CN102363760A - 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 prodn loss and influence human health, adds up according to FAO, and the financial loss that whole world agriculture prodn every year causes because of insect pest is up to 14%, and disease is with a toll of 12%, and crop smothering is with a toll of 11%.The amount of loss is equivalent to the half the of the Chinese agriculture gross output value, more than 4 times of Britain up to 1,260 hundred million dollars.The agriculture insect mainly concentrates among lepidopteran and the Homoptera, and part insect in the Coleoptera scarab beetle Superfamily is like grub; Also be important worldwide distribution subterranean pest-insect, endanger 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 influence quality.A large amount of investigation show that grub is caused harm and ranks first in subterranean pest-insect, account 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 is called for short Bt) is a kind of gram positive bacterium, and its distribution is very extensive;, gemma can form the parasporal crystal of forming by protein when forming with insecticidal activity; 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 insects such as multiple lepidopteran, Diptera, Coleoptera.In addition, Bt also has the effect of control evil to 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 in the control of agricultural pests, injurious forest-insect and sanitary insect pest, and Bt still be that transgenic pest-resistant engineered plant important function of gene is originated.
From strain HD-1Dipel, cloned 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, they are confirmed as different crowd, subgroup, class and subclass respectively according to the amino acid sequence coded homology.The Bt gene of finding so far that grub is had an insecticidal activity mainly contains genoid (Shu C. such as cry3, cry8, cry18, cry23, cry37, cry43; Et al.2009; Characterization of two novel cry8 genes from Bacillus thuringiensis strain BT185.Current Microbiology; 58 (4): 389-392.); Wherein studying maximum is the cry8 genoid; The cry8 genoid is made up of 1160-1210 amino acid, and molecular weight is 128-137kDa, and multiple coleopteran pests such as Scarabaeidae, Culculionidae, Chrysomelidae are had 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., et al.1994.Cloning in 1994; 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 tangible insecticidal activity (US Patent 5554534) (Tracy E.Michaels from isolating Cry8Aa1 of Bt bacterial strain PS50C and Cry8Ba1 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 screening successively 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 yellowish-brown rutelian (A.exoleta) and anomala corpulenta (A.corpulenta); The indoor biometrics mortality ratio all reach 100% (.2000. one strain such as Feng Shuliang has insecticidal activity to cockchafer subclass larva the new strain isolated of Bacillus thuringiensis. 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, make up and insect-resistant transgenic plants all has important significance for theories and using value for gene clone, engineering bacteria.
Find the history in existing so far more than 100 year of Tribactur from the beginning of this century, aspect the preventing and treating of farm crop and gardening plant insect, injurious forest-insect and sanitary insect pest, be widely used, also play good effect.But the inevitable outcome that makes single Bt insecticidal proteins continually 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 mainly is 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 receive the selective pressure of sterilant for a long time.1985, Mc Gaughey report warehouse grain pest Indian meal moth (Plodia interpunctella) under the selective pressure of Dipel (the commodity preparation of Bt subsp.kurstaik HD-1), bred for 15 generations after, 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 big Tanaka in Hawaii has first produced tangible 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,, found that the Bt sterilant obviously descends to the small cabbage moth control effect on China Application of B t sterilant time long Shenzhen and Guangzhou, Shanghai and other places; Mean resistance form (Feng Xia .1996. Guangdong small cabbage moth is to the resistance research of Bacillus thuringiensis. 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 at present in the laboratory and the field has at least tens kinds of insects that Bt and insecticidal crystal protein thereof have been produced resistance; 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 crucial 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 common micro-organisms center on June 12nd, 2010; Institute of Microorganism, Academia Sinica; Postcode 100101) preservation, classification called after bacillus thuringiensis (Bacillus thuringiensis), 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 usually or two or short chain cell exist, 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 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.
Discover that 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, the LC of the black gill cockchafer of Beijing University to China
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 for killing the sterilant of 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.
It is the application of preparation on agricultural of effective constituent that the present invention also provides with bacillus thuringiensis ST8.
Bacillus thuringiensis ST8 provided by the invention is a kind of newfound Bt resource; Contain anti insect gene, have the activity of killing Coleoptera Scarabaeidae insect preferably, use it for the preparation transgenic plant; Can the specific killing insect; And the usage quantity of reduction agricultural chemicals, reduce cost, reduce environmental pollution.Also there are not 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 value and application prospect, is fit to large-scale application in the insect-resistance that improves plant, can in agriculture prodn, promote the use of.
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 the genotypic PCR of cry identifies that wherein: M is DNA Marker 100bp in the bacillus thuringiensis ST8 bacterial strain, and 1 is cry8 genoid amplified production;
Fig. 4 is the pcr amplification product of cry8 full-length gene among the 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 to explain the present invention, but are not used for limiting scope of the present invention.Under the situation that does not deviate from the present invention's spirit and essence, modification or replacement to the inventive method, step or condition are done all belong to scope of the present invention.
If do not specialize, used chemical reagent is conventional commercial reagent among the embodiment, the conventional means that used technique means is well known to those skilled in the art among the embodiment.
Used soil sample in the present embodiment is from various places different plant foundation soil.
The 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-microbiotic partition method, take by weighing the 10g soil sample and put into the bottle that shakes that 50ml sodium-acetate substratum is housed, add each 400 μ g/ml of penicillin sodium salt and GT respectively, 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 the muddy liquid 2ml in upper strata in 65 ℃ of water-bath 15min, and the muddy liquid 0.1ml after the heat-obtaining processing is coated with flat board, flat board is put in 30 ℃ of incubators cultivated.Behind the 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).Warp is with opticmicroscope and electron microscope observation, and 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 usually or two or the existence of short chain cell, 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 that bacterial strain ST8 mainly produces the albumen (seeing accompanying drawing 2) of size about about 130KD.
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 the gel imaging system
The result shows that this bacterial strain belongs to bacillus thuringiensis.This bacillus thuringiensis ST8 is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on June 12nd, 2010 and (is called for short CGMCC; 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, classification called after bacillus thuringiensis (Bacillus thuringiensis).
The clone of cry8Kb1, cry8Pa1 and cry8Qa1 gene among the embodiment 2 bacterial strain ST8
Adopt genomic dna purification kit (available from match Parkson company) to extract total DNA of bacterial strain ST8; Design its full-length gene primer P1, P2 (primer sequence is following); With the total DNA of bacterial strain ST8 is template, with P1 and P2 amplification cry8 full-length gene, obtains being about the fragment (Fig. 4) of 3.5kb.PCR product behind the purifying is connected with the pGEM-T carrier, transforms, the picking positive colony, order-checking obtains sequence SEQ ID NO1, SEQ ID NO2 and SEQ ID NO3 respectively.
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; Be suspended in bacterial strain in the zero(ppm) water then, be 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; Uv lamp is irradiation 15min down, is cut into 2 * 2cm
2Size divides to be placed in the different concns bacterium liquid, soaks 5min; Take out drop and go excess liquid, be placed in the disinfectant petridish and dry, soak blade as contrast with the LB liquid nutrient medium, each petridish is put 4 blades; 30 of healthy 2-3 bollworms in age are put in each petridish choosing; Every processing repetition 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, it is active to show that bacterial strain does not have poisoning to this class pest.
To coleopteran pest: the Bt bacterial strain scrapes and is suspended in the sterilized water after cultivating 3d on the LB solid medium, adopts the method for lactose suspension acetone precipitation, and it is subsequent use to be prepared into pulvis.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 potato silk, as supplying examination worm master, each processing connects 30 of worms with 5-7 age in days Holotrichia parallela and holotrichia oblita; Repeat 3 times; With the processing that adds clear water as blank, infect raise 7d, 14d checks dead borer population, with SPSS 10.0 computed in software LC
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 (10)
1. bacillus thuringiensis (Bacillus thuringiensis) bacterial strain ST8, its deposit number is CGMCC No.3923.
2. bacillus thuringiensis ST8 according to claim 1 is characterized in that contain 3 kinds of anti insect gene cry8Kb1, cry8Pa1, cry8Qa1, its nucleotide sequence is respectively shown in SEQ ID NO1, SEQ ID NO2, SEQ ID NO3.
3. the microbial inoculum that contains the said bacillus thuringiensis ST8 of claim 1.
4. the described bacillus thuringiensis ST8 of claim 1 or its tunning or the described microbial inoculum of claim 3 application in improving plant resistance to insect.
5. the sterilant that contains the described bacillus thuringiensis ST8 of claim 1 or its tunning or the described microbial inoculum of claim 3.
6. the described bacillus thuringiensis ST8 of claim 1 or its tunning or the described microbial inoculum of claim 3 application in the preparation sterilant.
7. sterilant as claimed in claim 5 or the described application of claim 6 is characterized in that, described sterilant is for killing the sterilant of Coleoptera Scarabaeidae class insect.
8. the described bacillus thuringiensis ST8 of claim 1 or its tunning or the described microbial inoculum of claim 3 application in the preparation transgenic plant.
9. application as claimed in claim 8 is characterized in that described plant comprises cotton, oil crops, vegetables, flowers and/or grass.
10. the application of preparation on agricultural that is effective constituent with the said bacillus thuringiensis ST8 of claim 1.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103396977A (en) * | 2013-07-25 | 2013-11-20 | 中国科学院武汉病毒研究所 | Bacillus thuringiensis engineering bacterium for killing coleopteran pests as well as preparation method and application thereof |
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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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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Non-Patent Citations (2)
| Title |
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| CHANGLONG SHU ET AL: "Charaterization of a novel cry8 gene specific to Melolonthidae pests: Holotrichia oblita and holotrichia parallela", 《APPL MICROBIOL BIOTECHNOL》, vol. 84, no. 4, 28 April 2009 (2009-04-28), XP019737712, DOI: doi:10.1007/s00253-009-1971-2 * |
| HONG YU ET AL: "Characterization of Bacillus thuringiensis strain Bt185 toxic to the Asian cockchafer: Holotrichia parallela", 《CURR MICROBIOL》, vol. 53, no. 1, 9 June 2006 (2006-06-09), XP019423416, DOI: doi:10.1007/s00284-005-0097-8 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103396977A (en) * | 2013-07-25 | 2013-11-20 | 中国科学院武汉病毒研究所 | Bacillus thuringiensis engineering bacterium for killing coleopteran pests as well as preparation method and application thereof |
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