CN109810920A

CN109810920A - One thuringiensis strain bacillus and its application

Info

Publication number: CN109810920A
Application number: CN201910094086.1A
Authority: CN
Inventors: 丁学知; 刘卓林; 谢俊雁; 夏立秋; 孙运军; 胡胜标; 余子全; 黄伟涛; 胡益波
Original assignee: Hunan Normal University
Current assignee: Hunan Normal University
Priority date: 2019-01-30
Filing date: 2019-01-30
Publication date: 2019-05-28
Anticipated expiration: 2039-01-30
Also published as: CN109810920B

Abstract

One thuringiensis strain bacillus and its application, the thuringiensis of the present invention are thuringiensis X023,Bacillus thuringiensisX023, for the strain on May 16th, 2018 in China typical culture collection center preservation, culture presevation number is CCTCC NO:M 2018283.The invention also includes the application of thuringiensis, thuringiensis zymocyte liquid has insecticidal activity to bollworm and diamondback moth.

Description

One thuringiensis strain bacillus and its application

Technical field

It is right the present invention relates to a thuringiensis strain bacillus (Bacillus thuringiensis X023) and its application One plant height insecticidal toxicity wild type thuringiensis fermentation liquid of screening carries out biological characteristis.

Background technique

The insecticidal crystal protein (ICP) that thuringiensis (Bacillus thuringiensis, Bt) generates is one Kind Eco-friendly biological pesticide has efficiently specificity, is one of most widely used microbial insecticide.Su Yunjin at present Bacillus (Bt) insecticidal crystalline gene is successfully transferred in plant, as transformed variety increases and insecticidal preparation is applied With insect will inevitably enhance the resistance of insecticidal crystal protein.Constantly separate some new serotypes and height Virulence Bt new strains are then to solve the problems, such as most important most viable one of the method at present.

Earlier studies have shown that: Bt insecticidal crystal protein (ICP) is formed together with brood cell, therefore they are also designated as companion cell Crystal, it means that insecticidal crystal protein has certain to be associated with the formation of brood cell.There are several factors to will affect the generation of ICP, no Same insecticidal crystal protein desinsection toxicity has certain difference and has special insecticidal spectrum.

Summary of the invention

The technical problem to be solved by the present invention is to, overcome the deficiencies of the prior art and provide a thuringiensis strain bacillus and It is applied, and effectively carries out biological control to diamondback moth and bollworm.

The technical solution used to solve the technical problems of the present invention is that the thuringiensis of the present invention, is Su Yunjin Bacillus X023 (Bacillus thuringiensis X023, abbreviation Bt X023), which existed on May 16th, 2018 China typical culture collection center (abbreviation CCTCC, address: Wuhan, China Wuhan University) preservation, culture presevation number are CCTCC NO:M 2018283.

The thuringiensis 16S rRNA sequence of the present invention are as follows:

TACGGCTACCTTGTTACGACTTCACCCCAATCATCTGTCCCACCTTAGGCGGCTGGCTCCAAAAAGGTT ACCCCACCGACTTCGGGTGTTACAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCG CAGCATGCTGATCCGCGATTACTAGCGATTCCAGCTTCATGTAGGCGAGTTGCAGCCTACAATCCGAACTGAGAACG GTTTTATGAGATTAGCTCCACCTCGCGGTCTTGCAGCTCTTTGTACCGTCCATTGTAGCACGTGTGTAGCCCAGGTC ATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACT TAATGATGGCAACTAAGATCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACA ACCATGCACCACCTGTCACTCTGCTCCCGAAGGAGAAGCCCTATCTCTAGGGTTTTCAGAGGATGTCAAGACCTGGT AAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTC AGCCTTGCGGCCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAACTTCAGCACTAAAGGGCGGAAACCCTCTAACAC TTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCGCCTCAGTGT CAGTTACAGACCAGAAAGTCGCCTTCGCCACTGGTGTTCCTCCATATCTCTACGCATTTCACCGCTACACATGGAAT TCCACTTTCCTCTTCTGCACTCAAGTCTCCCAGTTTCCAATGACCCTCCACGGTTGAGCCGTGGGCTTTCACATCAG ACTTAAGAAACCACCTGCGCGCGCTTTACGCCCAATAATTCCGGATAACGCTTGCCACCTACGTATTACCGCGGCTG CTGGCACGTAGTTAGCCGTGGCTTTCTGGTTAGGTACCGTCAAGGTGCCAGCTTATTCAACTAGCACTTGTTCTTCC CTAACAACAGAGTTTTACGACCCGAAAGCCTTCATCACTCACGCGGCGTTGCTCCGTCAGACTTTCGTCCATTGCGG AAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTC GGCTACGCATCGTTGCCTTGGTGAGCCGTTACCTCACCAACTAGCTAATGCGACGCGGGTCCATCCATAAGTGACAG CCGAAGCCGCCTTTCAATTTCGAACCATGCGGTTCAAAATGTTATCCGGTATTAGCCCCGGTTTCCCGGAGTTATCC CAGTCTTATGGGCAGGTTACCCACGTGTTACTCACCCGTCCGCCGCTAACTTCATAAGAGCAAGCTCTTAATCCATT CGCTCGACTTGCATGTATTAGGCACGCCGCCAGC

GTTCATCCTGAGCCAGGATCAAACTCT

As

The separation of the thuringiensis X023 (Bt X023) of the present invention is identified: using heating water bath and dilution plate The methods of coating, one plant is directly isolated to obtain in the soil sample acquired from Xiangtan, Hunan Province area Xiangtan County gold rosy clouds mountain forest has Su Yun The bacterium of golden bacillus character, through colony morphological observation, Gram's staining, physiological and biochemical property, 16S rRNA DNA homolog Property analysis, identify that the bacterial strain is that Bacillus belongs to, thuringiensis kind is named as thuringiensis X023 (Bt X023)。

Insecticidal activity:

Thuringiensis X023 is inoculated into fermentation medium, 26-30 DEG C, 120-180rpm fermented and cultured 48- After 60h, zymocyte liquid is taken, raw survey of toxicity for carrying out bollworm and diamondback moth is tested.

The composition of fermentation medium is preferred are as follows: glucose 14-17g/L, peptone 12-15g/L, KH₂PO₄·3H₂O1.2- 1.6g/L, FeSO₄·7H₂O 0.02-0.03g/L, MnSO₄·H₂O 0.02-0.03g/L, MgSO₄·7H₂O 0.25-0.30g/ L, pH 6.8-7.2.

For thuringiensis as important biocontrol microorganisms, sphere of action covers a variety of Lepidopteras, coleoptera agricultural pests, The isolated thuringiensis of the present invention has good toxicity to the target insect diamondback moth of Lepidoptera and bollworm, right Agricultural production crop pest biological control has important application value.

Detailed description of the invention

Fig. 1 is phase contrast microscope morphological feature figure of the bacterial strain Bt X023 after isolating and purifying；

Fig. 2 is scanning electron microscope morphological feature figure of the bacterial strain Bt X023 brood cell's mixed crystal after isolating and purifying；

Fig. 3 is Bt X023 bacterial strain 16S rRNA sequence evolution tree；

Fig. 4 is the growth curve chart of Bt X023 bacterial strain in the fermentation medium；

Fig. 5 is to extract the result figure that holoprotein carries out SDS-PAGE after Bt X023 cultivates 36h；

M: albumen Marker；1-3: three groups of biology repeat.

Microbial strains preservation situation explanation

Thuringiensis X023 (Bacillus thuringiensis X023), the strain was on May 16th, 2018 In China typical culture collection center (abbreviation CCTCC, address: Wuhan, China Wuhan University) preservation, culture presevation number is CCTCC NO:M 2018283.

Specific embodiment

Below in conjunction with specific embodiment, invention is further described in detail.

Embodiment

1. the thuringiensis X023 (abbreviation Bt X023) of the present embodiment, the strain is on May 16th, 2018 in State's Type Tissue Collection (abbreviation CCTCC, address: Wuhan, China Wuhan University) preservation, culture presevation number are CCTCC NO:M 2018283.

2. the thuringiensis X023 strain isolation process of the present embodiment: from Xiangtan, Hunan Province area Xiangtan County Jin Xiashan Forest land acquires soil sample, and sterile water is added, and mixes, and 80 DEG C of heating 30min, gradient dilution takes 100 microlitres (μ L) to be spread evenly across LB It on plate, picks from the plate single colonie and dilutes again and line on LB plate, 48h is cultivated under the conditions of 26-30 DEG C, will repeat point Single colonie from purifying is inoculated into LB liquid medium shaking flask, 26-30 DEG C, 120-180rpm/min, cultivates 12h, 50% is sweet Oil mixes, long-term preservation in -80 DEG C of refrigerators.

3. optical microscopy and electron microscope observation:

Specific implementation process: taking bacterium solution 1.5mL, 9000rpm/min, the 3min of Bt X023 culture 36h, and centrifugation is gone Clearly, distilled water cleans twice, and thallus is resuspended with 200 μ L distilled waters, draws 10 μ L bacterium solutions drop in glass slide center, close the lid glass Piece, in the form of 100 times of oily microscopic observation thallus.Bt X023 is taken to cultivate 60h bacterium solution 1.5mL, 9000rpm/min, 3min, from The heart removes supernatant, is respectively cleaned 3 times with the acetone of physiological saline and 5%, and PBS buffer solution is washed 6-8 times, finally slow with 200 μ L PBS Thallus is resuspended in fliud flushing, and the 2.5% fixed 12h of glutaraldehyde fixer is added in the precipitating after washing, with sterile milli-Q water 2 times Afterwards, then successively it with the ethanol dehydration of 30%, 50%, 70%, 80%, 90% mass concentration gradient, needs to stand in each gradient 15min, dehydration finish, and are dehydrated 2 times with dehydrated alcohol, stand 15min every time, finally draw sample-alcohol suspension of mixing Drop observes thalli morphology on the sample stage for being covered with coverslip under a scanning electron microscope.

Fig. 1 is that bacterial strain Bt X023 is cultivated to the phase contrast microscope picture of 36h, shows that the bacterial strain is rod-shaped production brood cell in figure With the bacterium of rhomboidan.Fig. 2 is the scanning electron microscopic observation to brood cell's mixed crystal.

4. Bt X023 bacterial strain 16S rRNA DNA homolog sequence is analyzed:

Specific implementation process: from picking single colonie on LB plate, it is transferred to the 1.5mL containing 1.2mL LB liquid medium In Ep pipe, pipe cover with syringe needle prick aperture, 30 DEG C, 850rpm/min, shaken cultivation for 24 hours after, use bacterial gene Group DNA extraction kit carries out full-length genome extraction by operating procedure.Draw according to the design of bacterial 16 S rRNA gene order is general Object (Bf-F, AGAGTTTGATCCTGGCTCAG；Bf-R, ACGGCTACCTTGTTACGACTT) and by raw work (Shanghai) biological skill The synthesis of art Co., Ltd.

16S rRNA gene magnification is carried out by following reaction system and reaction condition:

Reaction system (20 μ L): aseptic double-distilled water, 12 μ L；5 × Buffer, 4 μ L；DNTP, 1.6 μ L；Bf-R (10 μM), 0.6μL；Bf-F (10 μM), 0.6 μ L；Genomic templates, 1 μ L；PrimerSTAR DNA Polymerase (Takara), 0.2 μ L；

Response procedures: 94 DEG C of 4min of initial denaturation are denaturalized 94 DEG C of 30s, and anneal 52 DEG C of 30s, extend 72 DEG C of 90s, 30 circulations, Extend 72 DEG C of 10min.

PCR product delivers Shanghai English fine horse biology skill through multifunctional dna purification and recovery kits together with appropriate primer The sequencing of art Co., Ltd.Sequencing result shows that the 16S rRNA gene order length of isolated strains Bt X023 is 1516bp.It will survey The 16S rRNA gene order of the Bt X023 obtained carries out online BLAST (NCBI, http://www.ncbi.nlm.nih.gov) Compare, the tetraploid rice of 16S rRNA gene order analysis shows that, the bacterial strain respectively with Bcillus thuringiensis knu-07(CP016588.1)、Bcillus thuringiensis C25(CP022345.1)、Bcillus thuringiensis YWC-28(CP013055.1)、Bcillus thuringiensis tolworthi(AP014864.1)、 The similitude of Bcillus thuringiensis HD-1 (CP010005.1) etc. is 99%.According to BLAST acquired results with And phase contrast microscope observation speculates that the bacterial strain belongs to Bcillus thuringiensis subspecies, is named as Bacillus Thuringiensis X023 and (Replications=1000, Bootstrap value take the phylogenetic tree for constructing such as Fig. 3 Percentage).

LB culture medium (/L): 10g sodium chloride, 10g peptone, 5g yeast powder, 20g agar (fluid nutrient medium is not added), pH 6.8-7.2；

The composition of fermentation medium are as follows: glucose 16g/L, peptone 14g/L, KH₂PO₄·3H₂O1.5g/L, FeSO₄· 7H₂O 0.02g/L, MnSO₄·H₂O 0.02g/L, MgSO₄·7H₂O0.25g/L, pH 6.8-7.2.

Specific implementation process: from picking single colonie on LB plate, it is transferred to the 1.5mL Ep containing 1.2mL LB culture medium Guan Zhong is covered in pipe and is pricked aperture with syringe needle, 26-30 DEG C, 850rpm/min, after shaken cultivation 12h, by 1% transfer in In 30mL fermentation medium, 26-30 DEG C, 120-180rpm shaken cultivation, every 2h sampling measures OD₆₀₀Value.

Thuringiensis X023 bacterial strain is as shown in Figure 4 in the growth curve of fermentation medium, the results showed that, same strain Bacterium lag phase is initial 2 hours, and 2h~16h of the logarithmic growth phase after fermentation starts, wherein thallus enters stationary phase Time 18h.Furthermore enter the time 36h of decline phase, but bacterial strain is consistent substantially in stationary phase duration.

6. the insecticidal activity assay of Bacillus thuringiensis X023 bacterial strain:

Specific implementation process: it after Bt X023 bacterial strain activates 12h in LB culture medium, is inoculated into respectively with 1% inoculum concentration It in fermentation medium, cultivates 48-60 hours, collects fermentation liquid.It takes a certain amount of fermentation liquid that fermentation medium dilution is added, makes its shape At with different diluted concentrations, the fermentation liquid that total system is 600 μ L, the man-made feeds of 30mL are added simultaneously in the fermentation liquid after dilution Be sufficiently mixed, finally make wherein fermentation liquid be diluted to 20,10,5, five kinds of 2.5,1.25uL/mL concentration gradients respectively.By 30mL Feed be averagely added to the raw drafting boards in 3 24 parallel holes, i.e. in 72 culture holes, add a larva in each hole, totally 72. It is fed in 28 ± 1 DEG C of incubator, the quantity of every dead worm of 24 hour record and total borer population, incubation time is no more than 96 hours. Record data calculate the LC of X023 fermentation liquid under different condition by the prohibit analysis of SPSS software₅₀(semilethal is dense Degree), use LC₅₀95% confidence interval of non-overlap as determine the significant difference of toxicity standard.

Table 1 is that Bt X023 cultivates the fermentation liquid after 60h in the fermentation medium and surveys result to the raw of diamondback moth

Table 2 is that Bt X023 cultivates the fermentation liquid after 60h in the fermentation medium and surveys result to the raw of bollworm

7. the holoprotein of Bacillus thuringiensis X023 bacterial strain extracts and SDS-PAGE:

Specific implementation process: the bacterium according to the growth curve of thuringiensis X023, after selecting fermented and cultured 36h Liquid, 9000rpm/min, 10min collect cell precipitation, are washed 2 times with PBS (20mM).Appropriate cell pyrolysis liquid (0.5M is added PH 8.0Tris-Cl, 8M urea, 65mM CHAPS, 75mM NaCl, 2M thiocarbamide, 1mM PMSF, 10mM protease inhibitors), Then carry out ultrasonication processing (130W, 3S, 3S, 10min), 13000r/min, 20min, obtain supernatant, by microplate reader into After row quantification of protein, SDS-PAGE detection is carried out.Fig. 5 is to extract Bt X023 holoprotein after cultivating 36h under the conditions of different fermentations SDS-PAGE is carried out, M. albumen Marker, 1-3 are that three biology repeat.

In BtX023 bacterial strain 36h holoprotein SDS-PAGE, there is the insecticidal crystal protein of obvious a large amount of 130KDa Band.

Sequence table

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<120>one thuringiensis strain bacillus and its application

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<212> DNA/RNA

<213>thuringiensis X023(Bacillus thuringiensis X023) (CCTCC NO:M 2018283)

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tacggctacc ttgttacgac ttcaccccaa tcatctgtcc caccttaggc ggctggctcc 60

aaaaaggtta ccccaccgac ttcgggtgtt acaaactctc gtggtgtgac gggcggtgtg 120

tacaaggccc gggaacgtat tcaccgcagc atgctgatcc gcgattacta gcgattccag 180

cttcatgtag gcgagttgca gcctacaatc cgaactgaga acggttttat gagattagct 240

ccacctcgcg gtcttgcagc tctttgtacc gtccattgta gcacgtgtgt agcccaggtc 300

ataaggggca tgatgatttg acgtcatccc caccttcctc cggtttgtca ccggcagtca 360

ccttagagtg cccaacttaa tgatggcaac taagatcaag ggttgcgctc gttgcgggac 420

ttaacccaac atctcacgac acgagctgac gacaaccatg caccacctgt cactctgctc 480

ccgaaggaga agccctatct ctagggtttt cagaggatgt caagacctgg taaggttctt 540

cgcgttgctt cgaattaaac cacatgctcc accgcttgtg cgggcccccg tcaattcctt 600

tgagtttcag ccttgcggcc gtactcccca ggcggagtgc ttaatgcgtt aacttcagca 660

ctaaagggcg gaaaccctct aacacttagc actcatcgtt tacggcgtgg actaccaggg 720

tatctaatcc tgtttgctcc ccacgctttc gcgcctcagt gtcagttaca gaccagaaag 780

tcgccttcgc cactggtgtt cctccatatc tctacgcatt tcaccgctac acatggaatt 840

ccactttcct cttctgcact caagtctccc agtttccaat gaccctccac ggttgagccg 900

tgggctttca catcagactt aagaaaccac ctgcgcgcgc tttacgccca ataattccgg 960

ataacgcttg ccacctacgt attaccgcgg ctgctggcac gtagttagcc gtggctttct 1020

ggttaggtac cgtcaaggtg ccagcttatt caactagcac ttgttcttcc ctaacaacag 1080

agttttacga cccgaaagcc ttcatcactc acgcggcgtt gctccgtcag actttcgtcc 1140

attgcggaag attccctact gctgcctccc gtaggagtct gggccgtgtc tcagtcccag 1200

tgtggccgat caccctctca ggtcggctac gcatcgttgc cttggtgagc cgttacctca 1260

ccaactagct aatgcgacgc gggtccatcc ataagtgaca gccgaagccg cctttcaatt 1320

tcgaaccatg cggttcaaaa tgttatccgg tattagcccc ggtttcccgg agttatccca 1380

gtcttatggg caggttaccc acgtgttact cacccgtccg ccgctaactt cataagagca 1440

agctcttaat ccattcgctc gacttgcatg tattaggcac gccgccagcg ttcatcctga 1500

gccaggatca aactct 1516

Claims

1. a kind of thuringiensis, which is characterized in that it is thuringiensis X023,Bacillus thuringiensis X023, the strain on May 16th, 2018 in China typical culture collection center preservation, protect by strain Hiding number is CCTCC NO:M 2018283.

2. the application of thuringiensis as described in claim 1, which is characterized in that its zymocyte liquid is to bollworm and small Diamond-back moth has insecticidal activity.

3. the application of thuringiensis according to claim 2, which is characterized in that the preparation method of zymocyte liquid: Thuringiensis X023 is inoculated into fermentation medium, 26-30 DEG C, 120-180 rpm fermented and cultured 48-60 h, To obtain the final product.

4. the application of thuringiensis according to claim 3, which is characterized in that the composition of fermentation medium are as follows: Glucose 14-17g/ L, peptone 12-15 g/ L, KH₂PO₄•3H₂O1.2-1.6 g/ L, FeSO₄•7H₂O 0.02-0.03 G/ L, MnSO₄•H₂O 0.02-0.03 g/ L, MgSO₄•7H₂O 0.25-0.30g/ L, pH 6.8-7.2.