An organophosphorus pesticide, ethoprophos, has been widely used in agriculture to control major insect pests. As ethoprophos is a well-known neurotoxin, its accumulation in soils and groundwater is concerning worldwide. In this study, we constructed an artificial ethoprophos-enrichment soil system, and its active concentration in soils was measured by gas chromatography on 15-days intervals during 90 days after ethoprophos treatment. Furthermore, the changes in bacterial community and microorganisms responsible for efficient bioremediation were investigated while ethoprophos was degraded in soils. From 15 to 60 days after the treatment, ethoprophos was actively degraded in soils and members of genera Collimonas and Sphingobium appeared dominantly in a natural microbial community especially in 60-days-after-treatment soil. We isolated a bacterium EP60845 from this soil sample, showing significant ethoprophos biodegradation activity in vitro. When we challenged EP60845 strain into ethoprophos-enrichment soils (250 mg/kg of soil), most ethoprophos was removed within 5-days. Phylogenetic 16S rRNA gene sequence analysis and biochemical properties by API 20GN kit demonstrated that the EP60845 strain was a novel Sphingobium sp., which could be used as an efficient ethoprophos- degrading agents for bioremediation purposes.

Riboswitches are structured RNA motifs that can directly bind specific metabolites. The binding of metabolites further regulates downstream metabolism eliminating the need for any regulatory proteins. We searched for novel bacterial vitamin B1 binding riboswitches in the metagenome of sun-dried saline soil. Soil microbial metagenomes were studied using NGS analysis. A total of approximately 50 Gb of the sequence data was obtained by Hi-seq and 454 GS FLX sequencing, and these sequences were subjected to riboswitch search. Hi-seq generated 614 contigs showing similarity to riboswitches, while 454-based sequencing generated 383 similar contigs. We matched whole metagenome contigs to local BLAST databases constructed using 91 previously known bacterial vitamin B1 thiamine pyrophosphate (TPP)-box motifs, and 11 SAM S-box motifs. Repetitive BLAST comparisons to local BLAST databases with nucleotide sequences from NGS identified 14 novel TPP-box motifs, and 7 S-box motifs respectively from the metagenome contigs. Further, RNA secondary structure analysis with public databases Rfam, and RibEx using these 21 riboswitch candidates revealed one contig, D8PYI to possess the most probable TPP-box structure. We constructed intragenic synthetic riboswitches to investigate whether the TPP-box motif region in D8PYI could harness gene expression in the presence of TPP. Construction of biosensors containing 100~400 bp fragments of D8PYI contigs, and in vivo imaging using the biosensors displayed TPP-specific changes in the expression of a green fluorescence protein reporter. In this regard, the adaptation of in silico riboswitch screening from environmental metagenomes could provide biosensors for detection of specific metabolites.

Plant-parasitic nematodes are the most devastating group of plant pathogens worldwide and are extremely challenging to control. In the present study, we have performed a genome wide analysis to identify common genes among four nematode species consisting of root-knot nematodes (Meloidogyne incognita and Meloidogyne hapla), cyst nematode (Heterodera glycines), and free living nematode (Caenorhabditis elegans) respectively. Using their whole genome sequences, we predicted 15,274 genes from M. incognita, 38,149 genes from M. hapla, 8,061 genes from H. glycines and 23,894 genes from C. elegans, where, among the predicted genes, 1,358, 1,350, 1,401, 1,365 respectively from each nematode, code for common groups of proteins. Further, 2,067, 2,086, 1,566, 2,903 genes were recollected using Clusters of Orthologous Groups (COG) database. Under our search criteria, a total of 800 common genes were identified in all the four studied nematode genomes. The most annotated conserved genes were obtained from four different species using Basic Local Alignment Searching Tool (BLAST). Uni- Prot Taxon identifier database was used to elucidate their taxonomic classification such as 698 genes under kingdom Metazoa, 660 genes confined to Nematoda, 290 genes in Chordata and 660 genes falling under class Chromadorea. The biochemical characterization of proteins expressed by these genes was examined using Pedant-Pro sequence analysis. The protein length, molecular weight, isoelectric point (pI), and transmembrane domain of the coded proteins were at a range of 300 to 999 amino acids (40.9%), molecular weight of over 100 kDa (96%), pI from 4.5 to 5.5 (27.6%) and 0 (56.6%), respectively. To classify protein function, the obtained BLAST hits were assigned to Gene Ontology classification scheme. The fractions of protein function were distributed as cellular component, biological processes and molecular function of the cell (22.2%), multicellular organism process (15.8%) and binding (48.3%), respectively. The current study provides an excellent resource for nematode functional genomics studies, which can be utilized further for studies on role of genes involved in nematode biological processes.

Human tissue-type plasminogen activator (t-PA) is responsible for fibrin-specific plasminogen activation and plays a key role in fibrinolysis thereby aiding breakdown of blood clots in the vasculature. In the present study, in order to develop a system for production of recombinant st-PA and t- PAHis6 proteins in transgenic rice seeds, a DNA fragment encoding t-PA gene was selected and cloned to a plant binary vector (pMJ21) harboring a rice GluB1 promoter, an N-terminal signal peptide of the rice glutelin B1 protein and a Pin II terminator. The constructed plasmid was transformed into Agrobacterium tumefaciens LBA4404 (pSB1) to facilitate introduction into rice callus. The insertion of the st-PA and t-PAHis6 genes into the genome of transgenic rice seeds and their transcripts were confirmed using PCR, and Southern blot as well as RT-PCR, respectively. The highest level of recombinant st-PA expression as determined by enzyme-linked immunosorbent assay (ELISA) was found to be 2,916 ng/total soluble protein (mg) in transgenic rice seeds. The amount of recombinant proteins expressed in transgenic plants was estimated to range from 634 ~ 2,916 ng/TSP mg (st-PA) and 925 ~ 2,640 ng/TSP mg(t- PAHis6), respectively. Immuno-blot analysis of transgenic rice seeds revealed single bands of approximately 68-kDa representing recombinant st-PA and t-PAHis6 proteins. These results demonstrate the expression and in vivo activity of recombinant st-PA and t-PAHis6 in transgenic rice seeds. This study is a promising endeavor for production of recombinant pharmaceutical proteins using rice seed system.

RNAi 기법은 예쁜꼬마선충을 비롯하여 초파리, 생쥐, 인체및 식물들을 포함한 다양한 생물종에서 작용기작이 잘 알려져 있고, 특정 유전자들의 발현을 제어하기 위해 사용되어 왔다.본 논문은 RNAi 기법을 통해 구축된 뿌리혹선충 저항성 형질전환식물체들의 개발현황, 발전 및 응용 가능성에 대해 고찰하고자 하였다. 지난 10여년에 걸쳐 진행된 연구들을 통해 다양한 분화단계의 뿌리혹선충으로부터 분석된 79,978개의 EST가 GenBank에등록되었고, 고구마뿌리혹선충(M. incognita)과 당근뿌리혹선충(M. hapla)의 전체 게놈 염기서열이 해독됨으로써 뿌리혹선충의 기생에 관련된 단백질들 및 식물세포벽 분해 관련 효소들에 대한 정보를 이용할 수 있게 되었다. RNAi의 기본 기작은 모든 진핵생물종에서 잘 보존되어 있고, 최근 식물기생선충들에서 RNAi 효과에 대한 연구결과들이 많이 발표되었다. 서로 다른 뿌리혹선충들에서 현재까지 22종 이상의 RNAi를위한 목표 유전자들이 보고되었다. 제2령 식물기생선충(second-stage juvenile of plant parasitic nematodes)에게dsRNA의 섭취를 유도하는 octopamine, resorcinol, serotonin등의 화합물들이 발견되었고, 이를 이용하여 선충 유전자의 발현제어를 쉽게 판별할 수 있는 새로운 기술의 활용이 가능하게 되었다. 최근에 RNAi 기술의 응용 및 발전을 통해 형질전환식물체에서 발현된 dsRNA에 의한 식물기생선충 유전자들의 발현제어가 증명되었고, 이를 위한 핵심 요소들로서 적절한 선충 표적 유전자의 선택, 식물체 내 높은 함량의 dsRNA의 발현 및선충이 섭취할 수 있는 충분한 양의 dsRNA의 운반 등이 중요하다는 것이 밝혀졌다. 특히, 비표적 유전자서열(off-target gene sequence)의 발현제어를 피하기 위해 다음과 같은 사항들이 고려되어야 한다. 1)비표적 유전자서열을 확인할 수 있는 소프트웨어의 개발 및 이를 통한 비표적 유전자서열을 제거한 RNAi 벡터를 제작하여야 한다. 2)식물과 동물에서 상동성이 높은 표적 유전자의 발현을 피해야 한다. 3)전사해석틀(open reading frames)의 염기서열들 보다 상동성이 낮은 5' 혹은 3'-비해석부위(untranslated regions)로부터 표적 유전자를설계하여야 한다.

선행 연구에서 형질전환 모상근 대량생산 최적배지 선발을 통해 WPM(Woody Plant basal salt Mixture) 배지가 형질전환 모상근 대량생산의 최적배지임을 확인할 수 있었다(Kim et al., 2012). 이에 당, 탄소원, pH, 무기염, 식물 호르몬 등이 형질전환 모상근의 수량성에 미치는 연구를 통해 최적의 형질 전환 모상근 대량생산 조건을 확립한 결과는 다음과 같다. 1. 형질전환 모상근 대량생산에 적합한 pH는 7.0이었으며, 최적의 탄소원과 농도는 1%의 sucrose임을 확인하였다. 2. 형질전환 모상근 대량생산에 적합한 적정 무기염들 (KH2PO4,NH4NO3, KNO3)의 농도는 KH2PO4 경우 MS배지와같은 농도인 0.94 mM에서 수량성이 303 g으로 가장 높았고, NH4NO3 및 KNO3 농도는 MS배지 농도의 1/4배인 3.87 mM, 3.52 mM 처리가 345 g과 358 g으로 수량성이 가장 높았다. 3. 형질전환 모상근 대량생산에 있어 식물생장조절물질 NAA, IBA의 최적 농도는 무처리 경우가 각각 595 g과 402 g으로 수량성이 가장 높아 대량생산에는 식물생장조절물질 처리가 필요 없을 것으로 판단되었다.