CN109810920A - A strain of Bacillus thuringiensis and its application - Google Patents
A strain of Bacillus thuringiensis and its application Download PDFInfo
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
- Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
一株苏云金芽胞杆菌及其应用，本发明之苏云金芽胞杆菌，为苏云金芽胞杆菌X023，Bacillus thuringiensisX023，该菌种于2018年5月16日在中国典型培养物保藏中心保藏，菌种保藏号为CCTCC NO：M 2018283。本发明还包括苏云金芽胞杆菌的应用，苏云金芽胞杆菌发酵菌液对棉铃虫和小菜蛾有杀虫活性。
A strain of Bacillus thuringiensis and its application, the Bacillus thuringiensis of the present invention is Bacillus thuringiensis X023, Bacillus thuringiensis X023, the strains were preserved in the China Center for Type Culture Collection on May 16, 2018, and the strain preservation number is CCTCC NO: M 2018283. The invention also includes the application of Bacillus thuringiensis, and the Bacillus thuringiensis fermented bacterial liquid has insecticidal activity on cotton bollworm and diamondback moth.
Description
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
<110>Hunan Normal University
<120>one thuringiensis strain bacillus and its application
<130>
<140>
<141>
<150>
<151>
<160> 1
<210> 1
<211> 1516
<212> DNA/RNA
<213>thuringiensis X023(Bacillus thuringiensis X023) (CCTCC NO:M 2018283)
<220>
<400>1
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 (4)
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| CN112920962A (en) * | 2020-12-04 | 2021-06-08 | 湖南师范大学 | ARTP-NTG composite mutagenesis bacillus thuringiensis mutant strain and application thereof |
| CN121136881A (en) * | 2025-11-12 | 2025-12-16 | 湖南师范大学 | A Bacillus thuringiensis species and its application in attracting adult fruit flies |
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