CN104593276A - Lecanicillium psalliotae AaIT genetic engineering strain with nematicidal function and application thereof - Google Patents
Lecanicillium psalliotae AaIT genetic engineering strain with nematicidal function and application thereof Download PDFInfo
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Abstract
Belonging to the technical field of biopesticide, the invention relates to a Lecanicillium psalliotae AaIT genetic engineering strain with nematicidal function and application thereof. According to the invention, a scorpion venom gene AaIT from Androctonus australis is transferred into a Lecanicillium psalliotae strain by a protoplast transformation technology so as to obtain the Lecanicillium psalliotae AaIT genetic engineering strain with high nematicidal activity and a preservation number of CCTCCM 2014629. Although the Lecanicillium psalliotae AaIT genetic engineering strain and the starting strain have the same growth rate and form, when the Lecanicillium psalliotae AaIT genetic engineering strain is culture in a solid CMA medium, the fatality rates on caenorhabditis elegans reach 61%, 80.5% and 96% respectively at 12h, 24h and 36h. After fermentation for 6 days in a PDA liquid medium, the fatality rates of the fermentation liquor on caenorhabditis elegans reach 71%, 84% and 94% respectively at 12h, 24h and 36h. The Lecanicillium psalliotae AaIT genetic engineering strain can be applied to preparation of nematicidal preparations.
Description
Technical field:
The present invention relates to a kind of cutter spore Verticillium engineering strain (Lecanicillium psalliotae AaIT) and the application thereof with nematode killing function, belong to biological pesticide technical field.
background technology:
Plant nematode is the Plant diseases generally occurred in world wide.According to statistics, the direct economic loss that the whole world is caused because of plant nematode is every year up to 1,570 hundred million dollars, wherein especially serious with root knot nematode, Cyst nematode.In China, the farm crop that plant nematode danger to vegetables, grain, tobacco, oil plant, fruit etc. are nearly all, have exceeded other Plant diseases to the harm of many important farm crop, and in the trend increased year by year.At present, the control of China to plant nematode depends on chemical pesticide.But chemical pesticide is poison is nematicide while, to the high toxicity of other biological, remaining in farm crop and edatope, all has a strong impact on food safety and Environment Ecological Safe.Along with Economic development, Chinese society is just changed to off type by simply having adequate food and clothing, and people's lives also change by having enough to eating, and healthy green food is the primary demand of the modern life.Therefore, research and develop GR and become the key issue that must solve in Sustainable Development of Modern Agriculture.And utilize nemic natural enemy to carry out to harmful nematode the concern that biological control is subject to people gradually.Although Nematophagous fungi has obtained investigation and application widely as important nematode biological and ecological methods to prevent plant disease, pests, and erosion resource, existing microorganism nematocides virulence is limited, preventive effect is unstable has become the main bugbear set up the effective prevention and control system of crop pathogenic nematode and face.Therefore, by strengthening the toxicity of nematicidal fungi, improving the nematicide ability of nematicidal fungi and bacterium, building efficient nematode engineering strain for biocontrol, become the new way effectively controlling pathogenic nematode.
Scorpion venom is a kind of neurotoxin, can as the ideal material of Study on Protein; Because scorpion toxin has high selectivity and specificity, and the gene of coding is less, is convenient to molecule manipulation, is therefore the ideal material of research egg neurobiology, immunology, white matter engineering and ionic channel.In recent years, many scholars have been had to utilize scorpion venom to take development of new biotic pesticide.Such as, insect-specific neurotoxin (neurotoxin) AaIT of one from the fertile tail scorpion of Huang (Androctonus australis) once proceeded in Metarhizium anisopliae (Metarhizium anisopliae) by Wang etc., promoted the toxicity of this fungus insecticide.Scorpion venom Gene A aIT successfully proceeds in insect pathogenic fungus beauveria bassiana Beauveria bassiana by the people such as Lu for 2008, substantially increases the toxic action of this bacterium to insect equally.Although scorpion venom gene is proceeded in insect pathogenic bacteria and improves the toxicity of insect pathogenic bacteria to insect and be reported that and so far, there is no the report utilizing the gene constructed biocontrol of nematodes engineering strain of scorpion venom.
In numerous Nematophagous fungi, Verticillium (Lecanicillium) is the very important biocontrol fungi resource of a class, and they can parasitize the ovum of free nematode (endoparasitism) and set nematode, female worm and cyst.Cutter spore Verticillium (Lecanicillium psalliotae), as Nematophagous fungi of paramount importance in Verticillium, has extremely strong nematicide effect.The present invention, to have the cutter spore Verticillium of better biocontrol effect as starting strain, using protoplast transformation technology by from importing in the scorpion toxin Gene A aIT of the fertile tail scorpion of Huang, obtaining the engineering strain with higher eelworm-killing activity.
By literature search, the open source literature report identical with the present invention is had no.
Summary of the invention:
The object of the present invention is to provide a kind of cutter spore Verticillium engineering strain (Lecanicillium psalliotae AaIT) and the application thereof with nematode killing function.
The present invention, by carrying out genetic modification to a strain cutter spore Verticillium bacterial strain, improves eelworm-killing activity, exploitation high-performance bio mematocide.The present invention is undertaken genetic engineering modified by tool setting spore Verticillium bacterial strain (China General Microbiological culture presevation administrative center (CGMCC) bacterial strain number: CGMCC1312), by protoplast transformation technology, the scorpion venom Gene A aIT coming from yellow fertile tail scorpion is transferred in cutter spore Verticillium bacterial strain, obtains the cutter spore Verticillium engineering strain (Lecanicillium psalliotae-AaIT) that a strain has higher eelworm-killing activity.This project bacterial strain is deposited in China typical culture collection center on December 5th, 2014; Depositary institution address: Wuhan University of Wuhan, China city; Preserving number is CCTCC M 2014629.
The gene order of the AaIT inserted is as follows:
CG
GGATCCTGTTCATGGAACATCACACTCGCTGACTCTGGACACCAACTG TATTCACTCGCTAATTCGTTCCTGGCTCAAATTCTTTTCCGTTCCCTAGACCAT CATGCGTGAACTTTCTTCGGTTCTCGCCCTTTCGGGCTTGCTGGCCCTGGCGTC GGCAAAGAAGAACGGCTACGCCGTCGATAGCAGCGGCAAGGCCCCCGAGTGC CTGCTGAGCAACTACTGCAACAACCAGTGCACCAAGGTCCACTACGCCGATA AGGGCTACTGCTGCCTGCTGAGCTGCTACTGCTTCGGCCTGAACGATGATAAG AAGGTCCTGGAGATCAGCGATACCCGTAAGAGCTACTGCGATACCACCATCAT CAACTAA
GGATCCCG.
Wherein:
gGATCCpart is BamHI endonuclease digestion site;
TGTTCATGGAACATCACACTCGCTGACTCTGGACACCAACTGTATTCACT CGCTAATTCGTTCCTGGCTCAAATTCTTTTCCGTTCCCTAGACCATC part is promotor;
The signal peptide of ATGCGTGAACTTTCTTCGGTTCTCGCCCTTTCGGGCTTGCTGGCCCTGGCG TCGGCA code segment AaIT;
The aminoacid sequence of AaIT is as follows:
MRELSSVLALSGLLALASAKKNGYAVDSSGKAPECLLSNYCNNQCTKVHYA DKGYCCLLSCYCFGLNDDKKVLEISDTRKSYCDTTIIN.
Wherein: MRELSSVLALSGLLALASA part is the signal peptide of AaIT.
The Nematophagous fungi cutter spore Verticillium engineering strain (Lecanicillium psalliotae AaIT) with nematode killing function of the present invention is for the preparation of the application in control Caenorhabditis elegans preparation.
Beneficial effect of the present invention is:
Cutter spore Verticillium engineering strain (Lecanicillium psalliotae AaIT) has higher eelworm-killing activity compared with wild-type starting strain.When strain culturing is on solid CMA substratum, genetic engineering bacterium reached more than 95% to the lethality rate of nematode at 36 hours, and the fermented liquid of bacterial strain reached 94% at 36 hours to the lethality rate of nematode, can apply preparing in mematocide.
accompanying drawing illustrates:
Fig. 1 showed cutter spore Verticillium starting strain and engineering bacterial strain and their fermented liquid at 12 hours, 24 hours and the comparison to the lethality rate of Caenorhabditis elegans in 36 hours.Wherein:
Fig. 1 (a) shows on CMA flat board, cutter spore Verticillium engineering strain and wild type strain at 12 hours, 24 hours and 36 hours lethality rates to Caenorhabditis elegans;
Fig. 1 (b) showed cutter spore Verticillium engineering strain and wild type strain fermented liquid at 12 hours, 24 hours and 36 hours lethality rates to beautiful hidden bar.
Embodiment:
Below in conjunction with accompanying drawing, the present invention is described in detail, but content of the present invention is not limited thereto.
One, cutter spore Verticillium engineering strain (Lecanicillium psalliotae AaIT) construction process
1. build gene transformation carrier;
The construction process of carrier is as follows: obtain scorpion venom Gene A aIT with primer AaIT-f and AaIT-r amplification, introduces BamHI restriction enzyme site at 5 ' end and 3 ' end simultaneously.With restriction enzyme BamHI, carrier pAN52-1N is cut, with ligase enzyme, the AaIT fragment obtained that increases is connected with linearizing pAN52-1N, builds and obtain pAN52-AaIT conversion carrier.
The relevant primer of AaIT gene 383bp fragment of being used for increasing is as follows:
AaIT-f:5 '-CG
gGATCCtGTTCATGGAACATC-3 '; Underscore represents at this gene fragment 5 ' end introducing BamHI restriction enzyme site
AaIT-r:5 '-CG
gGATCCgCGGCCGCTTAGTTGA-3 '; Underscore represents at this gene fragment 3 ' end introducing BamHI restriction enzyme site
2. Protoplast transformation
The preparation method of 2.1 protoplastiss
1) starting strain cutter spore Verticillium is inoculated on PDA or OA flat board, be positioned over after cultivating 8d in 28 DEG C of thermostat containers, the mycelia disk (preferably getting the mycelia block of colony edge) of cut-off footpath 0.5cm, be inoculated in 100ml TG liquid nutrient medium, 28 DEG C, 145rpm shake-flask culture 24-36h;
2) with the mycelia that the sterilized funnel collecting by filtration being lined with 6 layers of lens wiping paper is supported, and wash 2 mycelium with aqua sterilisa, then wash mycelia 2 times with MN buffer.
3) in 10ml cell wall degrading enzyme liquid, (10mg/ml snailase and the 10mg/ml cellulase of filtration sterilization in MN solution, is contained by mycelium suspended for the 0.5g collected,) in, 30 DEG C, 160rpm enzymolysis 3.5-4.5h, use sediments microscope inspection during this period, observe the concentration of cracking protoplastis out;
4) filter the mycelia of removing non-enzymolysis and falling with the sterilized funnel being lined with 4 layers of lens wiping paper, then transfer in the EP pipe of 1.5ml sterilizing by the solution containing protoplastis collected, centrifugal 8 minutes of 5000rpm, abandons supernatant.
5) add 5ml KTCbuffer washing precipitation, centrifugal 8 minutes of 5000rpm, abandons supernatant, is finally suspended in by protoplastis in 100 μ l KTC buffer, and with sediments microscope inspection, the protoplast concentration after concentrated is not less than 10
8/ ml.
2.2 protoplast transformation
This laboratory reference (1999) such as Tunlid, the REMI method for transformation that Zhang Lin (2008) and Turgeon etc. (2010) etc. are described, and in experimentation, carried out more improvement and simplification, concrete steps are as follows:
1) pAN52-AaIT and the SK-hph plasmid fragments that 30 μ l cut also purifying by linearizing enzyme is added in the 1.5mlEP pipe containing 100 μ l protoplastiss, it is mixed gently, then ice bath 40 minutes.
2) add equal-volume (135 μ l) PTC buffer, mix gently, place 60 minutes in 28 DEG C of constant incubators.
3) be suspended in liquid regeneration PDAS (10mL), coat containing on 800 μ g/ml Totomycin substratum after incubated overnight (about 12 hours) in 28 DEG C of constant incubators, 28 DEG C of constant temperature culture, just can see transformant colonies in about 6-9 days.
4) after transformant occurs, observe its growing state every day, according to the size of transformant timely picking list bacterium colony, then transfer 3 transformants continuously on the PDA flat board of 800 μ g/ml Totomycin.
3. screen transformant
The checking of cutter spore Verticillium scorpion toxin engineering strain transformant, apply the PCR checking that following two pairs of primers carry out transformant respectively:
1) checking of target gene fragment
PatYU：5’-ACTTTCTTCGGTTCTCGC-3’
PatYL：5’-AGAGCGGATTCCTCAGTC-3’
Transformant can be verified with PatYU and PatYL, can only amplify target gene fragment, and this designs, so can also verify that whether the direction of insertion is correct from the genomic dna inserting successful transformant from carrier to the downstream primer of primer.Positive transformant can clone the gene fragment of a 649bp.False positive transformant can not clone scorpion venom plain gene fragment.
2) hygromycin gene is verified
hphF：5’-GTCGGAGACAGAAGATGATATTGAAGGAGC-3’
hphR：5’-GTTGGAGATTTCAGTAACGTTAAGTGGAT-3’
Transformant can be verified with hphF and hphR, can only amplify hygromycin gene fragment from the genomic dna inserting successful transformant.Positive transformant can clone the hygromycin gene fragment of a 1.4kb.False positive transformant can not clone hygromycin gene fragment.
3) the Western blot of transformant verifies
By Epitope prediction, the peptide sequence DDKKVLEISDTRKSYC of synthesis AaIT protein antibodies, by injection rabbit, prepares polyclonal antibody.The synthesis of AaIT polypeptide and antibody preparation are completed by Sangon Biotech (Shanghai) Co., Ltd..In the fermented liquid of cutter spore Verticillium engineering strain, detect AaIT albumen by Western blot experiment, illustrate that AaIT albumen has successfully proceeded in cutter spore Verticillium engineering strain, and be secreted into extracellular.
Two, cutter spore Verticillium engineering strain (Lecanicillium psalliotae AaIT) cultural method:
1. test tube kind is cultivated
Culture medium prescription PDA substratum: 2% glucose; 1000ml murphy juice (200g potato add water 1200ml boil filtration), 1.8-2% agar.Cutter spore Verticillium engineering strain (Lecanicillium psalliotae AaIT) mycelium is inoculated on substratum, cultivates 7 ~ 10 days at 20-28 DEG C, obtain test tube kind.
2. culture dish enlarged culturing
Adopt aforementioned PDA substratum, diameter is 9 cm dishes, after inoculation YMF1.00118, cultivates 7 ~ 10 days, obtain the bacterial classification being used for fluid enlargement culture with after sealed membrane sealing at 20-25 DEG C.
3.CMA solid medium is cultivated
The formula of substratum is: 1000mL corn juice (Semen Maydis powder 20g add water 1200ml boil filtration in 30 minutes) 1.8-2% agar.
During slat chain conveyor, in each 9cm culture dish, pour 20ml sterilising medium into, after solidifying, inoculation is cultivated to CMA substratum.
When triangular flask cultivates (product spore), first at each 500ml triangular flask packing 110ml substratum, 121 DEG C of sterilizings 30 minutes, are directly inoculated into bacterial strain in triangular flask after solidifying.
4.PDA liquid culture
Liquid PDA substratum is composed as follows: 2% glucose, 1000ml murphy juice (100g potato add water 1200ml boil filtration), pH nature.Each 500ml triangular flask packing 150ml nutrient solution, 121 DEG C of sterilizings 20 minutes, access bacterium block, 28 DEG C of shaking tables (150rpm) cultivate 6 days.
Three, eelworm-killing activity test:
1, test organisms and contrast bacterium is prepared
1) test organisms is prepared: cultivate cutter spore Verticillium engineering strain (Lecanicillium psalliotae-AaIT) by aforementioned CMA slat chain conveyor method.
2) preparation contrast bacterium: cultivate cutter spore Verticillium starting strain (Lecanicillium psalliotae CGMCC1312) by aforementioned CMA slat chain conveyor method.
2, prepare test and use nematode
Caenorhabditis elegans nematode (C.elegans) is inoculated on the medium oatmeal of sterilizing, at 28 DEG C, cultivates 6-10 days, for subsequent use in 4 DEG C of Refrigerator stores.The required nematode graceful funnel method of shellfish washed out, be placed in 5ml centrifuge tube, add the washing of 5ml sterilized water, brief centrifugation, abandons supernatant, repeats to obtain clean confession for 5 times and tries nematode.With the nematode suspension of sterilized water by nematode dilution to be content be 15/μ 1.
3, test method
1) get the culture dish covering with CMA flat board for examination bacterium, add living nematode 100, keep flat, be placed in 28 DEG C, respectively at 12 hours, within 24 hours and 36 hours, observe under the microscope, the lethality rate of counting Caenorhabditis elegans nematode.Nonvaccinated CMA flat board is used to carry out control experiment.
Lethality rate %=
verge of death borer population× 100
100
Test establish three parallel, twice repetition.
2) 1cm will be got for examination bacterium
2in 150ml liquid PDA substratum, 28 DEG C of shaking tables (150rpm) cultivate 6 days, collect fermented liquid.Add living nematode 100 in 1ml fermented liquid, keep flat, be placed in 28 DEG C, respectively at 12 hours, within 24 hours and 36 hours, observe under the microscope, the lethality rate of counting Caenorhabditis elegans nematode.The PDA liquid of sterilizing is used to carry out control experiment.
Lethality rate %=
verge of death borer population× 100
100
Test establish three parallel, twice repetition.
4, test-results
The dull and stereotyped statistics of CMA adds Caenorhabditis elegans after 12 hours, and in wild type strain and engineering strain, the mortality ratio of nematode is 39.5% and 61% respectively; The nemic death rate of 24 hours is 50.5% and 80.5% respectively; The nemic death rate of 36 hours is respectively 72% and 96%.
Fermentation liquor treatment statistics is, adds Caenorhabditis elegans after 12 hours, and in the fermented liquid of wild type strain and engineering strain, the mortality ratio of nematode is 35% and 71% respectively; The nemic death rate of 24 hours is 61% and 84% respectively; The nemic death rate of 36 hours is respectively 83% and 94%.
Result shows: cutter spore Verticillium engineering strain is to the good nematicide effect of Caenorhabditis elegans nematode tool.After 36 hours, bacterial strain and the lethality rate of fermented liquid to Caenorhabditis elegans reach more than 94%.
SEQUENCE LISTING
<110> Yunnan University
<120> mono-kind has cutter spore Verticillium engineering strain and the application thereof of nematode killing function
<130> 2
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 383
<212> DNA
<213> Androctonus australis
<400> 1
cgggatcctg ttcatggaac atcacactcg ctgactctgg acaccaactg tattcactcg 60
ctaattcgtt cctggctcaa attcttttcc gttccctaga ccatcatgcg tgaactttct 120
tcggttctcg ccctttcggg cttgctggcc ctggcgtcgg caaagaagaa cggctacgcc 180
gtcgatagca gcggcaaggc ccccgagtgc ctgctgagca actactgcaa caaccagtgc 240
accaaggtcc actacgccga taagggctac tgctgcctgc tgagctgcta ctgcttcggc 300
ctgaacgatg ataagaaggt cctggagatc agcgataccc gtaagagcta ctgcgatacc 360
accatcatca actaaggatc ccg 383
<210> 2
<211> 89
<212> PRT
<213> Androctonus australis
<400> 2
Met Arg Glu Leu Ser Ser Val Leu Ala Leu Ser Gly Leu Leu Ala Leu
1 5 10 15
Ala Ser Ala Lys Lys Asn Gly Tyr Ala Val Asp Ser Ser Gly Lys Ala
20 25 30
Pro Glu Cys Leu Leu Ser Asn Tyr Cys Asn Asn Gln Cys Thr Lys Val
35 40 45
His Tyr Ala Asp Lys Gly Tyr Cys Cys Leu Leu Ser Cys Tyr Cys Phe
50 55 60
Gly Leu Asn Asp Asp Lys Lys Val Leu Glu Ile Ser Asp Thr Arg Lys
65 70 75 80
Ser Tyr Cys Asp Thr Thr Ile Ile Asn
85
Claims (2)
1. one kind has cutter spore Verticillium engineering strain (Lecanicilliumpsalliotae-AaIT) of nematode killing function, and its preserving number is CCTCC M 2014629.
2. cutter spore Verticillium engineering strain (Lecanicilliumpsalliotae-AaIT) with nematode killing function according to claim 1 is for the preparation of the application in control Caenorhabditis elegans preparation.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108018305A (en) * | 2017-12-01 | 2018-05-11 | 华南农业大学 | A kind of radopholus similes thorne control method mediated using radopholus similes thorne associated bacteria |
-
2014
- 2014-12-09 CN CN201410748810.5A patent/CN104593276A/en active Pending
Non-Patent Citations (2)
| Title |
|---|
| WEIGUO FANG ET AL.: "STRAIN IMPROVEMENT OF FUNGAL INSECTICIDES FOR CONTROLLING INSECT PESTS AND VECTOR-BORNE DISEASES", 《CURRENT OPINION IN MICROBIOLOGY》 * |
| ZHONGWEI GAN ET AL.: "Cloning of the gene Lecanicillium psalliotae chitinase Lpchi1 and identification of its potential role in the biocontrol of root-knot nematode Meloidogyne incognita", 《APPL MICROBIOL BIOTECHNOL》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108018305A (en) * | 2017-12-01 | 2018-05-11 | 华南农业大学 | A kind of radopholus similes thorne control method mediated using radopholus similes thorne associated bacteria |
| CN108018305B (en) * | 2017-12-01 | 2020-08-07 | 华南农业大学 | Method for preventing and treating radopholus similis by using radopholus similis accompanying bacteria mediation |
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Application publication date: 20150506 |