CN105557510B - The biological agent and method of preventing and controlling banana fusarium wilt - Google Patents
The biological agent and method of preventing and controlling banana fusarium wilt Download PDFInfo
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- CN105557510B CN105557510B CN201510952058.0A CN201510952058A CN105557510B CN 105557510 B CN105557510 B CN 105557510B CN 201510952058 A CN201510952058 A CN 201510952058A CN 105557510 B CN105557510 B CN 105557510B
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/06—Processes for producing mutations, e.g. treatment with chemicals or with radiation
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N57/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic phosphorus compounds
- A01N57/10—Biocides, pest repellants or attractants, or plant growth regulators containing organic phosphorus compounds having phosphorus-to-oxygen bonds or phosphorus-to-sulfur bonds
- A01N57/16—Biocides, pest repellants or attractants, or plant growth regulators containing organic phosphorus compounds having phosphorus-to-oxygen bonds or phosphorus-to-sulfur bonds containing heterocyclic radicals
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/10—Animals; Substances produced thereby or obtained therefrom
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/30—Microbial fungi; Substances produced thereby or obtained therefrom
Abstract
The invention discloses a kind of biological agent of preventing and controlling banana fusarium wilt and method.This method includes carrying out gene silencing regulation and control for the gene of GAP-associated protein GAP or its similar genes in the tropical No. 4 biological strain ergosterol biosynthetic processes of banana blight bacteria.Its active component of the biological agent of preventing and controlling banana fusarium wilt includes double-stranded RNA interference carrier, antisense RNA interference carrier or the microRNA interference carrier constructed by for the gene or its similar genes of GAP-associated protein GAP in the tropical No. 4 biological strain ergosterol biosynthetic processes of banana blight bacteria, and the double-stranded RNA interference carrier, antisense RNA interference carrier or microRNA interference carrier can cause target encoding gene silence.The present invention identifies and demonstrated the key protein of banana blight bacteria biological strain early development and important metabolic pathway, and the target site of disease-resistant new germ plasm is cultivated in exploitation and host plant induced gene silence that result of study can be as novel germicide.
Description
Technical field
The present invention relates to biological technical field, the biological agent and method of a kind of preventing and controlling banana fusarium wilt are specifically related to.
Background technology
Fragrant (big) any of several broadleaf plants is Monocotyledonae, ginger mesh, Musaceae, the plant of Musa, is worldwide widely distributed,
The tropical and subtropical zone area being distributed mainly between 20 ° of north and south latitude, share individual countries and regions plantation banana more than 130.Banana
(Musa spp.) is both important industrial crops and important cereal crops, is the second largest fruit that the whole world is only second to citrus；
Also in after rice, wheat, corn the fourth-largest cereal crops (Moffat, 1999；Huang Yonghong, 2011).By sharp spore sickle
Dao Jun Cuba specialized form (Fusarium oxysporum f.sp.cubense) fungus-caused droop (Fusarium
Wilt) in the large area outburst in the whole world and popular, serious threat banana industry and as restricting the main of its sustainable development
Factor.At present the disease the South Pacific Ocean, Asia, Africa, Australia, american torrid zone, subtropical zone banana plantation in it is universal
Occur (Butler, 2013；Hwang et al., 2004).Saved in Chinese Guangdong, Guangxi, Fujian, Yunnan, Taiwan and Hainan etc.
The banana main producing region such as (area) is on the rise.Banana blight is 10%-40% in the Jiao Yuan incidence of disease, and serious is reachable
More than 90%, so that Jiao Yuan lies waste (Liu Jingmei etc., 2006).
According to banana blight bacteria phyletic evolution EVOLUTION ANALYSIS with pathogenic it is demonstrated experimentally that banana blight bacteria is divided at present
3 biological strains, 24 trophosome compatibility groups (VCGs) (Ploetz, 2006).Wherein, No. 1 biological strain (Foc 1) is infected
" big close Kazakhstan " Gros michel, Silk, Taiwan Latundan, IC2 and dwarf banana, this microspecies is in worldwide distribution；No. 2 physiology
Microspecies (Foc 2) infect any of several broadleaf plants of cooking, and such as Triploid induction Bluggoe, and are limited to Central America；No. 4 biological strains (Foc 4)
Infect all Cavendish classes bananas, Taiwan Latundan, Gros Michel, Pisang Lilin and Bluggoe
(Hwang et al., 2004；Ploetz, 2000；Ploetz, 2005).In 3 biological strains, with No. 4 biological strain harmfulness
Maximum, all any of several broadleaf plants kinds can be almost infected, cause plant withered death.No. 4 biological strains can according to the adaptability difference of temperature
It is divided into two ecotypes：Tropical No. 4 microspecies (Tropical race 4, TR4) and the biological strain of subtropical zone 4
(Subtropical race 4, STR4), wherein the most destructive ecotype is tropical No. 4 microspecies (Foc TR4), infect
Current banana main breed Cavendish, its huge destructive power cause extensive concern (Ploetz, 2006).At present
No. 1 biological strain and No. 4 biological strains be present in China.The former infects dwarf banana, and the latter infects dwarf banana and banana, especially the torrid zone 4
Number biological strain, serious threat to China's banana industry.
Banana blight bacteria has three kinds of Spore Types：Macroconidium, microconidia and chlamydospore
(Ploetz, 2000).Banana blight is a kind of soil-borne disease, and its spore is hidden generally in soil, from perfume (or spice) under suitable condition
The root system intrusion bulb of any of several broadleaf plants or false stem, into plant vasular beam tissue, by blocking the keeping of moisture and nutrient, ultimately cause plant
The whole strain of strain is withered.And once soil is polluted by banana blight bacteria, in the case where lacking banana host, banana blight bacteria
It can be survived in soil up to 30 years as long as (Li et al., 2011), this just brings phase for effectively preventing and treating banana blight
When difficulty.And the prevention and treatment method of banana blight is mainly breeding resistant variety, chemical prevention, cultural control and biology at present
Preventing and treating etc..Although having done many explorations, up to the present still without effective preventing and controlling banana fusarium wilt occur chemical agent,
Therefore seed selection High quality and diseases resistance new varieties are most basic, maximally effective solution (Nel et al., 2007).However, banana
Cultivar is mostly triploid, and it is very difficult to cultivate new varieties by traditional banana cross breeding method.Excellent cultivar
It is middle to lack high anti-germ plasm resource, the disease-resistant variety planted at present in production mainly by screen somatic mutants cultivate and
Into mainly having " Formosana " (Hwang et al., 2004), " agriculture section No.1 " (Liu Shao admire etc., 2007) and " resisting withered No. 5 "
(Huang grasp intelligence etc., 2009), there is the shortcomings that such or such in these disease-resistant varieties, such as " anti-withered No. 5 " though disease resistance it is good,
Production cycle is long, and fruit ear commodity is bad, and mouthfeel is not good enough, deficiency in economic performance, is not received by consumers in general and banana peasant.Cause
This, it is significant to strengthen banana molecular breeding work.The developmental regulation mechanism of banana blight bacteria itself is failed to understand, constrains banana
The development of anti-blight molecular breeding work.To solve the problems, such as banana blight, it is necessary to growth to banana blight bacteria, development
Molecule mechanism studied, find new target site, screening, the effective chemistry of synthesis or biological control medicine.
The content of the invention
First purpose of the present invention is to provide the method for preventing and controlling banana fusarium wilt.
Specifically, realize that the technical scheme of the purpose is as follows.
A kind of method of preventing and controlling banana fusarium wilt, including for the tropical No. 4 biological strain ergosterols of banana blight bacteria
The gene of GAP-associated protein GAP or its similar genes carry out gene silencing regulation and control in biosynthetic process.
In one of the embodiments, the gene includes the encoding gene of GAP-associated protein GAP, the encoding gene front end
The non-coding region gene in non-coding region gene and/or encoding gene end 2000bp in 2000bp；The similar genes
For in random 50bp, with the corresponding encoding gene or the base of sequence similarity >=80% of the non-coding region gene
Cause.
In one of the embodiments, the GAP-associated protein GAP includes 3- hydroxy-3-methyl glutaryls-CoA-reductase
HMG1, C-24 sterol reductase ERG4, C-24 sterol methyl transferase ERG6, Cytochrome P450 lanosterol C-14 α-piptonychia
Base enzyme ERG11, Hydroxymethylglutaryl-CoA synthase ERG13, C-4 Sterol methyl oxidizing ferment ERG25 and C-3 sterol dehydrogenase
At least one of ERG26.
In one of the embodiments, the GAP-associated protein GAP is C-24 sterol methyl transferases ERG6 and/or cytochromes
P450 lanosterol C-14 α-demethylase ERG11.
In one of the embodiments, the coding gene sequence of the C-24 sterol methyl transferases ERG6 is SEQ ID
NO.1 or SEQ ID NO.2.
In one of the embodiments, the coding of the Cytochrome P450 lanosterol C-14 α-demethylase ERG11
Gene order is SEQ ID NO.3, SEQ ID NO.4 or SEQ ID NO.5.
In one of the embodiments, gene silencing regulation and control include double-stranded RNA interference, antisense RNA disturbs and small point
At least one of sub- RNA interference.
In one of the embodiments, the method for described preventing and controlling banana fusarium wilt comprises the following steps：
Specific nucleotide sequences fragment is chosen from the gene or its similar genes of at least one GAP-associated protein GAP to enter
The free tandem compound of row, construction of expression vector, the expression vector are double-stranded RNA interference carrier, antisense RNA interference carrier and small
At least one of molecule rna interference vector；
Expression vector containing the expression cassette is converted into banana plant, the expression double-strand of composition in banana plant
RNA, antisense RNA or microRNA；Or
By expression vector conversion banana endophyte or biocontrol microorganisms containing the expression cassette, then pass through the banana endophyte
Or biocontrol microorganisms carry out biological control to banana plant.
The present invention utilizes newest quantitative proteomicses technology and banana genetic transfoumation, identifies and demonstrate banana
The key protein of the wilt torrid zone 4 biological strain early development and important metabolic pathway, result of study can be used as and newly kill
The exploitation of microbial inoculum and host plant induced gene silence cultivate the target site of disease-resistant new germ plasm.Research finds ergosterol biology
The expression of GAP-associated protein GAP in route of synthesis and the development of tropical No. 4 biological strains of banana blight bacteria have important influence,
So as to carry out gene silencing regulation and control to the gene of the GAP-associated protein GAP or its similar genes, it can effectively suppress banana blight bacteria heat
Development with No. 4 biological strains, so as to have good effect to banana blight prevention and control.
It is a further object of the present invention to provide preventing and controlling banana fusarium wilt biological agent.According to more target site independent assortments,
In albumen that can be expressed by the target gene from tropical No. 4 biological strains of above-mentioned banana blight bacteria, single or multiple eggs are selected
White matter, design medicine combination, the chemical prevention for banana blight.Also can be from tropical No. 4 physiology of above-mentioned banana blight bacteria
In the encoding gene of the target proteinses of microspecies, the specific fragment of single or multiple genes is selected to carry out independent assortment, structure is posted
Double-stranded RNA interference carrier, antisense RNA interference carrier and the microRNA interference carrier of main plant induction gene silencing, enter to hold or participate in a prayer service at a temple
Any of several broadleaf plants genetic transformation cultivates resisting banana vascular wilt new lines, or structure produces the expression load of double-stranded RNA, antisense RNA or microRNA
Body, it is transferred in banana endophyte or biocontrol microorganisms, banana blight is carried out with the banana endophyte or biocontrol microorganisms of this Genetic Recombination
Biological control.It should be understood that, it is contemplated that the 5 ' non-coding regions and the non-coding region of end 3 ' of encoding gene front end can be adjusted
The expression of gene, the non-coding region in above-mentioned encoding gene front end and end 2000bp carry out gene regulation, fall within this
In the protection domain of invention.
Specifically, realize that the technical scheme of above-mentioned purpose is as follows.
A kind of biological agent of preventing and controlling banana fusarium wilt, its active component are included for tropical No. 4 lifes of banana blight bacteria
The double-stranded RNA interference in microspecies ergosterol biosynthetic process constructed by the gene or its similar genes of GAP-associated protein GAP is managed to carry
Body, antisense RNA interference carrier or microRNA interference carrier, the double-stranded RNA interference carrier, antisense RNA interference carrier or small
Molecule rna interference vector can cause target encoding gene silence.
In one of the embodiments, the gene includes the encoding gene of GAP-associated protein GAP, the encoding gene front end
The non-coding region gene in non-coding region gene and/or encoding gene end 2000bp in 2000bp；The similar genes
For in random 50bp, with the corresponding encoding gene or the gene of sequence similarity >=80% of non-coding region gene；
The encoding gene includes at least one of the gene of sequence as shown in SEQ ID NO.1-SEQ ID NO.5.
According to the biological agent of above-mentioned preventing and controlling banana fusarium wilt, it is new that banana genetic transformation cultivation resisting banana vascular wilt can be carried out
Strain, or carry out the new biocontrol microorganisms of banana endophyte and biocontrol microorganisms genetic transformation acquisition preventing and treating banana blight, its nucleotides sequence
Row, belong in protection scope of the present invention.It should be understood that, it is contemplated that highly similar nucleotide sequence also can initiator because heavy
It is silent, if for the similar nucleotide sequence of interference said gene expression, i.e., ORFs, front end or end with said gene
Noncoding region nucleotide sequence, in random 50bp, the nucleotide sequence of similarity >=80%, fall within the protection of the present invention
In the range of.
Brief description of the drawings
Fig. 1：The experimental design of the present invention and principle flow chart；
Fig. 2：The morphologic observation of Foc TR4 early growth and developments；
Fig. 3：3 different time points differentially expressed protein Vean diagrams；
Fig. 4：Differentially expressed protein GO function classifications, wherein, (A) raises difference table during Foc TR4 early developments
Up to the GO function classifications of albumen；(B) and up-regulation differentially expressed protein GO function classifications；
Fig. 5：Function classification of the Foc TR4 early developments differentially expressed proteins based on KOG databases；
Fig. 6：The differential protein of up-regulated expression in ergosterol biosynthesis pathway；
Fig. 7：Up-regulated expression albumen in Western blot verification portion ergosterol biosynthesis pathways；
Fig. 8：The Real Time PCR checking knots of 7 ergosterol biosynthesis pathway differentially expressed protein encoding genes
Fruit；
Fig. 9：Prochloraz and carbendazim suppress the In Vitro Bacteriostasis result of Foc TR4 growths, wherein, top is from left to right：Not
Add the control (CK) of epiphyte pharmaceutical, 41.7 μ g/mL Prochlorazs processing (A), 250 μ g/mL Prochlorazs processing (B) and 500 μ g/mL miaows are fresh
Amine handles (C)；Bottom is from left to right：Not plus epiphyte pharmaceutical control (CK), 83.3 μ g/mL carbendazim processing (D), 166.7 μ g/mL
Carbendazim handles (E) and 333.3 μ g/mL carbendazim processing (F).
Figure 10：500 μ g Foc TR4ERG6 double-stranded RNAs (SEQ ID NO.1 and 2 Combined expressions) suppress the body of its growth
Outer antibacterial result.In 250 μ L systems, spore quantity 1x10 is handled5Conidia/mL, with DEPC water polishing to 250 μ L, processing
Taken pictures after 12 and 24 hours.
Figure 11：500 μ g Foc TR4ERG11 double-stranded RNAs (Combined expression of SEQ ID NO.3,4 and 5) suppress its growth
In Vitro Bacteriostasis result.In 250 μ L systems, spore quantity 1x10 is handled5Conidia/mL, with DEPC water polishing to 250 μ L,
Processing is taken pictures after 12 and 24 hours.
Figure 12：Expression Foc TR4ERG6 double-stranded RNAs and expression Foc TR4ERG11 double-stranded RNAs transgenic bananas are being inoculated with
1x105After conidia/mL high concentration Spores, the phenotypic map of the 11st day.
Embodiment
For the ease of understanding the present invention, the present invention is carried out more fully below with reference to related specific embodiment and accompanying drawing
Description.Presently preferred embodiments of the present invention is given in accompanying drawing.But the present invention can realize in many different forms, and
It is not limited to embodiment described herein.On the contrary, the purpose for providing these embodiments makes to the disclosure
Understand more thorough and comprehensive.
Unless otherwise defined, all of technologies and scientific terms used here by the article is with belonging to technical field of the invention
The implication that technical staff is generally understood that is identical.Term used in the description of the invention herein is intended merely to description tool
The purpose of the embodiment of body, it is not intended that in the limitation present invention.Term as used herein "and/or" includes one or more phases
The arbitrary and all combination of the Listed Items of pass.
Banana blight of the present invention, refer to by No. 1 biological strain, tropical No. 4 biological strains or the life of subtropical zone 4
Manage microspecies and invade bulb or false stem from the root system of banana, into vascular tissue, block the keeping of moisture and nutrient, ultimately cause
The withered fungal disease of the whole strain of banana.
Embodiment 1
So far also without effective banana blight preventing control method, one to be that the pathogen has the reason for important extremely strong
Resistance.The early development of fungal spore is an important growth course in its history of life, and it colonizes host plant
The link of upper most critical, most of bactericide to fungal diseases of plants with effective preventive and therapeutic effect are sprouted both for fungal spore
The growth course of hair and early stage.In consideration of it, first identified of the present invention and demonstrating tropical No. 4 biological strains of banana blight bacteria
The key protein of early development and important metabolic pathway, the exploitation and host plant induction that result of study can be as novel germicide
Gene silencing cultivates the target site of disease-resistant new germ plasm.
1. the present invention is sent out using the newest quantitative proteomicses method analysis Foc TR4 early stages based on " Shotgun "
Educate the dynamic change of protein.Experimental design is as shown in Figure 1.Pass through the micro- sem observation of continuous 12 hours, it is determined that Foc
TR4 spores early development samples 4 time points：0h controls, the expansion of 3h conidiums and the appearance of germ tube, 7h conidiums are sprouted
Extension and the ripe mycelial appearance of 11h, the Morphology of four different phase spore/mycelia for sending out pipe are as shown in Figure 2.
Followed by the identification and functional analysis of large-scale spore early development differential protein, and the research knot to proteomics
The key protein related to early development process in fruit, verified using Real Time PCR in mRNA level in-site.Pass through
Western Western blottings further verify the key protein of up-regulated expression, and select corresponding bactericide for Foc TR4 early stages
The Key Metabolic approach of development carries out antibacterial experiment in vitro, the reliability of checking research result.
2.1 experiment material
With pathogenic No. 4 biological strain (the Fusarium oxysporum in the banana blight bacteria torrid zone of a plant height
F.sp.cubense tropical race 4, Foc TR4, VCG 01213/16) it is experiment material.
2.2Foc TR4 culture mediums
PDA：200g potatos, 20g glucose, 15g agar powders, deionized water are settled to 1000mL.
PDB：200g potatos, 20g glucose, deionized water are settled to 1000mL.
Complete medium (Complete Medium)：1% glucose sugar, 0.2% peptone (peptone), 0.1% yeast
Extract (yeast extract), 0.1% acid hydrolyzed casein (casamino acids), nitrate (6g NaNO3、0.52g
KCl、0.52g MgSO4·7H2O and 1.52g KH2PO4), trace element (2.2g ZnSO4·7H2O、1.1g H3BO3、0.5g
MnCl2·4H2O、0.5g FeSO4·7H2O、0.17g CoCl2·6H2O、0.16g CuSO4·5H2O、0.15g Na2MoO4·
2H2O and 5g Na2EDTA is dissolved in 1000 times of used time dilution in 100mL deionized waters) and vitamin (biotin (Biotin), dimension life
Plain B6 (Pyridoxin), vitamin B1 (Thiamine), vitamin B2 (Riboflavin), p-aminobenzoic acid (p-
Aminobenzoic acid, PABA) and nicotinic acid (Nicotinic acid) each 100mg be dissolved in 100mL deionized waters and using
When dilute 1000 times), pH6.0 (Seong et al., 2008).
2.3Foc TR4 spores and mycelia total protein extraction buffer solution：50mM Tris-HCl, pH 8.0,2%SDS, 10mM
DTT, 0.1mM EDTA, 0.2mM PMSF and protease inhibitors.
The cultivation of 2.4Foc TR4 spores and the morphologic observation of early development
No. four biological strains (Foc TR4, VCG 01213/16) in the banana blight bacteria torrid zone, are from Guangdong province, China Fanyu
Area infection banana blight symptom Cavendish banana caulos are separated to.Foc TR4 are inoculated in PDA culture medium, 28 DEG C
Light culture 7 days is to obtain conidium.Conidium is obtained with sterile water washing PDA plate, and passes through 4 layers of sterile gauze mistake
Filter, then filtered using 0.25 μm of cell filter, the spore concentration of filtering is finally calculated using cell counter.Will be dense eventually
Spend for 1 × 107Conidia/mL conidium is cultivated with complete medium in 28 DEG C, 150rpm shaking table, small at interval of 1
When take 1mL bacterium solutions in micro- sem observation, record the growth and development state of spore, Continuous Observation until it is ripe it is thread it is mycelial go out
Untill existing.The spore early development feature arrived according to the observation, it is determined that the sampling time point for collecting spore is 0,3,7 and 11 hour,
Collect spore/mycelium sample of Each point in time.
2.5Foc TR4 spores/mycelian protein matter extracting method
By repeated collection Foc TR4 in each 500mg of spore/mycelia of the four-stage of early development, first filled with liquid nitrogen
Divide grinding, then be transferred to 10mL centrifuge tubes, add 5mL protein extract buffers, concussion 30min is incubated at 4 DEG C, then by sample in 95
DEG C water-bath 10min, normal temperature, 12000rpm centrifugations 15min collect supernatant.5mL is added into remaining spore/mycelia fragment
Protein extract buffer, repeat the above steps.Merge the albumen supernatant obtained twice, add -20 DEG C of precoolings of 3 times of volumes
TCA acetone precipitations are stayed overnight, and centrifugation removes supernatant, and precipitation is washed 3 times (Taylor et al., 2008) with the acetone of precooling.With
Bradford methods determine protein concentration, recycle vacuum concentration system that gained precipitation is lyophilized into protein dry powder, ultralow in -80 DEG C
Saved backup in temperature refrigerator.
The 2.6 quantitative proteomicses analyses based on iTRAQ labelling techniques
2.6.1 the enzymolysis of protein and iTRAQ marks
First, lyophilized protein dry powder is added into appropriate 0.2mol/L TEAB buffer solutions according to its protein content
(pH 8.5) soluble protein, 20 μ L protein liquids (100 μ g) are taken, adding 1 μ L 2%SDS makes albuminous degeneration, then adds 2 μ L
50mmol/L TCEP reduce albumen, are eventually adding 1 μ L 200mmol/L MMTS to close cysteine residues.Will be through above-mentioned place
Protein sample is managed by enzyme to substrate 1:10 ratios add sequencing level trypsase and digested at 37 DEG C overnight.The enzymolysis, digestion of gained
(AB SCIEX, Foster City, CA) is marked according to iTRAQ kit operating procedures in peptide, respectively with iTRAQ reagents
114th, 115,116 and 117 marks utilized MALDI-TOF/TOF-MS corresponding to 0,3,7 and 11 hour of Foc TR4 early developments
5 different peptide ion labeling effciencies of (AB Sciex 4700) random detection.Successful four groups of samples will be marked to be blended in one
Rise, vacuum drying, the mark sample mixed first pass through strong cation post SCX cartridges (AB Sciex,
Framingham, MA) SDS is removed, and with Sep-Pak SPE cartridges (Waters, Milford, MA) desalting processing,
High pH reversed-phase liquid chromatographies separation is carried out again.
2.6.2 high pH reverse phase liquid chromatographies (hpRP) are grouped to total protein
High pH reversed-phase liquid chromatographies separation uses the high speed liquid chromatography systems of Dionex UltiMate 3000, its
There are built-in automatic sampler and UV-detector (Thermo-Dionex, Sunnyvale, CA) and XTerra MS C18
The chromatographic column of (3.5 μm, 2.1 × 150mm, Waters).Concrete operation step is as follows：What the iTRAQ through above-mentioned processing had been marked
Dissolved again with buffer A (20mmol/L ammonium formate, pH 9.5) after biased sample is lyophilized, in efficient liquid phase
Chromatographic system carries out reversed phase chromatography separation；With buffer A and buffer B (80%ACN/20%20mM NH4FA) it is eluent
(10% to 45%buffer B, 30min) (Yang et al., 2011) is eluted in 200 μ L/min flow velocitys Gradients, every
1min collects an elution fraction, will receive 48 components and merges into 12 parts of (Wang according to its absorption value under 214nm
Et al., 2011).20 μ L buffer C (2%ACN/0.5%NH are re-dissolved in after the sample of merging is freeze-dried4FA in),
Finally upper machine carries out nanoLC-MS/MS mass spectral analyses.
2.6.3nanoLC-MS/MS analysis
NanoLC-MS/MS mass spectral analyses employ electric field orbit trap cyclotron resonance combination mass spectrograph LTQ-Orbitrap
Velos (Thermo-Fisher Scientific, San Jose, CA), the mass spectrum be equipped with one " corconnex " and nanometer from
Source mass spectrometer (CorSolutions LLC, Ithaca, NY), contain (3 μm, 75 μm of an anti-phase nano-pillar of PepMap C18
× 15cm, Dionex) it is connected to 10 μM of analyte transmitters (NewObjective, Woburn, MA).Connect in the orbit trap
Mouth is connected with multidimensional liquid chromatography system UltiMate 3000MDLC (Dionex, Sunnyvale, CA).Each blending ingredients (5
μ L) it is injected into PepMap C18 trapping columns and is loaded with 20 μ L/min flow velocity, then using a gradient with 300nL/
0.1%FA of the min flow velocity from 5 to 38%ACN then carries out 5min slopes in the anti-phase nanometer post separation 120min of PepMap C18
95%ACN-0.1%FA and 5min are maintained at 95%ACN-0.1%FA in road.Pillar is rebalanced with 2%ACN-0.1%FA
Run next time again after 20min.Electron spray voltage is set in 1.5 by LTQ Orbitrap Velos in the positive-ion mode
Kilovolt, and carry out under 275 DEG C of source temperature eluting the detection of peptide fragment.The instrument is divided using Ultramark 1621 FT mass
Parser external calibration.Internal school is carried out as caliberator using background of the polysiloxanes ion signal in M/Z 445.120025
It is accurate.The instrument is operated in the case where data dependence gathers (DDA) pattern.In all experiments, full MS is scanned to obtain M/Z
400-1,400 mass range, the detection resolution of Orbitrap mass analyzers are arranged to 30,000.Pass through energetic encounter solution
The resolution ratio set from fragment ions spectrum caused by (HCD) in Orbitrap mass analyzers is 7,500, quality testing scope
For M/Z 100-2000.In each DDA analytical cycles, in following each scanning process, 10 most fierce multi-charges
Ion is selected as in the normalized fragment of 45% collision energy higher than 5000 threshold values.Dynamic excludes parameter setting
1 and continue to repeat 20s, the sizes of Exclude Lists excludes the width for repelling masses with ± 10ppm for 500, the 30s duration.
The HCD that soak time is 0.1ms is analyzed.Obtained using the softwares of Xcalibur 2.1 (Thermo-Fisher Scientific) analysis
Take all mass spectrometric datas.
2.6.4 MASS SPECTRAL DATA ANALYSIS
Orbitrap Velos are gathered using Proteome Discoverer 1.2 (PD 1.2, Thermo) software
Raw data file is converted to MGF files.Then using Mascot Daemon (version 2.3, Matrix Science,
Boston, MA) identification of the database to protein and iTRAQ quantitative analyses carried out based on II5 (Foc TR4) Protein Data Bank
Search, wherein II5 Protein Data Banks form (Fusarium by 16,446 genbank entries and 7,300,852 amino acid residues
Comparative Sequencing Project, Broad Institute of MIT and Harvard, http://
www.broadinstitute.org/).The search of Mascot acquiescences sets as follows：One trypsase for missing cracking site
Allow to modify with the MMTS that cysteine is fixed, fixed 4-plex iTRAQ modifications take off positioned at lysine (Lys) and N- ends
Amidatioon and methionine oxidized, asparagine (Asn) and glutamine (Gln) residue desamidization of variable modification, and 4-
Plex iTRAQ tyrosine (Tyr).The quality of peptide fragment and fragment ion is respectively that tolerance value is 20ppm and 0.1Da.In order to comment
Estimate false discovery rate (FDA) and identify deterministic scope to weigh, Elias and Gygi mesh is used in each repetition is set
Mark-bait strategy (Elias et al., 2007).Specifically, an automatic bait data library searching is to pass through Mascot
Selection check box in a bait random sequence database generation and test original spectrum and real database
Middle progress.In order to reduce the probability of wrong peptide fragment identification, only when peptide fragment meaning score value (>=20), and pass through Mascot probability point
Analyse (www.matrixscience.com/help/scoring_help.htmL#PBM) ability in the confidential interval more than 99%
Think to be accredited.It requires that during Mascot analyses each at least two, protein reliably identifying is unique
Peptide hop count.The protein found in same family constitutes the protein family in Mascot.In addition, it is contemplated that albumen quantifies,
One albumen will at least produce two complete iTRAQ reports ionization series.
2.6.5 the functional analysis of protein
In order to fully excavate the biological information that quantitative protein data are included, the present embodiment uses a variety of methods pair
The protein of Foc TR4 early developments carries out functional annotation and function classification.For the albumen of all identifications, using Blast2go
(Bioinformatics Department, CIPF, Valencia, Spain) carries out GO (Gene Onthology) functional annotation
(Conesa et al., 2008；Et al., 2008), and GO enrichment analyses are carried out to differential protein.COG(Cluster
Of Orthologous Groups of proteins, the adjacent class of albumen cluster) it is that ortholog classification is carried out to protein
Database.The albumen for forming each COG is assumed to come from same ancestral protein, and be orthologs or
It is paralogs.Orthologs refers to the albumen evolved by vertical family (species are formed) for coming from different plant species, and
And specifically retain and original protein identical function.Paralogs is that those derive from gene duplication in certain species
Albumen, it may evolve new with original relevant function.And KOG (eukaryotic orthologous groups, very
Core biology ortholog race) be ortholog race specific recognition eukaryotic protein sequence analytical database.Will identification
To protein and KOG databases be compared, predict the possible function of these protein and function statistic of classification (Li be to it
Et al., 2003).
The detection of 2.7Foc TR4 early development GAP-associated protein GAPs
Total protein (Fig. 1 and Fig. 2) in Foc TR4 spores/mycelia that four time points of extraction collect, passes through quantitative protein
3h, 7h and 11h are relative to the change in protein rule for compareing (0h) during group credit analysis Foc TR4 early developments.It is any to give
Ion ratio can be reported relative to the relative concentration change of control (0h) in 3h, 7h and 11h by iTRAQ 4-plex by determining albumen
Rate obtains.Experimental identification and quantitative protein are repeated by biology independent twice, 3659 albumen are identified altogether, wherein having
The individual albumen of 2966 (81%) is all detected in repeating to test twice.Repeat to identify quantitative values altogether in testing twice
3035, albumen, wherein to be all detected 2431 (80%) individual repeating experiment twice.As shown in figure 3, with reference to two secondary pollutants
The max-thresholds 2.0 for repeating and defining differential protein are learned, share 260,260 and 201 differential proteins respectively at 3,7 and 11 hours
Up-regulated expression, while also there are 265,480 and 429 differential proteins to lower expression.
The identification and functional analysis of 2.8Foc TR4 spore early development differential proteins
In order to obtain functional annotation information as much as possible, all differences at three time points are expressed using GO and KOG
Albumen carries out function classification.For the differentially expressed protein of identification, GO functions are carried out using bioinformatics tools Blast2go
Annotation, and differentially expressed protein classify in GO 3 main groups biological processes, molecular function, cellular component progress GO
Function enrichment analysis.The functional annotation of protein the based on GO the 3rd is horizontal in the present embodiment, and covers 20 functional categories.GO
Term fraction is calculated in each node according to formulaWherein seq is that annotation arrives not homotactic number, and
Dist is GO terms to the distance between sub- GO.The upper GO prediction results for reconciling downward differentially expressed protein are shown in Fig. 4.Research is found greatly
Part up-regulation Fig. 4 A or downward Fig. 4 B differentially expressed protein belongs to cellular process (cellular metabolic
Process), oxidation-reduction process (oxidation-reduction process), primary metabolite process (primary
Metabolic process), (small molecule metabolic process), biology close in small molecule metabolic process
Into metabolic process (the macromolecule metabolic of process (biosynthetic process), macromolecular
Process), nitrogen compound metabolic process (nitrogen compound metabolic process) etc..
Function classification based on KOG differentially expressed proteins is found：Share the individual differentially expressed protein of 747 (74%) and belong to KOG
Difference in functionality classification, the individual differentially expressed protein of also 262 (26%) not in KOG functional categories (Tatusov et al.,
2003；Tatusov et al., 2001).The KOG function classifications of differentially expressed protein, which are summarized, sees Fig. 5, almost comprising all work(
Can classification and important bioprocess (Leng et al., 2008).This shows the change of Foc TR4 early development protein group
Complexity (Fig. 5).KOG analysis determines that the related differentially expressed protein of Foc TR4 spores early development is most of
(60.4%) following 6 functional categories are belonged to：Carbohydrate and energetic supersession, cell wall synthesis and modification, protein
Biosynthesis, lipid and secondary metabolism (including ergosterol biosynthesis), the biosynthesis of signal transduction and nucleic acid, and these
Metabolic process always is where the target effect of current fungicide.It is worth noting that：It is different from other metabolic pathways,
In Foc TR4 spore early developments, all the 7 of the biosynthesis pathway of ergosterol is poor during participation lipid and secondary metabolism
Notable up-regulation, weight of the display ergosterol biosynthesis during Foc TR4 spore early developments is all presented in different expressing protein
The property wanted (Fig. 6).
The Western Blot checkings of 2.9Foc TR4 spore early development albumen
The all notable up-regulated expression of all differentially expressed proteins in the biosynthesis pathway of ergosterol, highlights ergot
The biosynthesis pathway of sterol is indispensable during Foc TR4 early developments.To verify above-mentioned Foc TR4 early developments
The expression pattern of the biosynthesis pathway differential protein of proteomics research result particularly ergosterol, using immuning hybridization
Carry out cross validation ERG4, ERG13 and ERG25 expression way (Fig. 7) as supplement verification method.Western Blot results of hybridization
Display：ERG4, ERG13 and ERG25 are steadily up-regulated expressions during Foc TR4 early developments, with quantitative protein
It is highly consistent that group learns result.
Checking of the differential protein of 2.10Foc TR4 spore early developments in mRNA is analyzed
As the premise of protein, Transcript abundance may largely directly affect egg in the regulation and control of transcriptional level
The expression of white matter.The change of one protein expression is probably the change due to its transcriptional level.In order to explore Foc TR4 spores
The biosynthesis of correlation, particularly ergosterol during early development between transcriptional level and quantitative protein group result
The expression pattern of approach differential protein encoding gene, the present embodiment are compiled to the biosynthesis pathway differential protein of 7 ergosterols
Code gene has carried out quantitative RT PCR analysis (Fig. 8).
Generally gene corresponding to differentially expressed protein is presented 2 kinds in the correlation of mRNA level in-site and quantitative protein abundance
Different type.ERG6, ERG11, ERG13 and ERG25 show that its protein abundance and gene expression amount are proportionate；And ERG4,
ERG26 and HMG1 genes and protein expression are negatively correlated, i.e., albumen is developing 3,7,11 hours up-regulated expressions, and gene deregulation table
Reach.Current many systems biology results of study show that mRNA expressions and the coefficient correlation of its protein expression level only have
0.4 to 0.6 (Lundberg et al., 2010；Nagaraj et al., 2011；Geiger et al., 2010).This part
MRNA expression data also further demonstrate the reliability of quantitative proteomicses result.
3 antibacterial experiment in vitro
Experimentation of the 3.1 different bactericide to Foc TR4 In Vitro Bacteriostasis
Five kinds of bactericide：Carbendazim (Bavistin, 10% suspension), the Prochloraz of various concentrations are used respectively
(Chronos, 25% wettable powder), S-Ethyl ethylthio sulfonate (Kebang chemistry, 80% missible oil), shenqinmycin (Nongle biological agents,
1% suspension) and albendazole (Daoyuan chemistry, 10% suspension) assess the influence that they grow to Foc TR4.
Bactericide for the inhibitory action of the radial growth of Foc TR4 mycelium is determined by the concentration of every kind of bactericide in PDA culture medium
Fixed.Purchased from the bactericide of commercial formulation directly with sterile deionized water dissolving, the PDA culture medium of respective concentration is configured to.
It is incubated at and the Foc TR4 mycelia of 7 days is grown in PDA culture medium, Foc TR4 mycelia blocks are taken with 10mm card punch, is placed in containing killing
The PDA plate center of microbial inoculum；Control takes Foc TR4 mycelia blocks to be placed in the PDA plate center without bactericide, in mold incubator
In 25 ± 2 DEG C of light cultures 5 days, upgrowth situation of the observation Foc TR4 bacterium colonies in PDA culture medium, in triplicate.
The inhibition that 3.2 different bactericide grow to Foc TR4
Prochloraz can trigger the endobacillary a variety of reactions of cause of disease as a kind of benzimidazole series bactericidal agent, including suppression
Ergosterol synthesis pathway, and there is efficient, wide spectrum and low toxicity (Siegel, 1981).Carbendazim (carbendazim)
And a kind of benzimidazole series bactericidal agent of widely used wide spectrum, the shape with micro-pipe during suppression fungi mitosis
Into, and then suppress fungi mitosis and cell proliferation.Experimental result shows that both epiphyte pharmaceuticals can significantly inhibit Foc TR4
Mycelial growth (Fig. 9).As long as 83.3 μ g/mL carbendazim leads to substantially reduce the growth (figure of mycelia on culture dish
9D).And compare, as long as the μ g/mL of Prochloraz 41.7 can more efficiently suppress the growth (Fig. 9 A) of mycelia.In order to compare it
The epiphyte pharmaceutical of its different target site is to Foc TR4 fungistatic effect, 3 kinds of epiphyte pharmaceutical S-Ethyl ethylthio sulfonates, shenqinmycin and rosickyite in addition
Imidazoles is used to test (using manufacturer's recommended concentration).Fungistatic effect to Foc TR4 is successively：Prochloraz>S-Ethyl ethylthio sulfonate>Shen piperazine
Mycin>Carbendazim>Albendazole>Control, it was observed that colony diameter be successively：1.2cm, 2.4 ± 0.4cm, 2.5 ± 0.3cm,
3.3 ± 0.2cm, 4.4 ± 0.1cm and8.0 ± 0.2cm.S-Ethyl ethylthio sulfonate is a kind of wide spectrum, efficient biological pesticide.It passes through
Carry out the normal physiological metabolism (Sun et al., 2014) of restricting bacterial with sulfydryl reaction.Shenqinmycin is by disturbing fungi line grain
Internal respiratory chain and oxidative phosphorylation effectively to suppress fungi growth (Li et al., 2010；Thomashow et al.,
2010).Albendazole is originally to be used to suppress nematode, and it optionally combines 'beta '-tubulin to suppress the poly- of its own
Micro-pipe is closed or be assembled into, therefore hinders the formation and cell division (Sun et al., 2014) of micro-pipe.From these different fungies
From the point of view of agent is to Foc TR4 fungistatic effect, Prochloraz is a kind of external extremely excellent epiphyte pharmaceutical for suppressing Foc TR4, and this also illustrates
It is a kind of effective means of suppression Foc TR4 early developments to disturb ergosterol biosynthesis pathway.Particularly the present embodiment reflects
Surely the differential protein of ergosterol biosynthesis pathway is arrived：3- hydroxy-3-methyl glutaryls-CoA-reductase HMG1 (HMG-
CoA reductase), C-24 sterol reductases ERG4 (C-24sterol reductase), C-24 sterol methyl transferases
ERG6 (C-24sterol methyltransferase), Cytochrome P450 lanosterol C-14 α-demethylase ERG11
(Cytochrome P450lanosterol C-14 α-demethylase), Hydroxymethylglutaryl-CoA synthase ERG13
(hydroxymethylglutaryl-CoA synthase), C-4 Sterol methyl oxidizing ferment ERG25 (C-4sterol methyl
Oxidase) and C-3 sterol dehydrogenases ERG26 (C-3sterol dehydrogenase) can be used as interference banana blight bacteria heat
Target site with No. 4 biological strain ergosterol biosynthesis pathways.
3.3Foc TR4ERG6 double-stranded RNAs (choose the expression of fragment tandem compound, fragment series connection from SEQ ID NO.1 and 2
The sequence of combination is as shown in SEQ ID NO.6), and Foc TR4ERG11 double-stranded RNAs (choose piece in SEQ ID NO.3,4 and 5
Section tandem compound expression, the sequence of fragment tandem compound is as shown in SEQ ID NO.7) In Vitro Bacteriostasis effect.
Foc TR4 are inoculated on PDB culture mediums to (200g potatos, 20g glucose, deionized water are settled to
1000mL), 28 DEG C of 180rpm shaken cultivations 2 days.Substantial amounts of thalline is obtained, and is filtered by 4 layers of sterile gauze, utilizes cytometer
Spore concentration after number device calculating filtering in 1mL nutrient solutions.Spore concentration is diluted to 1 × 105Conidia/mL, and should
1mL spore concentrations are 1 × 1055 EP pipes of conidia/mL separating devices, are separately added into 500 μ g ERG6, ERG11 and unloaded DI-
T-P double-stranded RNA, and with DEPC water polishing to same volume, the general volume that handles is 250 μ L.28 DEG C of 180rpm shaken cultivations,
Sampled at 12 and 24 hours, with fluorescence microscope spore growth situation and take pictures respectively.As shown in Figure 10 and Figure 11, pass through
Five secondary pollutants repeat test, it was demonstrated that Foc TR4ERG6 double-stranded RNAs, and Foc TR4ERG11 double-stranded RNAs show it is stronger
Inhibitory action.
It is understood that in other embodiments, DNA sequence dna corresponding to double-stranded RNA is not limited to SEQ ID NO.6 or SEQ ID
Shown in NO.7, it is appreciated that DNA sequence dna corresponding to double-stranded RNA can be the encoding gene and/or noncoding region base that slave phase closes albumen
Full-length gene or genetic fragment are chosen because in, or chooses multiple full-length genes and carries out free tandem compound, or chooses multiple genes
Fragment carries out the sequence that free tandem compound obtains.
Application of the embodiment 2 in resisting banana vascular wilt transgenic banana new lines are cultivated：
(1) structure and genetic transformation of Foc TR4ERG6 and Foc TR4ERG11 double-stranded RNA overexpression vectors
Overexpression vector skeleton is pYL-RNAi carriers (by Agricultural University Of South China's Life Science College Liu Yaoguang researcher's favour
Give), on this basis build Foc TR4ERG6 and Foc TR4ERG11 double-stranded RNA overexpression vector pYL-Ubi-ERG6 and
pYL-Ubi-ERG11。
The construction step of pYL-Ubi-ERG6 overexpression vectors is as follows：From two encoding genes of Foc TR4ERG6 albumen
Selection specific fragment, carries out artificial synthesized series connection fragment, the sequence such as SEQ ID for fragment of connecting in (SEQ ID NO.1 and 2)
Shown in NO.6, the multiple cloning sites 1 that are connected on pYL-RNAi carriers, then artificial synthesized series connection fragment is reversely connected to more
Cloning site 2, then the α of inverted Escherichia coli 5, choose single bacterium colony upgrading grain, and digestion verification recombinant plasmid is correct through sequencing identification
Afterwards, overexpression vector is formed.In host cell, the Foc TR4ERG6 connect fragment under maize ubiquitin promoter driving, group
Become second nature expression Foc TR4ERG6 double-stranded RNAs.
PYL-Ubi-ERG11 overexpression vectors are built：From three encoding gene (SEQ ID of Foc TR4ERG11 albumen
NO.3-5) in selection specific fragment, carry out artificial synthesized series connection fragment, the sequence for fragment of connecting as shown in SEQ ID NO.7,
Building process is same as above.
(2) freeze-thaw method conversion Agrobacterium EHA105
2 μ L overexpression vectors are taken to add into 100 μ L Agrobacterium EHA105 competent cells, after mixing, ice bath
30min.The quick-frozen 1min in liquid nitrogen, 37 DEG C of 3~5min of water-bath are transferred to immediately, after cell thawing, add the training of 1mL LB liquid
Base (tryptone 10g/L, yeast extract 5g/L and sodium chloride 10g/L) is supported, 28 DEG C, 180rpm shakes 2~4h.6000rpm from
Heart 5min, leave about 100 μ L supernatant suspension cell, and being coated on the LB flat boards containing 50mg/L Kan, 28 again
DEG C culture 2~3 days, choose single bacterium colony enter performing PCR detection, positive bacterium solution is saved backup in -70 DEG C.
Before subsequent transformation, the positive bacterium solution frozen is taken out, after defrosting, is coated on and is put down containing 50mg/L LB
On plate, culture is until grow single bacterium colony in 28 DEG C of constant temperature biochemical cultivation cases.Fallen within oese picking single bacterium containing 50mg/L
In Kan LB fluid nutrient mediums, 28 DEG C, 180rpm shakes bacterium and stayed overnight.6000rpm centrifuges 5min, (is contained with fresh LB fluid nutrient mediums
Have identical antibiotic concentration) suspend again, it is about 0.2 to adjust its OD600, and bacterium is shaken to exponential phase with identical condition
(OD600 is about 0.6~0.8).6000rpm centrifuges 10min, collects thalline, (MS is cultivated substantially with fresh M2 fluid nutrient mediums
Base, 2,4-D 1.0mg/L, biotin 1.0mg/L, inositol 100mg/L, glutamine 100mg/L, malt extract 100mg/L
And 45g/L sucrose, pH 5.3) suspension thalline, then it is about 0.2 to adjust OD600 again, and acetosyringone is added to final concentration
100 μm of ol/L, it is used to convert as engineering bacteria.
(3) conversion of the overexpression vector to banana variety
Take about 100mg subcultures 7d Grande Naine (Musa spp.Cavendish, cv Grande Naine, AAA
Type) embryonal suspension cell, it is transferred in 10mL engineering bacteria, under dark condition, 28 DEG C, 30min is infected in 45rpm vibrations.Then,
Agrobacterium is removed, adds 20mL M2 fluid nutrient medium (MS minimal mediums, 2,4-D 1.0mg/L, biotin 1.0mg/L, flesh
Alcohol 100mg/L, glutamine 100mg/L, malt extract 100mg/L, 45g/L sucrose, pH 5.3；Containing 100 μm of ol/L acetyl
Syringone) continue to co-culture 24h under the same conditions.Then, 28 DEG C, 110rpm are transferred to, is carried out under conditions of dark
Co-culture 3 days, if bacterial concentration is excessive during culture, then need to change M2 culture mediums to reduce Agrobacterium concentration.
After co-cultivation, the embryonal suspension cell infected is rinsed using M2 fresh cultures twice, is then contained in 20mL
There is culture screening 1 month in the M2 fluid nutrient mediums of 400mg/L cynnematins and 5mg/L hygromycin, change within every 2 weeks 1 culture
Base.After liquid screening, and embryonal suspension cell is gone to the M3 solid cultures of the antibiotic containing same concentrations and selective agent
(containing a great number of elements and trace element and molysite SH culture mediums (Schenk and Hildebrandt, 1972), MS vitamins on base
(Murashige and Skoog, 1962), biotin 1mg/L, glutamine 100mg/L, proline 230mg/L, malt extraction
Thing 100mg/L, NAA0.2mg/L, kinetin 0.1mg/L, inositol 1g/L, lactose 10g/L, sucrose 45g/L and plant gel
2.1g/L, pH 5.8) carry out embryo induction, cultivate 2 months maturations to embryo.
Choose maturation resistance embryo and be transferred in M4 embryo germination culture mediums (MS minimal mediums, 6-BA1mg/L,
NAA0.2mg/L, sucrose 30g/L and plant gel 2.1g/L, pH 5.8) induction embryo sprouting, antibiotic and sieve in culture medium
Agent concentration is selected suitably to reduce.After 1 month, by stalwartness sprouting embryo be transferred in root media RM (MS minimal mediums,
NAA0.1mg/L, sucrose 30g/L and plant gel 2.1g/L, pH 5.8) take root, obtain and completely intend transfer-gen plant, and in
It is potted plant in warmhouse booth.
The present embodiment is in conversion process, the genetic transforming method (Hu Chunhua etc., the agriculture bar that are mediated using Agrobacterium EHA105
The banana High-efficient Genetic Transformation system of bacterium mediation establishes Molecular Plant Breedings, 2010,8 (1):172-178) by above-mentioned super table
Up to vector introduction Grande Naine banana varieties, detected by hygromycin riddled basins HPT, thereby determine that and turned
Gene strain.
As shown in figure 12,1 parent (WT) and 4 overexpression transgenic banana strain (ERG6#8, ERG6#39,
ERG11#52 and ERG11#43) carry out tolerance to diseases test, the results showed that, the constructive expression Foc TR4ERG6 double-strands in banana
RNA and expression Foc TR4ERG11 double-stranded RNAs can effectively improve banana blight energy caused by the anti-Foc TR4 of transgenic banana
Power.
2nd, the application in preventing and treating banana blight biocontrol microorganisms is being cultivated：
(1) select the higher endophyte of content in host's banana or that has reported there is certain banana blight bacteria that suppresses to give birth to
Long biocontrol microorganisms, such as bacillus, pseudomonad, Trichoderma and actinomyces；
(2) expression vector for being adapted to banana endophyte or biocontrol microorganisms genetic transformation is selected, such as pET series；
(3) selection is adapted to the constitutive promoter in banana endophyte or biocontrol microorganisms strongly expressed；
(4) from the ORFs of corresponding gene of the present invention, front end or end non-coding region selected part specificity
Nucleotide sequence, the specific fragment of single or multiple genes carry out independent assortment, and structure produces double-stranded RNA, antisense RNA or small
Molecule RNA expression framework.Artificial synthesized nucleotide sequence may be selected in this part, to disturb banana blight bacteria one or several
Target gene is preferred.
(5) promoter and artificial synthesized nucleotide sequence in banana endophyte or biocontrol microorganisms strongly expressed will be adapted to, led to
After crossing genetic recombination, and convert into banana endophyte or biocontrol microorganisms, then by injecting, soaking, spraying, the measure such as pouring root enters
Row biological control.
Embodiment described above only expresses the several embodiments of the present invention, and its description is more specific and detailed, but simultaneously
Therefore the limitation to the scope of the claims of the present invention can not be interpreted as.It should be pointed out that for one of ordinary skill in the art
For, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to the guarantor of the present invention
Protect scope.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.
Claims (3)
- A kind of 1. method of preventing and controlling banana fusarium wilt, it is characterised in that including for tropical No. 4 biological strains of banana blight bacteria The gene of GAP-associated protein GAP carries out gene silencing regulation and control in ergosterol biosynthetic process, and the GAP-associated protein GAP is C-24 sterol first Based transferase ERG6, the C-24 sterol methyl transferases ERG6 coding gene sequence are SEQ ID NO.1 or SEQ ID NO.2, the gene silencing are regulated to double-stranded RNA interference, the RNA sequence such as SEQ ID NO:Shown in 6.
- 2. the method for preventing and controlling banana fusarium wilt as claimed in claim 1, it is characterised in that comprise the following steps：Construction of expression vector, the expression vector are double-stranded RNA interference carrier, and it contains such as SEQ ID NO:RNA shown in 6； The expression vector is converted into banana plant, the expression double-stranded RNA of composition in banana plant；Or by the expression vector Banana endophyte or biocontrol microorganisms are converted, then biological control is carried out to banana plant by the banana endophyte or biocontrol microorganisms.
- 3. a kind of biological agent of preventing and controlling banana fusarium wilt, it is characterised in that its active component includes being directed to banana blight bacteria Double-stranded RNA interference carrier in tropical No. 4 biological strain ergosterol biosynthetic processes constructed by the gene of GAP-associated protein GAP, institute Stating rna interference vector includes such as SEQ ID NO:RNA sequence shown in 6.
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