In the digestive gland cDNA library we generated, most of the unique sequences coded for enzymes (Fig. 1-3). Enzymes, the workhorses of the cell, are often overlooked in analysis of tissue expression patterns in favor of other groups, such as transcription factors and receptors (Wistow et al., 2002). However they are clearly of great importance and, for the digestive gland and gonad, they have special significance in some key areas. Morse et al., 1977 found that highly reactive and short-lived free radical oxidants could cause the induction of spawning in mollusks. Presence of these chemicals in excess can also cause gametes to be non-viable. Being a high metabolically active organ, as indicated by a large proportion of mRNA expressed being related to metabolism, digestive gland generates a large number of reactive oxygen species and it needs to detoxify these harmful by-products of aerobic respiration. Although cytochrome c oxidase and other proteins that reduce O 2 are remarkably successful in not releasing intermediates, small amounts of superoxide anion and hydrogen peroxide are unavoidably formed during respiration.
Pepper and Chory 1997; Chen and Ni 2006; Prestele et al.
In this study we describe the isolation and characterizationof ten genes encoding C3HC4-type RING zinc finger proteins from B. rapa. These genes were identified in the phy- logenetic relationship, motif compositions, and possible cis-elements. Also, the expression profiles of these genes in the development stage and under different stress treatment conditions were also analyzed using data from qRT-PCR and real-time PCR. Such a comprehensive analysis of these ten genes may provide important clues for understanding their diverse roles in the growth and development of the Chinese cabbage plant.
Establishment of RCVpW-based insecticide resistance detection method and characterizationofgenes responding to
sublethal doses of insecticides in thrips species
총채벌레류의 잔류접촉법 기반 살충제 저항성 검정체계 구축 및 살충제 아치사량 반응유전자 규명
항산화 효소들은 체내에서 생명 유지를 위해 산화 환원 반응을 조절하는 중 요한 역할을 한다. 이 논문에서는 까막전복으로부터 만들어진 cDNA library 로부터 중요한 항산화 효소로 알려진 catalase (aCAT), Cu,Zn-superoxide dismutase (aCu,Zn-SOD) 와 Mn-superoxide dismutase (aMn-SOD)에 대한 코딩 유전자의 서열을 분석하였다. 첫번째로, 전체 길이를 확인한 후 그 서열들은 기존에 data base 를 통해 비교되어졌고 구조와 기능이 유사한 다른 생물의 효소들과의 비교를 통해 보존된 서열이 확인되어졌다.
Department of Agricultural Biology, National Academy of Agricultural Science, RDA, Suwon, 441-100, Korea (Received October 7, 2012, Accepted 2012)
To find diapause-related genes, we were performed by differential hybridization with three types of [α- 32 P]dCTP-labeled total cDNA probes synthesized from diapause-prepared, diapause-maintained and diapause-activated stage of Bombus ignitus queen. Nine individual cDNA clones were found to be differentially expressed in diapause-maintained and dia- pause-activated stage. Among these clones, BIDC9(BIDC ; Bombus ignitus differentially expressed clone) was analyzed through full-length sequencing and expression pattern analysis. This clone was specifically expressed in the thorax organ. The effect of Juvenile hormone analog(JHA) and CO 2 treatment was examined. JHA treatment induced the expression of BIDC9 cDNA clone abruptly after 4 day of treatment. CO 2 treatment induced also the clone after 2 day of treatment. BIDC9 cDNA was identified as Bombus ignitus diapause gene contained an open reading frame of 1376 bp encoding 255 amino acids.
In molecular approach of candidated genes, the first candidate gene ankykorbin was named retinoic acid 14, and this gene is well known in genetics, the object of other species (Peng et al, 2000), which are places for more people to use genetic information obtained from the genome database, chimpanzees, mice, rat, avian, bet- ween the fish homology to verify. Compared to the human and chimps, dogs showed only 90 percent or more of the high homology. In fact, several species of these genes are preserved in the ability to perform reliably. The ankycorbin is highly concentrated at cor- tical actin cytoskeleton structures in terminal web and cell-cell adhesion sites and stress fibers. The ankycorbin appears to be an actin cytoskeleton associated protein
ginseng was evolutionarily conserved. In addition, the important functional domains and amino acid residues for protein interactions among active GA, GID1, SCF SLY1 , and RGA were also functionally conserved. Prediction and comparison of crystallographic structural similarities between PgGID1s and AtGID1a supported their function as GA receptors. Moreover, the subcellular localization and GA- dependent promotion of DELLA degradation in P. ginseng was similar to the canonical GA signaling pathways in other plants. Finally, we found that overexpression of PgRGA2 and PgSLY1-1 was sufficient to complement the GA-related phenotypes of atgid1a/c double- and rga quintuple-mutants, respectively. This critical information for these GA signaling
Ⅸ. Double KO mutant of MoAND1and MoAND2
To find relationship between MoAND1 and MoAND2, double KO mutant was constructed. Usually, the phenotype of the double mutants could be mainly predicted from the phenotypes of the single mutants, interesting interaction and more precise gene action can be revealed by double KO. The double KO mutant of MoAND1 and MoAND2 was produced by using targeted gene replacement method as described before. For double KO mutant, the MoAND2 gene deletion vector constructs were transformed into protoplasts of MoAND1 deletion mutant (Fig. 13). In the Double KO mutant, conidia morphology was similar with ΔMoand1 mutant, but hyphal growth was severely affected in complement and minimal agar media as shown in Figure 14. Especially, in minimal agar media, hyphal growth of double mutant reduced about three times compared with wild-type.
확인된 9개 형질전환 상추에서 brazzein, thaumatin 및 miraculin 유전자의 발현여부 확인은 각각의 잎으로부터 total RNA를 추출하여 RT-PCR 분석과 qRT-PCR 분석으로 수행하였다.
그 결과 WT에 비하여 형질전환 상추에서 유전자가 높게 발 현 되고 있음을 확인하였다. 그 중 brazzein에서는 B1, B3 개 체와 thaumatin에서는 T2개체, miraculin에서는 M1개체에서 Fig. 1 Amino acid sequence of sweet and taste modifying proteins. Sequence was collected from the Swiss-Prot biological database of proteins
Agrobacterium tumefaciens strain EHA105 harboring the binary vector pCAMBIA1300 that contained the miraculin gene and hygromycin as a selection marker were used. After 5 days of co-culture in a medium containing 100 µM acetosyringone, calluses were transferred to the liquid half EME medium with 15 mg/L hygromycin and 250 mg/L cefotaxime and then cultured for 2 weeks. Subsequently, the calluses were grown on a solid selection medium with 20 mg/L hygromycin for 4 weeks, followed by selection with 25 mg/L hygromycin for 4 more weeks. A Total 168 resistant embryo were then selected and transferred to the embryo maturation medium. After 3 weeks of culture, the heart-shaped embryos were transferred to MT medium containing 1 mg/L GA 3 , 20 ml/L coconut water, 20 mg/L NAA, and 14.6 mg/L coumarin for embryo germination. Finally, 135 germinated embryos were cultured on MT medium containing 30 g/L sucrose and 8 g/L agar and 115 normal plants recovered. The transformation procedure yielded 37 transgenic Miyagawa Wase plants containing the miraculin genes as verified by PCR amplification. Southern blot analyses of randomly selected 5 plants further confirmed that the miraculin transgene was stably integrated into the Miyagawa Wase genome.
Se Hyun Son, Kwang Won Seo, Yeong Bin Kim, Eun Bi Noh, Keun-Woo Lee, Tae-Ho Oh, Seung-Joon Kim, Jae-Chan Song, Tae-Wan Kim, Young Ju Lee*
College of Veterinary Medicine & Zoonoses Research Institute, Kyungpook National University, Daegu 41566, Korea Abstract: Edible offal is easily contaminated by Escherichia coli (E. coli) and fluoroquinolone (FQ)-resistant E. coli is considered a serious public health problem, thus, this study investigated the genetic characteristics of FQ-resistant E. coli from edible offal. A total of 22 FQ-resistant E. coli isolates were tested. A double mutation in each gyrA and parC led the highest MIC. Four (18.2%) isolates carried plasmid-mediated quinolone resistance genes. The fimH, eaeA, escV, astA, and iucC genes were confirmed. Seventeen isolates (77.3%) were positive for plasmid replicons. The isolates showed high genetic heterogeneity based on pulsed-field gel electrophoresis patterns.
clone present should reflect its expression level and concentra- tion in the allergen extract. For example, the allergenic potency of German cockroach extract is thought to be influenced by Bla g 3 and α-amylase because their concentration is very high in the extract. 19 The number of Bla g 3 clones was highest (48) in EST analysis, followed by Bla g 8 (23) and α-amylase (13) (Table 1). In 2005, Chung et al. 20 described the analysis of EST clones prepared from German cockroach midgut, and 154 clones showed significant homology with other database-registered genes among 363 ESTs generated from 465 clones. Among 154 clones analyzed, four allergen homologs were identified (4 clones of Bla g 1, 2 clones of amylase, 4 clones of trypsin and 1 clone of chymotrypsin). Bla g 3 was not identified since it is mainly expressed in hemolymph, not in midgut. 21 Moreover, ex- pression levels of Bla g 1 and Bla g 2 could be affected by the cul- ture conditions of cockroach, as their expression is known to be influenced by foods and insecticides. 22,23 A larger-scale EST anal- ysis and investigation of mRNA expression is necessary to better understand allergen expression.
Biotechnology Research Division, NIFS, Busan 46083, Korea
Macrophage Migration Inhibitory Factor (MIF) are well-defined role as unique cytokine and critical mediator in acute and chronic inflammatory diseases, autoimmune diseases. In this study, we isolated and characterized a full-length of MIF cDNA from the abalone (Haliotis discus hannai). The full-length cDNA of abMIF was of 1264 bp, consisting of a 5’-terminal UTR of 143 bp, an open reading frame of 360 bp and a 3-terminal UTR of 761 bp. The abalone MIF cDNA encodes a 119-amino acid polypeptide with a calculated molecular mass of 13.4 kDa and isoelectric point of 9.07. Multiple alignments and phylogenetic analysis with the deduced abalone MIF protein and showed strong homology with disk abalone (Haliotis discusdiscus). The deduced amino acid sequence of abMIF exhibited homology with other reported MIFs, such as 80%, with that of other disk abalone H. discus discus MIF gene. Quantitative real-time PCR (qRT-PCR) analysis indicated that abMIF was highly expression observed in hapatopacreas, intestine, foot, and gonad of normal conditioned abalone. Even though AbMIF mRNA level in hemocytes was low under the normal condition, it was sharply up-regulated and reached the maximum at 6 h post-infection with Vibrio parahaemolyticus, and then decreased at 24 h post-infection. This result indicates that abMIF plays an important role in responding in the innate immune system.
2 Imported Food Analysis Division, Center for Food and Drug Analysis, Busan Regional Ministry of Food and Drug Safety, Busan 608-829, Korea
In order to investigate environmental stress inducible genes in abalone, we analyzed differentially expressed transcripts from a Pacific abalone, Haliotis discus hannai, after exposure to heat-, cold- or hyposalinity-shock by suppression subtractive hybridization (SSH) method. 1,074 unique sequences from SSH libraries were composed to 115 clusters and 986 singletons, the overall redundancy of the library was 16.3%. From the BLAST search, of the 1,316 ESTs, 998 ESTs (75.8%) were identified as known genes, but 318 clones (24.2%) did not match to any previously described genes. From the comparison results of ESTs pattern of three SSH cDNA libraries, the most abundant EST was different in each SSH library: small heat shock protein p26 (sHSP26) in heat-shock, trypsinogen 2 in cold-shock, and actin in hyposalinity SSH cDNA library. Based on sequence similarities, several response-to-stress genes such as heat shock proteins (HSPs) were identified commonly from the abalone SSH libraries. HSP70 gene was induced by environmental stress regardless of temperature-shock or salinity-stress, while the increase of sHSP26 mRNA expression was not detected in cold-shock but in heat-shock condition.
Owning to its specific binding to carbohydrate, lectins playe important roles in pathogen recognition and clearance in invertebrates. In this study, a novel C-type lectin (designated CLHd) gene was isolated fromabalone, Haliotis discus discus, cDNA library. The complete cDNA sequence of the CLHd gene is 508 base pairs in length and encodes 151 amino acids. CLHd shares a highly conserved carbohydrate recognition domain with C-type lectins from mollusk and fish. However, the difference in functional amino acid residues relative to ligand binding suggested that CLHd possesses a novel specificity to carbohydrate recognition. Semi-quantitative reverse transcriptase polymerase chain reaction detected its mRNA expression in healthy and bacterial challenged abalones. CLHd mRNA transcription was up-regulated by Vibrio alginolyticus challenge and reached the maximum expression at 24 hr post the bacterial injection. For the understanding its biological activity, the recombinant CLHd gene was constructed and expressed in Escherichia coli. The recombinant CLHd specifically agglutinated V. alginolyticus at a concentration of 50 µg/ml in a calcium dependant way. Both the gene expression analysis and recombinant protein activity assay suggest that CLHd is an important immune gene involved in the recognition and elimination of pathogens in abalones.
Also, we identified 77 new microsatellite-containing sequences from the unigenes and classified the structure according to their repeat unit. According to homology search with BLASTX against the NCBI database, 63.1% of ESTs were homologous with known function and 22.2% of ESTs were matched with putative or unknown function. The remaining 14.6% of ESTs showed no significant similarity to any protein sequences found in the public database. Gene ontology (GO) classification showed that the most abundant GO terms were transport, nucleotide binding, plastid, in terms biological process, molecular function and cellular component, respectively. The sequence data will be used to characterize potential roles of new genes in Pinus and provided for the useful tools as a genetic resource.
The financial support given by The Korean Science and Engineering Foundation and the helps given by the staff of the Graduate School of Cheju National University is greatly appreciated.
I heartily admire Oh Cheol Hong and Kang Hyun Sil, for offering me their kind assistance to drive my work to success. I particularly appreciate all my lab members specially Wang Ning for sharing ideas and Wang Chung for his support. At the same time I make this opportunity to thank Park Ho Jin, Kang Kyong Im and all other Korean and international friends who supported me in many ways. I appreciate Yasantha and all other Sri Lankan friends in Cheju National University. My special thanks go to my friend Prashantha, who helped me by many ways to success my studies.