• Proceedings of the Korean Society of Plant Pathology Conference
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• 2003.10a
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• pp.77.1-77
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• 2003

We have isolated a full-length cDNA clone, CaCDPK4 encoding a typical calcium-dependent protein kinase (CDPK) from hot pepper cDNA library. Genomic southern blot analysis showed that it belongs to a multigene family, but represents a single copy gone in hot pepper genome. RNA expression pattern of this gene revealed that it is induced by infiltration of Xanthomonas axonopodis pv. glycines Bra into hot pepper leaves but not by water deficit stress. However, high salt treatment of NaCl (0.4 M) solution to hot pepper plants strongly induced CaCDPK4 gene. In addition, this gene is weakly responsive to the exogenous application of salicylic acid or ethephon. Biochemical study of the GST-CaCDPK4 recominant protein showed that it autophosphorylates in vitro and the presence of EGTA, a calcium chelater, eliminates the kinase activity of the recombinant protein. As a way to identify the in vivo function of CaCDPK4 in plants, VIGS (Virus-Induced Gene Silencing) was employed. Agrobacterium-mediated TRV silencing construct containing the kinase and calmodulin domain of CaCDPK4 resulted in cell death of Nicotiana benthamiana plants. A highly homologous H benthamiana CDPK gene, NbCDPK5, to CaCDPK4 was cloned from N. benthamiana cDNA library. VIGS of NbCDPK5 also resulted in cell death. The molecular characterization of this cell death phenotype is being under investigation.

• Plant Biotechnology Reports
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• v.4 no.2
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• pp.165-172
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• 2010

Genes that are expressed early in specific response to high salinity conditions were isolated from rapeseed plant (Brassica napus L.) using an mRNA differential display method. Five PCR fragments (DD1.5) were isolated that were induced by, but showed different response kinetics to, 200 mM NaCl. Nucleotide sequence analysis and homology search revealed that the deduced amino sequences of three of the five cDNA fragments showed considerable similarity to those of ${\beta}$-mannosidase (DD1), tomato Pti-6 proteins (DD5), and the tobacco harpin-induced protein hin1 (DD4), respectively. In contrast, the remaining clones, DD3 and DD2, did not correspond to any substantial existing annotation. Using the DD3 fragment as a probe, we isolated a full-length cDNA clone from the cDNA library, which we termed BnSWD1 (Brassica napus salt responsive WD40 1). The predicted amino-acid sequence of BnSWD1 contains eight WD40 repeats and is conserved in all eukaryotes. Notably, the BnSWD1 gene is expressed at high levels under salt-stress conditions. Furthermore, we found that BnSWD1 was upregulated after treatment with abscisic acid, salicylic acid, and methyl jasmonate. Our study suggests that BnSWD1, which is a novel WD40 repeat-containing protein, has a function in salt-stress responses in plants, possibly via abscisic acid-dependent and/or -independent signaling pathways.

• Asian-Australasian Journal of Animal Sciences
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• v.25 no.5
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• pp.606-612
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• 2012

Insulin-like growth factor-binding protein-5 (IGFBP-5) is one of the six members of IGFBP family, important for cell growth, apoptosis and other IGF-stimulated signaling pathways. In order to explore the significance of IGFBP-5 in cells of the Inner Mongolian Cashmere goat (Capra hircus), IGFBP-5 gene complementary DNA (cDNA) was amplified by reverse transcription polymerase chain reaction (RT-PCR) from the animal's fetal fibroblasts and tissue-specific expression analysis was performed by semi-quantitative RT-PCR. The gene is 816 base pairs (bp) in length and includes the complete open reading frame, encoding 271 amino acids (GenBank accession number JF720883). The full cDNA nucleotide sequence has a 99% identity with sheep, 98% with cattle and 95% with human. The amino acids sequence shares identity with 99%, 99% and 99%, respectively. The bioinformatics analysis showed that IGFBP-5 has an insulin growth factor-binding protein homologues (IB) domain and a thyroglobulin type-1 (TY) domain, four protein kinase C phosphorylation sites, five casein kinase II phosphorylation sites, three prenyl group binding sites (CaaX box). The IGFBP-5 gene was expressed in all the tested tissues including testis, brain, liver, lung, mammary gland, spleen, and kidney, suggesting that IGFBP-5 plays an important role in goat cells.

• BMB Reports
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• v.38 no.5
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• pp.602-608
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• 2005

As a crucial transcription factor family, heat-shock factors were mainly analyzed and characterized in tomato and Arabidopsis. In this study, we isolated two putative heat shock factors OsHSF6 and OsHSF12 that interact specifically with heat-shock element (HSE) from Oryza sativa L by yeast one-hybrid method. The full-length cDNA of OsHSF6 and OsHSF12 have 1074bp and 920bp open reading frame (ORF), respectively. Analysis of the deduced amino acid sequences revealed that OsHSF6 was a class A heat shock factor (HSF) with all the conserved sequence elements characteristic of heat stress transcription factor, while OsHSF12 was a class B HSF with C-terminal domain (CTD) lacking of AHA motif. Bioinformatic analysis showed that the sequences and structures of two HSFs' DNA binding domain (DBD) had a high similarity with LpHSF24. The results of RT-PCR indicated OsHSF6 gene was expressed immediately after rice plants exposure to heat stress, and the transcription of OsHSF6 gene accumulated primarily in immature seeds, roots and leaves. However, we did not find the transcription of OsHSF12 gene in different organs and growth periods. Our results implied that OsHSF6 might be function as a HSF regulating early expression of stress genes in response to heat shock, and OsHSF12 might be act as a synergistic factor to regulate the expression of down-stream genes.

• Proceedings of the Korean Society of Crop Science Conference
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• 2017.06a
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• pp.89-89
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• 2017

A gene encoding squalene synthase from grain amaranth was cloned and characterized. The full-length cDNA was 1805-bp long and contained a 1248-bp open reading frame encoding a protein of 416 amino acids with a molecular mass of 47.6 kDa. Southern blot analysis revealed that the A. cruentus genome contained a single copy of the gene. Comparison of the cDNA and genomic sequences indicated that the amaranth SQS gene had 12 introns and 13 exons. All of the exons contributed to the coding sequence. The predicted amino acid sequence of the SQS cDNA shared high homology with those of SQSs from several other plants. It contained conserved six domains that are believed to represent crucial regions of the active site. We conducted qRT-PCR analyses to examine the expression pattern of the SQS gene in seeds at different developmental stages and in several tissues. The amaranth SQS gene was low levels of SQS transcripts at the initial stage of seed development, but the levels increased rapidly at the mid-late developmental stages before declining at the late developmental stage. These findings showed that the amaranth SQS is a late-expressed gene that is rapidly expressed at the mid-late stage of seed development. In addition, we observed that the SQS mRNA levels in stems and roots increased rapidly during the four- to six-leaf stage of development. Therefore, our results showed that the expression levels of SQS in stem and root tissues are significantly higher than those in leaf tissues. In present study provides useful information about the molecular characterization of the SQS clone isolated from grain amaranth. Finally, a basic understanding of these characteristics will contribute to further studies on the amaranth SQS.

• Korean Journal of Environmental Biology
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• v.34 no.2
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• pp.97-106
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• 2016

$Na^+/K^+$-ATPase is a membrane protein and plays a key role in osmotic regulation in living organisms. In the present study, a cDNA sequence encoding the $Na^+/K^+$-ATPase alpha subunit from the monogonont rotifer, Brachionus koreanus was cloned by rapid amplification of cDNA ends technique. To investigate the role of this enzyme in osmotic stress, enzymatic activities of $Na^+/K^+$-ATPase were measured after exposure to different salinities for 48 h. The full-length Bk $Na^+/K^+$-ATPase cDNA was 3069 bp-long, encoding a 1022-amino acid polypeptide. Bk $Na^+/K^+$-ATPase possesses eight membrane spanning regions and five conserved domains. Phylogenetic analysis showed that Bk $Na^+/K^+$-ATPase had high identity with those of other species, and was closely clustered with other Brachionus sp. These findings indicate that this protein was conserved both structurally and functionally. B. koreanus $Na^+/K^+$-ATPase activity was stimulated in both hyposaline (6 psu) and hypersaline (32 psu) conditions, suggesting that this protein may play a role in osmoregulation. This study would provide better understanding of the physiology of B. koreanus and this enzyme may be useful as a molecular marker for evaluation of osmotic stress in aquatic environment.

• Asian-Australasian Journal of Animal Sciences
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• v.23 no.2
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• pp.272-278
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• 2010

In this study, we cloned, sequenced and characterized porcine y+L Amino Acid Transporter-1 (y+LAT1). By screening a translated EST database with the protein sequence of the human $y^{+}$LAT1 and by using rapid amplification of cDNA ends (RACE), the full-length cDNA encoding porcine $y^{+}$LAT1 was isolated from porcine intestine RNA. It was 2,111 bp long, encoding a 511 amino acid trans-membrane glycoprotein composed of 12 transmembrane domains. The predicted amino acid sequence was found to be 91%, 90%, 87% and 87% identical to those of cattle, human, mouse and rat $y^{+}$LAT1 respectively. Real-time RT-PCR results indicated that the small intestine had the highest $y^{+}$LAT1 mRNA abundance and the lung had the lowest $y^{+}$LAT1 mRNA abundance. Baby hamster kidney (BHK) cells transfected with green fluorescent protein (GFP) tagged porcine $y^{+}$LAT1 cDNA indicated that the cellular localization of the gene product in BHK was on the plasma membrane.

• Journal of Plant Biotechnology
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• v.39 no.3
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• pp.212-218
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• 2012

Glycosyltransferases are enzymes (EC 2.4) that catalyze the transfer of monosaccharide moieties from activated nucleotide sugar to a glycosyl acceptor molecule which can be a carbohydrate, glycoside, oligosaccharide, or a polysaccharide. In this study, a UDP-glucosyltransferase cDNA was isolated from Brassica rapa using a rapid amplification of cDNA ends (RACE) and subsequently named BrUGT. It has a full-length cDNA of 1,236 bp with 119 bp 5'-untranslated region (UTR), a complete ORF of 834 bp encoding a polypeptide of 277 amino acids (31.19 kDa) and a 3'-UTR of 283 bp. BLASTX analysis hits a catalytic domain of Glycos_transf_1 super family (cl12012) that belongs to the Glycosyltransferases group 1 with tetratricopeptide (TPR) regions located between 165 to 350 bp. Expression analysis showed high mRNA transcripts in pistil, followed by petal, seed and calyx of flower. Moreover, expression analysis of BrUGT in Chinese cabbage seedlings under stresses of cold, salt, PEG, $H_2O_2$, drought and ABA showed elevated mRNA transcript. Furthermore, when BrUGT gene was transformed into rice using pUbi-1 promoter, overexpression was evident among the $T_1$ plants. This study provides insights into the function of BrUGT in plants.

• Journal of Life Science
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• v.21 no.3
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• pp.459-463
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• 2011

The genome of the rhesus monkey has diverged as an average sequence identity of ~93%. The rhesus monkey has been widely used as a non-human primate in the field of biomedical and evolutional research. Insertion of transposable elements (TEs) induced several events such as transcriptional diversity and different expression in host genes. In this study, 112 transcripts were identified from a full-length cDNA library of brain tissues of the rhesus monkey. One transcript (R54) showed a different expression pattern between human and rhesus monkey tissues. This phenomenon can be an explanation that R54 transcript was acquired by splicing a donor site derived from exonization of the L2A element. Therefore, integration of TEs during primate radiation could contribute to transcriptional diversity and gene regulation.

• Fisheries and Aquatic Sciences
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• v.19 no.8
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• pp.33.1-33.7
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• 2016

Antifreeze proteins (AFPs) lower the freezing point but not the melting point of aqueous solutions by inhibiting the growth of ice crystals via an adsorption-inhibition mechanism. However, the function of type IV AFP (AFP IV) is questionable, as its antifreeze activity is on the verge of detectable limits, its physiological concentration in adult fish blood is too low to function as a biological antifreeze, and its homologues are present even in fish from tropic oceans as well as freshwater. Therefore, we speculated that AFP IV may have gained antifreeze activity not by selective pressure but by chance. To test this hypothesis, we cloned, expressed, and assayed AFP IV from cultured subtropical olive flounder (Paralichthys olivaceus), which do not require antifreeze protein for survival. Among the identified expressed sequence tags of the flounder liver sample, a 5'-deleted complementary DNA (cDNA) sequence similar to the afp4 gene of the longhorn sculpin was identified, and its full-length cDNA and genome structure were examined. The deduced amino acid sequence of flounder AFP IV shared 55, 53, 52, and 49 % identity with those of Pleuragramma antarcticum, Myoxocephalus octodecemspinosus, Myoxocephalus scorpius, and Notothenia coriiceps, respectively. Furthermore, the genomic structure of this gene was conserved with those of other known AFP IVs. Notably, the recombinant AFP IV showed a weak but distinct thermal hysteresis of $0.07{\pm}0.01^{\circ}C$ at the concentration of 0.5 mg/mL, and ice crystals in an AFP IV solution grew star-shaped, which are very similar to those obtained from other polar AFP IVs. Taken together, our results do not support the hypothesis of evolution of AFP IV by selective pressure, suggesting that the antifreeze activity of AFP IV may have been gained by chance.