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Molecular Characterization of the Mosquitocidal cry Genes from a Bacillus thuringiensis Serovar mogi
Qin Liu,Jae Young Choi,Xue Ying Tao,Joo Hyun Lee,Song Eun Kim,Zhenli Fu,Jae Su Kim,Yeon Ho Je 한국응용곤충학회 2012 한국응용곤충학회 학술대회논문집 Vol.2012 No.05
Plasmids from Bacillus thuringiensis have been implicated in pathogenicity as they carry the genes responsible for different types of diseases in mammals and insects. B. thuringiensis serovar mogi of a novel serogroup (H3a3b3d), which showed mosquitocidal activity against Anopheles sinensis and Culex pipiens pallens, was isolated from fallen leaves in Mungyeong city, Republic of Korea. In contrast to the complicated plasmid profiles of B. thuringiensis H3 serotype strains, the B. thuringiensis serovar mogi contained only megaplasmid (> 30 MDa) on which the toxin genes were occasionally located. Sequence analysis using 454-pyrosequencing revealed that the megaplasmid harbored at least seven putative cry genes, showing about 84%, 75%, 73%, 58%, 84%, 39% and 75% similarities in amino acid sequences with Cry27Aa, Cry19Ba, Cry20-like, Cry56Aa, Cry39ORF2, Cry8Ba and Cry40ORF2, respectively. These cry genes were cloned to the Escherichia coli-B. thuringiensis shuttle vector, pHT1K, and then introduced into an acrystalliferous B. thuringiensis Cry-B strain for further molecular characterization. This novel 3a3b3d type strain, B. thuringiensis serovar mogi, could be used as a good resource for studying unknown mosquitocidal cry genes.
Qin Liu,Jae Young Choi,Xue Ying Tao,Seok Hee Lee,Saes Byeol An,Song Eun Kim,Woo Jin Kim,Yeon Ho Je,Yeon Ho Je 한국응용곤충학회 2013 한국응용곤충학회 학술대회논문집 Vol.2013 No.04
Plasmids are crucial for determining the pathogenicity and host range of organisms of the Bacillus thuringiensis strains. In this research, a novel serogroup of B. thuringiensis serovar mogi (H3a3b3d), which showed mosquitocidal activity against Anopheles sinensis and Culex pipiens pallens, was isolated from fallen leaves in Mungyeong city, Republic of Korea. In contrast to the complicated plasmid profiles of B. thuringiensis H3 serotype strains, the B. thuringiensis serovar mogi contained two megaplasmids (> 30 MDa) on which the toxin genes were occasionally located. Sequence analysis using 454-pyrosequencing revealed that there are 7 putative cry genes, cry19Bb1, cry73Aa, cry40orf2, cry20Bb1, cry27Ab1, cry56Ba1 and cry39orf2, distributed on the two different megaplasmids, respectively. These cry genes were cloned to the Escherichia coli-B. thuringiensis shuttle vector, pHT1K under the control of its own promoter and p1KSD, which is a recombinant expression vector containing cyt1Aa promoter combined with the STAB-SD sequence, and then introduced into an acrystalliferous B. thuringiensis Cry-B strain for further molecular characterization. To investigate the role of these genes in crystal production, the expression profiles of these toxin genes were analyzed by quantitative PCR (qPCR) from the wild type strain. These results clearly indicate that the cry39orf2 was the dominant ingredient in the crystal. This novel 3a3b3d type strain, B. thuringiensis serovar mogi, could be used as a good resource for studying unknown mosquitocidal cry genes.
Xue Qin,Xie Guohua 한국정보디스플레이학회 2020 Journal of information display Vol.21 No.3
Organic light-emitting devices (OLEDs) on the silicon backplanes processed via the standard foundry complementary metal–oxide-semiconductor (CMOS) processes were developed with the state-of-the-art electrical doping technology to meet the optical and electrical requirements, resulting in high luminance (over 5000 cd/m2) for the green emissive OLED microdisplay. The unavoidable oxidized aluminium contact after the CMOS processes on the top layer of the pixel was found to significantly increase the driving voltage of the device (up to 1 V at 100 cd/m2 luminance). To aid in the extraction of the accurate device parameters for setting up an equivalent circuit, the reference top-emitting OLEDs without bottom metal contacts were deposited directly on interconnection-metal-only silicon substrates from the CMOS foundry. The distribution of the AlOx of the top-layer metal contact on silicon (the bottom anode of OLEDs) was confirmed by the X-ray photoelectron spectroscopy (XPS) depth profiles.
Xue Ying Tao,Jae Young Choi,Woo Jin Kim,Qin Liu,Song Eun Kim,Saes Byeol An,Seok Hee Lee,Soo Dong Woo,Byung Rae Jin,Yeon Ho Je 한국응용곤충학회 2013 한국응용곤충학회 학술대회논문집 Vol.2013 No.04
ORF78 (ac78) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is a baculovirus core gene of unknown function. To determine the role of ac78 in baculovirus life cycle, an ac78-deleted mutant AcMNPV, Ac78KO, was constructed. Quantitative PCR analysis revealed that ac78 is a late gene in the viral life cycle. After transfection into Spodoptera frugiperda cells, Ac78KO produced a single-cell infection phenotype indicating that no infectious budded viruses (BVs) were produced. The defection in BV production was also confirmed by both viral titration and Western blot. However, viral DNA replication is unaffected. Analysis of BV and occlusion derived virus (ODV) revealed that AC78 is associated with both forms of the virions and is a structural protein located to viral envelope. Electron microscopy showed that ac78 also plays an important role in embedding of ODV into occlusion body. This study therefore demonstrates that AC78 is a late virion associated protein and is essential for the viral life cycle.
Qin Liu,Jae Young Choi,Xue Ying Tao,Joo Hyun Lee,Song Eun Kim,Zhenli Fu,Woo Jin Kim,Yeon Ho Je 한국응용곤충학회 2012 한국응용곤충학회 학술대회논문집 Vol.2012 No.10
Bacillus thuringiensis serovar mogi of a novel serogroup (H3a3b3d), which showed mosquitocidal activity against Anopheles sinensis and Culex pipiens pallens, was isolated from fallen leaves in Mungyeong city, Republic of Korea. In contrast to the complicated plasmid profiles of B. thuringiensis H3 serotype strains, the B. thuringiensis serovar mogi contained only megaplasmid (> 30 MDa) on which the toxin genes were occasionally located. Sequence analysis using 454-pyrosequencing revealed that the megaplasmid harbored at least seven putative cry genes, showing about 84%, 75%, 73%, 58%, 84%, 39% and 75% similarities in amino acid sequences with Cry27Aa, Cry19Ba, Cry20-like, Cry56Aa, Cry39ORF2, Cry8Ba and Cry40ORF2, respectively. These cry genes were cloned to the Escherichia coli-B. thuringiensis shuttle vector, pHT1K, and then introduced into an acrystalliferous B. thuringiensis Cry-B strain for further molecular characterization. To investigate the role of these genes in crystal production, the expression profiles of these toxin genes were analyzed by quantitative real-time PCR (qrtPCR) from the wild type strain as well as transformant strains. The results clearly indicate that the cry39orf2 was the dominant ingredient in the crystal. This novel 3a3b3d type strain, B. thuringiensis serovar mogi, could be used as a good resource for studying unknown mosquitocidal cry genes.
Functional analysis of Autographa californica multiple nucleopolyhedrovirus ac78 and ac79
Xue Ying Tao,Jae Young Choi,Jae Su Kim,Qin Liu,Jong Bin Park,Joo Hyun Lee,Soo Dong Woo,Byung Rae Jin,Yeon Ho Je 한국응용곤충학회 2011 한국응용곤충학회 학술대회논문집 Vol.2011 No.10
Among 154 putative ORFs of Autographa californica multiple nucleopolyhedrovirus (AcMNPV), ac78 and ac79 are highly conserved genes in baculovirus, but their functions in the virus life cycle have been unknown so far. To determine their roles in AcMNPV replication, knockout mutants, ac78KO and ac79KO, were constructed using the plasmid capture system (PCS). Real-Time PCR analysis showed that both of ac78 and ac79 transcripts were first detected at 6 hours post-infection, and accumulated to maximum at 24 hours post-infection, suggesting that both of ac78 and ac79 are belong to late gene. When the genomic DNA of ac78KO was transfected into Sf9 cells, viral replication was restricted to a single cell infection. These results demonstrated that the ac78 play an important role in BV production, and therefore is essential for AcMNPV to mount a successful infection. Whereas Sf9 cells infected with the ac79KO showed normal viral symptoms such as rounding and swelling, OBs were not observed from majority of infected cells. These results suggested that the ac79 might play an important role in OB production.
Xue Ying Tao,Jae Young Choi,Woo Jin Kim,Qin Liu,Song Eun Kim,Saes Byeol An,Seok Hee Lee,Soo Dong Woo,Byung Rae Jin,Yeon Ho Je 한국응용곤충학회 2013 한국응용곤충학회 학술대회논문집 Vol.2013 No.04
ORF11 (ac11) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is a highly conserved gene of unknown function. To determine the role of ac11 in baculovirus life cycle, an ac11-knockout mutant AcMNPV, Ac11KO, was constructed. qPCR analysis revealed that ac11 is an early gene in the life cycle. After transfection into Spodoptera frugiperda cells, Ac11KO produced a single cell infection phenotype indicating that no infectious budded viruses (BVs) were produced. The defection in BV production was confirmed by both viral titration and Western blot. However, viral DNA replication is unaffected. Electron microscopy showed that ac11 is required for nucleocapsids envelopment to form ODV and their subsequent embedding into OB. This study therefore demonstrates that ac11 is an early gene which is essential for the viral life cycle.
Autographa californica Multiple Nucleopolyhedrovirus ac11 is Required for Virus Infection
Xue Ying Tao,Jae Young Choi,Qin Liu,Joo Hyun Lee,Song Eun Kim,Zhenli Fu,Byung Rae Jin,Yeon Ho Je 한국응용곤충학회 2012 한국응용곤충학회 학술대회논문집 Vol.2012 No.05
ORF11 (ac11) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is a highly conserved baculovirus gene whose homologs are found in all lepidoteran Group I NPV, but its function is unknown so far. To determine the role of ac11 in baculovirus life cycle, ac11 knock-out mutant, Ac11KO, was constructed using the plasmid capture system (PCS). Real-Time PCR analysis showed that ac11 transcript was first detected at 6 h post-infection (p.i.) and accumulated to maximum at 48 h p.i., indicating that ac11 is belong to late gene. When the genomic DNA of Ac11KO was transfected into Sf9 cells, viral replication was restricted to a cell transfected originally. While viral transmission of the Ac11KO was not observed in Sf9 cells, production of budded virus (BV) in Sf9 cells transfected with Ac11KO was observed by transmission electron microscopy (TEM). These results suggest that the ac11 is essential for AcMNPV to produce infective BV.