CN110437324A - Banana blight bacteria transcription factor FoRlm1 and its application - Google Patents

Abstract

The invention discloses a kind of banana blight bacteria transcription factor FoRlm1 and its application, the amino acid sequence of the banana blight bacteria transcription factor FoRlm1 is as shown in SEQ ID NO:1.The nucleotide sequence of the code area banana blight bacteria transcription factor FoRlm1 is as shown in SEQ ID NO:2.The present invention is by carrying out functional analysis to transcription factor FoRlm1, application of transcription factor FoRlm1 in terms of regulating cell wall integrity, Antioxidation Mechanism, beauvericin synthesis, fusarinic acid synthesis and bacterial strain is further clarified, the design for fungicide provides gene target and plant pest management aspect provides theoretical foundation and new thinking.

Description

Banana blight bacteria transcription factor FoRlm1 and its application

Technical field

The invention belongs to field of biotechnology more particularly to banana blight bacteria transcription factor and its applications.

Background technique

Banana (Musa spp.) is important one of the fruit in the world, and the fourth-largest after rice, wheat, corn Cereal crops (Wei Yuerong etc. 2005).In recent years, banana blight has become grave danger (Hwang&Ko of banana planting industry 2004；Ploetz 2006).According to banana blight bacteria (Fusarium oxysporum f.sp.cubense, FOC) to banana The Difference in Pathogenicity of kind can be divided into 4 biological strains, wherein No. 1 biological strain infects Gros Michel, No. 2 biological strains Hybrid triploid Bluggoe (ABB) is infected, No. 3 biological strains infect Heliconia spp., and No. 4 microspecies can infect Almost all of banana variety, caused by endanger maximum (Hwang&Ko 2004).The bacterium generate chlamydospore can soil, It survives in humus and non-host plant up to 30 years, is the primary source of infection of host plant morbidity, is difficult to eradicate crop rotation (Ploetz 2006)；When host plant plantation, after chlamydospore is by the signal induction of host plant root system metabolin, then Generation mycelia can be sprouted and infect host, to cause wilt disease that (Ohara&Tsuge 2004) occurs.

Fusariumsp (Fusarium spp.) MAPK signal path infects and pathogenic relevant multiple key elements to plant It has been accredited.Mitogen-activated protein kinase Fmk1 regulates and controls tomato wilt bacterium (Fusarium oxysporum F.sp.lycopersici infectivity) and pathogenic (Di Pietro et al., 2001).Kinases MGV1 (homologue of Saccharomyces cerevisiae Slt2) is female with Fusarium graminearum (Fusarium graminearum) Sexual development, heterocaryon are formed and plant infects related (Hou et al., 2002).Protein kinase FgOS-2 (S.cerevisiae The homologue of Hog1) it is important factor (the Van Thuat et for regulating and controlling Fusarium graminearum (F.graminearum) growth and development al.,2012).The transcription factor Mst12 (homologue of S.cerevisiae Ste12) in the downstream kinases Pmk1 is to participate in regulation rice Key element that pest bacterium (Magnaporthe grisea) is infected (Park et al., 2002).MAPK signal path downstream transcription Factor tomato wilt bacterium Ste12 (Rispail&Di Pietro, 2009) and Fusarium graminearum FgSte12 (Gu et al., 2014) its important virulence factor is had proved to be.Kinases MGV1 downstream transcription factor Mig1 be (S.cerevisiae Rlm1's Homologue) also assist in the intyrusive (Mehrabi et al., 2008) of regulation M.grisea.However, Fusarium oxysporum (Fusarium.oxysporum) report of MAPK signal path downstream transcription factor is also less.

F.oxysporum includes many nonpathogenic and a variety of Invisible element fungies that can infect plant, animals and humans. In phytopathogen, more than 150 pathological form F.oxysporum are recorded (Fourie et al., 2011).According to we institute Know, in F.oxysporum MAPK signal path downstream, only transcription factor Ste12 it is identified (Di Pietro et al., 2001).In banana blight bacteria, MAPK signal path downstream transcription factor FoRlm1 or the report of its homologous gene there is no. In view of more evolution sources (O'Donnell et al., 1998) of F.oxysporum, so comparing and identifying difference The critical function of the MAPK signal path downstream transcription factor of F.oxysporum, will be significantly.

Summary of the invention

The purpose of the present invention is to provide a kind of banana blight bacteria transcription factor FoRlm1 and its cell wall integrity, Antioxidation Mechanism, beauvericin synthesis, fusarinic acid synthesizes and the application of the pathogenic aspect of bacterial strain.

The technical solution adopted by the present invention is as follows:

The present invention provides a kind of banana blight bacteria transcription factor FoRlm1, amino acid sequence such as SEQ ID NO:1 Shown, the nucleotide sequence of code area is as shown in SEQ ID NO:2.

The present invention has also further clarified transcription factor FoRlm1 in the application of the following aspects by further investigation:

1, application of the banana blight bacteria transcription factor FoRlm1 in terms of regulation beauvericin synthesis gene.It is specific preferred To obtain the mutant of beauvericin synthetic gene expression level decline after knocking out FoRlm1 gene.

2, application of the banana blight bacteria transcription factor FoRlm1 in terms of the active oxygen degradation of regulating cell.

3, banana blight bacteria transcription factor FoRlm1 is in regulation bacterial strain to the application in terms of Congo red sensibility.

4, application of the banana blight bacteria transcription factor FoRlm1 in terms of regulation fusarinic acid synthesis gene.It is specific preferred To obtain the mutant of fusarinic acid synthetic gene expression level decline after knocking out FoRlm1 gene.

5, application of the banana blight bacteria transcription factor FoRlm1 at the regulation pathogenic aspect of bacterial strain.It is particularly preferred as, leads to After crossing knockout FoRlm1 gene, the mutant of pathogenic decrease is obtained.

Compared with prior art, the beneficial effects of the present invention are:

Banana blight bacteria transcription factor FoRlm1 has successfully been obtained in the present invention, carries out function to transcription factor FoRlm1 Analysis finds that it is relevant, including thin that transcription factor FoRlm1, which is with a series of regulation of physiological properties and toxin synthesis gene, Cell wall integrality, Antioxidation Mechanism, beauvericin and fusarinic acid synthesis.Also, compared with wild species bacterial strain, transcription factor Pathogenic decrease of the knockout mutations body of FoRlm1 to banana plant.The present invention for the design of fungicide provide gene target and Theoretical foundation and new thinking are provided in terms of plant pest management.

Detailed description of the invention

WT in Figure of description and text represents wild species strain X JZ2.

Fig. 1 is the functional domain of banana blight bacteria transcription factor FoRlm1 and the signal of exon/intron position Figure.

Fig. 2 is banana blight bacteria FoRlm1 and other disease fungus homologous protein chadogram relational graphs；With Clustal × 2.0 carry out the comparison of all protein sequences；The analysis of rear sequence is compared using 5.2 Poisson model method of MEGA.Step-length Value is repeated using 1000 step-lengths.

Fig. 3 is FoRlm1 gene knockout strategy schematic diagram；The restriction map and gene knockout policy map of genome area, The relative position of short primer is represented for gene knockout, transformant identification and Southern hybridization analysis；H:HindIII；E: EcoRV。

Fig. 4 is the PCR qualification result figure of mutant Δ FoRlm1；(A) it is expanded with primer pair ZJ-F1/ZJ-R1；(B) It is expanded with primer pair JC-F/R3；(C) it is expanded with primer pair ZJ-F2/ZJ-R2.

The Southern blot that Fig. 5 is mutant Δ FoRlm1 analyzes result figure；(A) with corresponding gene knockout area Amplified fragments are probe；(B) using the amplified fragments of corresponding gene flanking region as probe.

Fig. 6 is mutant Δ FoRlm1 to H₂O₂Cross sensibility analysis result figure；(A) figure on the left side indicates WT and mutant Δ FoRlm1 is containing 4mM H₂O₂The colonial morphology of 28 DEG C of culture 5d on MM plate；Right figure expression is grown in containing 4mM H₂O₂MM The radial growth inhibiting rate of corresponding indicator strain on plate；(B) peroxidase of WT and mutant Δ FoRlm1 is real-time Quantitative PCR analysis；Four peroxidase genes are FOIG_08821, FOIG_07465, FOIG_04532 and FOIG_09161； Each processing repeats three times；Error line indicates standard error.

Fig. 7 is the influence result figure of the cell wall integrity of FoRlm1 gene pairs banana blight bacteria；(A) indicator strain exists Containing on Congo red (40 μ g/mL) MM plate, the colonial morphology of 5d is cultivated at 28 DEG C；(B) containing Congo red (40 μ g/mL) MM The inhibiting rate of the radial growth of indicator strain on plate；Each processing repeats three times；Error line indicates standard error.

Fig. 8 is quantitative and chitin synthase 2 gene the expression result chart of mutant Δ FoRlm1 chitin；(A) WT and prominent The chitin content of variant Δ FoRlm1；(B) real-time quantitative PCR of the chitin synthase 2 gene of WT and mutant Δ FoRlm1 point Analysis；Each processing repeats three times；Error line indicates standard error.

Fig. 9 is the transcription result figure that transcription factor FoRlm1 regulates and controls that FOC beauvericin synthesizes gene；Use real-time quantitative PCR Analyze the gene expression dose relative to WT；Each processing repeats three times；Error line indicates standard error.

Figure 10 is the transcription result figure that transcription factor FoRlm1 regulates and controls FOC fusarinic acid synthetic gene cluster；Use real-time quantitative PCR analyzes the gene expression dose relative to WT；Each processing repeats three times；Error line indicates standard error.

Figure 11 is the Pathogenicity result figure of mutant Δ FoRlm1；(A) banana after investigation indicator strain inoculation 30d Seedling disease symptom；(B) disease incidence and disease index indicate the occurring degree of banana seedlings；Each processing repeats three times, each repetition 10 plants of banana seedlings；Error line indicates standard error.

Specific embodiment

Below by specific embodiment combination attached drawing, invention is further described in detail.

Experimental method used in the embodiment of the present invention is conventional method unless otherwise specified.

Material used in the embodiment of the present invention, reagent etc., are commercially available unless otherwise specified.The present invention Used in primer and application thereof be shown in Table 1.

The primer that 1 FoRlm1 gene knockout of table and PCR identification and real-time quantitative PCR use

The clone of 1 FoRlm1 gene of embodiment

The present invention obtains FoRlm1 gene by homologous comparison.In order to further confirm that the coding region sequence of gene, use The Trizol of Invitrogen extracts the total RNA of wild strain XJZ2 (Li Minhui etc., 2007), and reverse transcription, will at cDNA It expands the full length gene of FoRlm1, the purified clone of PCR product after reaction with primer Rlm1-F/Rlm1-R as template After be sequenced.

It learns that FoRlm1 gene is the sequence of a long 2122bp through sequencing, encodes 652 amino acid, included containing 2 Son.Using NCBI blastp by the amino acid alignment of FoRlm1 amino acid sequence and S.cerevisiae Rlm1, show 64% similitude (Fig. 1).Structural domain is analysis shows transcription factor FoRlm1 includes conservative MADS-box structural domain (Fig. 1).

Phylogenetic analysis shows tropical No. 4 microspecies of transcription factor FoRlm1 and F.oxysporum f.sp.cubense 54006MADS-box transcription factor EXM03441.1, Fusarium verticillioides 7600MADS-box transcription factor EWG41694.1, Fusarium oxysporum Fo5176 assume albumen FOXB_09439, Fusarium oxysporum F.sp.radicis-lycopersici 26381MADS-box transcription factor EXL58625.1, Fusarium oxysporum No. 1 58289 MADS of microspecies transcription factor RLM1ENH69552.1 and Fusarium fujikuroi IMI of f.sp.cubense Box family RLM1 transcription factor CCT64703.1 is in same branch.These the result shows that FOC FoRlm1 in Fusarium kind It is highly conserved, but the similitude of Rlm1 homologous protein corresponding with other fungies is lower, turns with yeast S.cerevisiae The affiliation for recording factor R lm1 is farther (Fig. 2).

The building of 2 gene knockout carrier of embodiment

According to the complete sequence of FoRlm1 gene, FoRlm1 gene knockout carrier is constructed.

Specific steps: using primer pair F1/R1 using XJZ2 genomic DNA as template, amplification contains the site SpeI-PstI FoRlm1 upstream region of gene segment obtains the FoRlm1 segments downstream containing XhoI-PstI with primer pair F2/R2, is connected respectively to Carrier pMD18-T, and be sequenced.The FoRlm1 upstream region of gene piece after PstI (T4DNA polymerase filling-in) and SpeI digestion Section is connected on the ClaI (T4DNA polymerase filling-in) and the site SpeI of carrier pCT74, obtains carrier pCT74- FoRlm1-up；Then the segments downstream of XhoI-PstI (T4DNA polymerase filling-in) digestion, it is connected to carrier XhoI (T4DNA polymerase filling-in) site of pCT74-FoRlm1-up, obtains gene knockout carrier pCT74-FoRlm1- KO。

The acquisition of 3 mutant of embodiment

The knockout carrier pCT74-FoRlm1-KO built with restriction enzyme XbaI enzyme cutting, makes its linearisation, with XJZ2 is F-strain, carries out the protoplast homologous recombination conversion of PEG mediation.The genomic DNA for obtaining transformant, using testing It demonstrate,proves primer pair F3/R3 and carries out PCR verifying, which can expand mould to a treaty 518bp tide from knockout mutations body Δ FoRlm1 Plain gene specific fragment cannot be expanded from wild mushroom to segment (Fig. 3, Fig. 4 A).It is carried out using verifying primer pair JC-F/R3 PCR verifying, which can expand from mutant Δ FoRlm1 to a treaty 2.8kb FoRlm1 gene specific segment, and from open country It cannot be expanded in non-hibernating eggs bacterial strain to segment (Fig. 3, Fig. 4 B).PCR verifying is carried out using verifying primer pair ZJ-F1/ZJ-R1, this draws Object can be expanded from wild species to a treaty 755bp FoRlm1 gene specific segment, and from knockout mutations body Δ FoRlm1 not It can expand and arrive segment (Fig. 3, Fig. 4 C).From 126 plants of transformants, 6 plants of FoRlm1 gene knockouts are identified altogether by verifying primer Mutant.

In order to further verify these mutant, whether FoRlm1 gene is knocked, and is largely extracted conversion subgenom DNA has carried out Southern hybridization verification.Knocking out area with the FoRlm1 gene itself that primer pair ZJ-F1/ZJ-R1 is expanded is Southern hybridization probe is digested base with HindIII using Roche company digoxin DNA marker and detection kit II Because of a group DNA.From the results, it was seen that the band of about 8.9kb occurs in banana blight bacteria wild species strain X JZ2, and knock out prominent Variant cannot detect any band (Fig. 5 A).

In order to further verify whether these knockout mutations bodies are single copy homologous replacement, with primer pair ZJ-F2/ZJ-R2 The FoRlm1 upstream region of gene segment of amplification is Southern hybridization probe, is tried using Roche company digoxin DNA marker and detection Agent box II is digested genomic DNA with EcoRV.From the results, it was seen that banana blight bacteria wild species strain X JZ2 occurs The band of about 2.0kb, and knockout mutations physical efficiency detects about 11.0kb band (Fig. 5 B) occur.Show that Δ FoRlm1 is base Because of single copy homologous recombination on the site FoRlm1.

The phenotypic analysis of 4 knockout mutations body Δ FoRlm1 of embodiment

(1) bacterium colony growthform

On PDA plate, mutant Δ FoRlm1 is compared with wild species strain X JZ2, and colonial morphology is similar, but mutant Aerial hyphae reduced.

(2) growth rate measurment

Wild-type strain XJZ2 and mutant are inoculated into PDA culture medium and cultivated, the results showed that, mutant Δ FoRlm1 Growth rate and wild-type strain XJZ2, be not present significant difference (table 2).

(3) mycelia and Observations On The Spore Morphology

Wild species strain X JZ2, mutant Δ FoRlm1 are inoculated on PDA plate respectively, in 28 DEG C of culture 3d, picking Mycelia is observed under an optical microscope, the results showed that, the mycelia of mutant is compared with spore shape with wild species, and there is no significant Difference.

(4) sporulation quantity measures

Determine the sporulation quantity of wild species strain X JZ2 and mutant Δ FoRlm1 on 28 DEG C of PDA plates, the results showed that, The sporulation quantity of mutant Δ FoRlm1 is not present significant difference (table 2) compared with wild species bacterial strain.

(5) mycelium morphology factor measures in YPD fluid nutrient medium

Wild species strain X JZ2 and mutant Δ FoRlm1 are determined in 28 DEG C, YPD fluid nutrient medium, 170rpm oscillation Dry mycelial weight after cultivating 3d, the results showed that, the dry mycelial weight of mutant Δ FoRlm1 substantially reduces compared with wild species bacterial strain (table 2).

The comparison of the increment and sporulation quantity of 2 banana blight bacteria wild species bacterial strain of table and mutant

Note:^aGrowth rate is calculated by calculating the indicator strain colony diameter of culture 5d on 28 DEG C of PDA plates.

^bIt is grown in the dry weight that the hypha,hyphae of 3d is cultivated in YPD culture medium.

^cWith the quantitative sporulation quantity for cultivating 5d indicator strain on 28 DEG C of PDA plates of blood counting chamber.

^dThe significant difference (P=0.05) of the different alphabet registration evidences of same file.Each processing repeats three times.Error Line indicates standard error.

Sensitivity analysis of 5 mutant of embodiment to hydrogen peroxide

The wild species strain X JZ2 and knockout mutations body Δ FoRlm1 of fresh acquisition spore (2 μ L, 10⁸Spore/mL) point It is inoculated with MM plate and contains hydrogen peroxide H₂O₂On the MM plate of (4mM), MM plate is as control.It is cultivated by calculating at 28 DEG C The colony diameter of 6d, come test mutant hydrogen peroxide sensibility.As shown in Figure 6A, with wild species strain X JZ2 and corresponding Complementing strain is compared, H of the mutant Δ FoRlm1 to 4mM₂O₂It is more sensitive.Real-time PCR Analysis shows and wild species bacterium Strain XJZ2 is compared, this four peroxidase genes FOIG_08821, FOIG_07465, FOIG_04532 and FOIG_09161 exist (Fig. 6 B) is significantly down-regulated in mutant Δ FoRlm1.These are the result shows that transcription factor FoRlm1 is dropped in the active oxygen of cell Solution aspect plays important regulating and controlling effect.It is well known that the generation of active oxygen is an important symbol of plant defense.

The analysis of 6 mutant cells wall integrity of embodiment

The wild species strain X JZ2 and knockout mutations body Δ FoRlm1 of fresh acquisition spore (2 μ L, 10⁸Spore/mL) point It is inoculated with MM plate and contains Congo red Congo red (CR, 40 μ g/mL) or fluorescent whitening agent calcofluor white On the MM plate of (CFW, 40 μ g/mL), MM plate is as control.The colony diameter of 6d is cultivated, at 28 DEG C by calculating to test Mutant is Congo red to cell wall inhibiting substances and the Susceptible change situation of fluorescent whitening agent.Mutant Δ FoRlm1 is containing (Fig. 7 A) is accelerated in growth on the MM plate of Congo red (40 μ g/mL), but on the MM plate containing fluorescent whitening agent (40 μ g/mL) Growth rate it is similar with wild species.Data analysis shows, such as Fig. 7 B, compared with wild species bacterial strain, being grown in for the mutant contains There is the inhibiting rate on the MM plate of Congo red (40 μ g/mL) to reduce.These results illustrate knockout mutations body Δ FoRlm1 to cell The Congo red resistance enhancing of wall inhibiting substances.

Chitin is the main component (Schoffelmeer et al., 1999) of F.oxysporum cell wall.Fungi Cell wall is extracted as described in forefathers (Yago et al., 2011).By the mycelia liquid nitrogen grinding of collection, then With addition 2mL Extraction buffer (50mM Tris-HCl pH 7.8；2%SDS；0.3M mercaptoethanol；1mM EDTA, pH 8.0) And 10min are centrifuged in 100 DEG C of processing 15min, 8000rpm, supernatant is outwelled, collects cell wall precipitating, and with the distilled water to sterilize Washing three times, is completely dried it, and with the distilled water of sterilizing, suspension cell wall is precipitated again, is then lyophilized, and weigh.Use 1mL The dissolving with hydrochloric acid 5mg cell wall of 6M, hydrolyzes at least 6h, 65 DEG C of drying samples, then dissolved with 1mL sterilizing distilled water by 100 DEG C.It takes The sample of 100 μ L dissolution, adds 100 μ L Solution A (containing 1.5M Na2CO3) in 4% acetylacetone,2,4-pentanedione, 100 after mixing DEG C 20min is boiled, then again plus 96% ethyl alcohol of 700 μ L, adds 100 μ L Solution B (30mL concentrated hydrochloric acid, 30mL second Pure and mild 1.6g dimethylaminobenzaldehyde), after 20 DEG C of culture 1h, with light absorption value of the spectrophotometer test sample at 520nm. The content of chitin is calculated by the amount of the aminoglucose of estimation chitin acid cleavage.The pure aminoglucose of the qualitative reference of aminoglucose exists The production of 0-250 μ g range standard curve (Kapteyn et al., 2000；Selvaggini et al.,2004).Each place Reason repeats three times.Chitin assay shows the chitin content of mutant Δ FoRlm1 less than wild species strain X JZ2 (figure 8A).Because the synthesis of chitin relies on the activity of chitin synthetase, carry out 7 chitins of Real-time PCR Analysis Synthesis gene (including FOIG_07229, FOIG_10825, FOIG_09216, FOIG_00580, FOIG_06735, FOIG_ 06738 and FOIG_06723) expression, as prediction, compared with wild species bacterial strain, this 7 chitin synthesis The expression of gene significantly declines (Fig. 8 B) in this mutant.

7 transcription factor FoRlm1 of embodiment is related to the beauvericin synthesis transcription of gene

Beauvericin is a kind of cyclohexadepsipeptide toxin with insecticidal activity and induction mammalian cell programmed cell death.For Better understand the influence that transcription factor FoRlm1 generates toxin, we banana blight bacteria genome identification with F.oxysporum f.sp.lycopersici beauvericin synthesizes three homologous genes of gene-correlation, including FOIG_ 15793, FOIG_15792 and FOIG_15791.FOIG_15793 encodes an enniatin and beauvericin synthesizes gene The homologous gene of beas, FOIG_15792 encode the homologous gene and FOIG_ of a 2- ketoisovalerate reductase gene kivr The homologous gene (Lopez-Berges et al., 2013) of 15791 one multidrug transporter abc3 of coding.Banana blight Bacterium beauvericin synthesizes the amino acid and F.oxysporum of gene FOIG_15793, FOIG_15792 and FOIG_15791 coding Corresponding amino acid is with the similitude for being more than 94% in f.sp.lycopersici.

Real-time quantitative PCR result has determined that transcription factor FoRlm1 rises in terms of regulation beauvericin synthesis genetic transcription The effect arrived.Wild species strain X JZ2 and mutant Δ FoRlm1 is inoculated in PD culture medium, then 28 DEG C of culture 2d collect bacterium Silk extracts total RNA；Total RNA reverse transcription is carried out at cDNA using Takara SYBR Premix Ex Taq Real-time quantitative PCR, the results showed that under the expression of the gene of Beas, kivr and abc3 is significant in mutant Δ FoRlm1 Drop, has dropped 5 times, 3 times and 2 times (Fig. 9) respectively.

8 transcription factor FoRlm1 of embodiment is related to the synthesis of fusarinic acid (fusaric aicd)

Fusarinic acid has middle hypotoxicity to animals and humans, but has plant compared with high toxicity, can cause various vegetables Fusariumsp wither here disease, damping off and root rot.By synthesizing gene (FUB1-FUB5) to F.verticillioides fusarinic acid Blastp compare analysis, we have found its corresponding homologous gene in banana blight bacteria genome.FOIG_16450 (FUB1) homologous gene of F.verticillioides PKS is encoded, the concentration that it can be catalyzed three acetic acid units is gone thoroughly 6 carbon polypeptide chain of formation reduced form；The homologous base of FOIG_16452 (FUB3) coding F.verticillioides kinase amino acid Cause, it may play important work in terms of absorption goes to form fusarinic acid from the nitrogen source of glutamine and oxaloacetate With；FOIG_16453 (FUB4) encodes the hydrolase homologous gene of F.verticillioides and FOIG_16454 (FUB5) is compiled The homologous gene of the acetyltranslocase of code F.verticillioides, it is and adds a first on fusarinic acid carboxylic acid aglucon Base generates that demethyl acetic acid is melon to wither here alcohol is related.In the amino acid and F.verticillioides of FUB1-FUB5 gene coding Corresponding amino acid is with the similitude for being more than 94%.

To determine that transcription factor FoRlm1 is related to fusarinic acid synthesis regulation.With Real-time PCR Analysis 5 and sickle Spore bacterium acid synthesizes the expression for having correlation gene (FUB1-FUB5), the results showed that, 5 fusarinic acid synthetic gene expressions are horizontal Significantly decline in the mutant, has dropped 3 times, 3 times, 3 times, 2 times and 4 times (Figure 10) respectively.

The pathogenic analysis of embodiment 9

Wild species strain X JZ2 and mutant Δ FoRlm1 is subjected to the pathogenic inoculation experiments of Brazilian any of several broadleaf plants (AAA) sand bed seedling. Hurt root inocalation method referring to Li Minhui etc. (2007).Selection growing way is consistent, and the Brazilian any of several broadleaf plants tissue culture sand bed seedling of the health of tool 4-5 piece leaf is used Tap water is rinsed well, and root is suitably hurt, and is 1 × 10 in concentration⁵Piece-root grafting kind 30-60min is soaked in the spore suspension of a/mL, is planted In sterilizing sand, positive and negative control, 30 plants of any of several broadleaf plants seedlings of every strain inoculated are made with wild strain XJZ2 and sterile water respectively.It is placed in In solarium, 32 DEG C of per day maximum temperature, is watered 1 time every other day by 18 DEG C of minimum temperature.False basal part of stem is cut after 30d observes result.

For the pathogenecity for objectively reflecting mutant, it is inoculated with the browning level for observing false stem after 30d respectively, referring to (Lee Clever etc., 2007) onset grade is divided into 4 grades according to false basal part of stem cross section browning degree, wherein 1 grade of table by grade scale Show false basal part of stem without brown, typical value 0；The false basal part of stem browning level of 2 grades of expressions only has eye of a needle size, typical value 1；3 Grade indicates that false basal part of stem browning level is less than the 1/2 of false stem diameter, typical value 2；The false basal part of stem browning level of 4 grades of expressions is greater than 1/2, typical value 3.

Knockout mutations body Δ FoRlm1 and wild species strain inoculated banana seedlings are subjected to pathogenic detection, cut after being inoculated with 30d False basal part of stem is opened, as a result such as Figure 11 A, the browning level of most knockout mutations bodies are significantly less than wild species bacterial strain.Knockout mutations body Disease index (21.67) and disease incidence (33.00%) be significantly less than wild species bacterial strain (respectively 71.11 and 93.00%) (figure 11B).These are the result shows that transcription factor FoRlm1 regulates and controls the pathogenic of banana blight bacteria.

The above content is specific embodiment is combined, further detailed description of the invention, and it cannot be said that this hair Bright specific implementation is only limited to these instructions.For those of ordinary skill in the art to which the present invention belongs, it is not taking off Under the premise of from present inventive concept, a number of simple deductions or replacements can also be made.

Sequence table

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645 650

<210> 2

<211> 2122

<212> DNA/RNA

<213> Fusarium oxysporum

<400> 2

atgggacgaa gaaagattga gattaaggcg atcaaagatg acaggaatcg ctctgtgtaa 60

gtaaacccca ggaacggcta caaatcacca gaccttgggg tcttcaagtt cgcgactaac 120

tggcgcacaa agcaccttcc tgaaacgcaa aggcggtctt ttcaagaagg ctcatgagct 180

ttccgtcctt tgctccgtcg atgtcgccgt cttcattttc ggtaacaaca agaaacttta 240

cgaatattcg tcaacggata tgcgagagct tatccaccga tatcaatatg tgagtcccac 300

ttctggaggt ggtcgcaacg cctctgaaca caggctgcga ctacgacgcg ctgtattatt 360

tgctaataga taatagcatg gcggtcccag tgaacacaaa ggtccttccg acttcaacgg 420

tggaaacgat gatgacgagg atgaggagaa cgacggcacc cctccccatg gtcctgaagt 480

tgtcgagaac cagatgatgc ctccccatgc ttacggacaa catcaacctc ctttcccaca 540

gattcggcat catacacctt cagcatcacc acctattggc aatggcggtc cattccaagc 600

gcatcctggt catcctatcc agcgacaaca tactccgcaa ccatcgatcg gctcacgacc 660

ggcatctcga acggatatgc gccgcatggg tcccggcatg gtccagcctc cacctccggc 720

cggccctccg catcccggaa tgaattacat gcccaacccg cctatctaca actcgcctca 780

tcctcccggt ctaatacctc aacatggtcc tcattctcaa tatgcttatc atcaacagcc 840

tagccacatg cagcaaccag gaccgtacat ggatgaccgc aggtccccga tgccctctcc 900

gatgcctccc gcttacactt cgcaaccgcc gtcacaacca atccaagctc ctgttcgtcc 960

aacgccatcg cctcaaccac ctcaacaaca gcttcctcca aacatgtcac aaatgtcacc 1020

tccgccgccc cagcctgaac gtcgacttca tgatcctccg ccaccgcctc ccgtggaacc 1080

caagacggaa ccacaagaac gccctcagcc gccattactg aacacggaca gtgccattaa 1140

gaagctacct caaagaaaat ctcacagtat cttcactccc atcgaagaga accggtctat 1200

tttgtcccag catctggcat cttttacttc cgagtcgaac aaatcagagt ctgctgctgc 1260

cgctgccgct gctaatgctg caaacgccgc caaccgttca caatcagttg atgtggctgc 1320

actgaaccgg gccgcggatg ggcccaaatc atcaccccat ttgccacagc gtgccagtac 1380

tcagaccgat gaaaagtctc gaacggtatc gctgtcgtca ataccagaaa ccactttgac 1440

tccgccttcg cgttctaaca gtgcaaaggc cggcggtccc ggaggagctc gacctcgtgg 1500

ccctcgctta acggtgcaga ttccggatgg cggatctgaa ggcggcggta gcgcacgaac 1560

ggcagagtca aactcgccgc gggttgccac agaaactaca acgcaggcgc cccagcgcca 1620

caactctcag tcatcacttg tgcttcctcc tccctctccg tctgcgtcag caatattgtc 1680

cgcaggtgct actggtccgc caaatccttt tgcccggccg cctcctcagc agaatatgaa 1740

tggtgatact cctgtctctg ccttgccatc acgtttcttg acaaatgaac tccttccgag 1800

cccaagtagc ttctatcccg attggaactt ccggggaggt gacagtaaca cactaccaag 1860

tccgctgaac tttgcaacgc cagttgtcgg ttcaggccct agtttcctca gagatgacct 1920

gaatacgaca ccaaatgcaa actcaaacgc tttcaaggac agagatcctc tgcctaatgc 1980

caatgggagc tcaggtcaaa acttgaatgt agctccaagc aattctactg caacgaaacg 2040

aaagactcct gagccaggag ccacaacgca gagcgatact gcggaggagt cagaaccaaa 2100

gcggttgaag gtggacgagt aa 2122

<210> 3

<211> 21

<212> DNA/RNA

<213>artificial sequence (Artificial Sequence)

<400> 3

atgggacgaa gaaagattga g 21

<210> 4

<211> 20

<212> DNA/RNA

<213>artificial sequence (Artificial Sequence)

<400> 4

ttactcgtcc accttcaacc 20

<210> 5

<211> 29

<212> DNA/RNA

<213>artificial sequence (Artificial Sequence)

<400> 5

aaaactagtc gtgaagagac ttggagttg 29

<210> 6

<211> 29

<212> DNA/RNA

<213>artificial sequence (Artificial Sequence)

<400> 6

aaactgcagt tgtgcagttc aagttggag 29

<210> 7

<211> 29

<212> DNA/RNA

<213>artificial sequence (Artificial Sequence)

<400> 7

aaactcgagg cctatctaca actcgcctc 29

<210> 8

<211> 29

<212> DNA/RNA

<213>artificial sequence (Artificial Sequence)

<400> 8

aaactgcagg ctgcctcaaa acaagagac 29

<210> 9

<211> 20

<212> DNA/RNA

<213>artificial sequence (Artificial Sequence)

<400> 9

gagttgcttt agcctcgttt 20

<210> 10

<211> 20

<212> DNA/RNA

<213>artificial sequence (Artificial Sequence)

<400> 10

gcaagacctg cctgaaaccg 20

<210> 11

<211> 20

<212> DNA/RNA

<213>artificial sequence (Artificial Sequence)

<400> 11

ggtcaagacc aatgcggagc 20

<210> 12

<211> 20

<212> DNA/RNA

<213>artificial sequence (Artificial Sequence)

<400> 12

catgggacga agaaagattg 20

<210> 13

<211> 20

<212> DNA/RNA

<213>artificial sequence (Artificial Sequence)

<400> 13

gggcatgtaa ttcattccgg 20

<210> 14

<211> 20

<212> DNA/RNA

<213>artificial sequence (Artificial Sequence)

<400> 14

cactgtccaa gttccatccc 20

<210> 15

<211> 20

<212> DNA/RNA

<213>artificial sequence (Artificial Sequence)

<400> 15

gaagcgaagg tggtttggct 20

<210> 16

<211> 21

<212> DNA/RNA

<213>artificial sequence (Artificial Sequence)

<400> 16

gttggacttg gggttgatgg g 21

<210> 17

<211> 23

<212> DNA/RNA

<213>artificial sequence (Artificial Sequence)

<400> 17

caagcgtggt attctcactc tgc 23

<210> 18

<211> 21

<212> DNA/RNA

<213>artificial sequence (Artificial Sequence)

<400> 18

ttgctctttg ggagggattt t 21

<210> 19

<211> 24

<212> DNA/RNA

<213>artificial sequence (Artificial Sequence)

<400> 19

gaacagtttc ttaccgcctt tacc 24

<210> 20

<211> 20

<212> DNA/RNA

<213>artificial sequence (Artificial Sequence)

<400> 20

ggaaatgccc agtgaacagc 20

<210> 21

<211> 23

<212> DNA/RNA

<213>artificial sequence (Artificial Sequence)

<400> 21

ccgatacagc cataccaagg ata 23

<210> 22

<211> 22

<212> DNA/RNA

<213>artificial sequence (Artificial Sequence)

<400> 22

tgtcgtcagt gatggtcgtt cc 22

<210> 23

<211> 24

<212> DNA/RNA

<213>artificial sequence (Artificial Sequence)

<400> 23

tgagtctgcg tggtgtattc gtaa 24

<210> 24

<211> 23

<212> DNA/RNA

<213>artificial sequence (Artificial Sequence)

<400> 24

cgctcgttct cattctttca gtt 23

<210> 25

<211> 23

<212> DNA/RNA

<213>artificial sequence (Artificial Sequence)

<400> 25

cttgtttcgt ttcctacggt cag 23

<210> 26

<211> 21

<212> DNA/RNA

<213>artificial sequence (Artificial Sequence)

<400> 26

accgttcctc cgatgcgtta c 21

<210> 27

<211> 24

<212> DNA/RNA

<213>artificial sequence (Artificial Sequence)

<400> 27

gcaatccgtt ctcagtgtca atac 24

<210> 28

<211> 20

<212> DNA/RNA

<213>artificial sequence (Artificial Sequence)

<400> 28

catcccaggt gccacagact 20

<210> 29

<211> 20

<212> DNA/RNA

<213>artificial sequence (Artificial Sequence)

<400> 29

ctgacagcgg gtggagtttc 20

<210> 30

<211> 20

<212> DNA/RNA

<213>artificial sequence (Artificial Sequence)

<400> 30

agttgtggcc gaatgagatg 20

<210> 31

<211> 24

<212> DNA/RNA

<213>artificial sequence (Artificial Sequence)

<400> 31

gttgatttgt cacgacttgg taga 24

<210> 32

<211> 24

<212> DNA/RNA

<213>artificial sequence (Artificial Sequence)

<400> 32

actcagtatg gtttcaccca ggtc 24

<210> 33

<211> 21

<212> DNA/RNA

<213>artificial sequence (Artificial Sequence)

<400> 33

cgaggttggc tttcgtgcta t 21

<210> 34

<211> 21

<212> DNA/RNA

<213>artificial sequence (Artificial Sequence)

<400> 34

gtgaggacga cggcaagata a 21

<210> 35

<211> 22

<212> DNA/RNA

<213>artificial sequence (Artificial Sequence)

<400> 35

tgaccaaggg ctgaagatga ct 22

<210> 36

<211> 20

<212> DNA/RNA

<213>artificial sequence (Artificial Sequence)

<400> 36

ataccgtgcc caagagcgtg 20

<210> 37

<211> 19

<212> DNA/RNA

<213>artificial sequence (Artificial Sequence)

<400> 37

gaaccgagcc cgtgaaatg 19

<210> 38

<211> 22

<212> DNA/RNA

<213>artificial sequence (Artificial Sequence)

<400> 38

cgctttcctg gtcctaagat tg 22

<210> 39

<211> 19

<212> DNA/RNA

<213>artificial sequence (Artificial Sequence)

<400> 39

tgtccataac gcccaaccc 19

<210> 40

<211> 20

<212> DNA/RNA

<213>artificial sequence (Artificial Sequence)

<400> 40

catcaacagt cccgccagtg 20

<210> 41

<211> 21

<212> DNA/RNA

<213>artificial sequence (Artificial Sequence)

<400> 41

cggagtttgc gagcgaagat a 21

<210> 42

<211> 20

<212> DNA/RNA

<213>artificial sequence (Artificial Sequence)

<400> 42

ccacagcact gccgaaaatg 20

<210> 43

<211> 21

<212> DNA/RNA

<213>artificial sequence (Artificial Sequence)

<400> 43

tgacgaagaa gccgtgagac a 21

<210> 44

<211> 21

<212> DNA/RNA

<213>artificial sequence (Artificial Sequence)

<400> 44

gcaaagcaaa ggacaaaatg g 21

<210> 45

<211> 20

<212> DNA/RNA

<213>artificial sequence (Artificial Sequence)

<400> 45

gcagcagcct cgtggaagaa 20

<210> 46

<211> 20

<212> DNA/RNA

<213>artificial sequence (Artificial Sequence)

<400> 46

cgagaagccc cagacaccat 20

<210> 47

<211> 20

<212> DNA/RNA

<213>artificial sequence (Artificial Sequence)

<400> 47

tccccaagcc caactacagc 20

<210> 48

<211> 21

<212> DNA/RNA

<213>artificial sequence (Artificial Sequence)

<400> 48

tgctacatcg ccctcaccaa c 21

<210> 49

<211> 21

<212> DNA/RNA

<213>artificial sequence (Artificial Sequence)

<400> 49

cacaagcgta ggctgctcaa t 21

<210> 50

<211> 24

<212> DNA/RNA

<213>artificial sequence (Artificial Sequence)

<400> 50

agtcactcgt tctttcggtc tagg 24

<210> 51

<211> 22

<212> DNA/RNA

<213>artificial sequence (Artificial Sequence)

<400> 51

gcaggagttc ggttgatggt at 22

<210> 52

<211> 21

<212> DNA/RNA

<213>artificial sequence (Artificial Sequence)

<400> 52

aagcggtgtt gtggcaatat g 21

<210> 53

<211> 20

<212> DNA/RNA

<213>artificial sequence (Artificial Sequence)

<400> 53

aggtcctttc ctgagcgtcc 20

<210> 54

<211> 22

<212> DNA/RNA

<213>artificial sequence (Artificial Sequence)

<400> 54

cagagtgttt ccagtggtcg tg 22

<210> 55

<211> 20

<212> DNA/RNA

<213>artificial sequence (Artificial Sequence)

<400> 55

taacctccgc cattcgtcag 20

Claims (10)

1. banana blight bacteria transcription factor FoRlm1, which is characterized in that its amino acid sequence is as shown in SEQ ID NO:1.

2. banana blight bacteria transcription factor FoRlm1 according to claim 1, which is characterized in that the nucleosides of its code area Acid sequence is as shown in SEQ ID NO:2.

3. banana blight bacteria transcription factor FoRlm1 of any of claims 1 or 2 is in terms of regulation beauvericin synthesis gene Application.

4. application according to claim 3, which is characterized in that after knocking out FoRlm1 gene, obtain beauvericin and close The mutant declined at gene expression dose.

5. banana blight bacteria transcription factor FoRlm1 of any of claims 1 or 2 is in terms of the active oxygen degradation of regulating cell Application.

6. banana blight bacteria transcription factor FoRlm1 of any of claims 1 or 2 is in regulation bacterial strain to Congo red sensibility The application of aspect.

7. banana blight bacteria transcription factor FoRlm1 of any of claims 1 or 2 is in terms of regulation fusarinic acid synthesis gene Application.

8. application according to claim 7, which is characterized in that after knocking out FoRlm1 gene, obtain fusarinic acid and close The mutant declined at gene expression dose.

9. banana blight bacteria transcription factor FoRlm1 of any of claims 1 or 2 is in the application of the regulation pathogenic aspect of bacterial strain.

10. application according to claim 9, which is characterized in that after knocking out FoRlm1 gene, obtain pathogenic decrease Mutant.