Bt Strain Registration

Bt S2160-1, a High Mosquitocidal Strain of Bacillus thuringiensis  

Xuanjun Fang , Wenfei Zhang
Hainan Institute of Tropical Agricultural Resources, Sanya, 572025, Hainan
Author    Correspondence author
Bt Research, 2012, Vol. 3, No. 5   doi: 10.5376/2012.bt.03.0005
Received: 03 Sep., 2012    Accepted: 26 Sep., 2012    Published: 08 Oct., 2012
© 2012 BioPublisher Publishing Platform
This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Preferred citation for this article:

Fang and Zhang, 2011, Bt S2160-1, a High Mosquitocidal Strain of Bacillus thuringiensis, Bt Research, Vol.3, No.5 29-32 (doi: 10.5376/bt.2012.03.0005)

Abstract

Bt strain S2160-1 was found to produce crystalline toxins with high insecticidal effects against mosquitoes (Culex quinquefasciatus and Aedes albopictus), which was characterized from the Bt isolate (Isolate No: 20070720S2160) from soil samples collected from Guangxi Dawangling Natural Reserves of Guangxi Province of China in 2007. Bt strain S2160-1 was stocked in the HITAR Bacillus Collections with Accession No 20070720S2160 and owned by HITAR. Scanning electron microscope observation showed that the spherical shape crystals of Bt S2160-1 with size at one fourth volume of spore to that of reference strain of Bti. SDS-PAGE analysis showed that the intact parasporal protein of Bt S2160-1 containing four major polypetide of about 140kDa, 130kDa, 75kDa and 30kDa was different from that of Bti. Furthermore, bioassays exhibited high toxicity to mosquito larva of Culex quinquefasciatus and Aedes albopictus. There are a great of differences in terms of the large plasmid patterns, parasporal proteins profiles and cry genes contents between Bt S2160-1 and Bti. The cloned genes of cry50Ba1 and cry54Ba1 expressed in BL21 (DE3) cells, but not success in Cry 30Ea1 and Cry 30Ga. Thus, it is believed that Bt S2160-1 could be applied into mosquito control as the potential alternative to prevent mosquito from developing the resistance to the toxins of Bti.

Keywords
Bacillus thuringiensis; Bt isolate 20070720S2160; Bt Strain S2160-1; Cry30Ea1; Cy30Ga; cry50Ba1; cry54Ba1; Mosquito

1. General description

Bt strain: S2160-1

Isolate: 20070720S2160

Isolate source: Soil

Collected by: Wenfei Zhang and Liu Xie et al.

Collection date: July-20-2007

Collection site: Guangxi Dawangling Natural Reserves of Guangxi Province, Southwest China

Subspecies: Unknown

Serovariety: Unknown

Identified by: Xuanjun Fang, Wenfei Zhang, Liu Xie

Deposited at:The HITAR Bacillus Collections with Accession No 20070720S2160

Owned by: Hainan Institute of Tropical Agricultural Resources

 

2. Molecular Features

Crystal shape: Spherical

Protein profile: 140 kDa, 130 kDa, 75 kDa and 30 kDa

Plasmid profile: the plasmid profile of Bt S2160-1 was significantly different to that of Bti, and the other strains

Genotype: in contrast to Bti no cry4 or cry10 genes were identified using the S5un4/S3un4 primers (Table 1) and also that no PCR products were obtained with primers designed to amplify cry11 and cyt1 genes

Cloned gene: Cry 30Ea1 (Accession number EU503140.1); Cry 30Ga (Accession number HQ638217.1); Cry 50Ba1 (Accession number GU446675.1); Cry 54Ba1 (Accession number GU446677.1)

Target insect: Mosquitoes (Culex quinquefasciatus and Aedes albopictus)

 

3. Featured Photos or Table

Figure 1: Scanning electron micrograph of spores and crystals from Bt S2160-1 (Zhang et al., 2012).

 

Figure 2: Profiles of SDS-PAGE of Bt S2160-1 (Zhang et al., 2012).

 

Figure 3: PFGE banding patterns of mega plasmid from 2160-1 (Zhang et al., 2012).

 

Figure 4: PCR–RFLP patterns of cry-type genes from Bt S2160-1 (Zhang et al., 2012).

 

Figure 5: SDS-PAGE gel electrophoresis analysis of genes of cry50Ba and cry54Ba expressed in BL21 (DE3) cells using pET expression system.

 

  

Figure 1 Scanning electron micrograph of spores and crystals from Bt S2160-1

Note: SP is a spore formed during the stationary phase of growth, next to crystal protein is CP produced as regular crystal is related to spore formation

 

  

Figure 2 SDS-PAGE analyses of crystal proteins from Bt S2160-1 and Bti

Note: Bt S2160-1 and Bti were cultured in G-Tris medium, The samples for SDS-PAGE analysis taken before and after sporation respectively and crystal proteins digested with 1uM trypsin were analyzed together. PM1: protein molecular weight marker, land 1, 4: strain proteins before sporation from Bti and Bt S2160-1 respectively; lane 2, 5: crystal proteins after sporation from Bti and Bt S2160-1 respectively; lane 3, 6: crystal proteins from Bti and Bt S2160-1 were treated with 1ug/ml trypsin

 

  

Figure 3 PFGE patterns of larger plasmids from Bt strains

Note: lane1: B. thuringiensis subsp kurstaki HD73; lane 2: B. thuringiensis subsp kurstaki HD-1; lane 3: B. thuringiensis subsp israelensis AND508; lane 4: Bt S2160-1; B. thuringiensis subsp israelensis AND508 harboring the large plasmids of pXO16 (350 kb) and pBtoxis (128 kb), was used as size marker

 

  

Figure 4 PCR–RFLP patterns of cry-type genes from Bt S2160-1

Note: M. DNA molecular weight marker; lane1, 2, 3 are PCR amplification using primer pairs S5un4/S3un4, up30-5/up30-3 and up39.40-5/up39.40-3 respectively; lane4, 5, 6. were RFLP patterns of the corresponding PCR production digested with BanI/ HaeⅢ , BglⅡ/PstI and HindIII/HaeIII restriction enzymes

 

  

Figure 5 SDS-PAGE gel electrophoresis analysis of genes of cry50Ba and cry54Ba expressed in BL21 (DE3) cells

Note: PM2: protein molecular weight marker; lane 1: E. coli BL21 (DE3) cell harboring pET-30a incubated with IPTG; lane 2, 5: E. coli BL21 (DE3) cell harboring pETcry54Ba and pETcry50Ba incubated without IPTG; lane 3, 6: E. coli BL21 (DE3) cell harboring pETcry54Ba

 

  

Table 1 Larvicidal activity of spore/crystal mixtures from Bt S2160-1 and Bti against Plutella xylostella and mosquitoes Note: a 95% FL are the 95% fiducial limits in parentheses, as determined by probit analysis

 

4. Related Publications

Zhang W, Crickmore N, George Z, Xie L, He YQ, Li Y, Tang JL, Tian L, Wang X, Fang X (2012) Characterization of a new highly mosquitocidal isolate of Bacillus thuringiensis--an alternative to Bti? J Invertebr Pathol 109 (2):217-222. doi:10.1016/j.jip.2011.11.003

 

5. Filed Patent or Related to This Strain

No.

 

References 

Zhang W, Crickmore N, George Z, Xie L, He YQ, Li Y, Tang JL, Tian L, Wang X, Fang X (2012) Characterization of a new highly mosquitocidal isolate of Bacillus thuringiensis--an alternative to Bti? J Invertebr Pathol 109 (2):217-222. doi:10.1016/j.jip.2011.11.003

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