Molecular Cloning of Two cDNAs Encoding an Insecticidal Toxin from the Spider, Araneus ventricosus, and Construction of a Recombinant Baculovirus Expressing a Spider Toxin

Chung, Eun-Hwa,Lee, Kwang-Sik,Han, Ji-Hee,Je, Yeon-Ho,Chang, Jin-Hee,Roh, Jong-Yul Korean Society of Sericultural Science 2002 International Journal of Industrial Entomology Vol.4 No.1

We have cloned cDNAs encoding toxin from the spider, Araneus ventricosus, and constructed a recombinant baculovirus expressing the insecticidal toxin. The cDNAs encoding toxin were cloned from the cDNA library of A. ventricosus. Sequence analysis of the cDNAs encoding the toxin of A. ventricosus revealed that the 240 bp cDNA for AvTox-1 and 192 bp cDNA for AvTox-2 have an open reading frame of 80 and 64 amino acid residues, respectively. The deduced protein sequence of the toxin genes of AvTox-1 and AvTox-2 was aligned to that of the snack Anemonia sulcata and scorpion Centruroides limpidus limpidus, respectively. Northern blot analysis indicated that AvTox-2 toxin gene showed a fat body-spe-cific expression pattern at the transcriptional level. Furthermore, we have explored the possibility of improving baculovirus by incorporating the A. vontricosus toxin gene into Bombyx mori nuclear polyhedrosis virus genome under the control of polyhedrin promoter, The AvTox-2 toxin gene was expressed as approximately 5.8 kDa band in the recombinant baculovirus-injected silkworm larvae. Bioassays with the recombinant virus expressing AvTox-2 on 5th instar silkworm larvae demonstrated a decrease in the time to kill $(LT_{50} days)$ compared to wild-type BmNPV-Kl $(LT_{50} 6.72 days)$ in the injection of 10 viruses. These results indicate that A. ventricosus toxin is a novel member of the spider toxin family, suggesting that the toxin gene can be used in recombinant baculoviruses to reduce insect feeding damage and increase the speed of insect kill.

Biochemical and Pathological Characters of Different Bt Cry Toxins

Eom, Seonghyeon,Kim, Yonggyun 한국응용곤충학회 2013 한국응용곤충학회 학술대회논문집 Vol.2013 No.10

An entomopathogenic bacterium, Bacillus thuringiensis (Bt), is Gram-positive and undergoes sporulation along with the production of the insecticidal crystal toxins in a paraspore form. This study investigated the biochemical and insecticidal activities of Cry toxins of various Bt strains of Bt aizawai, Bt kurstaki, Bt tenebrionis, and Bt israelensis. Bt aizawai for a Cry1C (135 kDa), Bt kurstaki for a Cry1A (133 kDa), Bt tenebrionis for a Cry3 (73 kDa), Bt israelensis for a Cry4A (134 kDa) and Cry4B (128 kDa). To look for insect pest spectra of these four Cry toxins against four different insects, Cry1A was the most potent to Plutella xylostella, Cry1C to Spodoptera exigua, Cry3 to Tribolium castaneum, and Cry4A to Drosophila melanogaster. To further analyze the differential insecticidal activities of Cry toxins, two Cry toxin genes were expressed using baculovirus expression system. Both Cry1Ac and Cry1Ca toxins significantly enhanced the baculoviral pathogenicity against P. xylostella and S. exigua. However, the synergistic effects were different depending on the type of Cry toxins. These results suggest that the different insecticidal spectra of different Bts are explained by the different Cry toxins produced by the bacteria.

Bacillus thuringiensis serovar. darmstadiensis의 곤충치사독소 유전자분리 및 구조해석

김도영,구본성,도대홍 한국식품영양학회 1996 韓國食品營養學會誌 Vol.9 No.4

지금까지 많은 연구가 되어 있지 않던 Bacillus thuringiensis serover. darmstadiensis의 내독소를 Rengorafin-76 단계적 기울기 원심분리로 분리하여 전자 현미경으로 관찰하여 이중피라미드 구조를 가진 독소 단백질을 확인하였으며 B. thuringiensis serover. kurstaki HD1의 독소 생성유전자와 B. thuringiensis serovar. darmsladiensis의 유전자가 유사성이 있다는 보고를 근거로 하여 B. thuringiensis serovar. HD1의 독소생성유전자를 가진 프로브(pUYBT 9044)로 이용하여 colony hybridization 및 southern hybridization한 결과 2.6Kb EcoRI 단편 및 Southern hybridizationg한 결과 2.6Kb EcoRI 단편 및 3.6Kb HindⅢ 단편을 선발할 수 있었다. 이들 단편들은 B. thuringiensis serovar.kurstaki HD1 독소 유전자와 hybridization시 유사성이 있었다. 특히 3.5Kb HindⅢ 단편은 2.6Kb EcoRI 단편에 클로닝되어 있는 1.8Kb의 HD1 독소유전자와 유사성이 있는 부분을 공유하고 있었으며 1.0Kb정도의 EcoRI-HindⅢ 부분이 더 삽입한 것을 알 수 있었다. Bacillus thuringiensis serovar. darmstadiensis produced bipyramidal endo-toxin. The toxin protein was purified by Renografin-76 step gradient centrifugation and investrigated by electron microscope. Analysis of totoal plasmid DNA patterns showed that four different size of plasmids existed in wild type B. thuringiensis serovar. darmstadiensis. Total plasmids DNA was isolated and transformed into pst I site of pBR322 cloning vector. Ten clones containing crystal toxin gene were forst screened colony hybridization by using PUYBT 9044 probe ontained B. thuringiensis kurskaki HD 1 toxin gene. Cloned-DNA was digested with EcoR1 and HindⅢ and transformed to pIBI30 sequencing vector. Finally, 2.6kb and 3.6kb size fragments contatined toxin-gene were cloned with restriction analysis.

Molecular cloning and characterization of two peptide toxins from the spider Araneus ventricosus

Hu Wan,이광식,추영무,제연호,Jianhong Li,진병래 한국응용곤충학회 2013 Journal of Asia-Pacific Entomology Vol.16 No.1

Spider toxins have great potential in the development of biopesticides. Here, we report the molecular cloning and characterization of two peptide toxins from the spider Araneus ventricosus. Two cDNAs encoding peptide toxins were cloned from A. ventricosus. Analysis of the cDNA sequence shows that the mature peptides of AvT-39 and AvT-48 consist of 39-amino acid residues and 48-amino acid residues, respectively. Both of the mature peptides include six conserved cysteine residues and a principal structural motif typical of spider toxins. The AvT-39 and AvT-48 cDNAs, which encode the mature peptide, were expressed in baculovirusinfected insect cells. AvT-39 and AvT-48 expression in insect cells significantly decreased cell viability. Additionally,the median lethal time (LT50) of Spodoptera exigua larvae inoculated with recombinant AcNPV expressing AvT-48 was approximately 1 day shorter than that of larvae expressing wild-type AcNPV, demonstrating that the recombinant virus reduced LT50 by approximately 25%. Taken together, our findings describe the molecular characterization of two peptide toxins from A. ventricosus and demonstrate the potential for these toxins to be used as biopesticides.

Molecular Characterization of a Novel Vegetative Insecticidal Protein from Bacillus thuringiensis Effective Against Sap-Sucking Insect Pest

( Sattar Sampurna ),( Mrinal K. Maiti ) 한국미생물 · 생명공학회 2011 Journal of microbiology and biotechnology Vol.21 No.9

Several isolates of Bacillus thuringiensis (Bt) were screened for the vegetative insecticidal protein (Vip) effective against sap-sucking insect pests. Screening results were based on LC50 values against cotton aphid (Aphis gossypii), one of the dangerous pests of various crop plants including cotton. Among the isolates, the Bt#BREF24 showed promising results, and upon purification the aphidicidal protein was recognized as a binary toxin. One of the components of this binary toxin was identified by peptide sequencing to be a homolog of Vip2A that has been reported previously in other Bacillus spp. Vip2 belongs to the binary toxin group Vip1-Vip2, and is responsible for the enzymatic activity; and Vip1 is the translocation and receptor binding protein. The two genes encoding the corresponding proteins of the binary toxin, designated as vip2Ae and vip1Ae, were cloned from the Bt#BREF24, sequenced, and heterologously expressed in Escherichia coli. Aphid feeding assay with the recombinant proteins confirmed that these proteins are indeed the two components of the binary toxins, and the presence of both partners is essential for the activity. Aphid specificity of the binary toxin was further verified by ligand blotting experiment, which identified an~50 kDa receptor in the brush border membrane vesicles of the cotton aphids only, but not in the lepidopteran insects. Our finding holds a promise of its use in future as a candidate gene for developing transgenic crop plants tolerant against sap-sucking insect pests.

총설 : 곤충살충성 세균 Photorhabdus의 Insecticidal Toxin과 연구동향

장은경 ( Eun Kyung Jang ),신재호 ( Jae Ho Shin ) 한국미생물 · 생명공학회 2010 한국미생물·생명공학회지 Vol.38 No.2

Oral Toxicity of Symbiotic Bacteria of Entomopathog Nematode to the Sweetpotato Whitefly, Bemisia tabaci

Yam Kumar Shrestha,Hong-Soo Choi,Kwan-Suk Lee,Chang-Suk Kim,Sukchan Lee,Kyeong-Yeoll Lee 한국응용곤충학회 2010 한국응용곤충학회 학술대회논문집 Vol.2010 No.10

The oral toxicities of symbiotic bacteria Photorhabdus temperata ssp temperata (Ptt), mutually associated with entomopathogenic nematode Heterorhabditis megidis, and P. luminescens ssp. laumondii (TT01) with H. bacteriophora, were demonstrated to adults of the sweetpotato whitefly Bemisia tabaci. Sucrose solution (25%) containing bacteria-free supernatant of culture media of symbiotic bacteria was ingested into adult whiteflies within the glass tube. Whitefly mortalities were shown similar patterns against two bacterial media. Mortalities were significantly increased to 60-64% at 36 hours and almost 100% at 60 hours after treatments. In addition, We demonstrated the effect of oral ingestion of symbiont culture media on the gene expression of B. tabaci. Several genes fluctuated those expression levels. Our results suggest that oral ingestion of symbiont culture media of entomopathogenic nematodes significantly changed metabolic rates and highly lethal to whiteflies. The use of symbiotic bacteria of entomopathogenic nematodes provides a great potential as an alternative genetic resource of Bacillus thuringiensis, a major resource of microbial insecticide.

Insecticidal activity of recombinant baculovirus co-expressing Bacillus thuringiensis crystal protein and Kunitz-type toxin isolated from the venom of bumblebee Bombus ignitus

최재영,정명표,Xue Ying Tao,진병래,제연호,박홍현 한국응용곤충학회 2013 Journal of Asia-Pacific Entomology Vol.16 No.1

To improve the insecticidal activity of Autographa californica nucleopolyhedrovirus (AcMNPV), using co-expression of Bacillus thuringiensis crystal protein and a Kunitz-type toxin isolated from bumblebee Bombus ignitus venom, a recombinant AcMNPV, ApPolh5-3006BiKTI, expressing Bi-KTI under the control of early promoter from Cotesia plutellae bracovirus (CpBV) was constructed. In this recombinant virus, B. thuringiensis cry1-5 crystal protein gene was introduced into the genome by the fusion of polyhedrin-cry1-5 under the control of polyhedrin gene promoter. RT-PCR analysis indicated that both Bi-KTI and polyhedrin-cry1-5 fusion protein were successfully expressed from the infected cells. In addition, SDS-PAGE revealed that polyhedrin-cry1-5 fusion protein expressed by recombinant viruses was occluded into the polyhedra. ApPolh5-3006BiKTI showed an improved insecticidal activity against larvae of Plutella xylostella and Spodoptera exigua. At lowdosage rates, it wasmore effective against S. exigua than on P. xylostella, but more rapid insecticidal activity was shown in P. xylostella. These results strongly suggest that co-expression of Bt toxin and Kunitz-type toxins could be successfully applied to improve the insecticidal activity of baculoviruses.

Insecticidal activity of recombinant baculovirus expressing both spider toxin isolated from Araneus ventricosus and Bacillus thuringiensis crystal protein fused to a viral polyhedrin

정명표,최재영,Xue Ying Tao,진병래,제연호,박홍현 한국곤충학회 2012 Entomological Research Vol.42 No.6

A novel recombinant Autographa californica nucleopolyhedrovirus (AcMNPV), ApPolh5‐3006AvTox2, co‐expressing two insecticidal toxins, one isolated from the spider Araneus ventricosus, and the other from Bacillus thuringiensis, was constructed to improve the insecticidal activity of AcMNPV. The recombinant virus was designed to express insect‐specific spider toxin, Av‐Tox2, under control of the early promoter from Cotesia plutellae bracovirus (CpBV). In addition, the B. thuringiensis cry1‐5 crystal protein gene was introduced into the genome of this recombinant virus by fusing it with the viral polyhedrin gene, thus creating a hybrid polyhedrin‐cry1‐5 gene under control of the polyhedrin gene promoter. Reverse transcription‐polymerase chain reaction (RT‐PCR) analysis revealed that both Av‐Tox2 and Polyhedrin‐Cry1‐5 fusion protein were successfully expressed in the infected cells. In addition, SDS‐PAGE revealed that Polyhedrin‐Cry1‐5 fusion protein expressed by recombinant viruses occluded the polyhedra. ApPolh5‐3006AvTox2 showed significantly reduced LD50 and ST50 values against both Plutella xylostella and Spodoptera exigua larvae. These results strongly suggested that coexpression of spider and B. thuringiensis insecticidal toxins could be successful in improving the insecticidal activity of baculoviruses.

Insecticidal Activities of Recombinant Baculviruses Expressing Kunitz-type Toxin Isolated from Insect Venoms

Jae Young Choi,Yong Wang,Xue Ying Tao,Jae Su Kim,Jong Yul Roh,Byung Rae Jin,Yeon Ho Je 한국응용곤충학회 2011 한국응용곤충학회 학술대회논문집 Vol.2011 No.10

Although baculoviruses have a long history of safe use as specific, environmentally benign insect control agents, their use has been limited by several factors, especially their slow speed of action. In this study, we intended to improve the insecticidal activities of Autographa californica nucleopolyhedrovirus (AcMNPV) by expressing Kunitz-type toxin isolated from venoms of Bombus ignitus or Araneus ventricosus. For this, recombinant AcMNPVs, AcBi-KTT, AcAv-Tox1 and AcAv-Tox2 expressing Bi-KTT, Av-Tox1 and Av-Tox2, respectively, under the control of p10 gene promoter were constructed. While polyhedra produced by these recombinant viruses were identical to those of the wild-type AcMNPV in shape, their sizes were relatively smaller than those of the AcMNPV. Among recombinant viruses, AcBi-KTT and AcAv-Tox2 showed significant reduction in median lethal time (LT50) against Spodoptera exigua larvae. Especiaaly, these two viruses showed about 6.2~10-folds higher polyhedra production rate compared to that of the AcMNPV. These results suggested that Kunitz-type toxins from insect venom could be successfully applied to improve insecticidal activity of baculoviruses.