain and other multiple grass, causes seasonal febrile diseases.Especially this bacterium is infected the rice blast that paddy rice causes all there is generation every year in each cultivated rice district in the world, harm extensively, serious.Generally speaking, the harm of rice blast can make paddy rice underproduction 5-10%, and the grave illness field can cause the paddy rice total crop failure.Rice blast was once repeatedly popular in China, also was one of main disease of China paddy rice.1993, rice blast was very popular in China, still underproduction over ten billion jin under the situation of control comprehensively.2004, provinces and cities such as Chongqing and Sichuan broke out large-area rice blast, and only the Hechuan city (county-level city) in Chongqing just has 20,000 mu of paddy rice that rice blast seriously takes place, wherein nearly 5000 mu of paddy rice total crop failure.Magnaporthe grisea also can infect the turfgrass that Herba Stenotaphri helferi, Bermuda grass, eremochloa ophiuroides etc. belong to, and causes big area withered, is the common disease of turfgrass.
The infection processs of many plant pathogenic fungis comprises that the contacting of conidium and host surface, spore germination, appressorium and infectivity nail form, infect links such as mycelia expansion.Wherein, appressorium is that form in many plant pathogenic fungi pathogenic courses such as Magnaporthe grisea a kind of infects essential structure.The Magnaporthe grisea appressorium is a kind ofly to be hemispherical, to deposit the melanic structure that infects all around.Melanic deposition makes when appressorium can be born infection process because huge turgescence (8.0Mpas or 1161Psi that the glycerine accumulation produces; Howard, 1991), initiatively puncture host's epidermis thereby order about to infect to follow closely, differentiate blister primary infection mycelia and secondary invader silk then, in host cell, spread with the intercellular substance.Lack the appressorium forfeiture infiltration capability of melanin deposition, pathogenic thereby pathogenic bacteria is lost.
The growth of appressorium is an extremely complicated process with maturation.That has cloned at present forms relevant gene with the Magnaporthe grisea appressorium, comprise: MPG1 (Talbot et.al.1993), CPKA (Mitchel and Dean, 1995), MAGB (Liu and Dean.1997), PMK1 (Xu and Hamer 1996), ACR1 (Lau and Hamer, 1998), MAC1 (Chaoi and Dean 1997); PTH11 (DeZwaan et.al.1999) and CBP1 (Kamakura et.al.2002).Wherein the function of gene products such as MPG1, MAGB, PTH11 and CBP1 is the perception outer signals, the hydrophobin of MPG1 coding and the signaling molecule of rice leaf surface are done mutually, the signal that produces is passed to the α subunit on the heterotrimeric G protein of MagB coding, G protein alpha subunit activates the adenylate cyclase by the MAC1 coding, thereby cAMP signal transduction path (Liu and Dean, 1997 have been activated; Choi andDean, 1997).CAMP makes it discharge catalytic subunit, the target protein in catalytic subunit phosphorylation downstream in conjunction with the adjusting subunit of PKA.The appressorium of Δ cpka mutant forms postpones, and does not have pathogenicly, but can invade from wound, causes morbidity.Also work by the PTH11p of PTH11 coding in the upstream that appressorium forms approach, PTH11p may irritation cell in the degraded of lipid, discharge triglyceride, triglyceride is as the morphogenesis (DeZwaan et al., 1999) of signaling molecule stimulation appressorium.The appressorium rate of formation of ACR1 deletion mutant reduces (Lau andHamer, 1998).PMK1 regulates the initial of appressorium differentiation, and Δ pmkl can not form appressorium on any contact surface, does not directly also have spore injection rice tissue pathogenic; Though external source cAMP can make mutant form the early stage hook formation of appressorium, can not form sophisticated appressorium, does not also produce turgescence, thereby lost pathogenic (Xu and Hamer 1996).
In addition, also have some appressoriums to form affected genetic locus and identified, but gene clone also, as CON1 and CON7 (Shi and Leung 1995); APF1 (Silue et.al.1998); APP1, APP2 andAPP3 (Zhu et.al.1996); APP5 (Chun and Lee 1999).However, differentiation of Magnaporthe grisea appressorium and formation mechanism are still very undistinct.
The present invention has cloned new gene M gPTH2 who controls appressorium maturation and virulence in the Magnaporthe grisea by inserting the sudden change approach.The disappearance of this gene causes Magnaporthe grisea can not form the appressorium of normal morphology and melanism, and has significantly reduced the formation ability that infects nail, and the virulence on susceptible rice varieties significantly reduces.Therefore, the expression of this gene and proteins encoded thereof and modification can be used as the target of design, screening antifungal drug.In addition, the present invention also finds, gibberella saubinetii (Gibberella zeae), corn stalk rot disease bacterium (beading gibberella, Gibberellamoniliformis), also exist in the Sclerotinia sclerotiorum plant pathogenic fungis such as (Sclerotinia sclerotiorum) with Magnaporthe grisea MGPTH12 and be higher than homologous protein more than 40% at consensus amino acid sequence, may have similar biological function, so the expression and the modification of homologous protein also can be used as the target sieving antifungal drug in these fungies.
Summary of the invention
Of the present invention to be intended to the Magnaporthe grisea be pattern, and the gene of the ripe and virulence of new control fungi appressorium is provided.This gene is characterized by at Magnaporthe grisea called after MgPTH12: shown in SEQ ID NO:1 the 1st to the 5526th nucleotide sequence.The feature of this gene also be the cDNA of its coding and protein have respectively shown in the SEQ ID NO:2 the 1st to the 2164th nucleotide sequence and SEQ ID NO:3 shown in the 1st to the 470th aminoacid sequence.The promotor of this gene shown in SEQ ID NO:1 the 1st to the 1940th nucleotide sequence.MgPTH12 encoded protein MGPTH12 has Homeobox, but does not have tangible similarity with the albumen of present known function on amino acid levels.Therefore, MgPTH12 is a new gene.
Gene M gPTH12 provided by the present invention clones from Magnaporthe grisea by inserting the sudden change approach.Detailed process comprises: at first, obtain the mutant that virulence reduces by screening Magnaporthe grisea REMI transformant storehouse; Then, determine that by genetic cross and Southrn hybridization analysis the virulence of mutant reduces mutant phenotype and is divided into from situation with the heredity of inserting mark; Further, obtain to be divided into the flanking sequence that in mutant, inserts mark, and be that probe screening-gene group TAC library and suitable enzyme are cut the dna fragmentation that obtains to contain goal gene with this flanking sequence by plasmid rescue; At last, adopt complementation test, knock out functional verification and note that experiment and isolating cDNA carry out goal gene.The present invention has obtained a mutant X3073 by screening from Magnaporthe grisea REMI transformant storehouse.
Magnaporthe grisea REMI transformant storehouse involved in the present invention is meant by the plasmid integration technology of restriction enzyme mediation (Restrict Enzyme Mediated Integration, REMI) Magnaporthe grisea bacterial strain colony that make up, that contain several ten thousand independent transformant.
Screening mutant involved in the present invention mainly comprises two aspects: the one, spore inoculating on the onion entocuticle, is observed its infection processs; Another aspect be with spore inoculating on susceptible rice leaf, observe its virulence on rice varieties.To the mensuration of this two aspect, the present invention has adopted the method for conventional spray inoculation.The mutant X3073 that the present invention obtained in the appressorium rate of formation, infect on nail rate of formation and the virulence and compare with wild-type, have obvious defects.
Hereditary coseparation analysis among the present invention is meant to analyze and inserts being divided into from situation of mark hygromycin gene and mutant phenotype in the mutant.The method that is adopted is the method for genetic cross.Be about to mutant and do not have mutant phenotype and hygromycin resistance with one and bacterial strain that mating type is opposite is hybridized, analyze its thecaspore offspring spore germination rate, appressorium rate of formation, infect and follow closely rate of formation, virulence and hygromycin resistance.If wherein all offsprings that do not have a hygromycin resistance are wild-type, illustrate that then the mutant phenotype of this mutant is isolating altogether with inserting the mark hygromycin gene, promptly this mutant is for inserting mutant.The mutant X3073 that the present invention obtained is isolating altogether in appressorium rate of formation, the defective phenotype that infects on nail rate of formation and the virulence with inserting mark.
Southern hybridization analysis involved in the present invention, be meant with the selective marker of inserting in the plasmid (be hygromycin gene in the present invention) and inserted mutant and carry out genome Southern hybridization obtaining, with insertion number of sites and the integration situation such as copy number of analysis insertion plasmid in the Magnaporthe grisea genome for probe.By analysis, the mutant X3073 that the present invention obtained has the series connection of the selective marker of multiple copied and inserts on MgPTH12.
Goal gene clone involved in the present invention at first is according to hereditary coseparation analysis and Southern hybridization analysis result plasmid rescue to be carried out in target insertion site.At first, choose suitable digestion with restriction enzyme mutant gene group DNA, carry out recirculation, be connected containing the dna fragmentation that inserts plasmid and part flanking sequence, and transformed into escherichia coli, to obtain and to be determined at the genome sequence that inserts the site side.Further, with obtaining flanking sequence be the genomic library of probe screening Magnaporthe grisea.Used genomic library is the TAC library of Magnaporthe grisea wild type strain Y34 among the present invention, and its average cloned sequence size is 50kb, covers complete genomic 16-18 altogether doubly.Among the present invention, sieve the storehouse with the side sequence, obtain 6 positive colonies, they all contain the nucleotide sequence just like the 1st to the 5526th shown in the SEQ ID NO:1.
Gene function involved in the present invention is confirmed by complementation test and knock out experiment to carry out, and specifically comprises: the structure of complementary carrier, knockout carrier and carrier imported in the Magnaporthe grisea obtains the recombinant conversion body of Magnaporthe grisea.The structure of complementary carrier is meant that the dna segment that contains functional target gene full length sequence of will be cloned links to each other with a carrier that has new selective marker (being neomycin resistance gene in the present invention).In the present invention, foreign vector being imported the Magnaporthe grisea preferred method is protoplast transformation.In the present invention, the Magnaporthe grisea bacterial strain that complementary carrier imported is the resulting insertion mutant of the present invention X3073.The structure of gene knockout carrier is meant that two flank sequence orientations with this gene of the prediction of pcr amplification gained are connected in the carrier, and this carrier has been connected into a neomycin resistance gene in advance, is connected into hygromycin gene between two flank sequences.The Magnaporthe grisea bacterial strain that gene knockout carrier imported is wild-type Magnaporthe grisea P131, gene knockout carrier passes through the flanking sequence and the genomic corresponding sequence generation of the wild type strain homologous recombination of gene both sides, thereby coding region gene sequence and the hygromycin gene of corresponding site MgPTH12 in the genome are replaced.The present invention proves by gene complementation: the ectopic integration of MgPTH12 in mutant X3073 genome makes the mutant phenotype of this mutant recover normal; Simultaneously, gene knockout also proves: the form that the coding region gene sequence of MgPTH12 and hygromycin gene PERMUTATION OF SEQUENCES cause MgPTH12 to knock out the appressorium that body produces has the phenotype defective the same with said mutation body X3073.
The gene M gPTH12 of control fungi appressorium maturation provided by the present invention and virulence, it is characterized by: the dna sequence dna that has shown in SEQ ID NO:1 sequence or have 70% above sequence identity and have identity function with SEQ ID NO:1, this gene can come from Magnaporthe grisea, also can come from other plant pathogenic fungi.
The present invention also provides the cDNA of MgPTH12 gene, the dna sequence dna that it has shown in SEQ ID NO:2 sequence or has 70% above sequence identity and have identity function with SEQ ID NO:2, this gene can come from Magnaporthe grisea, also can come from other plant pathogenic fungi.
The present invention is also according to the cDNA sequence of MgPTH12 gene, the albumen of control fungi appressorium maturation with the virulence coded by said gene is provided, the albumen that it is characterized in that having shown in SEQ ID NO:3 sequence or on aminoacid sequence, have 40% above consistence and have identity function with SEQ ID NO:3, this albumen can come from Magnaporthe grisea, also can come from other plant pathogenic fungi.
It inserts gene involved in the present invention sudden change and causes Magnaporthe grisea can not form the appressorium of normal morphology and melanism, and the virulence on susceptible rice varieties significantly weakens.Therefore, the most important purposes of the present invention is: use above-mentioned achievement, design and screening can destroy the compound of the expression of this genetic expression, shearing and proteins encoded thereof; Or design and screen and to modify or to block the compound that it carries out cellular localization to this proteic aminoacid sequence; Thereby the antifungal medicament of development of new.Perhaps with above-mentioned institute's clone gene and proteins encoded thereof be expressed as reporter gene or albumen, design, screening related compound are blocked the signal pathway that this gene and albumen thereof participate in, thus the antifungal medicament of development of new; Perhaps to have shown in SEQ ID NO:3 sequence or to have 40% above sequence identity with SEQ ID NO:3 and have the aminoacid sequence design polypeptide in arbitrary zone in the albumen of identity function; and preparation antibody is used for detecting proteic expression under the compound treatment situation, all belongs within protection scope of the present invention.In addition, purposes of the present invention also comprises: in Magnaporthe grisea again separate this gene as probe or according to the DNA of this gene or the sequences Design primer of cDNA by PCR with the DNA of this gene or a certain section of cDNA, perhaps isolating in other fungi, with this gene have the sequence of certain sequence homology.
The invention still further relates to and come from gibberella saubinetii (Gibberella zeae), corn stalk rot disease bacterium (beading gibberella, Gibberella moniliformis), Sclerotinia sclerotiorum plant pathogenic fungis such as (Sclerotinia sclerotiorum), on aminoacid sequence, have and be higher than 40% conforming homologous protein with Magnaporthe grisea MgPth12, they may have identical biological function with Magnaporthe grisea MgPTH12.Therefore, the promotor of these homologous proteins and encoding gene thereof also can be used as target site, is used to design and screen the medicine of anti-these fungies.
Below by description of drawings further to understand the present invention.
Description of drawings
Accompanying drawing 1.Wild type strain P131 and mutant X3073, complementary transformant CX8, knock out transformant K75 appressorium form relatively.Illustrate: A is P131, and B is X3073, and C is CX8, and D is K75.Inoculating spores concentration is 5 * 10
5Individual/milliliter, inoculate photomicrograph in back 12 hours, amplify 400 times.Scale length is 20 microns in the diagram.
The appressorium rate of formation of accompanying drawing 2. Magnaporthe grisea wild type strain P131 and its mutant X3073 different time sections on onion epidermis relatively.Illustrate: inoculating spores concentration is 5 * 10
5Individual/milliliter.
Accompanying drawing 3. Magnaporthe grisea wild type strain P131 and its mutant X3073 compare the virulence of paddy rice.Illustrate: a left side is wild-type P131, and the right side is mutant X3073.Inoculating spores concentration is 5 * 10
5Individual/milliliter, inoculate back 72 hours and take pictures.
Accompanying drawing 4. complementary vector construction synoptic diagram.PCR forward primer design enzyme cut SmaI restriction enzyme site 5 '-GAGCCCGGGATCCATTACAAAA-3 ', reverse primer design enzyme cuts the EcoRI restriction enzyme site and is 5 '-GCAGAATTCAATCGTCAGCAGTAT-3 ', from wild bacterium P131, amplify 5.5kb and comprise MgPTH12 coding region nucleotide sequence, connect the KN carrier, constitute complementary carrier.
The appressorium rate of formation of complementary transformant CX8 different time sections on onion epidermis of accompanying drawing 5. Magnaporthe grisea wild type strain P131 and its mutant X3073 relatively.Illustrate: inoculating spores concentration is 5 * 10
5Individual/milliliter.
The complementary transformant CX8 of accompanying drawing 6. Magnaporthe grisea wild type strain P131 and its mutant X3073 compares the virulence of paddy rice.Illustrate: a left side is wild-type P131, and the right side is complementary transformant CX8.Inoculating spores concentration is 5 * 10
5Individual/milliliter, inoculate back 72 hours and take pictures.
Accompanying drawing 7: the vector construction and the proof of Magnaporthe grisea paddy rice wild type strain P131 MgPth12 gene knockout body.Left arm forward primer 5 '-GCGGAATTCTTTGGTATGTG-3 ', it 5 ' has the EcoRI restriction enzyme site, and reverse primer is 5 '-GTCAAGCTTATCGGAACAGG-3 ', its 5 ' end has the HindIII restriction enzyme site; Right arm forward primer 5 '-TAAGTCGACCGATGTTGCTG-3 ' its 5 ' have the XhoI restriction enzyme site, reverse primer is 5 '-TAGACCGTGCTGCAGATTG-3 ', connect the KN carrier respectively, connect hygromycin gene at last in the SalI site, constitute knockout carrier.Figure below is the Southern hybridization affirmation figure that knocks out body, and lower-left figure be to be the Southern hybridization figure of probe with MgPth12, and bottom-right graph is to be the Southern hybridization figure of probe with the metathetical hygromycin gene.
The appressorium rate of formation that accompanying drawing 8. Magnaporthe grisea wild type strain P131 and its MgPTH12 knock out body K75 different time sections on onion epidermis compares.Illustrate: inoculating spores concentration is 5 * 10
5Individual/milliliter.
Accompanying drawing 9. Magnaporthe grisea wild type strain P131 and its MgPTH12 knock out the virulence comparison of body K75 to paddy rice.Illustrate: a left side is wild-type P131, and is right for knocking out body K75.Inoculating spores concentration is 5 * 10
5Individual/milliliter, inoculate back 72 hours and take pictures.
Accompanying drawing 10. comes from Magnaporthe grisea (Magnaporthe grisea, Mg), gibberella saubinetii (Gibberella zeae, Gz), corn stalk rot disease bacterium (Gibberella moniliformis, Gm), Sclerotinia sclerotiorum (Sclerotinia sclerotiorum, the sibship of PTH12 proteinoid Ss).
Embodiment
For a better understanding of the present invention, illustrate further by the following examples, but be not limitation of the present invention.
Embodiment 1: mutant choice and evaluation.
1, conidial preparation
The mycelia of each REMI transformant of Magnaporthe grisea bacterial strain P131 is fully interrupted, be uniformly applied on Tomato juice's medium oatmeal (every liter contain 150ml Tomato juice, 30-50 gram rolled oats boiled crossed leaching filtrate, 20 gram agar in 30 minutes) flat board, 26 ℃ of-28 ℃ of cultivations, when the visible newborn mycelia of naked eyes grows media surface, gently mycelia is washed with cotton swab, and water is rinsed well, cover the individual layer gauze, in 26 ℃ of-28 ℃ of illumination cultivation 48 hours, at the promptly visible a large amount of Magnaporthe grisea spore of media surface.
2. spore suspension preparation: 1 described spore is washed the back filter in the centrifuge tube of 50ml with double-deck lens wiping paper, 4000rpm room temperature collection in centrifugal 5 minutes spore is suspended in 0.25% the polysorbas20 then.
3. appressorium morphologic variation mutant choice and pathogenic mensuration:
Respectively with the spore suspension of wild type strain and mutant equably spray inoculation in the face of blade of five leaves, one heart stage rice seedling, dark, preserve moisture and cultivate continuous illumination after 36 hours, in the susceptible scab number of back 72 hours statistical unit length blades of inoculation.Using the same method prepares conidial suspension, adjusts conidium concentration to every milliliter 5 * 10
5Spore, spray inoculation is in the front of fresh onion entocuticle equably, be total to the 5-10ml spore suspension of spraying approximately, observe conidial sprouting in microscopically, appressorium, infect nail and the processes such as formation of infectivity mycelia, and calculate conidial germination rate, appressorium, infect the rate of formation of nail and infectivity mycelia.
In magnification is that 10 * 20 microscopically is observed, the morphological differences of Magnaporthe grisea wild type strain and the appressorium of REMI transformant relatively, obtained an appressorium on onion epidermis, infect follow closely and the ratio of infectivity mycelia than the mutant X3073 of the obvious reduction of wild-type.See accompanying drawing 1, wherein wild-type P131 and mutant X3073 form on onion epidermis shown in the ratiometer 1 that infects nail and infectivity mycelia.
Nail is infected in table 1.P131 and X3073 formation in 24,36 and 48 hours behind the inoculation onion epidermis and extendability infects the ratio of mycelia
| The inoculation back time (hour) | Infect nail rate of formation (/ 100 appressorium) | Infectivity mycelia rate of formation (/ 100 appressorium) | ||
| P131 | X3073 | P131 | X3073 | |
| 24 36 48 | 53.7±5.0 80±3.1 94.7±0.3 | 11.8±2.2 14.9±0.3 15.9±0.5 | 21.6±3.5 57.8±2.1 82±3.2 | 4.0±1.1 6.2±0.5 6.4±0.6 |
Simultaneously, on the affine kind Lijiang xintuanheigu of paddy rice, the virulence of mutant is also lower than the wild-type, and wild strain P131 and mutant 3073 form the scab situation as shown in Figure 3 on affinity paddy rice Lijiang xintuanheigu blade.
Embodiment 2 mutation type surfaces change and insert the mark coseparation analysis
Method being divided into genetic cross to insertion mark hygromycin gene and mutant phenotype in the mutant from analyzing, mutant and one do not had mutant phenotype and hygromycin resistance, and the Magnaporthe grisea bacterial strain S1528 that mating type is opposite is hybridized, analyze its thecaspore offspring's appressorium form and hygromycin resistance situation, concrete grammar is as follows:
With said mutation body X3073 with do not have the cultivation that on the tomato medium oatmeal, stands facing each other of hygromycin resistance, the normal Magnaporthe grisea bacterial strain of appressorium form S1528.At first be cultured to colony edge and be about to receive together, then it is moved to 20 ℃ of illumination cultivation, about about 20 days perithecium in the intersection formation black projection of bacterium colony in 25 ℃.The sophisticated perithecium of picking squeezes gently in sterilized water and breaks, and discharges the ascus in the shell, and ascus suspension is coated on the water agar plate, and the single mycelia of picking ascus endoascus spore germination is cultivated on the tomato medium oatmeal after 24 hours.Add up these thecaspores offspring's hygromycin resistance and appressorium form.The result is as shown in table 2, shows that the normal offspring of appressorium form does not have hygromycin resistance in the thecaspore offspring who measures, and illustrates that the phenotype of the appressorium shape sudden change of this mutant is isolating altogether with inserting the mark hygromycin gene.In the filial generation, the offspring of moisture resistance mycin is mutant phenotype entirely, illustrates that inserting the mark hygromycin gene among the mutant X3073 only is inserted in a genomic site.
Table shape and the hygromycin resistance of the filial generation of table 2 mutant and S1528
| Offspring's sum | The moisture resistance mycin | Moisture resistance mycin not | |||||
| Wild-type | Mutant | Add up to | Wild-type | Mutant | Add up to | ||
| X3073×S1528 | 54 | 0 | 33 | 33 | 21 | 0 | 21 |
The clone of the gene M gPTH12 of embodiment 3 control Magnaporthe grisea appressorium maturations and morphologic variation.
1. plasmid rescue
By to inserting the rescue of plasmid in the mutant, obtained the genome sequence of corresponding insertion site side, and determined the on position of plasmid in the mutant.Concrete grammar is as follows:
Choose and insert plasmid pUCATPH (Lu, S., Lyngholm, L., Yang, G., Bronson, C., Yoder, O.C.1994.Taggedmutants at the Toxl locus of Cochliobolus heterostrophus by restriction enzyme-mediated integration.Proc.Natl.Acad.Sci.USA91:1264-12653) in pUC18 partly do not have the restriction enzyme XhoI of restriction enzyme site to clear up the genome of mutant X3073 fully.Carry out from connecting the competent cell JM109 of transformed into escherichia coli with the T4DNA ligase enzyme the refining back of ethanol sedimentation.Extract the plasmid of transformant, and carry out enzyme and cut evaluation.Enzyme is cut in the correct plasmid of evaluation and the sequence of pUC18 part, also contain the genomic sequence that part is inserted the site side in being contained pUCATPH.In database, retrieve then identifying that correct plasmid checks order, analyze near the scope of the gene in insertion site and possible exon.The result shows that in mutant X3073, plasmid is inserted in corresponding to the 174th and 1678 among the SEQ ID NO.1, and middle portion may lack.The genome database comparison of the Magnaporthe grisea that the official website (http://www.riceblast.org/) of the side sequence that obtains and international rice blast fungus genome association has been announced, these two are inserted sites and lay respectively at chromosomal contig2.1168 I number, on 2.1169.
2, genome TAC library screening
Dna segment (SEQ ID NO.4) with one section 621bp between these two sites is a probe, the genome TAC library of screening Magnaporthe grisea P131, obtain 6 positive colonies, they all comprise the genomic gene (nucleotide sequence of the 1st to the 5526th shown in the SEQ ID NO.1) of MgPTH12.
Restriction enzyme that relates in the plasmid rescue process and T4DNA ligase enzyme are produced by precious biotechnology (Dalian) company limited, consult and use specification sheets and use.
Embodiment 4:MgPTH12 biological function is confirmed
Comprise the experiment of complementation test and gene knockout.
1, the structure of complementary carrier and knockout carrier
A. the structure of complementary carrier
At first the primer of the restricted property of anamorphic zone restriction endonuclease XbaI recognition sequence amplifies the restriction enzyme that neomycin phosphotransferase gene is connected into pBlueScriptKS (+) and cuts between the XbaI of site the plasmid vector called after KN that obtains from cloned plasmids carrier pS65T-C1 (gi:1019893).Full length sequence with this gene of the prediction of pcr amplification gained is connected among the KN then.Restriction enzyme that relates in the building process and ligase enzyme, the end-filling enzyme is produced by precious biotechnology (Dalian) company limited, consults and uses specification sheets and uses.
B. the structure of knockout carrier (gene replacement vector).
Structure with reference to the exon-intron of the genomic gene of predicting among the FGENESH, two flank sequence orientations of this gene of the prediction of pcr amplification gained are connected in the carrier, this carrier has been connected into a neomycin resistance gene in advance, is connected into hygromycin gene between two flank sequences.
2, the conversion of Magnaporthe grisea.
Protoplast transformation is adopted in the conversion of Magnaporthe grisea.Adopt and do not destroy the intragentic restriction enzyme of carrier, transform the protoplastis of Magnaporthe grisea complementary carrier linearizing.The preparation and the method for transformation of protoplastis are as follows:
A. the preparation of protoplastis
500 milliliters of triangular flasks, 150 milliliters of liquid CM substratum (yeast extract 0.1%, enzymic hydrolysis casein food grade 0.05%, the glucose 1% of packing into, nitrocalcite 0.1%, potassium primary phosphate 0.02%, sal epsom 0.025%, sodium-chlor 0.015%), insert 1 * 10 of Magnaporthe grisea P131, X3073 respectively
6Individual conidium, at 26-28 ℃, shake training 30-32 hour under 100 rev/mins of conditions, three layers of sterilization lens wiping paper filter collects mycelium, mycelium is transferred in 50 milliliters of centrifuge tubes of sterilization after washing with the 0.7M sodium chloride solution, the enzyme penetrating fluid that per 1 gram mycelia adding is 1 milliliter (contains 20 mg/ml driselases, with the preparation of 0.7M sodium-chlor), 26-28 ℃, under 100 rev/mins of conditions after enzymolysis 3-4 hour, with 0.7M sodium-chlor washing mycelium, filter through three layers of sterilization lens wiping paper, collect protoplastis, 4,000 rev/min centrifugal 15 minutes, earlier with 25 milliliters of STC (1.2M sorbyl alcohol, 10mM Tris-Cl, pH7.5,50mM calcium chloride) the washing protoplastis is once washed 2 times with 10 milliliters of STC respectively then, with STC protoplastis concentration is transferred to 0.5-1 * 10 after the centrifugation
8Individual/milliliter.
B. Magnaporthe grisea transforms
Respectively with Magnaporthe grisea P131, the X3073 protoplastis is sub-packed in 50 milliliters of centrifuge tubes of sterilization, every pipe 150 microlitres, add isopyknic linearizing carrier (about 2 micrograms respectively, add with restriction enzyme KpnI linearizing gene replacement vector pKO8 in the centrifuge tube of Magnaporthe grisea P131, add linearizing complementary carrier pXC5.5 in the centrifuge tube of Magnaporthe grisea X3073 respectively with restriction enzyme EcoRI) and the STC mixed solution, placed on ice 20 minutes, dropwise slowly add 2 milliliters/pipe PTC solution (60% poly-hexylene glycol 3350 then, 10mM Tris-pH7.5,50mM calcium chloride), static on ice 20 minutes, add the ice-cold STC of 25 milliliters/pipe, slow mixing, 4,000 rev/min, 4 ℃ centrifugal 15 minutes, remove supernatant, every then pipe adds 3 milliliters LR substratum (0.1% yeast extract, 0.1% enzymic hydrolysis casein food grade, 1M sucrose), room temperature leaves standstill cultivated after 12-13 hour, change culture dish over to, add 12 milliliters of SR (LR+1.6% agar) that are cooled to 50 ℃, mixing, treat its solidify dry up after, 0.7% top-layer agar of spreading 12 milliliters of one decks above (is cooled to 50 ℃, contains the G418 (complementation test) of 400 mcg/ml or the Totomycin of 300 mcg/ml (U.S. Roche company produces, the gene substitution test).Cultivated 4-6 days for 28 ℃, (the complementary transformant of Magnaporthe grisea mutant X3073 is respectively CX8 with the transformant that occurs; The gene substitution transformant K75 of Magnaporthe grisea P131) goes to solid CM substratum (0.6% yeast extract, 0.3% enzymic hydrolysis casein food grade, 0.3% acid hydrolysis casein food grade, 1% sucrose, 1.6% agar) (contain on the G418 (complementation test) of 400 mcg/ml or the Totomycin of 250 mcg/ml (gene substitution test), after the postsearch screening single bacterium colony changed on the rolled oats tomato substratum and cultivate, and carry out monospore and separate.
The result of complementation test shows that the ectopic integration of complementary carrier in Magnaporthe grisea mutant X3073 genome makes the appressorium of mutant recover wild form.The appressorium of the complementary transformant CX8 of mutant and the wild-type bacteria P131 rate of formation that infects nail and infectivity mycelia on onion epidermis is more as shown in table 3
Table 3.P131, CX8 forms the ratio that infects nail and infectivity mycelia on onion epidermis
| The inoculation back time (hour) | Infect nail rate of formation (/ 100 appressoriums) | Infectivity mycelia rate of formation (/ 100 appressoriums) | ||
| P131 | CX8 | P131 | CX8 | |
| 24 36 48 | 53.7±5.0 80.0±3.1 94.7±0.3 | 51.3±4.4 82.4±3.8 87.4±1.8 | 21.6±3.5 57.8±2.1 82.0±3.2 | 20.1±3.1 58.7±3.1 76.9±8.3 |
On affinity rice varieties Lijiang xintuanheigu blade, form the scab situation as shown in Figure 7.
The result of genetic knock-out experiment shows, the portion gene group sequence and the hygromycin gene PERMUTATION OF SEQUENCES of MgPTH12 gene cause the shape of the appressorium that transformant K75 produced have with the present invention in the same variation of mutant X3073.The coding region gene sequence of MgPTH12 and hygromycin gene PERMUTATION OF SEQUENCES also cause the virulence of transformant to weaken, be included in the rate of formation (seeing Table 4) that infects nail and infectivity mycelia on the onion epidermis, and the quantity (as shown in Figure 11) that on affinity rice varieties Lijiang xintuanheigu blade, forms scab.
Table 4. gene knockout transformant K75 and wild-type bacteria P131 formed the ratio that infects nail and infectivity mycelia in back 24 hours, 36 hours and 48 hours in inoculation
| The inoculation back time (hour) | Infect nail rate of formation (/ 100 appressorium) | Infectivity mycelia rate of formation (/ 100 appressorium) | ||
| P131 | K75 | P131 | K75 | |
| 24 36 48 | 53.7±5.0 80.0±3.1 94.7±0.3 | 11.6±1.2 13.9±1.1 15.9±1.3 | 21.6±3.5 57.8±2.1 82.0±3.2 | 4.3±0.6 5.6±0.5 6.6±1.0 |
The cDNA clone of embodiment 5:MgPTH12
1, the prediction of MgPTH12 cDNA
Predict to Softberry (http://www.softberry.com/) that with the complementation test gene nucleotide series prediction of result is that a cDNA length is 1344 Nucleotide, 447 proteinic ORF encode.
2, the RT-PCR of prediction MgPTH12 cDNA.Extract total RNA by guanidinium isothiocyanate (GT) method, at first the total length ORF design two ends primer according to prediction carries out RT-PCR.Then, carry out two ends RACE with 3 '-Full RACE Core Set and 5 '-Full RACE Core Set, and carry out sequencing respectively.At last, again according to two ends RACE result, design primer full-length cDNA, and check order, the full length sequence that is obtained is seen SEQ ID NO:2.By software analysis, possible ORF length is 1410 Nucleotide, 470 the amino acid whose protein of encoding.
RT-PCR reagent, RACE test kit etc. are produced by precious biotechnology (Dalian) company limited, consult and use specification sheets and use.
Embodiment 6: the bioinformatic analysis of control appressorium maturation and virulence albumen PTH12 in the plant pathogenic fungi genome.
At first, retrieve, analyze at NCBI (http://www.ncbi.nlm.nih.gov/), the albumen that may exist in the important existing genomic data of pathogenic fungi such as gibberella saubinetii (Gibberella zeae), corn stalk rot disease bacterium (beading gibberella, Gibberella moniliformis), Sclerotinia sclerotiorum (Sclerotinia sclerotiorum) is analyzed, is predicted.Found that: in the Magnaporthe grisea MGPTH12 albumen in these several pathogenic bacterias, all have a correspondence, consensus amino acid sequence is higher than 40% homologous protein.The albumen of this genes encoding is made up of 470 amino acid in the Magnaporthe grisea, and the homologous protein in other fungal pathogen is then distinguished called after GzPTH12, GmPTH12, SsPTH12.The proteic sibship of PTH12 as shown in Figure 12 in the several plant pathogenic fungi.
Sequence table
＜110〉China Agricultural University
＜120〉come from the control fungi appressorium maturation of Magnaporthe grisea and gene M gPTH12 of virulence and uses thereof
<160>7
<210>1
<211>5526
<212>DNA
＜213〉Magnaporthe grisea (Magnaporthe grisea)
<400>1
TATTATACTG TATACCTTTA CCGCATTTTG CAGATCAGTG TCTGCCTCTG GCCCTTGTCC 60
ATTTTCGTCT TCCGAACTCC TTTACCAGGA AGCACGCTGT TCGGCACCAA GGAGTGAGTT 120
CCTGCTGTAC AGTACCGTAG GTAGAGTGGT AAGGTAGTTG ATCAGATCCT GGAGCTCCAG 180
GTCCCGAATG AATTGGATCT CTTGGGGAAA GCTTTCGTAC GGTTTGGTTG AGAAGGGGGG 240
GGGGCAGCTC TAGAGCAAGC AATTAGCAGT CATTCAGAGA CAAGCAAGCC ACACACGCCA 300
CAGCCCGGCT AGCAGTAGCG TATTTGCTTT GTCCCTTTTT TTCTTGGGCT TTTTTTTTTT 360
TTTTTTTTTT TTTTTTTTTG ACGGCCGTTG TCAACGGCTG CATTTCCAGC GTGGTTGAGT 420
TGGCAGGATA CTGTACTCAT GTCCAACAAC CTTAGAGCTT CCACGGACTC GTAGCACGCA 480
CGCACTCACA CCTGCACGCA CACTCCTCTT TCGTCTCGCT CTCTTACTAG CAAACCAGAC 540
TGTCCCACTG GCCCCCTACC GATACCAACC ATTCAATCGA CAAGTACGGT ACGATGTACT 600
GCGTACAATG TGCAACCAAC CTTGTTGATT CTCATATATC CCTGCATGCT TGTGGGTCTC 660
GAGTCGACGG GGACTTGCCC ACATCCATAA CCAACTGCAT CCCTGACCGA TGCAGGAATC 720
GACACAGTAC CAGATTGCAT CTGCTATGTA TGTCATCCTT ACCAAACCCT CGACCCATGT 780
TCATATTTCC TGTCCCCTAC CCTTGAATTT TGAATAATAA TAATAATGAT AATATTAAAT 840
AAATTCTCAT TTATTATTTC GTGCTCGCGT CCTCAGGCTA CTTTCGGATT AGTTTCTAGC 900
TTTACCCCCG GTAGACCGTG CTGCAGATTG TATAAACTGC TTTTTTACCT ACCTGCTCCT 960
TCCGCAATTC TCCCGCGCCT GGGGTACCTG GACTTGTGTT TTTCTTTGGT CAGTCATTCC 1020
GCAACGGCCT CTCCAAGAAA AAAACGAAGA AGAAAAAAGC AAACCAAGTC CTTCCGCTAG 1080
AGCTACGGCG GTAGCAGTTT CACCTAGCAC ATCGTCGTGC TGAAGAACTG TCCTAATTCT 1140
TTTTGACTCG GGCCTGTTTA CACATGCTCC CCCAAGTCTA CTAGAACTTC CCTTTTTGGC 1200
CACAACATCT GTGCACCTTG CCCGTTGGAT TCCCACACCA CGAACTAGGT CATCCATCCT 1260
ACCCCATCCG CATACAGAGA GGGTCCCGGC ACTTGCGTTT CTACAAAAGA ACACCATCAC 1320
CCTCACCCCA TTCGACGTTG CAGCACTCGT CTTATTCCTA ACCCATCTCT TGATCAACAC 1380
CAAAGTGCCA GGTTGCTGTC AACTTTTGGT CCTCCCTTTC ACTTCTTGTT TTTCTTTTAT 1440
TTGCGTGAAA TACGGATAGA CTTCACTCTC TCGTGACCGA CCTCGTCCAC CATTTGGCAC 1500
TCGCAACATC TTAAATCTTT AGCGTCCTCG ACAAACTGCA GGAAGACATT CGCTTCTTGC 1560
TATAACGCAA CACCAGCAGT GGTCAGGAGG TACTCAGCAT CGACTCGACC GCCATTACGC 1620
ATTCCCGCCG CCTCGGTTCA TTGACCGACA CTAGGGCTGA ACCATTCGAC GCTGTTCATA 1680
ATCCTGTCAG CTGGAGGCCA ACGCCAGCTA CATTTTATCA TTCGCCGCCG GACGCGTTAC 1740
TAGTTGGGAC ACAAACCCCC ACACAACCTT TCGTTAACAA CAAGCTGCCT TCTTTTTAAA 1800
GTATGTCGCT GCTGCCACAC CATCGAAGCC AGCAGAGCTA AGGGCTTCTT GACCGCTCTG 1860
CGTCTAAGTC ACATCTTGTA CTGACCCCAG ATCCAGCTAT TTGTAACTGA GCTGGCGCTG 1920
CTACTCCGTC AACTTCCACT CGAATAGGGC ATGGAATATA CCTTGCCAAA CCACCGTTAC 1980
TCTCACAGCA ACATCGGTCA ACGAAGCAAT TCACCCACAA TGTCCACACT CGCGATGGCT 2040
GCACCATCCC CGCATGCTCC TTTCAAGAAG GAGTACTTCT CCAACTGGGA CGACCGCCGG 2100
CAAGCGGATT ATGCTCCTTC CAGGCCAAGG AGTGATCCCG AGAGGATAGC GCTACCCTCG 2160
ATTCGCCAGG TATGTTTCTG ATGCTACACC TTTGCAGGGC CGTTGTATGT CTAATACCCT 2220
GGGTGTACAG GCTTTCCCCG ATCTCCTTCG CCTCCGTGTT ATCCCCCAAG ATGGCCAAAC 2280
CAGCACACCA TCCTCCACCA CATCACCGAT ATCAGGACCA CCTGGCACCC TGACACCACC 2340
CGAATATGTT CACTCCCCGA CCAGCCAGAA CAAGCGCAGG AGGTTATCGT TCGGTGACGA 2400
CCAGGAGGAA AGGGTCAGCC AGGTTCCAAG GCTCTACACC AGCCAGTCCC AGGCATACCA 2460
ACGACGTGAC AACCGTCCTC TTTCTCCAGT CACCCTAAGT GGCGGGCCCC GTTCTAGCAC 2520
CTCCGATAGC TGGGCTGGAT CCTCCCGAAC GAGCCCGTTC CTTCCTGGTA CCAGCACTTC 2580
GACCTTGAGA TCACCGGCTG CTATTGAGCA AAACGACCCA ATGGACAGGC AGCGCCCGAC 2640
GCTACCAAGC CTTCCTCATC TGAACTTCGA CAGGTCACCA ATGGAATCCC AGTCTCACAT 2700
GCACCACCAC CATCAACAGC ACCCTCACCA GCAGCAGCAG CAGCAGCACA GCCACCACGT 2760
GAGGGCAATG TCTGGGGACG AGTACATGAT GGAGCAGCAC CGCGGAATGG TCCAGCAGCA 2820
CCCACACTCT GCCGTAGAGC CTGGATACCG TCAGCCCGCA CCAAGCGGCT ACCCATACCC 2880
GTATCATCAC CCCTCGCGGA GCCAGTCCCT TTCAATCGGT GCCATGCCCC CGATGGATCG 2940
CATGCCATTT TCGCCTTCTG GCTACAGCGC CTACCACGGC GACTACATGA GGATGGGTGA 3000
CATGGGTGGC ATGGGCTTCA ATGGAGACAA CAAGCAACGC AAGCGCAGAG GCAACCTGCC 3060
TAAGGAGACA ACCGACAAGC TTCGCGCTTG GTTCTTGGCA CACCTCTCAC ACCCGTACCC 3120
AACTGAGGAC GAGAAGCAGG AGCTGATGAG GCAGACCGGC CTTCAGATGA GTAAGTTTAG 3180
ATCTCTTGGC AACAATCACC ACAATGAATA TCATGTCCTA ACACTTTTGG TTTCTCTAGA 3240
CCAAATCTCC AACTGGTTCA TCAACGCTCG AAGACGCCAG CTCCCAGCCA TGATCAACAA 3300
CGCCCGCGCC GAGTCAGATG CTCTTGCCCA AGGGCGCGGC CTCTCGGACG GCTCCAAGAT 3360
TCTCCTCTCA ACTGAGCGGT CAGACTACGA CAGCGATAAG AGGGGTAGCC CTATCAGCGA 3420
CGATGGCGCC AGCCACCTTT ATCACGATGA CATGCACAGA CGTGCCGTTG GCATGAAGCG 3480
TGGCAGCGTC TAAACACGCT ACTATCACAC TCTTTTTTTT TCTTTTTTTT TACACATCCC 3540
TTTTTTATAG CTCCTTCCAC TGTACGAACA TATCTTGTTG GGAATACTGC TGTTTTTTAT 3600
TTAGTATGTT TATGTCTGCC GATTCACAAG TACGGATGGA TTTCTTCTTT TCTCGACATG 3660
TATTATTTCG TATATACCAC TTGCAATGGG GGGATGGGCT CTGACATTTG ACACGACATT 3720
TTCCTTTACT CATATTTGCT TTTTGATTAC CCCCCAACTG TACAAATTTT TAGACACCTG 3780
GCACATGGAG TTTTTTTTTA TTACTACCTG GAAGAGAGAA AGACAGCGGC ACGGAAGAAA 3840
AAAAGGAGTT TTTCTTATCG GAACAGGCCA ACAGGGCACG AAGCATTCAG CGGGCATGAA 3900
GGAACTGGAA CTGGTCTAGA AGGAGGAAAA CACCTTAGGT AGTCTCACCC GCCCACATCC 3960
CTTGCAATCG AATAGCGATG CTCGGTCAGA CGAGCGGGGG AGAGGACACA ATGAGAAAAA 4020
GGAACATTTC CAAAATAAGT TAATTCCGAG ATGATGTGCA TTGGACCCTC CAGTGGCCCC 4080
CACAAAGCAC CTCGTGCAAA AAGAAATGGG AAAACGCAGC CAGGACTACA CTAACAACGC 4140
AGCCTGCATA CAGACACAGG AATCTAAGTC CCAGGGCTTC GGGTCGATCA CGGGCCCGTC 4200
CGTGAGGCGC CATATTTTCT TCTTTTCTTC TATTTTTTCC CTGGTTGACT ATCTAGGTCA 4260
GATGTGGTAC TTGGATGAAG AGCGGTATGC AGCAAAACGG ATATGACCGA GGAGTTTGAA 4320
TCGGAGGCAC GACTAAGAAG AGGCTTCGTT AGCGTTGTTT TTGTTTCCTA GTTTATTGCT 4380
TGCCCTGTTC CTTTGTACTT GTCTTTGTTC TTTTGCTTTT AGTATGGGAG GGGGTGGCGG 4440
CATCAACTTG TCATTGCATT GACACTAGTC TCAACGGCGG TTTAACGGTT CCCCTCCAAA 4500
AACGTGATAG GGAATGATCA CAACGACGAA GTTCAAAGGC GGTGAATCGC CAAGAGGGTG 4560
TGTGCGTATT TTTTTTTGTT TGTGTATGTG TGTCTCTCTC TCTCTCTCTC TCTCTCTCTC 4620
TCTCTCTCTC TCTCTCACAC ACACACACTC TCTCTCTCTT TTTGCCGGTA GGACAAAATA 4680
GTCAATATGT TTCTATTCAA CATGAAGCCT GTCTACAGGA CCCTTGTTTC TTGGGAACGT 4740
GGGCCAACAA CTGAGACAAG AACATGGGGG CCATTGGCAT TGCTCTGCAA GTACGTGTGT 4800
AGTTCAAATC ACTTGTCAGT AGGGGACAAA AGAATGACGT GCGACTTGTT CATTTTGACT 4860
TTGAGCGAGA CAGGGAACGT GATGCAAGTG CCTGGCCTGA AGGCGTGGTC CGAGAGGCAA 4920
AAAGGACTCA AGGACAAGTC AAGATTTGAG AGGAAAAAGG GAAACAGACG ACGTCGCTGA 4980
CATGGCACAT GGTAAGCATG TTGGAAAGAG AAAAAAAAAC AGGGGACTAA GGTTCAGATG 5040
GATGCGCAGC TCTCGAGTCG ATACGAAAGA AATAAGTACC GAGCCCACTT TGTTGTTGAC 5100
GGCCGGCACA TACCAAAGAA AATGCTACCC ACTTACAATA CAGTCAAGTA GCCGTGTAAG 5160
TTGAGCCAAG GCAGACAGAT AGCGTAGGCC AAGGAACACC GAAGATCTGA CACACATAGG 5220
AACCGACATC CCCCCGACTT ACTCGACAAA ACAGTCCCCG GCGATTTTAC CCCGCGAGTA 5280
GTGATGAATG GCGAGCTTGG TTCGAATCAG TGAAGAATAG GACCGAAAAG TTGTCGTTTG 5340
CCCGCCGCCG GGCCCCCCAT CTGTCCCACC AGACGTGACC CTGCAAGTCG CTCTTTTTGA 5400
TCAGAAAGAA AGAGGCGTAT CCTCTCGTAA ATGACCGATA CGATGATCTA TTGCGGGTGT 5460
TGGTTGTGTC GCGCGGCGAC AGATTAAGAA AAAGAACGCC CACACCCCAC TTTTGTAATG 5520
GATCCC 5526
<210>2
<211>2164
<212>DNA
＜213〉Magnaporthe grisea (Magnaporthe grisea)
<220>
<221>CDS
<222>(333)..(1745)
<400>2
CGTCCTCGAC AAACTGCAGG AAGACATTCG CTTCTTGCTA TAACGCAACA CCAGCAGTGG 60
TCAGGAGGTA CTCAGCATCG ACTCGACCGC CATTACGCAT TCCCGCCGCC TCGGTTCATT 120
GACCGACACT AGGGCTGAAC CATTCGACGC TGTTCATAAT CCTGTCAGCT GGAGGCCAAC 180
GCCAGCTACA TTTTGCCATT CGCCGCCGGA CGCGTTACTA GTTGGGACAC AAACCCCCAC 240
ACAACCTTTC GTTAACAACA AGCTGCCTTC TTTTTAAACT ATTTGTAACT GAGCTGGCGC 300
TGCTACTCCG TCAACTTCCA CTCGAATAGG GCATGGAATA TACCTTGCCA AACCACCGTT 360
ACTCTCACAG CAACATCGGT CAACGAAGCA ATTCACCCAC AATGTCCACA CTCGCGATGG 420
CTGCACCATC CCCGCATGCT CCTTTCAAGA AGGAGTACTT CTCCAACTGG GACGACCGCC 480
GGCAAGCGGA TTATGCTCCT TCCAGGCCAA GGAGTGATCC CGAGAGGATA GCGCTACCCT 540
CGATTCGCCA GGCTTTCCCC GATCTCCTTC GCCTCCGTGT TATCCCCCAA GATGGCCAAA 600
CCAGCACACC ATCCTCCACC ACATCACCGA TATCAGGACC ACCTGGCACC CTGACACCAC 660
CCGAATATGT TCACTCCCCG ACCAGCCAGA ACAAGCGCAG GAGGTTATCG TTCGGTGACG 720
ACCAGGAGGA AAGGGTCAGC CAGGTTCCAA GGCTCTACAC CAGCCAGTCC CAGGCATACC 780
AACGACGTGA CAACCGTCCT CTTTCTCCAG TCACCCTAAG TGGCGGGCCC CGTTCTAGCA 840
CCTCCGATAG CTGGGCTGGA TCCTCCCGAA CGAGCCCGTT CCTTCCTGGT ACCAGCACTT 900
CGACCTTGAG ATCACCGGCT GCTATTGAGC AAAACGACCC AATGGACAGG CAGCGCCCGA 960
CGCTACCAAG CCTTCCTCAT CTGAACTTCG ACAGGTCACC AATGGAATCC CAGTCTCACA 1020
TGCACCACCA CCATCAACAG CACCCTCACC AGCAGCAGCA GCAGCAGCAC AGCCACCACG 1080
TGAGGGCAAT GTCTGGGGAC GAGTACATGA TGGAGCAGCA CCGCGGAATG GTCCAGCAGC 1140
ACCCACACTC TGCCGTAGAG CCTGGATACC GTCAGCCCGC ACCAAGCGGC TACCCATACC 1200
CGTATCATCA CCCCTTGCGG AGCCAGTCCC TTTCAATCGG TGCCATGCCC CCGATGGATC 1260
GCATGCCATT TTCGCCTTCT GGCTACAGTG CCTACCACGG CGACTACATG AGGATGGGTG 1320
ACATGGGTGG CATGGGCTTC AATGGAGACA ACAAGCAACG CAAGCGCAGA GGCAACCTGC 1380
CTAAGGAGAC AACCGACAAG CTTCGCGCTT GGTTCTTGGC ACACCTCTCA CACCCGTACC 1440
CAACTGAGGA CGAGAAGCAG GAGCTGATGA GGCAGACCGG TCTTCAGATG AACCAAATCT 1500
CCAACTGGTT CATCAACGCT CGAAGACGCC AGCTCCCAGC CATGATCAAC AACGCCCGCG 1560
CCGAGTCAGA TGCTCTTGCC CAAGGGCGCG GCCTCTCGGA CGGCTCCAAG ATTCTCCTCT 1620
CAACTGAGCG GTCAGACTAC GACAGCGATA AGAGGGGTAG CCCTATCAGC GACGATGGCG 1680
CCAGCCACCT TTATCACGAT GACATGCACA GACGTGCCGT TGGCATGAAG CGTGGCAGCG 1740
TCTAAACACG CTACTATCAC ACTCTTTTTT TTTTCTTTTT TTTTACACAT CCCTTTTTTA 1800
TAGCTCCTTC CACTGTACGA ACATATCTTG TTGGGAATAC TGCTGTTTTT TATTTAGTAT 1860
GTTTATGTCT GCCGATTCAC AAGTACGGAT GGATTTCTTC TTTTCTCGAC ATGTATTATT 1920
TCGTATATAC CACTTGCAAT GGGGGGATGG GCTCTGACAT TTGACACGAC ATTTTCCTTT 1980
ACTCATATTT GCTTTTTGAT TACCCCCCAA CTGTACAAAT TTTTAGACAC CTGGCACATG 2040
GAGTTTTTTT TTATTGCTAC CTGGAAGAGA GAAAGACAGC GGCACGGAAG AAAAAAAGGA 2100
GTTTTTCTTA TCGGAACAGG CCAACAGGGC ACGAAGCATT CAGCGGGCAT GAAGGAACTG 2160
GAGC 2164
<210>3
<211>470
<212>PRT
＜213〉Magnaporthe grisea (Magnaporthe grisea)
Met Glu Tyr Thr Leu Pro Asn His Arg Tyr Ser His Ser Asn Ile Gly
1 5 10 15
Gln Arg Ser Asn Ser Pro Thr Met Ser Thr Leu Ala Met Ala Ala Pro
20 25 30
Ser Pro His Ala Pro Phe Lys Lys Glu Tyr Phe Ser Asn Trp Asp AsP
35 40 45
Arg Arg Gln Ala Asp Tyr Ala Pro Ser Arg Pro Arg Ser Asp Pro Glu
50 55 60
Arg Ile Ala Leu Pro Ser Ile Arg Gln Ala Phe Pro AsP Leu Leu Arg
65 70 75 80
Leu Arg Val Ile Pro Gln Asp Gly Gln Thr Ser Thr Pro Ser Ser Thr
85 90 95
Thr Ser pro Ile Ser Gly Pro pro Gly Thr Leu Thr Pro pro Glu Tyr
100 105 110
Val His Ser Pro Thr Ser Gln Asn Lys Arg Arg Arg Leu Ser phe Gly
115 120 125
Asp Asp Gln Glu Glu Arg Val Ser Gln Val Pro Arg Leu Tyr Thr Ser
130 135 140
Gln Ser Gln Ala Tyr Gln Arg Arg Asp Asn Arg Pro Leu Ser Pro Val
145 150 155 160
Thr Leu Ser Gly Gly Pro Arg Ser Ser Thr Ser Asp Ser Trp Ala Gly
165 170 175
Ser Ser Arg Thr Ser Pro Phe Leu Pro Gly Thr Ser Thr Ser Thr Leu
180 185 190
Arg Ser Pro Ala Ala Ile Glu Gln Asn Asp Pro Met Asp Arg Gln Arg
195 200 205
Pro Thr Leu Pro Ser Leu Pro His Leu Asn Phe Asp Arg Ser Pro Met
210 215 220
Glu Ser Gln Ser His Met His His His His Gln Gln His Pro His Gln
225 230 235 240
Gln Gln Gln Gln Gln His Ser His His Val Arg Ala Met Ser Gly Asp
245 250 255
Glu Tyr Met Met Glu Gln His Arg Gly Met Val Gln Gln His Pro His
260 265 270
Ser Ala Val Glu Pro Gly Tyr Arg Gln Pro Ala Pro Ser Gly Tyr Pro
275 280 285
Tyr Pro Tyr His His Pro Leu Arg Ser Gln Ser Leu Ser Ile Gly Ala
290 295 300
Met Pro Pro Met Asp Arg Met Pro Phe Ser pro Ser Gly Tyr Ser Ala
305 310 315 320
Tyr His Gly Asp Tyr Met Arg Met Gly Asp Met Gly Gly Met Gly Phe
325 330 335
Asn Gly Asp Asn Lys Gln Arg Lys Arg Arg Gly Asn Leu Pro Lys Glu
340 345 350
Thr Thr Asp Lys Leu Arg Ala Trp Phe Leu Ala His Leu Ser His Pro
355 360 365
Tyr Pro Thr Glu Asp Glu Lys Gln Glu Leu Met Arg Gln Thr Gly Leu
370 375 380
Gln Met Asn Gln Ile Ser Asn Trp Phe Ile Asn Ala Arg Arg Arg Gln
385 390 395 400
Leu Pro Ala Met Ile Asn Asn Ala Arg Ala Glu Ser Asp Ala Leu Ala
405 410 415
Gln Gly Arg Gly Leu Ser Asp Gly Ser Lys Ile Leu Leu Ser Thr Glu
420 425 430
Arg Ser Asp Tyr Asp Ser Asp Lys Arg Gly Ser Pro Ile Ser Asp Asp
435 440 445
Gly Ala Ser His Leu Tyr His Asp Asp Met His Arg Arg Ala Val Gly
450 455 460
Met Lys Arg Gly Ser Val
<210>4
<211>621
<212>DNA
＜213〉Magnaporthe grisea (Magnaporthe grisea)
CTGCAGATTG TATAAACTGC TTTTTTACCT ACCTGCTCCT TCCGCAATTC TCCCGCGCCT 60
GGGGTACCTG GACTTGTGTT TTTCTTTGGT CAGTCATTCC GCAACGGCCT CTCCAAGAAA 120
AAAACGAAGA AGAAAAAAGC AAACCAAGTC CTTCCGCTAG AGCTACGGCG GTAGCAGTTT 180
CACCTAGCAC ATCGTCGTGC TGAAGAACTG TCCTAATTCT TTTTGACTCG GGCCTGTTTA 240
CACATGCTCC CCCAAGTCTA CTAGAACTTC CCTTTTTGGC CACAACATCT GTGCACCTTG 300
CCCGTTGGAT TCCCACACCA CGAACTAGGT CATCCATCCT ACCCCATCCG CATACAGAGA 360
GGGTCCCGGC ACTTGCGTTT CTACAAAAGA ACACCATCAC CCTCACCCCA TTCGACGTTG 420
CAGCACTCGT CTTATTCCTA ACCCATCTCT TGATCAACAC CAAAGTGCCA GGTTGCTGTC 480
AACTTTTGGT CCTCCCTTTC ACTTCTTGTT TTTCTTTTAT TTGCGTGAAA TACGGATAGA 540
CTTCACTCTC TCGTGACCGA CCTCGTCCAC CATTTGGCAC TCGCAACATC TTAAATCTTT 600
AGCGTCCTCG ACAAACTGCA G 621
<210>5
<211>453
<212>PRT
＜213〉corn stalk rot disease bacterium (beading gibberella, Gibberella moniliformis)
<400>5
Met Ser Met Leu Ala Thr Ala Ser Pro Ser Pro His Pro Ser Ser Phe
1 5 10 15
Gly Met Pro Arg Pro Trp Glu Thr Asn Arg Cys Pro Asp Tyr Ser Leu
20 25 30
Arg Pro Arg Thr Glu Asn Asp Lys Val Ala Leu Pro Ser Ile Arg Gln
35 40 45
Val His Asn Ile Asp Ser Thr Ser Lys Pro Leu Cys Leu Ala Asn Ile
50 55 60
Thr Gln Ala Phe Pro Glu Leu Gln Leu Gln Thr Gln Pro Pro His Asp
65 70 75 80
Leu Asn Thr Lys pro Pro Ser Thr Gly Pro Pro Leu Gly Ala Pro Pro
85 90 95
Leu Pro Ala Ala Ser Pro Gln Tyr Ile His Ser Pro Asn Ser Asn Lys
100 105 110
Arg Arg Arg Leu Ser Ile Glu Arg Glu Val Glu Met Glu Arg Val Arg
115 120 125
Gln Val Pro Arg Arg Tyr Ser pro Asp Arg Ala Ser Pro Arg Gln Ile
130 135 140
Ser Pro His Leu Pro Ile Gln Gly Gly Ser Gln Glu Asn Trp Ala Ala
145 150 155 160
Pro Thr Arg Thr Ser Pro phe Leu Thr Asn Gly Ser Asn Ser His Ser
165 170 175
Val Ser Ile Glu Val Ser Glu Arg Ala Glu Ser Arg Ser Thr Leu Pro
180 185 190
Ser Leu Pro pro Pro Arg Ser Leu Glu Arg Glu Leu Pro Val Gly Arg
195 200 205
Gly Thr Ala Pro Ala Asp Gly Tyr Arg Pro Pro Gln Gln Ser Met Pro
210 215 220
His Ser Arg Thr Pro Ile Ser Glu Pro Gly Val Ser Pro Tyr Arg Glu
225 230 235 240
Asn Gly Tyr Gly Tyr Pro Tyr His His Pro Thr Arg Tyr Gln Ser Leu
245 250 255
Ser Thr Gly Ser Ala His Ser Tyr Asp Arg Thr Pro Phe Thr Pro Gly
260 265 270
Thr Tyr Asn Thr Pro Tyr Gln Asp Phe Val Arg Phe Gly Asp Met Ser
275 280 285
Ser Ala Ser Leu Ser Gly Asp Asn Lys Gln Arg Lys Arg Arg Gly Asn
290 295 300
Leu Pro Lys Glu Thr Thr Asp Lys Leu Arg Ala Trp Phe Val Ala His
305 310 315 320
Leu Gln His Pro Tyr Pro Thr Glu Asp Glu Lys Gln Asp Leu Met Arg
325 330 335
Gln Thr Gly Leu Gln Met Ser Lys Phe Gly Arg Thr Lys Pro Arg Thr
340 345 350
Glu Lys Pro Ser Gln Asn Trp Lys Leu Thr Cys Pro Pro Thr Asp Gln
355 360 365
Ile Ser Asn Trp Phe Ile Asn Ala Arg Arg Arg Gln Leu Pro Ala Met
370 375 380
Ile Asn Asn Ala Arg Ala Glu Thr Asp Ala Met Thr Gly Ala Arg Gly
385 390 395 400
Gly Asp Leu Lys Val Leu Ala Thr Thr Glu Arg Gly Asp Phe Asp His
405 410 415
Ser Lys Arg Glu Pro Ala Gly Pro Leu Ser Asp Gly Glu Gly Ala Thr
420 425 430
Tyr Asp Glu Glu Leu Glu Ala Leu Ser Gln Arg Arg Pro Gly Thr Ile
435 440 445
Gly Arg Gly Ser Val
450
<210>6
<211>463
<212>PRT
＜213〉gibberella saubinetii (Gibberella zeae)
<400>6
Met Glu Asp Ser Arg Ser Leu Ser Lys Gln Ser Asp Thr Lys Leu Gln
1 5 10 15
Gln Pro Ala Ser Gln Leu Pro Asn Met Ser Met Ile Ala Thr Ala Ser
20 25 30
Pro Ser Pro His Pro Ser Ser Phe Gly Met Pro Arg Pro Trp Glu Thr
35 40 45
Asn Arg Cys Pro Asp Tyr Ser Leu Arg Pro Arg Thr Glu Asn Asp Lys
50 55 60
Val Ala Leu Pro Ser Ile Arg Gln Ala Phe Pro Glu Leu Gln Leu Gln
65 70 75 80
Thr Gln Pro Pro His Asp Leu Asn Thr Lys Pro Leu Val Thr Gly Pro
85 90 95
Pro Leu Gly Thr Ala Pro Leu Ala Ala Ala Ser Pro Gln Tyr Val His
100 105 110
Ser Pro Asn Ser Ser Lys Arg Arg Arg Leu Ser Met Glu Arg Glu Met
115 120 125
Glu Ser Glu Arg Val Arg Gln Val Pro Arg Leu Cys Tyr Ser Pro Asp
130 135 140
Arg Glu Ser Pro Arg Gln Ile Ser Pro Asn Leu Ala Ile Gln Ala Gly
145 150 155 160
Thr Arg Glu Asn Trp Thr Ala Pro Thr Arg Thr Ser Pro Tyr Leu Thr
165 170 175
Asn Gly Ala Thr His His Ser Val Pro Met Glu Val Gly Glu Arg Leu
180 185 190
Glu Ala Arg Pro Ala Leu Pro Ser Leu Pro Pro Pro Arg Ser Leu Glu
195 200 205
Arg Glu Ala Val Leu Val Ser Arg Gly Pro Ala Pro Ala Pro Val Pro
210 215 220
Thr Pro Val Pro Thr Pro Ala Pro Ala Pro Ala Ser Ile Pro Ala Ser
225 230 235 240
Val Ser Ala Ser Ala Pro Ala Pro Ala Pro Thr Glu Gly Tyr Arg Ser
245 250 255
Ser His Gln Pro Met Ser His Ser Arg Thr Pro Ile Pro Glu Ser Gly
260 265 270
Val Ser Pro Tyr Arg Glu Ser Ser Tyr Gly Tyr Pro Tyr His His Pro
275 280 285
Thr Arg Tyr Gln Ser Leu Ser Ala Gly Ser Ala His Ser Phe Asp Arg
290 295 300
Thr Pro Phe Thr Pro Gly Ala Tyr Asn Ala Pro Tyr Gln Asp Phe Val
305 310 315 320
Arg Phe Gly Glu Met Gly Pro Ala Ser Leu Ser Gly Asp Asn Lys Gln
325 330 335
Arg Lys Arg Arg Gly Asn Leu Pro Lys Glu Thr Thr Asp Lys Leu Arg
340 345 350
Ala Trp Phe Val Ala His Leu Gln His Pro Tyr Pro Thr Glu Asp Glu
355 360 365
Lys Gln Asp Leu Met Arg Gln Thr Gly Leu Gln Met Asn Gln Ile Ser
370 375 380
Asr Trp Phe Ile Asn Ala Arg Arg Arg Gln Leu Pro Thr Met Ile Asn
385 390 395 400
Asn Ala Arg Ala Glu Thr Asp Ala Met Ser Ser Ala Arg Gly Gly Asp
405 410 415
Met Lys Val Leu Ala Thr Thr Glu Arg Gly Asp Phe Asp His Gly Lys
420 425 430
Arg Glu Pro Val Gly Pro Leu Ser Asp Gly Glu Gly Ala Thr Tyr Glu
435 440 445
Glu Glu Leu Glu Ala Leu Ser Gln Arg Arg Pro Gly Thr Met Gly Arg
450 455 460
Gly Ser Val
<210>7
<211>329
<212>PRT
＜213〉Sclerotinia sclerotiorum (sclerotinia sclerotiorum)
<400>7
Met Thr Glu Arg Thr Tyr Asn Thr Ser Pro Glu Ser Asn Lys Arg Arg
1 5 10 15
Ile Ser Ser Asn Ser Ser Tyr Asp Asp Glu Ile Gln Gln Gln Arg Gly
20 25 30
Arg Tyr Gln Ser Val Ser Ser Ser Arg Ser Pro Met Thr Tyr His His
35 40 45
Asp Ser Gly Ala Ala Ser Pro Val Gly Pro Ile Ala Arg Arg Glu Ser
50 55 60
Leu Arg Ser Ile Pro Glu Arg Arg Ala Pro Phe Ser Asp His Thr Ser
65 70 75 80
Arg Pro Pro Phe His Ser Pro Ile Ser Glu Pro Ser Ser Thr Ile Tyr
85 90 95
Arg Gln Asn Leu Pro Gly Val Ala Thr Lys Asp Arg Ser Tyr Asp Arg
100 105 110
Pro Tyr His Gln Ile Pro Ala Gly His Ser His Asp Tyr Pro Arg Ser
115 120 125
Glu Tyr Ser Leu Glu Ala Cys Arg Pro Tyr Gln Gln His Asn Tyr Ala
130 135 140
Pro Ala His Asp Thr Ser Tyr Ala Pro Ser Gln Ser Asp Tyr Gly Tyr
145 150 155 160
Gln Gln Pro Arg Asn Gln Ala Tyr Pro Gly His Pro Pro Tyr Gln Leu
165 170 175
Asn Gln Gly Gln Thr Pro Phe Thr Glu Gly His His Ser Gly Asn Tyr
180 185 190
Ser Ser Ala Ala Tyr Gln Ala Gln Asp Met Asp Ser Lys Pro Arg Lys
195 200 205
Arg Arg Gly Asn Leu Pro Lys Pro Thr Thr Asp Ile Leu Thr Thr Trp
210 215 220
Phe Ile Asn His Leu Glu His Pro Tyr Pro Asn Glu Glu Glu Lys Gln
225 230 235 240
Leu Leu Met Val Gln Thr Gly Leu His Leu Asn Gln Ile Ser Asn Trp
245 250 255
Phe Ile Asn Ala Arg Arg Arg Lys Leu Pro Ala Leu Gln Asn Asn Ala
260 265 270
Arg Ala Glu Asn Ala Ala Arg Ser His His Asn Arg Met His Ser Ala
275 280 285
Asp Asp Glu Gln Ser Pro Met Ser Leu Cys Asn Ser Asp Ala Gly Leu
290 295 300
Ser Pro Arg Asp Ala Ser Thr Phe Asn Glu Gly Trp Ser Ser Arg Gln
305 310 315 320
Glu Glu Arg His Asn Gln His His Tyr
325
Claims (11)
1, a kind of albumen of controlling appressorium maturation and virulence in the fungi, called after MGPTH12 in Magnaporthe grisea, the protein that it is characterized in that having one of following amino acid residue sequences:
1) the SEQ ID № in the sequence table: 3;
2) with SEQ ID № in the sequence table: 3 amino acid residue sequence is through the replacement of one or several amino-acid residue and/or the protein of disappearance and/or interpolation and appressorium formation of control fungi and virulence.
3) with sequence table in SEQ ID №: 3 have consensus amino acid sequence be higher than 40% as the protein of corn stalk rot disease bacterium (Gibberella moniliformis), gibberella saubinetii (Gibberella zeae), Sclerotinia sclerotiorum pathogenic fungies such as (Sclerotiniasclerotiorum), SEQ ID № in its aminoacid sequence such as the sequence table: 5,6,7.
2, the proteic encoding gene of described fungi control appressorium maturation of claim 1 and virulence.This gene is characterised in that to have one of following nucleotide sequence:
1) SEQ ID № in the sequence table: 1 dna sequence dna;
2) SEQ ID № in the code sequence tabulation: the polynucleotide of 3 protein sequences;
3) with sequence table in SEQ ID №: 1 dna sequence dna that limits has 80% above consistence, and the identical function protein DNA sequence of encoding.
3, the cDNA of the proteic gene of the described control of claim 2 fungi control appressorium maturation and virulence is characterized in that having one of following nucleotide sequence:
1) SEQ ID № in the sequence table: 2 dna sequence dna;
2) SEQ ID № in the code sequence tabulation: the polynucleotide of 3 protein sequences;
3) with sequence table in SEQ ID №: 2 dna sequence dnas that limit have 80% above homology, and the identical function protein DNA sequence of encoding.
4, the cDNA of the proteic gene of the described control of claim 3 fungi control appressorium maturation and virulence, its feature is that also the sequence of coding region is SEQ ID №: the 333rd at 5 ' end of 2 is to 1745 bit bases.
5, utilize the proteic gene of claim 2,3,4 described control fungi control appressorium maturations and virulence and expression vector, clone and the host bacterium that cDNA makes up thereof.
6, the promotor of the described control of claim 2 fungi appressorium maturation and the proteic encoding gene MgPTH12 of virulence has the № from sequence SEQ ID: the 1st nucleotide sequence to 1940 bit bases of 5 ' end of 1.
7, the described fungi of claim 1 control appressorium ripe with the proteic MGPTH12 of virulence and the consistent expression that is higher than 40% homologous protein thereof as target in the utilization that designs and screen in the antifungal drug.
8, the described fungi of claim 1 control appressorium ripe with the proteic MGPTH12 of virulence and the consistent modification that is higher than 40% homologous protein thereof as target in the utilization that designs and screen in the antifungal drug.
9, the shearing of the transcription product of the described fungi control of claim 2 appressorium maturation and the proteic gene M gPTH12 of virulence is as the utilization of target in design and screening antifungal drug.
10, the promotor of the described control of claim 6 fungi appressorium maturation and the proteic encoding gene MgPTH12 of virulence and conjugated protein as the utilization of target in design and screening antifungal drug.
11, claim 7,8,9,10 described antifungal medicines are the medicament of anti-Magnaporthe grisea (Magnaporthe grisea), gibberella saubinetii (Gibberella zeae), corn stalk rot disease bacterium (Gibberella moniliformis), Sclerotinia sclerotiorum pathogenic fungies such as (Sclerotinia sclerotirum).
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB200510109444XA CN100404550C (en) | 2005-10-20 | 2005-10-20 | Gene MgPTH12 for controlling mature and pathogenicity of fungi appressorium derived from Magnaporthe grisea and its uses |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB200510109444XA CN100404550C (en) | 2005-10-20 | 2005-10-20 | Gene MgPTH12 for controlling mature and pathogenicity of fungi appressorium derived from Magnaporthe grisea and its uses |
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| Publication Number | Publication Date |
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| CN1951957A true CN1951957A (en) | 2007-04-25 |
| CN100404550C CN100404550C (en) | 2008-07-23 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110257402A (en) * | 2019-07-10 | 2019-09-20 | 吉林大学 | A kind of southern corn leaf blight ChCDC10 gene and its application |
| CN113584077A (en) * | 2021-08-31 | 2021-11-02 | 西南大学 | Silencing vector for silencing cupula mori key pathogenic gene CsGPA1 and application and method thereof |
| CN113699181A (en) * | 2021-08-31 | 2021-11-26 | 西南大学 | Silencing vector for silencing cupula mori G protein alpha subunit coding gene CsGPA3 and application and method thereof |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1293094C (en) * | 2005-06-06 | 2007-01-03 | 中国农业大学 | Fungus conidium shape protein associated with pathogenicity and genes encoding same and use thereof |
| CN1821409A (en) * | 2005-09-12 | 2006-08-23 | 中国农业大学 | Fungus virulence new gene MgKIN17 coming from pyricularia gisea and its use |
-
2005
- 2005-10-20 CN CNB200510109444XA patent/CN100404550C/en not_active Expired - Fee Related
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110257402A (en) * | 2019-07-10 | 2019-09-20 | 吉林大学 | A kind of southern corn leaf blight ChCDC10 gene and its application |
| CN110257402B (en) * | 2019-07-10 | 2021-11-26 | 吉林大学 | Maize small leaf spot pathogen ChCDC10 gene and application thereof |
| CN113584077A (en) * | 2021-08-31 | 2021-11-02 | 西南大学 | Silencing vector for silencing cupula mori key pathogenic gene CsGPA1 and application and method thereof |
| CN113699181A (en) * | 2021-08-31 | 2021-11-26 | 西南大学 | Silencing vector for silencing cupula mori G protein alpha subunit coding gene CsGPA3 and application and method thereof |
| CN113584077B (en) * | 2021-08-31 | 2023-09-15 | 西南大学 | Silencing vector for silencing of Leptosphaeria multocida key pathogenic gene CsGPA1, application and method thereof |
| CN113699181B (en) * | 2021-08-31 | 2023-09-15 | 西南大学 | Silencing vector for silencing of calixania cerealis G protein alpha subunit coding gene CsGPA3, application and method thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN100404550C (en) | 2008-07-23 |
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