Co-reporter:Qi Yang;Sijia Tang;Jie Rang;Mingxing Zuo;Xuezhi Ding
Current Microbiology 2015 Volume 70( Issue 4) pp:457-463
Publication Date(Web):2015 April
DOI:10.1007/s00284-014-0747-9
Bacillus thuringiensis is a kind of insecticidal microorganism which can produce a variety of toxin proteins, it is particularly important to find an effective strategy to identify novel toxin proteins rapidly and comprehensively with the discovery of the wild-type strains. Multi-dimensional high-performance liquid chromatography combined with mass spectrometry has become one of the main methods to detect and identify toxin proteins and proteome of B. thuringiensis. In this study, protein samples from B. thuringiensis strain 4.0718 were analyzed on the basis of two-dimensional liquid chromatography–tandem mass spectrometry (2D-LC–MS/MS), and tryptic peptides of whole cell from the late sporulation phase were eluted at different concentration gradients of ammonium chloride and followed by secondary mass spectrum identification. 831 and 894 proteins were identified from two biological replicates, respectively, while 1,770 and 1,859 peptides were detected correspondingly. Among the identified proteins and peptides, 606 proteins and 1,259 peptides were detected in both replicates, which mean that 1,119 proteins and 2,370 peptides were unique to the proteome of this strain. A total of 15 toxins have been identified successfully, and seven of them were firstly discovered in B. thuringiensis strain 4.0718 that were Crystal protein (A1E259), pesticidal protein (U5KS09), Cry2Af1 (A4GVF0), Cry2Ad (Q9RM89), Cry1 (K4HMB5), Cry1Bc (Q45774), and Cry1Ga (Q45746). The proteomic strategy employed in the present study has provided quick and exhaustive identification of toxins produced by B. thuringiensis.
Co-reporter:Yunjun Sun;Qiang Zhao;Dasheng Zheng;Xuezhi Ding;Jingfang Wang
Biotechnology Letters 2014 Volume 36( Issue 1) pp:105-111
Publication Date(Web):2014 January
DOI:10.1007/s10529-013-1330-3
Three structural domains of mosquitocidal Cry11Aa and Cry11Ba from Bacillus thuringiensis were exchanged to produce interdomain chimeras [BAA (11Ba/11Aa/11Aa), ABA (11Aa/11Ba/11Aa), AAB (11Aa/11Aa/11Ba), ABB (11Aa/11Ba/11Ba), BAB (11Ba/11Aa/11Ba), BBA (11Ba/11Ba/11Aa]. Chimeras BAB, BAA, BBA, and AAB formed inclusion bodies in the crystal-negative B. thuringiensis host and produced expected protein bands on SDS-PAGE gel. However, no inclusion body or target protein could be found for chimeras ABA and ABB. In bioassays using the fourth-instar larvae of Culex quinquefasciatus and Aedes aegypti, AAB had ~50 % lethal concentrations of 4.8 and 2.2 μg ml−1, respectively; however, the rest of chimeras were not toxic. This study thus helps to understand the domain-function relationships of the Cry11Aa and Cry11Ba toxins. The toxic chimera, AAB, might be a candidate for mosquito control as its amino acid sequence is different from the two parental toxins.
Co-reporter:Fu Yan;Xing Cheng;Xuezhi Ding;Ting Yao;Hanna Chen;Wenping Li
Current Microbiology 2014 Volume 68( Issue 5) pp:604-609
Publication Date(Web):2014 May
DOI:10.1007/s00284-013-0516-1
Av3, a neurotoxin of Anemonia viridis, is toxic to crustaceans and cockroaches but inactive in mammals. In the present study, Av3 was expressed in Escherichia coli Origami B (DE3) and purified by reversed-phase liquid chromatography. The purified Av3 was injected into the hemocoel of Helicoverpa armigera, rendering the worm paralyzed. Then, Av3 was expressed alone or fusion expressed with the Cry1Ac in acrystalliferous strain Cry−B of Bacillus thuringiensis. The shape of Cry1Ac was changed by fusion with Av3. The expressed fusion protein, Cry1AcAv3, formed irregular rhombus- or crescent-shaped crystalline inclusions, which is quite different from the shape of original Cry1Ac crystals. The toxicity of Cry1Ac was improved by fused expression. Compared with original Cry1Ac expressed in Cry−B, the oral toxicity of Cry1AcAv3 to H. armigera was elevated about 2.6-fold. No toxicity was detected when Av3 was expressed in Cry−B alone. The present study confirmed that marine toxins could be used in bio-control and implied that fused expression with other insecticidal proteins could be an efficient way for their application.
Co-reporter:Shengbiao Hu;Xu Zhang;Yusheng Li;Xuezhi Ding;Xiaofeng Hu
Biotechnology Letters 2013 Volume 35( Issue 7) pp:1045-1051
Publication Date(Web):2013 July
DOI:10.1007/s10529-013-1171-0
A triple recombineering technique was used with plasmid pHT315 to produce pHTEC, a construct carrying chitinase and cry2Aa genes from Bacillus thuringiensis subsp. kurstaki 4.0718. Transformation of wild-type B. thuringiensis strain HD73 and the acrystalliferous strain Cry-B with pHTEC resulted in the recovery of recombinant strains that expressed Cry2Aa as cubic crystals in the cell pellet and soluble chitinase protein. The toxicity of HD73 (pHTEC) against Helicoverpa armigera larvae increased sevenfold when compared with HD73 (pHT315) harboring pHT315 vector. The triple recombineering protocol was optimized by comparing recombination efficacy mediated by RecE/RecT and Redα/Redβ and by using single-strand DNA as substrate.
Co-reporter:Hailong Wang;Ting Yao;Mei Cai;Xiuqing Xiao;Xuezhi Ding
Biotechnology Letters 2013 Volume 35( Issue 2) pp:279-284
Publication Date(Web):2013 February
DOI:10.1007/s10529-012-1076-3
To identify the transposon insertion sites in a soil actinomycete, Saccharopolyspora spinosa, a genome walking approach, termed SPTA-PCR, was developed. In SPTA-PCR, a simple procedure consisting of TA cloning and a high stringency PCR, following the single primer-mediated, randomly-primed PCR, can eliminate non-target DNA fragments and obtain target fragments specifically. Using SPTA-PCR, the DNA sequence adjacent to the highly conserved region of lectin coding gene in onion plant, Allium chinense, was also cloned.
Co-reporter:YuShuang Luo;XiaoXiao Kou;XueZhi Ding;ShengBiao Hu
Science China Life Sciences 2012 Volume 55( Issue 2) pp:172-180
Publication Date(Web):2012 February
DOI:10.1007/s11427-012-4276-0
To promote spinosad biosynthesis by improving the limited oxygen supply during high-density fermentation of Saccharopolyspora spinosa, the open reading frame of the Vitreoscilla hemoglobin gene was placed under the control of the promoter for the erythromycin resistance gene by splicing using overlapping extension PCR. This was cloned into the integrating vector pSET152, yielding the Vitreoscilla hemoglobin gene expression plasmid pSET152EVHB. This was then introduced into S. spinosa SP06081 by conjugal transfer, and integrated into the chromosome by site-specific recombination at the integration site ΦC31 on pSET152EVHB. The resultant conjugant, S. spinosa S078-1101, was genetically stable. The integration was further confirmed by PCR and Southern blotting analysis. A carbon monoxide differential spectrum assay showed that active Vitreoscilla hemoglobin was successfully expressed in S. spinosa S078-1101. Fermentation results revealed that expression of the Vitreoscilla hemoglobin gene significantly promoted spinosad biosynthesis under normal oxygen and moderately oxygen-limiting conditions (P<0.01). These findings demonstrate that integrating expression of the Vitreoscilla hemoglobin gene improves oxygen uptake and is an effective means for the genetic improvement of S. spinosa fermentation.
Co-reporter:Feng Wu;Xinmin Zhao;Yunjun Sun;Wenping Li
Current Microbiology 2012 Volume 64( Issue 2) pp:106-111
Publication Date(Web):2012 February
DOI:10.1007/s00284-011-0038-7
The 20 kb DNAs are associated with crystals in many subspecies of Bacillus thuringiensis. We isolated 20 kb DNAs from crystals of B. thuringiensis strain 4.0718, then constructed a gene library using DNA fragments of Sau3AI partial digestion and pbluescriptIISK(+) vector. We screened out 440 recombinants, yielding a genomic coverage of ten and including 99% sequence of DNA which achieved the required theoretical value to construct the gene library. Through NCBI Blast and homology analysis, the sequencing results proved that the DNA came from the chromosome of B. thuringiensis. Moreover, we have completed the multiple alignment of homologous ropB protein sequences and phylogenetic analysis using bioinformatic software. For further investigation of the interactions between 20 kb DNAs and protoxins, molecular docking has also been done.
Co-reporter:Shipinga Shan;Xuezhia Ding;Youminga Zhang;Shengbiaoa Hu;Yunjuna Sun;Ziquana Yu;Lizhenb Han
Chinese Journal of Chemistry 2011 Volume 29( Issue 3) pp:427-432
Publication Date(Web):
DOI:10.1002/cjoc.201190099
Abstract
Alkaline phosphatases (ALPs) attached to the midgut membrane with glycosyl phosphotidyl inositol (GPI) have been proposed as the putative Cry1Ac toxin receptor in Helicoverpa armigera. Activated toxins bind to ALP receptors on the brush border membrane vesicle (BBMV) of the midgut epithelium, which activates intracellular oncotic pathways and leads to cell death. However, with the long-term use of Cry toxin, insects can develop a strong resistance to insecticidal delta-endotoxins. Although the molecular mechanism of insect resistance has not been fully understood, insects develop resistance to biopesticides due to changes of toxins binding to midgut receptors. So, it is a good idea to investigate the molecular mechanism of insect resistance by analyzing ALP receptor from Helicoverpa armigera (Ha-ALP). Based on crystal structure of shrimp alkaline phosphatase, the three-dimensional structure of the Cry1Ac toxin-binding Ha-ALP receptor was obtained by homology modeling and the model was further evaluated using PROSA energy and ERRAT. The important role of binding of toxin to GalNAc on Ha-ALP was discussed in the aspect of Cry1Ac toxicity. Specific recognition sites of the binding of oligosaccharides to Ha-ALP were predicted. Post-translational modification of ALP provides insights into the functional properties of ALP and leads to profound understanding of receptor and toxin interactions.
Co-reporter:Shiping Shan;Youming Zhang;Xuezhi Ding;Shengbiao Hu;Yunjun Sun
Current Microbiology 2011 Volume 62( Issue 2) pp:358-365
Publication Date(Web):2011 February
DOI:10.1007/s00284-010-9714-2
Cry1Ac insecticidal crystal proteins produced by Bacillus thuringiensis (Bt) have become an important natural biological agent for the control of lepidopteran insects. In this study, a cry1Ac toxin gene from Bacillus thuringiensis 4.0718 was modified by using error-prone PCR, staggered extension process (StEP) shuffling combined with Red/ET homologous recombination to investigate the insecticidal activity of delta-endotoxin Cry1Ac. A Cry1Ac toxin variant (designated as T524N) screened by insect bioassay showed increased insecticidal activity against Spodoptera exigua larvae while its original insecticidal activity against Helicoverpa armigera larvae was still retained. The mutant toxin T524N had one amino acid substitution at position 524 relative to the original Cry1Ac toxin, and it can accumulate within the acrystalliferous strain Cry-B and form more but a little smaller bipyramidal crystals than the original Cry1Ac toxin. Analysis of theoretical molecular models of mutant and original Cry1Ac proteins indicated that the mutation T524N located in the loop linking β16–β17 of domain III in Cry1Ac toxin happens in the fourth conserved block which is an arginine-rich region to form a highly hydrophobic surface involving interaction with receptor molecules. This study showed for the first time that single mutation T524N played an essential role in the insecticidal activity. This finding provides the biological evidence of the structural function of domain III in insecticidal activity of the Cry1Ac toxin, which probably leads to a deep understanding between the interaction of toxic proteins and receptor macromolecules.
Co-reporter:Yong Le Liu;Qin Yun Wang;Fa Xiang Wang;Xue Zhi Ding;Li Qiu Xia
The Protein Journal 2010 Volume 29( Issue 6) pp:440-444
Publication Date(Web):2010 August
DOI:10.1007/s10930-010-9271-3
A unique residue W544 in the β18–β19 loop of the Bacillus thuringiensis Cry1Ac toxin has been implicated in its toxicity. In this study, the effects of mutations at this residue on protein stability during protease treatment, UV irradiation, and preservation were examined. Residue 544 of Cry1Ac was involved in maintaining structural stability, and substitution of a polar group at this position was unfavorable to protein stability. One mutant, W544F, produced larger crystals and was more stable. This mutant showed greater resistance to UV radiation than the wild type Cry1Ac but retained equal toxicity. This is the first report showing that residue 544 in the Cry1Ac domain III plays a significant role in toxin structural stability. Our W544F mutant is a significant development in terms of field applications of Cry1Ac toxin.
Co-reporter:Xinming Zhao;Xuezhi Ding;Ziquan Yu;Yuan LÜ;Wenna Tao
Chinese Journal of Chemistry 2009 Volume 27( Issue 10) pp:2085-2089
Publication Date(Web):
DOI:10.1002/cjoc.200990350
Abstract
Cyt2Ca1 is an insecticidal crystal protein produced by Bacillus thuringiensis ET29 during its stationary phase, and this δ-endotoxin demonstrates remarkable insecticidal activity against not only insects of the order Coleoptera, but also against fleas, and in particular the larvae of the cat flea, Ctenocephalides felis. The first theoretical model of the three-dimensional structure of Cyt2Ca1 was predicted and compared with Cyt2Aa, which is lethal to insect larvae. The three-dimensional structure of the Cyt2Ca1 was obtained by homology modeling on the structures of the Cyt2Aa protein. The deduced model resembles previously reported Cyt2Aa toxin. A binding mode of inositol monophosphate as a polar head group of the putative membrane phospholipid ligand to Cyt2Ca1 was presented using molecular docking. The residues of Leu9, Glu21, Tyr23 and Gln110 of the Cyt2Ca1 toxin are responsible for the interactions with inositol monophosphate via eight hydrogen bonds. Those residues could be important for receptor recognition. This binding simulation will be helpful for the design of mutagenesis experiments aimed at the improvement of toxicity, and lead to a deep understanding of the mechanism of action of Cyt toxins.
Co-reporter:S. B. Hu;P. Liu;X. Z. Ding;L. Yan;Y. J. Sun
Applied Microbiology and Biotechnology 2009 Volume 82( Issue 6) pp:1157-1167
Publication Date(Web):2009 April
DOI:10.1007/s00253-009-1910-2
Previous studies revealed that chitinase could enhance the insecticidal activity of Bacillus thuringiensis and it has been used in combination with B. thuringiensis widely. However, the expression of B. thuringiensis chitinase is rather low and needs induction by chitin, which limits its field application. It would make sense to constitutively express the chitinase at a sufficiently high level to offer advantages in biological control of pests. In this study, a signal peptide-encoding sequence-deleted chitinase gene from B. thuringiensis strain 4.0718 under the control of dual overlapping promoters plus Shine–Dalgarno sequence and terminator sequence of cry1Ac3 gene was cloned into shuttle vector pHT315 and introduced into an acrystalliferous B. thuringiensis strain Cry−B. The recombinant plasmid was stably maintained over 240 generations in Cry−B. Chitinase was overexpressed within the sporangial mother cells in the form of spherical crystal-like inclusion bodies. The chitinase inclusions could be solubilized and exhibit chitinolytic activity in 30 mmol l−1 Na2CO3–0.2% β-mercaptoethanol buffer at a wide range of alkaline pH values, and what’s more, the chitinase inclusions potentiated the insecticidal effect of Cry1Ac protoxin when used against larvae of Spodoptera exigua and Helicoverpa armigera.
Co-reporter:Zhao Xin-Min;Xia Li-Qiu;Ding Xue-Zhi;Wang Fa-Xiang
The Protein Journal 2009 Volume 28( Issue 2) pp:104-110
Publication Date(Web):2009 February
DOI:10.1007/s10930-009-9169-0
Cry5Aa is a crystal protein produced by Bacillus thuringiensis serovar. damstadiensis during its stationary phase, this δ-endotoxin is active against nematodes and has great potential for nematodes control. The theoretical model of the three-dimensional structure of Cry5Aa was predicted by homology modeling on the structures of the Cry1Aa which is specific to Lepidopteran insects. The structure of the Cry5Aa resembles previously reported Cry toxin structures but shows the following distinctions. Cry5Aa has a long insertion in α2 of domain I. Some loops in the domain II and III of Cry5Aa are exposed to the solvent. In this work we give a brief description of our model and hypothesize the residues of the Cry5Aa that could be important in receptor recognition and pore formation. This model will be helpful for the design of mutagenesis experiments aimed to the improvement of toxicity, and lead to a deep understanding of the mechanism of action of nematicidal toxins.
Co-reporter:LiQiu Xia;XiaoShan Long;XueZhi Ding;YouMing Zhang
Current Microbiology 2009 Volume 58( Issue 1) pp:52-57
Publication Date(Web):2009 January
DOI:10.1007/s00284-008-9265-y
A fusion gene was constructed by combining the cry1Ac gene of Bacillus thuringiensis strain 4.0718 with a neurotoxin gene, hwtx-1, which was synthesized chemically. In this process, an enterokinase recognition site sequence was inserted in frame between two genes, and the fusion gene, including the promoter and the terminator of the cry1Ac gene, was cloned into the shuttle vector pHT304 to obtain a new expression vector, pXL43. A 138-kDa fusion protein was mass-expressed in the recombinant strain XL002, which was generated by transforming pXL43 into B. thuringiensis acrystalliferous strain XBU001. Quantitative analysis indicated that the expressed protein accounted for 61.38% of total cellular proteins. Under atomic force microscopy, there were some bipyramidal crystals with a size of 1.0 × 2.0 μm. Bioassay showed that the fusion crystals from recombinant strain XL002 had a higher toxicity than the original Cry1Ac crystal protein against third-instar larvae of Plutella xylostella, with an LC50 (after 48 h) value of 5.12 μg/mL. The study will enhance the toxicity of B. thuringiensis Cry toxins and set the groundwork for constructing fusion genes of the B. thuringiensiscry gene and other foreign toxin genes and recombinant strains with high toxicity.
Co-reporter:LiQiu Xia;FaXiang Wang;XueZhi Ding;XinMin Zhao;ZuJiao Fu
Science Bulletin 2008 Volume 53( Issue 20) pp:3178-3184
Publication Date(Web):2008 October
DOI:10.1007/s11434-008-0391-5
The β18–β19 loop in domain III of Cry1Ac toxin is unique among Bacillus thuringiensis Cry proteins. In this study, the role of the loop structure in insecticidal activity of Cry1Ac toxin was investigated. Alanine scanning mutations within the loop were initially generated and most mutants were over-expressed and reduced toxicity at different degrees, except mutant N546A that showed almost 2 times enhanced toxicity against Helicoverpa armigera larvae. Further mutagenic analysis of N546 revealed that a charged amino acid in this position would cause very unfavorable influence on insecticidal activity. In addition, the deletion of N546 led to protein instability because of destruction of the loop integrity. Besides, mutant W544F was much more toxic than W544Y, indicating that hydrophobic nature of the position was important for maintaining the stability and activity of Cry1Ac protein. These findings are the first biological evidence for a structural function of β18–β19 loop in insecticidal activity of the Cry1Ac toxin.
Co-reporter:Li-Qiu Xia;Xin-Min Zhao;Xue-Zhi Ding;Fa-Xiang Wang
Journal of Molecular Modeling 2008 Volume 14( Issue 9) pp:843-848
Publication Date(Web):2008 September
DOI:10.1007/s00894-008-0318-8
Cry5Ba is a δ-endotoxin produced by Bacillus thuringiensis PS86A1 NRRL B-18900. It is active against nematodes and has great potential for nematode control. Here, we predict the first theoretical model of the three-dimensional (3D) structure of a Cry5Ba toxin by homology modeling on the structure of the Cry1Aa toxin, which is specific to Lepidopteran insects. Cry5Ba resembles the previously reported Cry1Aa toxin structure in that they share a common 3D structure with three domains, but there are some distinctions, with the main differences being located in the loops of domain I. Cry5Ba exhibits a changeable extending conformation structure, and this special structure may also be involved in pore-forming and specificity determination. A fuller understanding of the 3D structure will be helpful in the design of mutagenesis experiments aimed at improving toxicity, and lead to a deep understanding of the mechanism of action of nematicidal toxins.
Co-reporter:Zujiao Fu;Yunjun Sun;Xuezhi Ding
Applied Microbiology and Biotechnology 2008 Volume 79( Issue 5) pp:875-880
Publication Date(Web):2008 July
DOI:10.1007/s00253-008-1488-0
Assessment of protoxin composition in Bacillus thuringiensis parasporal crystals is principally hampered by the fact that protoxins in a single strain usually possess high sequence homology. Therefore, new strategies towards the identification of protoxins have been developed. Here, we established a powerful method through embedding solubilized protoxins in a polyacrylamide gel block coupled to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of in-gel-generated peptides for protoxin identification. Our model study revealed that four protoxins (Cry1Aa, Cry1Ab, Cry1Ac and Cry2Aa) and six protoxins (Cry4Aa, Cry4Ba, Cry10Aa, Cry11Aa, Cyt1Aa, and Cyt2Ba) could be rapidly identified from B. thuringiensis subsp. kurstaki HD1 and subsp. israelensis 4Q2-72, respectively. The experimental results indicated that our method is a straightforward tool for analyzing protoxin expression profile in B. thuringiensis strains. Given its technical simplicity and sensitivity, our method might facilitate the present screening program for B. thuringiensis strains with new insecticidal properties.
Co-reporter:Xuezhi Ding;Zhaohui Luo;Bida Gao;Yunjun Sun
Current Microbiology 2008 Volume 56( Issue 5) pp:442-446
Publication Date(Web):2008 May
DOI:10.1007/s00284-008-9112-1
In order to improve the insecticidal activity, the chitinase gene from tobacco (Nicotiana tabacum) endochitinase and the cry1Ac gene from Bacillus thuringiensis were cloned into the vector pHT315 and designated as pHUAccB5 plasmid. The constructed transcriptional fusion was attempted under the control of the native cry1Ac promoter. Plasmid pHUAccB5 was introduced into B. thuringiensis acrystalliferous by electroporation. Analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot, the transformant XBU-HUAccB5 produced 130–kDa Cry1Ac protein and 30-kDa chitinase protein. During the chitinase active analysis, the transformant, XBU-HUAccB5 chitinase active, reached 7.5 U/mL at 72 h, and was 5 times higher than the HTX-42 and 6 times higher than the parent strains. When the insecticidal activity of the transformant was evaluated against Helicoverpa armigera Hubner, the XBU-HUAccB5 toxicity was 11.30 times higher than the transformant HTX-42 expressed single cry1Ac at 48 h and was 18.76 times higher at 72 h.
Co-reporter:Yunjun Sun;Wei Wei;Xuezhi Ding;Zhiming Yuan
Archives of Microbiology 2007 Volume 188( Issue 4) pp:327-332
Publication Date(Web):2007 October
DOI:10.1007/s00203-007-0252-7
The association of 20 kb heterologous DNA fragments with the parasporal crystals from native and recombinant Bacillus thuringiensis strains was analyzed, respectively. The cry2Aa10 gene cloned in plasmid pHC39 was transformed into B. thuringiensis subsp. kurstaki strains Cry¯B and HD73, producing recombinant strains Cry¯B(pHC39) and HD73(pHC39). SDS-PAGE and scanning electron microscopy analyses demonstrated that the recombinant Cry¯B(pHC39) produced cuboidal crystals of Cry2Aa10 protoxin, while recombinant HD73(pHC39) produced both bipyramidal crystals of Cry1Ac1 protoxin and cuboidal crystals of Cry2Aa10 protoxin. Bioassay results proved that recombinant HD73(pHC39) showed higher insecticidal activity to Helicoverpa armigera than Cry¯B(pHC39). It was found that 20 kb DNA fragments were present in bipyramidal and cuboidal crystals from both native and recombinant strains, and the 20 kb heterologous DNAs contained chromosome-specific and resident large plasmid-borne DNA fragments, suggesting the 20 kb heterologous DNA fragment embodied in crystals came randomly from the bacterial chromosomal and plasmid genome. This was the first investigation devoted exclusively on the origin of 20 kb DNA fragments in the parasporal crystals of B. thuringiensis. The data provides a basis for further investigation of the origin of 20 kb DNAs in the crystals and the interaction of DNA and protoxins.
Co-reporter:Yunjun Sun, Zujiao Fu, Xiaohong He, Chunhua Yuan, ... Liqiu Xia
Journal of Invertebrate Pathology (March 2016) Volume 135() pp:60-62
Publication Date(Web):1 March 2016
DOI:10.1016/j.jip.2015.02.005
•Intact cry1Ac from Bacillus thuringiensis and hwtx-XI from spider were constructed as transcriptional fusion.•Fusion protein of Cry1Ac and HWTX-XI formed parasporal inclusion in B. thuringiensis.•The Cry1Ac-HWTX-XI fusion exhibited enhanced toxicity against Helicoverpa armigera and Spodoptera exigua larvae.•Bi-functional HWTX-XI might be a candidate for enhancing the toxicity of B. thuringiensis.In order to assess the potency of bi-functional HWTX-XI toxin from spider Ornithoctonus huwena in improving the insecticidal activity of Bacillus thuringiensis, a fusion gene of cry1Ac and hwtx-XI was constructed and expressed in an acrystalliferous B. thuringiensis strain Cry-B. Western blot analysis and microscopic observation revealed that the recombinant strain could express 140-kDa Cry1Ac-HWTX-XI fusion protein and produce parasporal inclusions during sporulation. Bioassay using the larvae of Helicoverpa armigera and Spodoptera exigua showed that the Cry1Ac-HWTX-XI fusion was more toxic than the control Cry1Ac protoxin, as revealed by 95% lethal concentration. Our study indicated that the HWTX-XI from spider might be a candidate for enhancing the toxicity of B. thuringiensis products.Download full-size image
Co-reporter:Xiang-li YE, Li-qiu XIA
Agricultural Sciences in China (January 2011) Volume 10(Issue 1) pp:92-100
Publication Date(Web):January 2011
DOI:10.1016/S1671-2927(11)60311-8
Co-reporter:Xin Min ZHAO, Pan Deng ZHOU, Li Qui XIA
Biomedical and Environmental Sciences (2012) Volume 25(Issue 5) pp:
Publication Date(Web):1 January 2012
DOI:10.3967/0895-3988.2012.05.014
ObjectiveTo investigate the theoretical model of the three-dimensional structure of mosquitocidal Cry30Ca2 and its molecular docking with N-acetylgalactosamine.MethodsThe theoretical model of Cry30Ca2 was predicted by homology modeling on the structure of the Cry4Ba. Docking studies were performed to investigate the interaction of Cry30Ca2 with N-acetylgalactosamine on the putative receptor.ResultsCry30Ca2 toxin is a rather compact molecule composed of three distinct domains and has approximate overall dimensions of 95 by 75 by 60Å. Domain II is a helix bundle, Domain II consists of three antiparallel β-sheets, Domain III is composed of two β-sheets that adopt a β-sandwich fold. Residue 321Ile in loop1, residues 342Gln 343Thr and 345Gln in loop2, residue 393Tyr in loop3 of Cry30Ca2 are responsible for the interactions with GalNAc via 7 hydrogen bonds, 6 of them were related to the oxygen atoms of hydroxyls of the ligand, and one to the nitrogen of the ligand.ConclusionThe 3D structure of Cry30Ca2 resembles the previously reported Cry toxin structures but shows still some distinctions. Several residues in the loops of the apex of domain II are responsible for the interactions with N-acetylgalactosamine.
Co-reporter:W.P. Li, L.Q. Xia, X.Z. Ding, Y. Lv, Y.S. Luo, S.B. Hu, J. Yin, F. Yan
Gene (1 May 2012) Volume 498(Issue 2) pp:323-327
Publication Date(Web):1 May 2012
DOI:10.1016/j.gene.2012.01.034
In order to assess possible enhancement of biopesticide activity, the fusion gene of crystal protein gene cry1Ac with the insect-specific neurotoxin ω-ACTX-Hv1a gene and egfp was expressed in Bacillus thuringiensis acrystalliferous strain Cry-B under the control of the native gene expression system. The fusion recombinant Cry-B(1Ac-ACTX-EGFP) generally produced two or three small crystal-like inclusion bodies in each cell and the GFP signal could be clearly observed. A 166 kDa full-length fusion protein was identified by immunoblot analysis. Virulence of the fusion inclusions was at least fivefold higher toward larvae of Spodoptera exigua. These results demonstrated that a foreign protein could be expressed and accumulate as parasporal inclusions in B. thuringiensis by C-terminal fusion with the native endotoxin while retaining partial insecticidal activity.Highlights► The fusion protein Cry1Ac-ω-ACTX-Hv1a-EGFP was successfully expressed in B. t. ► Heterogeneous toxins formed parasporal inclusion bodies together with endotoxins. ► Virulence of the fusion inclusions was significantly improved against S. exigua. ► It is a promising approach for potent biopesticides with lower resistance potential.
