CN102329760B - New bacterial strain of Bacillus thuringiensis for killing grub pest and pest killing protein thereof - Google Patents
New bacterial strain of Bacillus thuringiensis for killing grub pest and pest killing protein thereof Download PDFInfo
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- CN102329760B CN102329760B CN 201110318767 CN201110318767A CN102329760B CN 102329760 B CN102329760 B CN 102329760B CN 201110318767 CN201110318767 CN 201110318767 CN 201110318767 A CN201110318767 A CN 201110318767A CN 102329760 B CN102329760 B CN 102329760B
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Abstract
The invention relates to a new bacterial strain of Bacillus thuringiensis for killing grub pest and a pest killing protein thereof. The new bacterial strain has the collection number of CCTCC (China Center For Type Culture Collection) No. M2011267. The bacterial strain and the pest killing protein provided by the invention can be used for preparing the preparation capable of preventing and controlling grub pests. The bacterial strain provided by the invention has specific insecticidal activity on the grub pests. The pest killing crystal protein provided by the invention has good prevention and control effect on main soil insects such as Holotrichia oblita and Holotrichia parallela in China.
Description
Technical field
The invention belongs to the biological control of insect pests technical field, be specifically related to a kind of new bacterial strain of bacillus thuringiensis and insecticidal proteins thereof that kills the grub category insect, i.e. the new bacterial strain B-Y7-1 of bacillus thuringiensis and insecticidal proteins thereof.
Background technology
Grub is the common name of Coleoptera scarab beetle Superfamily larva, and the plants such as various farm crop, fruit tree, forest and herbage of causing harm are most species in the subterranean pest-insect, the widest and the heaviest large monoid of causing harm that distributes, and also are dwelling property of the soil insects that generally acknowledged both at home and abroad difficulty is prevented and treated.What statistics according to investigations, foot end were injured 86% is caused by grub harm.Approximately 100,000,000 mu of annual national grub generation areas, the serious time once reached 300,000,000 2 thousand ten thousand mu, and production loss reaches 20%～40%, and the whole nation is injured the most serious especially with Huanghuai Area.The grub kind that China has put down in writing has more than 100 to plant, wherein common are more than 30 and plant, wherein occur general and serious with holotrichia oblita (Holotrichia oblita Faldermann), Holotrichia parallela (Holotrichia parallela Motschulsky), anomala corpulenta (Anomala corpulenta Motschlsky) etc.Chemical pesticide has been brought into play certain effect in the control of grub, but chemical pesticide easily causes serious environmental pollution and the destruction of the eubiosis, the simultaneously residual and accumulation of chemical pesticide in food not only is detrimental to health, and is unfavorable for the Sustainable development of society.And that biological control has is free from environmental pollution, person poultry safety and insect are difficult for producing the advantages such as resistance, and therefore, developing new biological prevention is current integrated pest management problem demanding prompt solution.
Bacillus thuringiensis (Bacillus thuringiensis, be called for short Bt) be extremely widely gram positive bacterium of a kind of distribution, it is when forming gemma, can produce the parasporal crystal that various insects, mite class and protozoon etc. is had the property of protein of specific activity, because its insecticidal crystal protein (Insecticidal crystal proteins, be called for short ICPs) efficiently single-minded to target pest, to non-target organism safety and environmentally safe.Therefore, Bt has been widely used in the insect of the aspects such as control agricultural, warehousing and health, is the microbial pesticide that output is maximum both at home and abroad at present, range of application is the widest, and its insecticidal crystal protein also is to be most widely used at present and the pest-resistant albumen of a most potential class.Before the eighties in 20th century, about the research of Bt with use in the control that mostly concentrates on lepidoptera pest, after the screening such as Ohba (1992) has obtained the new bacterial strain Buibui of Bt that a strain has special insecticidal activity to multiple chafer larva, in recent years the research and development of the activated Bt bacterial strain of cockchafer subclass larva tool caused people's concern.At present in succession separate in the world some and killed the Bt bacterial strain of chafer class pest, separated the Bt strain isolated HBF-1 that Anomala exoleta (Anomala exoleta) and anomala corpulenta etc. is had insecticidal activity such as (2000) such as Feng Shuliang, Yu etc. (2006) have separated the activated bacterial strain Btl85 of Holotrichia parallela tool.But the Bt bacterial strain of present successful control of grubs class pest and products thereof quantity is also relatively less, therefore, screening and separating goes out to have the more bacillus thuringiensis of efficient insecticide effect, excavates the insecticidal proteins of novel, efficient and wide spectrum, and is significant for the control of grub category subterranean pest-insect.
Summary of the invention
The purpose of this invention is to provide a kind of new bacterial strain of bacillus thuringiensis and insecticidal proteins thereof that kills the grub category insect, i.e. bacillus thuringiensis bacterial strain B-Y7-1, and insecticidal crystal protein is to remedy the deficiencies in the prior art.
One aspect of the present invention relates to the new bacterial strain Bacillus of bacillus thuringiensis thuringiensisB-Y7-1, and it is preserved in Chinese Typical Representative culture collection center on July 26th, 2011, and deposit number is CCTCC No.M2011267.
The bacterial strain of the present invention's screening is for the preparation of the active bacteria formulation of control of grubs class pest.
Another aspect of the present invention relates to a kind of insecticidal crystal protein that separates from new bacterial strain, the aminoacid sequence SEQ ID NO:1 of its albumen.
Insecticidal proteins of the present invention is for the preparation of the preparation of control of grubs class pest.
Strains separation of the present invention is from Yantai Area of shandong Province soil, the grub category insect had the specificity insecticidal activity, its insecticidal crystal protein is different from domestic and international report, effective object is not identical yet, and the main subterranean pest-insect holotrichia oblita of China and Holotrichia parallela are all had preferably prevention effect.
Description of drawings
Fig. 1: bacillus thuringiensis bacterial strain B-Y7-1 insecticidal crystal protein SDS-PAGE figure of the present invention,
Wherein swimming lane 1 is the molecular weight of albumen standard, and swimming lane 2 is B-Y7-1.
Embodiment
Separation and the biological characteristics of embodiment 1 bacillus thuringiensis bacterial strain B-Y7-1:
1.1 soil sample source
Soil sample is collected in the various places, Shandong Province, selects the soil sampling of the bare places such as wild grass ground, forest land, gets approximately 50g of the following 5～10em soil in earth's surface, pack sealing, numbering and registration collecting location.
1.2 substratum
Strains separation adopts NGKG selective medium (NISSUI company, Japan), 3.975g NGKG substratum adds 135mL distilled water, sterilization 20min, add egg yolk liquid 15mL (3mL egg yolk liquid+12mL 0.85% physiological saline) after being cooled to 50 ℃, be down flat plate.Strain culturing adopt the LB substratum (NaCl 1% for peptone 1%, yeast powder 0.5%, pH7.0) or beef-protein medium (pH7.0), solid medium adds 1.3%～1.5% agar for extractum carnis 0.3%, peptone 0.5%.
1.3Bt the separation of bacterial strain
The 1g soil sample is suspended in the 10mL sterilized water, and then concuss 20min leaves standstill 5min.With 100 times of upper strata suspension dilutions, get 80 μ L and be applied on the NGKG selective medium flat board, be inverted for 30 ℃ and cultivate 72h.Choose outward appearance like the single bacterium colony of genus bacillus with transfering loop, after the dyeing of PHENOL 99.8 MIN ((CARBOLIC ACID)) azaleine, microscopy has or not parasporal crystal to produce.With microscopy to the bacterium colony streak inoculation that parasporal crystal is arranged behind LB plate culture medium purifying, obtain bacillus thuringiensis bacterial strain.
1.4Bt the cultivation of bacterial strain and preservation
With single colony inoculation on LB or beef-protein medium inclined-plane, 30 ℃ cultivate 3～4d after, place 4 ℃ of refrigerators, but short-term preservation three months is to half a year.Maybe will cultivate the gemma that produces behind 3～4d and make spore suspension through aseptic technique, get 100 μ L and add in the sandy soil pipe of sterilizing, the rearmounted vacuum drier inner drying of abundant mixing, the mouth of pipe seals with paraffin, be put in the container that fills the calcium chloride siccative, but prolonged preservation.During use, from the switching of original preservation of bacteria strain, be inoculated on LB or the beef-protein medium inclined-plane and activate.
1.5Bt the biological characteristics of bacterial strain B-Y7-1
Bt bacterial strain B-Y7-1 separates from Yantai Area of shandong Province, and rounded, the wax shape of bacterium colony, edge are rough on LB or beef-protein medium, all can grow between 15～40 ℃, and optimum growth temperature is 27～32 ℃.It is shaft-like that nourishing body is, and size is 3.2～5.2 μ m * 1.2～2.0 μ m, single giving birth to or the one-tenth chain, Gram-positive, the paramethyl red positive reaction, acid is produced in acetyl methyl carbinol (V-P reaction) positive reaction in glucose, fructose, maltose and Zulkovsky starch.The brood cell's capsule that forms is long oval, slightly expands than nourishing body; Discharge free gemma and parasporal crystal after brood cell's capsule breaks, gemma is oval, and size is 2.0 μ m * 0.8 μ m, and parasporal crystal is closely square, differs in size, and the parasporal crystal protein molecular weight is 130kDa.
Preparation and the biological activity determination of embodiment 2Bt bacterial strain B-Y7-1 bacterium liquid
2.1 sample preparation
The single bacterium colony of picking bacterial strain B-Y7-1, be inoculated in the LB liquid nutrient medium of 5mL, 30 ℃ of 200r/min shaking culture 16～17h, get in the LB liquid nutrient medium that the 1mL nutrient solution is inoculated in 100mL, 30 ℃ of 200r/min shaking culture are 72h approximately, test under microscope, after gemma and crystal separation, the centrifugal 10min abandoning supernatant of 10000r/min with the thalline that sterilized water suspends and collects, is made the bacterium liquid formulation for subsequent use.
2.2 biological activity determination
Do contrast with sterilized water, 5mL bacterium liquid and 10g feed (thin cooking shredded potato) and 90g sterilization soil are mixed, put into diameter and be 12cm and highly be the plastics casing of 9cm, every box accesses 5 of the grubs of 3 ages in days, each processes 6 boxes (totally 30 cephalonts), repeats 3 times, places under the room temperature and raises, check dead worm and the borer population of living respectively at 7d and 14d, the calculation correction mortality ratio.The result shows, Bt bacterial strain B-Y7-1 all has higher insecticidal activity to holotrichia oblita and Holotrichia parallela larva, when concentration is 6.7 μ g/g (protein content with soil mass ratio), the insecticidal effect of holotrichia oblita and Holotrichia parallela larva 14d is reached respectively 86.7% and 76.7%.
The biological activity of embodiment 3Bt bacterial strain B-Y7-1 parasporal crystal protein
3.1 the preparation of protein sample
Choose the single bacterium colony of bacterial strain B-Y7-1, be inoculated in the LB liquid nutrient medium, 30 ℃ of 200r/min cultivate approximately 3～4d, after gemma and crystal separation, and the purification parasporal crystal protein, the sequence that records is SEQ ID NO:1.With Bradford determination of protein concentration kit measurement protein concentration, protein sample is stored in-20 ℃, for biological activity determination behind the albumen alkaline hydrolysis.
3.2 the biological activity of parasporal crystal protein
The parasporal crystal protein of purifying is diluted to respectively different concentration, respectively the protein solution of 5mL different concns and 10g feed (thin cooking shredded potato) and 90g sterilization soil are mixed, put into diameter and be 12cm and highly be the plastics casing of 9cm, every box accesses 5 of the grubs of 3 ages in days, each processes 6 boxes (totally 30 cephalonts), repeats 3 times, places under the room temperature and raises, check dead worm and the borer population of living respectively at 7d and 14d, calculate median lethal concentration(LC﹠-{50}) (LC
50).Determination of activity the results are shown in Table 1.
Table 1 parasporal crystal protein is to the biological activity determination of holotrichia oblita and Holotrichia parallela
The result shows, parasporal crystal protein of the present invention all has preferably insecticidal effect to holotrichia oblita and Holotrichia parallela, to the LC of holotrichia oblita 14d
50Be 5.7 μ g/g, to the LC of Holotrichia parallela 14d
50Be 2.0 μ g/g.Therefore, the new bacterial strain of the bacillus thuringiensis that the present invention filters out and insecticidal proteins thereof have obvious insecticidal effect, have broad application prospects.
Claims (2)
1. a bacillus thuringiensis (Bacillus thuringiensis) B-Y7-1 bacterial strain, its deposit number is CCTCC No. M2011267.
2. bacteria preparation includes the viable bacteria of the described bacterial strain of claim 1.
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| CN106116877A (en) * | 2016-06-29 | 2016-11-16 | 固镇县华丰蔬菜专业合作社 | A kind of microbial manure preventing and treating Cucurbita pepo L. Holotrichia diomphalia Bates insect pest |
| CN106905421B (en) * | 2017-04-06 | 2020-10-09 | 中国农业科学院植物保护研究所 | Novel insecticidal protein and nucleotide sequence thereof |
| CN107912448B (en) * | 2017-12-04 | 2021-04-02 | 青岛农业大学 | Grub-killing bacillus thuringiensis microcapsule suspending agent and preparation method thereof |
| CN111235088B (en) * | 2020-03-15 | 2022-07-26 | 青岛农业大学 | Holotrichia parallela embryonic cell line and application thereof |
| CN113337448B (en) * | 2021-07-27 | 2022-04-19 | 中国农业科学院植物保护研究所 | Citrobacter and application thereof in preventing and treating scarab beetles |
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| US5554534A (en) * | 1991-12-16 | 1996-09-10 | Mycogen Corporation | Bacillus thuringiensis toxins active against scarab pests |
| US5185148A (en) * | 1991-12-16 | 1993-02-09 | Mycogen Corporation | Process for controlling scarab pests with Bacillus thuringiensis isolates |
| CN1181203C (en) * | 2001-08-20 | 2004-12-22 | 中国农业科学院植物保护研究所 | Bt gene with high toxicity to Lepidoptera and Coleoptera insects, expression vector and engineering bacteria |
| CN100532555C (en) * | 2004-11-16 | 2009-08-26 | 中国农业科学院植物保护研究所 | Highly efficient cry8F gene for Coleoptera pest, expression protein and application thereof |
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| CN101050449A (en) * | 2007-03-16 | 2007-10-10 | 中国农业科学院植物保护研究所 | Engineering bacterium UV173A of Bacillus thuringiensis, preparation method and application |
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