CN102329760A

CN102329760A - New bacterial strain of Bacillus thuringiensis for killing grub pest and pest killing protein thereof

Info

Publication number: CN102329760A
Application number: CN 201110318767
Authority: CN
Inventors: 李长友; 郑桂玲; 耿以龙; 周洪旭; 张媛; 初立良
Original assignee: Qingdao Agricultural University
Current assignee: Qingdao Agricultural University
Priority date: 2011-10-19
Filing date: 2011-10-19
Publication date: 2012-01-25
Anticipated expiration: 2031-10-19
Also published as: CN102329760B

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

Kill new bacterial strain of bacillus thuringiensis and the insecticidal proteins thereof of grub category insect

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. 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 big 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.About 100,000,000 mu of area takes place in annual national grub, and 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 the Yellow River and Huai He River basin.Kind surplus the grub kind that China has put down in writing has 100; Wherein common have 30 surplus kind, wherein take place 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 function in the control of grub; But chemical pesticide is prone to cause serious environmental to pollute and ecological balance damage; Residual and the accumulation of chemical pesticide in food simultaneously 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 advantages such as resistance, and therefore, developing new biological control technology is current insect comprehensive regulation problem demanding prompt solution.

Bacillus thuringiensis (Bacillus thuringiensis; Be called for short Bt) be a kind of distribution gram positive bacterium extremely widely; It can produce the parasporal crystal that various insects, mite class and protozoon etc. is had the property of protein of specific activity when forming gemma, 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 aspects such as control agricultural, warehousing and health, is the microbial pesticide that at present domestic and international output is maximum, range of application is the widest, and its insecticidal crystal protein also is to use at present the most extensively and the most potential one type of pest-resistant albumen.Before the eighties in 20th century; About the research of Bt with use and concentrate on mostly in the control to lepidoptera pest; After screening such as Ohba (1992) had obtained the new bacterial strain Buibui of Bt that a strain has special insecticidal activity to multiple chafer larva, the research and development that in recent years cockchafer subclass larva is had an active Bt bacterial strain had caused people's attention.At present separate some in the world in succession and killed the Bt bacterial strain of chafer class pest; Separated the Bt strain isolated HBF-1 that yellowish-brown rutelian (Anomala exoleta) and anomala corpulenta etc. is had insecticidal activity like (2000) such as Feng Shuliang, Yu etc. (2006) have separated Holotrichia parallela have been had active bacterial strain Btl85.But the Bt bacterial strain of present successful control of grubs class pest and products thereof quantity is also less relatively; 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 deficiency of 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 culture collection center on July 26th, 2011, and deposit number is CCTCC No.M2011267.

The bacterial strain of the present invention screening is used to prepare the active bacteria formulation of control of grubs class pest.

Another aspect of the present invention relate to a kind of from new bacterial strain the isolated pesticidal crystallin, its proteic aminoacid sequence SEQ ID NO:1.

Insecticidal proteins of the present invention is used to prepare the preparation of control of grubs class pest.

Strains separation of the present invention is soil from area, Yantai, Shandong Province; The grub category insect had the specificity insecticidal activity; Its insecticidal crystal protein is different with domestic and international report; Effective object is also inequality, and main subterranean pest-insect holotrichia oblita of China and Holotrichia parallela are all had the better 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 bare places such as wild grass ground, forest land, gets the about 50g of 5～10em soil below the face of land, and pack is sealed, numbering and registration collecting location.

1.2 substratum

Strains separation adopts NGKG selective medium (NISSUI company, Japan), and 3.975g NGKG substratum adds 135mL zero(ppm) water, and sterilization 20min adds egg yolk liquid 15mL (3mL egg yolk liquid+12mL 0.85% saline water) after being cooled to 50 ℃, falls dull and stereotyped.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 Carnis Bovis seu Bubali cream 0.3%, peptone 0.5%.

1.3Bt the separation of bacterial strain

The 1g soil sample is suspended in the 10mL sterilized water, and concuss 20min leaves standstill 5min then.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 the single bacterium colony of outward appearance like 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

Single colony inoculation on LB or beef-protein medium inclined-plane, behind 30 ℃ of cultivation 3～4d, place 4 ℃ of refrigerators, but short-term is preserved three months to half a year.Maybe will cultivate the gemma that produces behind 3～4d and process 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,, be inoculated on LB or the beef-protein medium inclined-plane and carry out activation from original preservation bacterial classification switching.

1.5Bt the biological characteristics of bacterial strain B-Y7-1

Bt bacterial strain B-Y7-1 separates from area, Yantai, Shandong Province, and rounded, the wax shape of bacterium colony, edge are rough on LB or beef-protein medium, between 15～40 ℃, all can grow, 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, and Gram-positive, the paramethyl red positive reaction, acid is produced in acetyl methyl carbinol (V-P reaction) positive reaction in glucose, fructose, SANMALT-S 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.

The preparation and the biological activity determination of embodiment 2Bt bacterial strain B-Y7-1 bacterium liquid

2.1 specimen preparation

The single bacterium colony of picking bacterial strain B-Y7-1 is 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 about 72h of 200r/min shaking culture, test under microscope; After treating gemma and crystal separation; The centrifugal 10min abandoning supernatant of 10000r/min, with the thalline that sterilized water suspends and collects, it is subsequent use to process the bacterium liquid formulation.

2.2 biological activity determination

Do contrast with sterilized water, 5mL bacterium liquid and 10g feed (fine earth beans silk) and 90g sterilization soil are mixed, put into diameter and be 12cm and highly be the plastics casing of 9cm; Every box inserts 5 of the grubs of 3 ages in days; Each handles 6 boxes (totally 30 cephalonts), repeats 3 times, places under the room temperature and raises; Check dead worm and the borer population of living, calculation correction mortality ratio respectively at 7d and 14d.The result shows; Black gill cockchafer of Bt bacterial strain B-Y7-1 Beijing University to China and Holotrichia parallela larva all have higher insecticidal activity; When concentration was 6.7 μ g/g (protein content and native mass ratio), the insecticidal effect of black gill cockchafer of Beijing University to China and Holotrichia parallela larva 14d reached 86.7% and 76.7% respectively.

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 about 3～4d, treat gemma and crystal separation after, 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 ℃ behind the albumen alkaline hydrolysis, supplies biological activity determination to use.

3.2 the biological activity of parasporal crystal protein

The parasporal crystal protein of purifying is diluted to different concentration respectively, respectively the protein solution of 5mL different concns and 10g feed (fine earth beans silk) and 90g sterilization soil is mixed, put into diameter and be 12cm and highly be the plastics casing of 9cm; Every box inserts 5 of the grubs of 3 ages in days; Each handles 6 boxes (totally 30 cephalonts), repeats 3 times, places under the room temperature and raises; Check the dead worm and the borer population of living respectively at 7d and 14d, calculate median lethal concentration(LC&-{50}) (LC ₅₀).The result of determination of activity sees table 1.

The biological activity determination of black gill cockchafer of table 1 parasporal crystal protein Beijing University to China and Holotrichia parallela

The result shows that black gill cockchafer of parasporal crystal protein of the present invention Beijing University to China and Holotrichia parallela all have insecticidal effect preferably, the LC of the black gill cockchafer 14d of Beijing University to China ₅₀Be 5.7 μ g/g, to the LC of Holotrichia parallela 14d ₅₀Be 2.0 μ g/g.Therefore, new bacterial strain of the bacillus thuringiensis that the present invention filters out and insecticidal proteins thereof have tangible insecticidal effect, have broad application prospects.

Claims

1. A novel strain of Bacillus thuringiensis, whose preservation number is CCTCC No.M2011267.

2. A bacterial preparation comprising live bacteria of the strain described in claim 1.

3. from the insecticidal crystal protein isolated from the bacterial strain described in claim 1, the amino acid sequence of its protein is SEQ ID NO:1.

4. A preparation for controlling grub class pests, comprising the insecticidal crystal protein according to claim 3.