CN103004765B - Application of Bt-06A crystal protein for killing pine wood nematode - Google Patents
Application of Bt-06A crystal protein for killing pine wood nematode Download PDFInfo
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Abstract
The invention discloses an application of Bt-06A crystal protein for killing pine wood nematode, belonging to a biologic prevention technique of plant diseases. The pine wood nematode is fed by using transgenosis Botrytis cinerea which can express the Bt-06A crystal protein, so that the Bt-06A crystal protein which is expressed by the positive converter of the Botrytis cinerea brings the lethal effect to be exerted in the body of the pine wood nematode. A good research platform is provided for fields of biological medicine and microorganism insecticide.
Description
Technical field
The present invention relates to the biological prevention of plant disease, being specifically related to a kind of Bt-06A crystalline protein for killing the purposes of pine wood nematode.
Background technology
Pine wood nematode, formal name used at school Bursaphelenchus xylophilus (Steiner & Buhrer), different name Bursaphelenchus lignicolus Mamiya & Kiyohara, originates from North American continent (Wingfield M J, 1983; Mamiya Y, 1987), this damage by disease and insect is distributed in North America, Northeast Asia and Some European area (Xie Bingyan, 2009) in the world at present.China from nineteen eighty-two after this damage by disease and insect of Late Cambrian of the Zhongshan Tomb, Nanjing, expand year by year, generating region at county level was doubled every 5 years, at the bottom of calendar year 2001, pine wood nematode generating region at county level, the whole nation has reached 81, and in July, 2007, propagate, be diffused into 12 (districts of province of China, city) (district of 113 counties, city), accumulative lethal pine tree more than 5,000 ten thousand strain, to 2009, this disease has spread 192 administrative areas at the county level to 14 provinces of China (municipality directly under the Central Government of autonomous region), 674 small towns, within 2010, be diffused into 197 administrative areas at the county level, the administrative areas at the county level of morbidity in 2010 are 197, to 2011 Nian You186Ge administrative areas at the county level, reduce by 27 epidemic-stricken areas at county level, newly increase 16 epidemic-stricken areas simultaneously, great economic loss and ecological disruption are caused.At present, its mechanism of causing a disease still imperfectly understands, and still do not have approach very effectively to the prevention and control of pine wood nematode, situation is still very severe simultaneously.
For many years, to the control of forest pest mainly based on chemical control means, chemical pesticide, while kill pests, has has also killed and wounded natural enemy and other useful species of insect, has destroyed the ecological balance.Compared with chemical control, biological control has safe, effective, lasting feature, and avoids above-mentioned series of problems.Therefore biological prevention has become the focus that people pay close attention to.In biological insecticides, bacillus thuringiensis is the quasi-microorganism insecticide that purposes is the widest in the world, output is maximum at present.
Bacillus thuringiensis (Bacillus thuringiensis, be called for short Bt), it is a kind of distribution Gram-positive bacteria extremely widely, its trophosome is shaft-like, later stage starts in its one end or central authorities to form an ovate gemma, also known as interior raw spore, it is formed in this process, in thalline, one or both ends form the consistent or different parasporal crystal of one or more shape (parasporal crystal) as cry5, cry6, cry12, cry13 and cry14, to various insects and nematode, mite class and some other protozoa have special insecticidal activity, and Bt crystalline protein does not have toxicity (Betz et al. to mammal and other vertebrates, 2000), based on this thuringiensis (Baclillus thuringiensis, Bt) crystal toxin albumen is widely used in and controls hexapeopoda pest (Cannon, 1996, Schnepf et al., 1998, Maagd et al., 1999), current Bt is the important gene source of transgenic pest-resistant engineered plant, Bt became the strong substitute of chemical synthetic pesticide, and the cultivated area turning the plant of Bt crystal protein gene increases year by year, by 2005, turn the cultivated area of bt gene cotton more than 2,600 ten thousand hectares of (James, 2005), at present, cultivated area still expands in continuation.
At present, (the G.Borgonie such as G.Borgonie, M.Claeys, F.Leyns, G.Arnaut, D.De Waele, A.V.Coomans, Effect of nematicidal Bacillus thuringiensis strains on free-livingnematodes.1.Light microscopic observations, species and biological stage specificity and identification of resistant mutants of Caenorhabditis elegans, Fundamental and Applied Nematology, Vol.19 (1996), pp.391-398) prove that Bt crystalline protein has lethal effect to pattern nematode, (the Xiang-Qian Li such as Xiang-Qian Li, Jun-Zhi Wei, Anderson Tan and Raffi V.Aroian.Resistance to root-knot nematode in tomato roots expressing a nematicidal Bacillus thuringiensis crystal protein Plant Biotechnology Journal (2007) 5, pp.455-46) describe Bt crystalline protein, to root-knot nematode, there is resistance, but, up to the present, do not see the report of this crystalline protein in the insecticidal activity of pine wood nematode, do not meet the application of this respect.
Summary of the invention
The present invention, according to the blank in above-mentioned field and demand, provides the novelty teabag of Bt-06A crystalline protein, and a kind of new method killing pine wood nematode, and the overexpression vector PBI-GOH-BT06A of Bt-06A gene that this method relates to.For biological medicine, microbial insecticide field provide good research platform.
Bt-06A crystalline protein is for killing the purposes of pine wood nematode, and the nucleotide sequence of described Bt-06A crystalline protein is as shown in Seq ID No.1.
A kind of method killing pine wood nematode, it is characterized in that: with transgenosis the pathogen of Botrytis cinerea (Botrytis cinerea) pine wood nematode of feeding can expressing Bt-06A crystalline protein, the Bt-06A crystalline protein that described the pathogen of Botrytis cinerea positive transformant is expressed plays lethal effect in pine wood nematode body, and the nucleotide sequence of described Bt-06A crystalline protein is as shown in Seq ID No.1.
The transgenosis the pathogen of Botrytis cinerea (Botrytis cinerea) of described expression Bt-06A crystalline protein, for having the Agrobacterium-Mediated Transformation of Bt-06A crystalline protein expression vector by transforming and obtain.
Described Bt-06A crystalline protein expression vector refers to PBI-GOH-BT06A.
Transform the strain of transgenosis Agrobacterium tumefaciems or the transgenosis Botrytis cinerea bacteria strain that there are Bt-06A crystalline protein expression vector.
The strain of above-mentioned transgenosis Agrobacterium tumefaciems or transgenosis Botrytis cinerea bacteria strain are killing the application in pine wood nematode.
The invention provides Bt-06A crystal and kill the novelty teabag on pine wood nematode.Confirm by experiment, transform and have the botrytis cinerea of the gene of Bt-06A crystalline protein to feed pine wood nematode, increased considerably the lethality of pine wood nematode.Present invention also offers the Bt-06A crystalline protein overexpression vector for transforming botrytis cinerea.
Accompanying drawing explanation
Fig. 1 is PBI-121 collection of illustrative plates
Fig. 2 is Psilent-1 collection of illustrative plates
Fig. 3 is PBI-GRH collection of illustrative plates
Fig. 4 is that carrier PBI-GR Cla I enzyme cuts detection
Fig. 5 is that carrier PBI-GOH Kpn I enzyme cuts detection
Fig. 6 is that carrier PHD-OH-BT06A and PHD-OH-sGFP enzyme cut qualification (M:Maker trans 2K lus, 1:PHD-OH-sGFP, 2:PHD-OH-BT05)
Fig. 7 is for turning the fluoroscopic examination of sGFP gene grey mold
Fig. 8 identifies (M:Maker Trans 2K plus, 1-10: transformant, CK: non-transgenosis grey mold) for turning BT crystalline protein 6A gene grey mold PCR
Fig. 9 is Southern hybridization analysis (CK: non-transgenosis grey mold; 1-1,2-1,7-1,8-1: genomic DNA is cut through EcoR I enzyme; 1-2,2-2,7-2,8-2: genomic DNA is cut through BamH I enzyme)
Figure 10 analyzes (M:Maker trans 2K plus for turning Bt-06A gene grey mold RT-PCR; 2: transformant 2; 7: transformant 7; 8: transformant 8; CK: non-transgenosis grey mold)
Embodiment
By following examples, the present invention is described in further detail.
Biomaterial:
Pine wood nematode (Bursaphelenchus xylophilus) (US1) is from Zhejiang, and Secondary Culture has for many years been carried out in this laboratory.
The pathogen of Botrytis cinerea (Botrytis cinerea), is provided by Institute of Microorganism, Academia Sinica.
Agrobacterium tumefaciems (Agrobacterium tumefaciens) bacterial strain EHA105, is purchased.
Agrobacterium-mediated plant conversion carrier PBI-121 (see Fig. 1), known carrier, is purchased; Psilent-1 (see Fig. 2), is existing known carrier, is so kind as to give by Institute of Microorganism, Academia Sinica Liu Xingzhong researcher.
Applicant states, above biomaterial all has preservation in applicant laboratory, can provide for demonstration test from the applying date in Two decades years to the public.
Instrument
Olympus stereomicroscope, U.S. Polyscience refrigerated centrifuge (9500), constant incubator, refrigerator, electronic balance, high-pressure sterilizing pot, PCR instrument, electrophoresis apparatus, gel imaging system, water-bath, pipettor, homogenizer, centrifuge tube, rifle head, beaker and pH meter etc.
Reagent:
Trizol Reagent, RT-PCR Reverse Transcription box is purchased from Invitrogen company;
Southern hybridization kit is purchased from ROCHE company;
SanPrep pillar DNA in a small amount extraction agent box and SanPrep pillar DNA glue reclaims kit all purchased from Sheng Gong biotechnology Co., Ltd;
Tag archaeal dna polymerase, DNA Marker 2000 and clone competent escherichia coli cell used all purchased from Quan Shijin biotech firm;
TA Cloning Kit, restriction enzyme HindIII, Bgl II, Xba I and Spe I etc. are all purchased from Takara biotech firm.
TAE buffer solution (50 ×): Tris alkali 242g, glacial acetic acid 57.1ml, 0.5mol/L EDTA (pH8.0) 100ml.
Potato medium (PDA), for cultivating the pathogen of Botrytis cinerea: take 100g peeled potatoes, chopping, adds 500ml distilled water, boils 30min, and with gauze elimination potato, supply 500ml and load triangular flask, adjusted to ph is 6.0.Take 10g glucose again, 7.5g agar powder, shake up after adding triangular flask.121 DEG C, after 20min high pressure steam sterilization, pour flat board into.
LB medium, for the blue hickie screening of Escherichia coli.Dusty yeast 5g/L, peptone 10g/L, NaCl 10g/L.
YEB medium, is divided into solid and liquid two kinds, the activation for Agrobacterium thalline: dusty yeast 0.2g, mannitol 5g, MgSO47H2O 0.1g, K2HPO4 0.25g, NaCl 0.05g, agar 6g, water-soluble, be settled to 500ml (liquid nutrient medium does not add agar).
MM medium, minimal medium used when cultivating Agrobacterium tumefaciems for expanding, the formula of 500ml is as follows: 5ml K-buffer (pH7.0): KH2PO4 145g/L, K2HPO4 200g/L; 10ml M-N:NaCl 7.5g/L, MgSO4.7H2O15g/L; 5ml glucose 20g/L (filtration sterilization is placed on 4 DEG C of refrigerator storage); 5ml FeSO4 0.01g/L (filtration sterilization is placed on 4 DEG C of refrigerator storage); 0.5ml CaCl2.2H2O 1g/L; 2.5ml Spore Elements (CuSO4.5H2O0.1g/L, ZnSO4.7H2O 0.1g/L, Na2MoO4.2H2O0.1g/L, MnSO4.H2O 0.1g/L, H3BO30.1g/L); 1.25ml, NH4NO3 20g/L; 470.75ml sterile water.
IM medium, for Fiber differentiation Agrobacterium tumefaciems, the formula of 1L: 0.8ml 1.25M K-buffer, (pH is adjusted to 4.9); 20ml M-N:NaCl 15g/L, MgSO4.7H2O 30g/L; 1ml CaCl
2.2H
2o 2g/L; 10ml 50% glycerine; 2.5ml NH
4nO
320g/L; 5ml Spore Elements; 40ml MES 1M (pH is adjusted to 5.5), with NaOH adjust ph (filtration sterilization, 4 DEG C of storages); 10ml FeSO4 0.01g/L (filtration sterilization, 4 DEG C of storages); 10ml glucose 20g/L (filtration sterilization); 2ml AS 100mM (with anhydrous alcohol solution, being positioned over-20 DEG C); 898.7ml water.
Dual culture medium (Co-IM), carries out Dual culture for the pathogen of Botrytis cinerea and Agrobacterium tumefaciems.During preparation, the glucose consumption in inducing culture IM is reduced by half.
Kanamycin: soluble in water, compound concentration is 10mg/ml, is stored in-20 DEG C;
Rifampin: be dissolved in methyl alcohol, compound concentration is 10mg/ml, is stored in-20 DEG C;
Hygromycin: 50mg/ml, lucifuge is stored in 4 DEG C;
Cephalosporin: soluble in water, 200mg/ml, is stored in-20 DEG C;
MES buffer solution (pH5.5): MES 0.15M, DTT 4Mm, EDTA 4mM.
Embodiment 1: the carrier-PBI-GOH building agriculture bacillus mediated filamentous fungi overexpression
Agrobacterium Jie Genetic Transformation in Higher Plants carrier PBI-121 (Fig. 1), protoplast transformation filamentous fungi RNAi carrier Psilent-1 (Fig. 2) (Institute of Microorganism, Academia Sinica Liu Xingzhong researcher is so kind as to give).The contaminated area of PBI-121 is contained can infect plant and the required toxalbumin of expressing of fungi.
The structure of carrier PBI-GOH is mainly skeleton with PBI-121, its T-DNA district is transformed, synthesized section of DNA sequence (Seq ID No.5) by Shanghai bio-engineering corporation, its structure is Pme I, Kpn I restriction enzyme site, aspergillus nidulans trp promoter, multiple clone site, aspergillus nidulans tryptophan terminator, Cla I restriction enzyme site (5 '------3 ') with reference to Psilent-1.First PBI-121 is transformed, respectively in upstream and downstream design primer (121-F, 121-R) of the T-DNA left margin (LB) of PBI-121, upstream primer is that Cla I restriction enzyme site is added in LB front end, downstream primer design is at Eaml105 I place, amplification is to the fragment (Seq ID No.6) of 612bp, then the fragment substituted in PBI-121 between these two restriction enzyme sites is connected, by its called after PBI-G.Be connected on PBI-G by the gene of synthesis at Pme I and ClaI restriction enzyme site place, called after PBI-GO, enzyme cuts qualification as Fig. 4; Carrier PBI-GO adds hygromycin gene as selected marker.Design primer HYG-F and HYG-R (Kpn I restriction enzyme site is added at two ends) with carrier Psilent-1 for template amplification obtains hygromycin gene HPH, amplified production is connected to carrier PMD-18T, enzyme is cut and is identified and check order and confirm to be inserted into the Kpn I restriction enzyme site place of carrier PBI-GO, namely carrier PBI-GOH (Fig. 3) is obtained, enzyme cuts qualification PBI-GOH (Fig. 5) positive colony and sequence confirms, PBI-GOH sequence is shown in Seq ID No.2, and it is correct that sequencing result display builds position.
121-F：CGGAATTCCG?CTTTTGATTTATAAGG
121-R：TCGACAGCCTGTCACGGTTAAGCGAG
HYG-F：5’-T
GGTACCTAGACGTTAACTGATATTGAA-3’
HYG-R：5’-T
GGTACCTAAACCCAGGGCTGGTGACGGA-3’
Embodiment 2: build the overexpression vector containing genes of interest
The gene order (Seq ID No.1) of coding BT crystalline protein 6A is synthesized by Shanghai biotechnology company, connect with on PUC57 carrier, the gene of BT crystalline protein 6A is connected to Xba I and the BamH I restriction enzyme site place of PUC57 with 5 ' to 3 ' direction, Apa I is positioned at PUC57 carrier B amH I downstream (3 ' direction).
The BT crystalline protein 6A gene order of synthesis is connected to Xba I, the Apa I restriction enzyme site place of PBI-GOH, namely obtains the overexpression vector PBI-GOH-BT06A containing genes of interest.SGFP gene is as genetically modified crt gene, amplification is from carrier PCH-sGFP, HindIII, Xba I restriction enzyme site is added at two ends, primer is GFP-F, GFP-R, PCR primer is after cloning and sequencing checking, be connected to carrier PBI-GOH in HindIII, Xba I site, obtain carrier PBI-GOH-sGFP.PHD-OH-BT06A and PHD-OH-sGFP cuts (Fig. 6) after qualification through enzyme, sequence verification, PHD-OH-BT06A sequencing result (Seq ID No.3), PHD-OH-sGFP sequencing result (Seq ID No.4).
GFP-F：CCAAGCTTGGATGGTGAGCAAGGGCGAGGAG
GFP-R：GCTCTAGAGCTTACTTGTACAGCTCGTCCAT
Embodiment 3: the genetic transformation of grey mold
This experiment take Agrobacterium tumefaciems as mediation, the T-DNA fragment (with hygromycin resistance) expressing BT crystalline protein 6A is transformed in the pathogen of Botrytis cinerea, have successfully been obtained grey mold transformant.
3.1 Agrobacterium competence make:
(1) the mono-bacterium colony of picking Agrobacterium EHA105, be inoculated in 20ml liquid YEB medium (rifampin containing 50mg/L), 28 DEG C, 200rpm cultivates 48h;
(2) the 500 μ l that transfer shake in EHA105 to 50ml liquid YEB (rifampin containing the 50mg/L) medium of training, and 28 DEG C, it is about 0.6 that 200rpm is cultured to OD600 value;
(3) by bacterium liquid ice bath 30min, 4 DEG C, the centrifugal 10min of 5000rpm, abandons supernatant;
(4) use the glycerine suspension thalline of 10% of 50ml precooling, 4 DEG C, 4500rpm, centrifugal 10min, abandons supernatant, collects thalline;
(5) use the glycerine suspension thalline of 10% of 25ml precooling, 4 DEG C, 4500rpm, centrifugal 10min, abandons supernatant, collects thalline;
(6) use the glycerine suspension thalline of 10% of 10ml precooling, 4 DEG C, 4500rpm, centrifugal 10min, abandons supernatant, collects thalline;
(7) use the glycerine suspension thalline of 10% of 1-2ml precooling, packing 50 μ l/ manages ,-80 DEG C of preservations after quick-frozen in liquid nitrogen.3.2 electric robin transformation Agrobacterium:
1. 1 μ l PBI-GOH-BT06A and PBI-GOH-sGFP plasmid are joined respectively in 50 μ l Agrobacterium competent cells, transfer in electric shock cup in centrifuge tube after mixing, ice bath 1min;
2. electric shock cup is put in position, pin shock button and shock by electricity;
3. add 500 μ l not containing any antibiotic YEB liquid nutrient medium, 28 DEG C, 200rpm, shaken cultivation 3h, activation thalline;
4. with sterilizing rifle head, bacterium liquid is mixed, draw 60 μ l and be evenly applied on YEB solid culture medium (blocking that 50mg/L and rifampin 50mg/L), cultivate 2 days for 28 DEG C.
5. extract Agrobacterium plasmid carry out enzyme cut qualification or detected by bacterium liquid PCR.
3.3 preparations of infecting bacterium liquid:
1. get the Agrobacterium streak inoculation after conversion on YEB solid culture medium, cultivate 40h for 28 DEG C, occur to single bacterium colony;
2. picking list colony inoculation is in YEB liquid nutrient medium, 28 DEG C, 200rpm, shaken cultivation, stops when OD600 reaches about 0.6;
The centrifugal 10min of 3.4500rpm, abandons supernatant, and sediment is the thalline of collection;
4. add appropriate IM medium, with rifle head pressure-vaccum, thalline is suspended.
3.4 for the acquisition of the pathogen of Botrytis cinerea mycelia of agrobacterium mediation converted
Be coated with the Botrytis cinerea mycelia on disconnected PDA solid culture medium with oese, be inoculated in 200mlPDA liquid nutrient medium.25 DEG C, 150rpm shaken cultivation 2 days.Mycelia is collected by the filtered through gauze of sterilizing.Grind in the mortar of sterilizing, after fully grinding, add appropriate IM medium.
3.6 Dual culture and screening process
1. Dual culture base is through carrying out high pressure steam sterilization, to be cooledly adds MES, AS, glucose, FeSO4 to when about 50 DEG C, is down flat plate.By the time, after culture medium solidifying, in super-clean bench, the sterilizing miillpore filter with the equal size of culture dish is spread, catch up with most bubble;
2. the Agrobacterium after the pathogen of Botrytis cinerea mycelia fragment and induction is mixed by 4: 1, get 400 μ l and be evenly coated onto on solid co-cultivation medium;
3. lucifuge, cultivates 3 days for 23 DEG C;
4. the miillpore filter on medium is peeled, reverse side is taped against on PDA medium (cephalosporin 300 μ g/mL, hygromycin 50 μ g/mL), be placed in 25 DEG C and cultivate 2 days;
5. miillpore filter is peeled from medium, culture dish is put in 25 DEG C and cultivates 1-3 days, the bacterium colony grown is the bacterium colony of energy hygromycin;
6. single bacterium colony of hygromycin of growing of picking, in the upper screening further of PDA medium (containing cephalosporin).For ensureing the validity of hygromycin, every plate needs to make negative and positive control.
Embodiment 4: the qualification of transgenosis grey mold
The extraction of grey mold DNA:
1. with liquid nitrogen ash fully grinding grey mold mycelia in sterile mortar;
2. getting the ground sample accounting for 1/5 volume joins in 2ml centrifuge tube, and in pipe, then add 800 μ l FPCB (being purchased from the raw work in Shanghai), vortex mixes, and 55 DEG C of water-bath 30min, shake frequently;
3. add 800 μ l phenol: chloroform (1: 1) solution, mixing; 4 DEG C of centrifugal 10min of 12000rpm;
4. get supernatant to new 2ml centrifuge tube, add 800 μ l chloroforms; 4 DEG C of centrifugal 10min of 12000rpm;
5. get supernatant to new 1.5ml centrifuge tube, add 550 μ l isopropyl alcohols, mixing, room temperature leaves standstill 10min, 4 DEG C of centrifugal 10min of 12000rpm;
6. abandon supernatant, add the ethanol rinse of 1ml 70%, 4 DEG C of centrifugal 5min of 7500rpm;
7. abandon supernatant, after alcohol volatilizes, DNA is dissolved in 20 μ l TE ,-20 DEG C of storages;
8.1% agarose gel electrophoresis detects content and the quality of DNA.
After hygromycin selection, the grey mold turning sGFP gene, directly with fluorescence microscope qualification, turns the grey mold of BT-06 gene, extracts genomic DNA, carry out PCR qualification respectively
(PCR primer: Bt-06-F ATGGCCATCGATAGCAAGACT and Bt-06-R GGATCCTTATGGAGGGTTGTTG), Southern hybridize qualification
(probe primer: Bt-06-SF ATGGCCATCGATAGCAAGA and Bt-06-SR TCTAGCCTTGAAATCCTTGAT), method of operating method is see kit specification (Roche).
The transformant of picking conversion sGFP gene carries out fluorescence sight and looks under wavelength 488nm, compare, wild strain unstressed configuration, and the bacterial strain transforming sGFP gene sends green fluorescence (Fig. 7) with wild-type strain simultaneously.Picking has the transformant turning BT-06A gene of hygromycin resistance, extract DNA, carry out PCR qualification (Fig. 8), there are 9 transformants after pcr amplification, obtain the band of corresponding size in 10 transformants of random picking, and PCR result is checked order, determine that BT-06A gene integration is in grey mold.
The transformant 1,2,7,8 that random picking PCR identifies carries out southern hybridization analysis, carry out enzyme with EcoRI and BamHI to its genomic DNA respectively to cut, with digoxin labelled probe, southern result display (Fig. 9), except transformant 1 is two copy, all the other transformants are all incorporated in grey mold genome with single copy.
Embodiment 5: transgenosis grey mold RT-PCR verifies
Expression detection is carried out to single copy 3 transformants 2,7,8 be incorporated in grey mold, extract the total serum IgE of wild type grey mold and these 3 transformants respectively and reverse transcription is cDNA, carry out RT-PCR analysis (primer: Bt-06-RTF ACATCTCTCGATCAGTTCTTAC and Bt-06-RTR TGGTCCTAACAAGAAGCTATAC), result shows (Figure 10), and Bt-06 all has expression in 3 transformants.
Embodiment 6:Bt-06A crystalline protein is on the impact of the Population breeding amount of pine wood nematode
Get about 200 pine wood nematodes just eluted from grey mold, be inoculated in turn sGFP gene and Bt-06A gene grey mold on, with not genetically modified grey mold for blank, each process 3 repeat 2 times parallel, the nematode of each process is placed in after 25 DEG C of constant incubators cultivate 7 days, adds up the quantity of the nematode of each process and dead nematode population.
The grey mold of managing throughout cultivates nematode, within 7 days, collect nematode afterwards and add up, result display (table 1), non-transgenosis grey mold and the grey mold turning sGFP gene, its population growth amount and dead nematode population difference not remarkable, and with the grey mold turning Bt-06A gene and non-transgenosis grey mold and the grey mold population growth amount and the equal significant difference of mortality that turn sGFP gene, population growth amount is less than and mortality is greater than non-transgenosis grey mold and turn the grey mold of sGFP gene.
Table .1 transgenosis grey mold is on the impact of nematode population and dead worm amount
Claims (1)
1. kill the method for pine wood nematode for one kind, it is characterized in that: with transgenosis the pathogen of Botrytis cinerea (Botrytis cinerea) pine wood nematode of feeding can expressing Bt-06A crystalline protein, the Bt-06A crystalline protein that described transgenosis the pathogen of Botrytis cinerea is expressed plays lethal effect in pine wood nematode body, and the nucleotide sequence of described Bt-06A crystalline protein is as shown in Seq ID No.1;
The transgenosis the pathogen of Botrytis cinerea (Botrytis cinerea) of described expression Bt-06A crystalline protein, for having the Agrobacterium-Mediated Transformation of Bt-06A crystalline protein expression vector by transforming and obtain;
Described Bt-06A crystalline protein expression vector refers to PBI-GOH-BT06A, and its nucleotide sequence is as shown in Seq ID No.3.
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