CN102363760B - Bacillus thuringiensis ST8, insecticidal genes thereof and applications thereof - Google Patents
Bacillus thuringiensis ST8, insecticidal genes thereof and applications thereof Download PDFInfo
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Abstract
The invention provides a Bacillus thuringiensis strain ST8, which has a preservation number of CGMCC No.3923. The strain ST8 has three insecticidal genes which are cry8Kb1, cry8Pal and cry8Qal respectively, and nucleotide sequences of the three insecticidal genes are respectively represented by SEQ ID NO1, SEQ ID NO2 and SEQ ID NO3. The strain ST8 provided by the invention has a high killing activity on coleoptera soil insects which are harmful to grains, cash crops of cotton, oil plants, vegetables and the like, and various plants of flowers, lawns and the like. The invention also provides applications of the strain ST8 in the preparation of plant insecticides, the improvement of the insecticidal activity of plants, and the cultivation of transgenic plants.
Description
Technical field
The present invention relates to biological technical field, particularly, relate to a kind of bacillus thuringiensis ST8 and killing gene and application.
Background technology
In the human being's production process, insect pest is the important factor that causes agriculture production loss and affect human health, and according to the FAO statistics, the financial loss that whole world agriculture production every year causes because of insect pest is up to 14%, and the disease loss reaches 12%, and crop smothering loss reaches 11%.The amount of loss is equivalent to half of the Chinese agriculture gross output value up to 1,260 hundred million dollars, more than 4 times of Britain.The agriculture insect mainly concentrates among lepidopteran and Homoptera, Some Insects in Coleoptera scarab beetle Superfamily, as grub, be also important worldwide distribution subterranean pest-insect, endanger the seed and seedling that dicotyledonous and unifacial leaf food crop, various vegetables, oil plant, taro, cotton, herbage and flowers and fruit, woods etc. are sowed, and the phase of causing harm be long, all can cause harm from being seeded into results, cause the disconnected ridge that is short of seedling, even total crop failure, also can affect quality.Investigation shows in a large number, and grub is caused harm in subterranean pest-insect and ranks first, and accounts on the 70-80% of total subterranean pest-insect amount.Mainly contain Holotrichia parallela (Holotrichia parallela Motschulsky), holotrichia oblita (Holotrichia oblita faldermann), anomala corpulenta (Anomala carpulenta Motsch) etc., harm is comparatively serious on China North China such as Hebei, Shandong and other places.
Bacillus thuringiensis (Bacillus thuringiensis, be called for short Bt) be a kind of gram positive bacterium, its distribution is very extensive, can form the parasporal crystal that is formed by protein with insecticidal activity in sporulation, have another name called insecticidal crystal protein (Insectididal crystal proteins is called for short ICPs), ICPs is by the cry genes encoding, this albumen has strong toxicity to sensitive insect, and to higher animal and people's nontoxicity.In recent decades, Bt has been widely used in controlling the insects such as multiple lepidopteran, Diptera, Coleoptera.In addition, Bt also has the effect of control evil to the various pests such as Hymenoptera, Homoptera, Orthoptera, Mallophaga and plant pathogeny line insect, mite class, protozoon.At present Bt has become the strong substitute of chemical synthetic pesticide, Bt or the important gene source of transgenic pest-resistant engineered plant in the control of agricultural pests, injurious forest-insect and sanitary insect pest.
Cloned from strain HD-1Dipel since first can express the gene of insecticidal activity from Schnepf in 1981, people separating clone the gene of more than 470 kind of ICPs, according to the coding amino acid sequence homology they be defined as respectively different group, subgroup, class and subclass.the Bt gene that grub is had an insecticidal activity of finding so far mainly contains cry3, cry8, cry18, cry23, cry37, the genoids such as cry43 (Shu C., et al.2009, Characterization of two novel cry8 genes from Bacillus thuringiensis strain BT185.Current Microbiology, 58 (4): 389-392.), wherein most study is the cry8 genoid, the cry8 genoid is comprised of 1160-1210 amino acid, molecular weight is 128-137kDa, to Scarabaeidae, Culculionidae, the multiple coleopteran pest such as Chrysomelidae has special insecticidal activity (Crickmore N., et al.1998.Revision of the nomenclature for the Bacillus thuringiensis pesticidal crystal proteins.Microbiol Mol Biol Rev, 62:807-813).1992, Ohba etc. filter out new bacterial strain (Bt.subsp.japonensis BuiBui) (the Ohba M. that the chafer larva is had special insecticidal activity in the world first from the Bt bacterial strain, et al., 1992.Aunique isolate of Bacillus thuringiensis serovar japonensis with a high larvicidal activity specific for scarabaeid beetles, Letters in Applied Microbiology, 14:54-57), therefrom cloned a kind of new killing gene cry8Ca (Sato R. in 1994, et al.1994.Cloning, heterologous expression, and localization of a novel crystal protein gene from Bacillus thuringiensis serovar japonensis strain buibui toxic to scarabaeid insects.Curr.Microbiol.28:15-19).U.S. Mycogen company has obvious insecticidal activity (US Patent 5554534) (Tracy E.Michaels from Cry8Aa1 and the Cry8Ba1 that Bt bacterial strain PS50C separates to brevitarsis Cotinis sp, Kenneth E.Narva, Foncerrada, 1994.Bacillus thuringiensis toxins active against scarab pests.USP5554534.).The U.S. has separated two kinds of gene cry8Bb1 and cry8Bc1 gene from the Bt bacterial strain, discovery is to colorado potato bug, the west corn root leaf A, the chrysomelid exploitation (Abad, et al.2002.Genes encoding novel proteins with pesticidal activity against Coleopterans.WO 02/34774 A2) that has significant insecticidal effect and be used for transgenic insect-resistant corn of southern corn root.In China, the successively screening in recent years of Plant Protection Office of Hebei Academy of Agricultural Sciences and Agricultural University Of Hebei obtains the Bt bacterial strain that many strains have special insecticidal activity to the larva of Anomala exoleta (A.exoleta) and anomala corpulenta (A.corpulenta), the indoor biometrics mortality ratio all reach 100% (.2000. one strain such as Feng Shuliang has the new strain isolated of Bacillus thuringiensis of insecticidal activity to cockchafer subclass larva. Chinese biological control, 16 (2): 74-78).The Bt185 of plant protection institute of the Chinese Academy of Agricultural Sciences had separated a strain to the efficient (LC50 of Holotrichia parallela larva in 2004; 0.94cfu/ml) bacterial strain Bt185, and therefrom cloned four cry8 genoids that kind is new: cry8Ea1, cry8Ea2; cry8Fa1, cry8Ha1.This type of killing gene of the overwhelming majority has all been carried out patent protection by research mechanism of external section or seeds company at present; Therefore, these Bt resources are excavated and exploitation is a urgent and valuable job, had independent intellectual property right, build and insect-resistant transgenic plants all has important theory significance and using value for gene clone, engineering bacteria.
Find Tribactur from the beginning of this century history of existing more than 100 year so far is widely used aspect the preventing and treating of farm crop and gardening plant insect, injurious forest-insect and sanitary insect pest, and also plays good effect.But the inevitable outcome that makes continually single Bt insecticidal proteins is the adaptation because of insect produces the resistance to the Cry proteinoid; At present, many insect populations are producing resistance to insecticidal crystal protein in succession in varying degrees.Begin mid-term 80 year last century, resistance problem constantly is confirmed in laboratory and field test (Mc GaugheyW.H.1985.Insect resistance to the biological insecticide Bacillus thuringiensis.Science.229:193-195), and reason is mainly continue to use single variety and inferiorly cause the Bt of dosage and the application of Bt transgenic anti-insect plants causes insect population to be subject to for a long time the selective pressure of sterilant.1985, Mc Gaughey report warehouse grain pest Indian meal moth (Plodia interpunctella) was under the selective pressure of Dipel (the commodity preparation of Bt subsp.kurstaik HD-1), and after breeding for 15 generations, resistance increases by 97 times; Under the high dosage selective pressure, resistance can increase by 250 times.nineteen ninety, the small cabbage moth that confirms first large Tanaka in Hawaii has produced obvious resistance (Tabashnik B.E to the Bt sterilant, et al.1994.Reversal of resistance to Bacillus thuringiensisin Plutella xylostella.Proc.Natl.Acad.Sci.USA.91:4120-4124), since the nineties in last century, use long Shenzhen of Bt sterilant time in China, Guangzhou, the ground such as Shanghai, find that the Bt sterilant obviously descends to the small cabbage moth prevention effect, mean resistance form (the resistance research of Feng Xia .1996. Guangdong small cabbage moth to Bacillus thuringiensis. the insect journal, 39 (3): 238-244, Hofte H., et al.1988.Monoclonal antibody analysis and insecticidal spectrum of three types of lepidopteran-specific insecticidal crystal proteins of Bacillus thuringiensis.Appl.Environ.Microbiol.54:2010-2017).Find in the laboratory at present and the field has at least tens kinds of insects to produce resistance to Bt and insecticidal crystal protein thereof, arrive with the selective pressure mathematical model prediction, under the condition of Bt transgenic anti-insect plants selective pressure, insect will produce resistance (Schnepf, E., et al.1998.Bacillus thuringiensis and its pesticidal Crystal proteins.Microbiol.Mol.Biol.Rev.65 (3): 775-806).
Be the loss of avoiding resistant insects to cause, seeking new high virulence Bt bacterial strain and genetic resources is the effective way that addresses this problem, and this biological control to China also has very important meaning.
Summary of the invention
The object of the present invention is to provide a kind of bacillus thuringiensis and killing gene and application with insecticidal activity.
Bacterial strain provided by the invention is bacillus thuringiensis ST8, this bacterial strain has been deposited in (address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, China Committee for Culture Collection of Microorganisms's common micro-organisms center on June 12nd, 2010, Institute of Microorganism, Academia Sinica, postcode 100101) preservation, Classification And Nomenclature is bacillus thuringiensis (Bacillus thuringiensis), and preserving number is CGMCC No.3923.
This bacterial strain ST8 cell is shaft-like, and the blunt circle in two ends can form the brood cell, can form spherical parasporal crystal simultaneously, sees accompanying drawing 1.The bacterial strain size is the μ m of (1.2-1.5) μ m * (3.5-4.4), and single or two or short chain cell exist usually, and a vegetative cell is a sporocyst, each sporocyst contains a gemma, inferior end is given birth to, and the other end has a parasporal crystal, and sporocyst does not expand.
The present invention finds that this bacterium contains 3 kinds of anti insect gene cry8Kb1, cry8Pa1, cry8Qa1, and its nucleotide sequence is respectively as shown in SEQ ID NO1, SEQ ID NO2, SEQ ID NO3.
The invention provides the microbial inoculum that contains bacillus thuringiensis ST8.
The application of the microbial inoculum that the invention provides bacillus thuringiensis ST8 or its tunning or contain bacillus thuringiensis ST8 in improving plant resistance to insect.
Research finds, bacterial strain of the present invention has higher insecticidal activity to coleopteron, wherein to the LC of Holotrichia parallela insecticidal activity
50Be 3.06 * 10
8Cfu/g is to the LC of holotrichia oblita
50Be 1.14 * 10
8Cfu/g.The invention provides bacillus thuringiensis ST8 or its tunning or contain the sterilant of this bacteria agent.
The application of the microbial inoculum that the invention provides bacillus thuringiensis ST8 or its tunning or contain this bacterium in the preparation sterilant.
Described sterilant is the sterilant that kills Coleoptera Scarabaeidae class insect.
The application of the microbial inoculum that the invention provides bacillus thuringiensis ST8 or its tunning or contain this bacterium in cultivating transgenic plant.
Described transgenic plant can be transgene cotton, oil crops, vegetables, flowers and/or grass.
The present invention also provides the application of the preparation take bacillus thuringiensis ST8 as effective constituent on agricultural.
Bacillus thuringiensis ST8 provided by the invention is a kind of newfound Bt resource, contain anti insect gene, has the activity of killing preferably Coleoptera Scarabaeidae insect, use it for the preparation transgenic plant, can the specific killing insect, and the usage quantity of reduction agricultural chemicals, reduce costs environmental contamination reduction.Also do not have at present insect or insect this bacterial strain to be produced the report of resistance, therefore, bacillus thuringiensis ST8 of the present invention has important economic worth and application prospect, is fit to large-scale application in the insect-resistance that improves plant, can promote the use of in agriculture production.
Description of drawings
Fig. 1 is circular parasporal crystal, the gemma (5000 *) of bacillus thuringiensis ST8 bacterial strain;
Fig. 2 is bacillus thuringiensis ST8 bacterial strain SDS-PAGE electrophoretic analysis, and wherein: M is protein Marker;
Fig. 3 is that in bacillus thuringiensis ST8 bacterial strain, the genotypic PCR of cry identifies, wherein: M is DNA Marker 100bp, and 1 is cry8 genoid amplified production;
Fig. 4 is the pcr amplification product of cry8 full-length gene in bacillus thuringiensis ST8, and wherein: M is DNA Marker 100bp, and 1 is the PCR product of cry8 full-length gene.
Embodiment
Following examples are used for explanation the present invention, but are not used for limiting the scope of the invention.Without departing from the spirit and substance of the case in the present invention, modification or replacement to the inventive method, step or condition are done all belong to scope of the present invention.
If do not specialize, in embodiment, chemical reagent used is conventional commercial reagent, the conventional means that in embodiment, technique means used is well known to those skilled in the art.
Soil sample used in the present embodiment is from the different plant root soil in various places.
Separation and Culture and the evaluation of embodiment 1 bacillus thuringiensis ST8 bacterial strain
(1) separation and Culture of bacterial strain
Soil picks up from area, Wenjiang, Chengdu, Sichuan Province.Adopt sodium-acetate-Antibiotics separation method, take the 10g soil sample and put into the shaking flask that 50ml sodium-acetate substratum is housed, add respectively each 400 μ g/ml of penicillin sodium salt and gentamicin sulphate, shaking table is cultivated (200r/min, 30 ℃) 4h.The earth suspension 10ml that fetches earth after cultivate finishing adds the aseptic centrifugal 15min of centrifuge tube 3000r/min, gets upper strata dirty solution 2ml in 65 ℃ of water-bath 15min, and the dirty solution 0.1ml after heat-obtaining is processed is coated with flat board, flat board is put in 30 ℃ of incubators cultivated.After 48h from the flat board the bacterial strain smear of the similar Bt of picking.Find that a strain contains the Bt bacterial strain of spherulite form (seeing accompanying drawing 1).Through using opticmicroscope and electron microscope observation, this strain cell is shaft-like, the blunt circle in two ends, the bacterial strain size is 1.2-1.5 μ m * 3.5-4.4 μ m, single or two or the existence of short chain cell usually, and a vegetative cell is a sporocyst, each sporocyst contains a gemma, inferior end is given birth to, and the other end has a parasporal crystal, and sporocyst does not expand.
(2) evaluation of bacterial strain
The SDS-PAGE electrophoresis shows, bacterial strain ST8 mainly produces the approximately albumen (seeing accompanying drawing 2) of 130KD left and right size.
5’-GTCCGAATAATCAGAATGAATATG-3’
5’-CGTTTTGCCTCTCTCACTGCATC-3’
Amplification system
Amplification condition
The amplification of cry8 gene:
The amplified reaction product is electrophoresis on 1% sepharose, puts and observes pcr amplification result (seeing accompanying drawing 3) in gel imaging system
Result shows that this bacterial strain belongs to bacillus thuringiensis.This bacillus thuringiensis ST8 has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (abbreviation CGMCC on June 12nd, 2010, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101), preserving number is CGMCC No.3923, and Classification And Nomenclature is bacillus thuringiensis (Bacillus thuringiensis).
Cry8Kb1, cry8Pa1 and cry8Qa1 gene cloning in embodiment 2 bacterial strain ST8
Adopt genomic dna purification kit (available from matching Parkson company) to extract total DNA of bacterial strain ST8; Design its full-length gene primer P1, P2 (primer sequence is as follows); Take the total DNA of bacterial strain ST8 as template, with P1 and P2 amplification cry8 full-length gene, obtain being about the fragment (Fig. 4) of 3.5kb.PCR product after purifying is connected with the pGEM-T carrier, transforms, the picking positive colony, order-checking obtains respectively sequence SEQ ID NO1, SEQ ID NO2 and SEQ ID NO3.
P1：5’ATGAGTCCAAATAATCAAAAT?3’
P2：5’TTACTCTACGTCAACAATCAAT?3’
The insecticidal activity of embodiment 3 bacterial strain ST8 detects
To lepidoptera pest: with bacterial strain ST8 in liquid LB substratum 30 ℃, 200r/min shaking culture 30h; Centrifugal collection thalline (12,000r/min, 15min, 4 ℃), Bechtop is air-dry, metering thalline weight; Then bacterial strain is suspended in distilled water, is mixed with 6 different concns from 1 μ g/ml to 100ng/ml; The old tender moderate Caulis et Folium Brassicae capitatae blade of choosing is cleaned, and dries; Shine 15min under ultraviolet lamp, be cut into 2 * 2cm
2Size divides to be placed in different concns bacterium liquid, soaks 5min; Taking-up drains unnecessary liquid, is placed in the culture dish of sterilization to dry, and soaks blade in contrast with the LB liquid nutrient medium, and each culture dish is put 4 blades; 30 of healthy 2-3 bollworms in age are put in each culture dish choosing; Every processing repeats 3 times, put indoor, in 3d " Invest, Then Investigate " larva death condition, with SPSS 10.0 computed in software LC
50Result such as table 1 show that bacterial strain is active without poisoning to this class pest.
To coleopteran pest: the Bt bacterial strain scrapes and is suspended in sterilized water after cultivating 3d on the LB solid medium, adopts the method for lactose suspension acetone precipitation, is prepared into pulvis standby.Above-mentioned pulvis is diluted according to 2 times of differential gradient concentrations of geometric ratio.With diluent, join mixing in the sterilization fine earth of even thickness cooking shredded potato, try the worm master with 5-7 age in days Holotrichia parallela and holotrichia oblita as confession, each processing connects 30 of worms, repeat 3 times,, infect and raise 7d, 14d and check dead borer population, with SPSS 10.0 computed in software LC as blank with the processing that adds clear water
50Result such as table 1 show that bacterial strain is to Holotrichia parallela and holotrichia oblita tool cytotoxicity.
The insecticidal activity of table 1 ST8
Claims (7)
1. bacillus thuringiensis (Bacillus thuringiensis) bacterial strain ST8, its deposit number is CGMCC No.3923.
2. the microbial inoculum that contains the described bacillus thuringiensis ST8 of claim 1.
3. bacillus thuringiensis ST8 claimed in claim 1 or its tunning or microbial inoculum claimed in claim 2 application in improving plant resistance to insect.
4. the sterilant that contains bacillus thuringiensis ST8 claimed in claim 1 or its tunning or microbial inoculum claimed in claim 2.
5. sterilant as claimed in claim 4, is characterized in that, described sterilant is the sterilant that kills Coleoptera Scarabaeidae class insect.
6. bacillus thuringiensis ST8 claimed in claim 1 or its tunning or microbial inoculum claimed in claim 2 application in the preparation sterilant.
7. application as claimed in claim 6, is characterized in that, described sterilant is the sterilant that kills Coleoptera Scarabaeidae class insect.
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| CN101113424A (en) * | 2007-07-04 | 2008-01-30 | 中国农业科学院植物保护研究所 | Coleoptera pest efficient Bacillus thuringiensis cry8G gene, protein and uses thereof |
| CN101130762A (en) * | 2007-08-07 | 2008-02-27 | 中国农业科学院植物保护研究所 | Efficient bacillus thuringiensis cry8H gene, protein for vaginata destructive insect and uses of the same |
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| CN101113424A (en) * | 2007-07-04 | 2008-01-30 | 中国农业科学院植物保护研究所 | Coleoptera pest efficient Bacillus thuringiensis cry8G gene, protein and uses thereof |
| CN101130762A (en) * | 2007-08-07 | 2008-02-27 | 中国农业科学院植物保护研究所 | Efficient bacillus thuringiensis cry8H gene, protein for vaginata destructive insect and uses of the same |
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| Title |
|---|
| Changlong Shu et al.Charaterization of a novel cry8 gene specific to Melolonthidae pests: Holotrichia oblita and holotrichia parallela.《Appl Microbiol Biotechnol》.2009,第84卷(第4期), |
| Characterization of Bacillus thuringiensis strain Bt185 toxic to the Asian cockchafer: Holotrichia parallela;Hong Yu et al;《Curr Microbiol》;20060609;第53卷(第1期);13-17 * |
| Charaterization of a novel cry8 gene specific to Melolonthidae pests: Holotrichia oblita and holotrichia parallela;Changlong Shu et al;《Appl Microbiol Biotechnol》;20090428;第84卷(第4期);701-707 * |
| Hong Yu et al.Characterization of Bacillus thuringiensis strain Bt185 toxic to the Asian cockchafer: Holotrichia parallela.《Curr Microbiol》.2006,第53卷(第1期), |
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