Rice stripe virus (RSV) is one of the serious plant pathogenic viruses for rice transmitted by small brown planthopper, SBPH, Laodalphax striatellus. RNA interference (RNAi) is an universal gene-knockdown mechanism in eukaryotic organisms which includes insects，and has been considered as an alternative strategy to control insect pests. Hence, we applied this technique to interfere the translation of target RNA genes to knockdown the virus gene on RSV-viruliferous L. striatellus. Three out of seven RSV genes, RdRp, NS3, and NCP were used as target genes and each dsRNA targeting the viral genes were delivered to the insects indirectly through the rice leaves by irrigation. As a result, not only the relative expression level of target genes decreased but also those of non-target genes and the replication of RSV genome as well. In summary, leaf-mediated dsRNA feeding methods would be useful in the knockdown of target genes on piercing-sucking insects. The genes used in this experiment can be utilized for the development of pest-resistant transgenic plants based on RNAi.

Crystals of proteinaceous insecticidal proteins, Cry proteins, produced by Bacill us thuringiensis (Bt) have been generally used used to control insect pests. In this st udy, through the 3D structure prediction and accompanying mutagenesis study for the Mod-Cry1Ac, 7 and 16 amino acid residues from domain I and II, respectively, responsible for its insecticidal activity against larvae of Plutella xylostella, Spodopt era exigua and Ostrinia furnacalis were identified. To construct novel cry genes wi th enhanced insecticidal activity, we randomly mutated these 24 amino acid sequen ces by in vitro muti site-directed mutagenesis, resulting in totally 34 mutant cry gen es. For further characterization, these mutant cry genes were expressed as a fusion protein with polyhedrin using baculovirus expression system. SDS-PAGE analysis of the recombinant polyhedra revealed that expressed Cry proteins was occluded in to polyhedra and activated stably to 65 kDa by trypsin. When the insecticidal activit ies of these mutant Cry proteins against to larvae of P. xylostella, S. exigua, and O. furnacalis were assayed, they showed higher or similar insecticidal activity compar ed to those of Cry1Ac and Cry1C. Especially, among them Mutant-N16 showed th e highest insecticidal activity against to both of P. xylostella, S. exigua and Ostrinia furnacalis. Therefore, Mutant-N16 is estimated to have the potential for the efficac ious bioagent.

The Asian honeybee, Apis cerana, is a native honeybee species in Korea which is important in agriculture for pollination and honey production. For better understanding of the physiology of A. cerana, high-throughput Illumina transcriptome sequencing was performed to analyze the gene expression profiles of queen, worker and larva. A total of 219,799,682 clean reads corresponding to 22.2Gb of nucleotide sequences was obtained from the whole body total RNA samples. The Apis mellifera reference mRNA sequence database was used to measure the gene expression level with Bowtie2 and eXpress software and the Illumina short reads were mapped to 11,459 out of 11,736 A. mellifera reference genes. Total of 9,221 genes with FPKM value greater than 5 of each sample group were subjected to evolutionary genealogy of genes: Non-supervised Orthologous Groups (eggNOG) with BLASTX for gene ontology analysis. The differential gene expression between queen and worker, and worker and larva were analyzed to screen the overexpressed genes in each sample group. in the queen and worker sample group, total of 1,766 genes were differentially expressed with 887 and 879 genes overexpressed over two folds in queen and worker, respectively. In the worker and larva sample group, total of 1,410 genes were differentially expressed with 1,009 and 401 genes overexpressed over two folds in worker and larva, respectively.

Rice stripe virus (RSV) is one of the serious plant pathogenic viruses for rice and mediated by small brown planthopper, Laodalphax striatellus. So far, the studies have been mainly focused on the interaction between the host plant and the virus. In this study, for better comprehension of the interactions among Rice stripe virus, rice and small brown planthopper, transcriptomes of the RSV-viruliferous (RVLS) and non-viruliferous L. striatellus (NVLS) were comparatively analysed. For this, non-viruliferous L. striatellus were collected from non-infected rice field and fed RSV-infected rice for 5 days. With the RNAs prepared from the RSV-viruliferous and the non-viruliferous small brown planthoppers, we conducted Illumina RNA sequencing (Hiseq 2000) and then two transcriptome databases were generated from RVLS and NVLS, respectively. The transcriptome of RVLS and NVLS were campared to figure out how the gene expression of the insects affected by Rice Stripe Virus. RSV-dependently regulated genes analysed from this study may have important functions in the transmission and replication of RSV.

Sacbrood virus (SBV) is one of the most fatal pathogens against Asian honeybee, Apis cerana. This virus cause failure of the insect larvae to pupate and death of the adult insects. This study has analyzed the host genes affected by viral infection, by comparing the expression level of host transcripts infected with or without SBV. As a first step, we sequenced the cDNA libraries of Asian honeybee by using illumina RNA sequencing. The sequences were de novo assembled to acquire honeybee transcriptome sequences. The transcriptome was annotated by the sequence comparison to known protein sequences by BLASTX and evolutionary genealogy of genes: Non-supervised Orthologous Groups (eggNOG) database with functional categories and description. By mapping the RNA-seq data to de novo assembled transcripts, we characterized the differentially expressed transcripts between SBV-infected and non-infected Asian honeybee.

The novel serogroup of Bacillus thuringiensis serovar mogi (H3a3b3d) was isolated from fallen leaves, sampled in a forest region of the city of Mungyeong, Korea. Plasmids from B. thuringiensis have been implicated in pathogenicity as they carry the genes responsible for different types of diseases in mammals and insects. In this study, the genome sequence of the strain was determined. The 6.0-Mb genome of B. thuringiensis mogi contains three replicons: a circular chromosome (5.40-Mb) encoding 5,652 predicted open reading frames (ORFs), and two megaplasmids, pMOGI364 (364 564 bp) and pMOGI222 (222 348 bp). The G+C contents of these replicons ranged from 31.3% to 34.2% for pMOGI364 and pMOGI222, respectively. There are six putative cry genes, cry19Bb1, cry73Aa, cry20Bb1, cry27Ab1, cry4Aa and cry56Ba1, distributed on these two megaplasmids. 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. Also, these cry genes were cloned to the Escherichia coli-B. thuringiensis shuttle vector, pHT1K under the control of its own promoter and then introduced into an acrystalliferous B. thuringiensis Cry-B strain for further molecular characterization.

Rice stripe virus disease (RSVD), one of the most serious disease of rice is mediated through the sucking by small brown planthopper, Laodalphax striatellus. So far, the studies have been mainly focused on the interaction between the host plant and the virus. In this study, for better comprehension of the interactions among the host plant, vector insect and plant-pathogenic virus, we investigated transcriptome of the vector insect and the differences between viruliferous and naïve L.striatellus. For this, naïve L. striatellus were collected from non-infected rice field and 50 L.striatellus of them were fed RSV-infected rice for 5 days. With the RSV-viruliferous and the naïve insects, we conducted Illumina RNA sequencing (Hiseq 2000) and obtained 175,243,488 and 146,031,348 reads from viruliferous and naïve L.striatellus, respectively. These reads were assembled into contigs and two transcriptome databases were generated. The transcriptome of naïve and RSV-viruliferous L. striatellus were campared to figure out up-regulated or down-regulated genes. These RSV-dependently regulated genes may have important function in the behavior of planthoppers or the transmission of RSV.

Proteinaceous insecticidal proteins, Cry proteins, from Bacillus thuringiensis (Bt) are insecticidal proteins that are highly active against several species of Lepidoptera. Thus, cry genes encoding these Cry proteins have been widely applied for construction of transgenic crops resistant to pest insects. In this study, through the 3D structure prediction and accompanying mutagenesis study for the Mod-Cry1Ac, 7 and 16 amino acid residues from domain I and II, respectively, responsible for its insecticidal activity against larvae of Spodoptera exigua and Ostrinia furnacalis were identified. We used site-directed mutagenesis to improve the insecticidal activity of Mod-Cry1Ac, resulted 31 mutant cry genes. These mutant cry genes encodes potent insecticidal proteins in the form of crystalline protoxins of 95 kDa. SDS-PAGE analysis of the recombinant polyhedra revealed that expressed Cry proteins was occluded into polyhedra and activated stably to 65 kDa by trypsin. When the insecticidal activities of these mutant Cry proteins against to larvae of P. xylostella, S. exigua and O. furnacalis were assayed, they showed higher or similar insecticidal activity compared to those of Cry1Ac and Cry1C. Especially, Mutant-N16 is considered to have the potential for the efficacious biological insecticide since it showed the highest insecticidal activity.

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.

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.

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.

Crystals of proteinaceous insecticidal proteins, Cry proteins, produced by Bacillus thuringiensis (Bt) have been generally used to control insect pests. In this study, through the 3D structure prediction and accompanying mutagenesis study for the Mod-Cry1Ac, 7 and 16 amino acid residues from domain I and II, respectively, responsible for its insecticidal activity against larvae of Spodoptera exigua and Ostrinia furnacalis were identified. To construct novel cry genes with enhanced insecticidal activity, we randomly mutated these 23 amino acid sequences by in vitro muti site-directed mutagenesis, resulting in totally 24 mutant cry genes. For further characterization, these mutant cry genes were expressed as a fusion protein with polyhedrin using baculovirus expression system. SDS-PAGE analysis of the recombinant polyhedra revealed that expressed Cry proteins was occluded into polyhedra and activated stably to 65 kDa by trypsin. When the insecticidal activities of these mutant Cry proteins against to larvae of P. xylostella and S. exigua were assayed, they showed higher or similar insecticidal activity compared to those of Cry1Ac and Cry1C. Especially, among them Mutant-N16 showed the highest insecticidal activity against to both of P. xylostella and S. exigua. Therefore, Mutant-N16 is considered to have the potential for the efficacious biological insecticide.

The baculovirus expression system is one of the most popular methods used for the production of recombinant proteins but has several complex steps which have proved inherently difficult to meet a multi-parellel process. We have developed a novel recombinant bacmid, bEasyBm that enabling easy and fast generation of pure recombinant virus without any purification step. In the bEasyBm, attR recombination sites were introduced to facilitate the generation of recombinant viral genome by in vitro transposition. Moreover, extracellular RNase gene from bacillus amyloliquefaciens, barnase, was expressed under the control of Cotesia plutellae bracovirus early promoter. Therefore, only when the barnase gene was replaced to gene of interest, the bEasyBm could replicate in host insect cells. When the bEasyBm was transposed with pDualBac-EGFP and pDualBac-LUC respectively, there were no non-recombinant backgrounds were detected from unpurified BmEasy-EGFP or BmEasy-LUC stocks. In addition, the resulting recombinant virus, BmEasy-EGFP, showed comparable level of EGFP expression efficiency with the plaque-purified recombinant virus, BmEGFP, which was constructed using bBmGOZA system. Based on these results, high-throughput condition for generation of multiple recombinant viruses in a time was established.

Varieties of Bacillus thuringiensis (Bt) crystal proteins, Cry proteins, have so far been found as one of the most successful biological control agents which are safe to natural environments for a long time. Recently, cry genes encoding these Cry proteins have been widely applied for construction of transgenic crops resistant to pest insects. In this study, through the 3D structure prediction and accompanying mutagenesis study for the Mod-Cry1Ac, 7 and 16 amino acid residues from domain I and II, respectively, responsible for its insecticidal activity against larvae of Spodoptera exigua and Ostrinia furnacalis were identified. To construct novel cry genes with improved insecticidal activity, we randomly mutated these 23 amino acid sequences by in vitro muti site-directed mutagenesis, resulting in totally 24 mutant cry genes. For further characterization, these mutant cry genes were expressed as a fusion protein with polyhedrin using baculovirus expression system. SDS-PAGE analysis of the recombinant polyhedra revealed that expressed Cry proteins was occluded into polyhedra and activated stably to 65 kDa by trypsin. In the further study, we plan to investigate their insecticidal activity against Plutella xylostella, S. exigua and O. furnacalis larvae.