UDP-glycosyltransferases (UGT) catalyze the conjugation of a range of diverse small lipophilic compounds with sugars to produce glycosides, playing an important role in the detoxification of xenobiotics and in the regulation of endobiotics in insects. Recent progress in genome sequencing has enabled an assessment of the extent of the UGT multigene family in insects. Here we report over 310 putative UGT genes identified from genomic databases of eight different insect species together with a transcript database from the lepidopteran Helicoverpa armigera. Phylogenetic analysis of the insect UGTs showed Order-specific gene diversification and inter-species conservation of this multigene family. Only one family (UGT50) is found in all insect species surveyed (except the pea aphid) and may be homologous to mammalian UGT8. Three families (UGT31, UGT32, and UGT305) related to Lepidopteran UGTs are unique to baculoviruses. A lepidopteran sub-tree constructed with 40 H. armigera UGTs and 44 Bombyx mori UGTs revealed that lineage-specific expansions of some families in both species appear to be driven by diversification in the N-terminal substrate binding domain, increasing the range of compounds that could be detoxified or regulated by glycosylation. By comparison of the deduced protein sequences, several important domains were predicted, including the N-terminal signal peptide, UGT signature motif, and C-terminal transmembrane domain. Furthermore, several conserved residues putatively involved in sugar donor binding and catalytic mechanism were also identified by comparison with human UGTs. Many UGTs were expressed in fat body, midgut, and Malpighian tubules, consistent with functions in detoxification, and some were expressed in antennae, suggesting a role in pheromone deactivation. Transcript variants derived from alternative splicing, exon skipping, or intron retention produced additional UGT diversity. These findings from this comparative study of two lepidopteran UGTs as well as other insects reveal a diversity comparable to this gene family in vertebrates, plants and fungi and show the magnitude of the task ahead, to determine biochemical function and physiological relevance of each UGT enzyme.

Capsaicin β-glucoside was isolated from the feces of Helicoverpa armigera, H. assulta and H. zea that fed on capsaicin-supplemented artificial diet. The chemical structure was identified by NMR spectroscopic analysis as well as by enzymatic hydrolysis. The excretion rates of the glucoside were different among the three species; those in the two generalists, H. armigera and H. zea, were higher than in a specialist, H. assulta. UDP-glycosyltransferases (UGT) enzyme activities measured from the whole larval homogenate of the three species with capsaicin and UDP-glucose as substrates were also higher in the two generalists. Compared among five different larval tissues (labial glands, testes from male larvae, midgut, the Malpighian tubules, and fatbody) from the three species, the formation of the capsaicin glucoside by one or more UGT is high in the fat body of all the three species as expected, as well as in H. assulta Malpighian tubules. Optimization of the enzyme assay method is also described in detail. Although the lower excretion rate of the unaltered capsaicin in H. assulta indicates higher metabolic capacity toward capsaicin than in the other two generalists, the glucosylation per se seems to be insufficient to explain the decrease of capsaicin in the specialist, suggesting H. assulta might have another important mechanism to deal with capsaicin more specifically.

Background : Glycosylation of natural compounds results in great diversity of secondary metabolites. Glycosylation steps are implicated not only in plants growth and development but also in plant defense responses to various environmental stresses. This process is mediated by members of a multigene superfamily glycosyltransferase (GT), which catalyze the transfer of single or multiple activated sugars to a wide range of substrates, thus influences their chemical property and bioactivity. Although its activity has been recognized for a long time and genes coding UGTs in several higher plants have been identified, specific function of GTs in detail still remains elusive. Methods and Results : Spatial and temporal expression patterns of a ginseng UDP-dependent glycosyltransferase, was analyzed by qRT-PCR. It was expressed highly in rhizome, upper root and youngest leaf compared that of other organs. Spacial expression was observed by GUS histochemical assay after generating promoter::GUS fusion. Noticeably, it expressed axillary branch as well as other organs tested by qRT-PCR. Overexpression of PgUGT in Arabidopsis resulted in fused organ in axillary branch. Stress responsiveness against various abiotic stresses and subcellular localization in Arabidopsis are also addressed. Conclusion : PgUGT phylogenetically closed to PgUGT71A27 involved in ginsenoside compound K (C-K) production. Considering that the C-K is not reported in raw ginseng material, further characterization of this gene may shed light on the biological function of C-K in ginseng growth and development. Organ fusion phenotype could be caused by defective growth of cells in boundary region, commonly regulated by phytohormones such as auxins or brassinosteroids, which in needs to be analyzed further.

Background : Platycodon grandiflorum is a perennial plant and a member of Camanulaceae family. Since ancient times, they have been using P. grandiflorum as an important medicinal plant in Korea. Platycodin D is the most abundant saponin derived from P. grandiflorum and pharmacologically active component. UDP-glycosyltransferases (UGTs) are important enzymes in the saponin biosynthesis. UGT is a glycosyltransferase and act on the final step of the secondary metabolite biosynthesis. Methods and Results : We tried to identify UGT genes related to saponin biosynthesis of P. grandiflorum through RNA-seq analysis. The sequencing was performed using Illumina Hi-Seq platform after cDNA library preparation. The produced reads were assembled using CLC Genomics Workbench software (CLC Bio, Inc.). We obtained 122,663 contigs and found 137 putative UGT genes. Familes of UGT71, UGT73, and UGT74 were selected as putative saponin biosynthesis related gene families using phylogenetic relationship analysis. qPCR condition about UGT73 is preheating 94℃ 180 sec, denaturation 94℃ 60 sec, annealing 53℃ 60 sec, extension 72℃ 90 sec, final extension 72℃ 600 sec, 45 cycles repeated. Conclusion : The results in this study could help to find the UGTs related to saponin biosynthesis pathway of P. grandiflorum.

UDP-glucose 4-epimerase (UGE; EC 5.1.3.2) is an enzyme that plays an essential role in the interconverts UDP-D-glucose (UDP-Glc) and UDP-Dgalactose (UDP-Gal). Five members of the Chinese cabbage (Brassica rapa) UDP-glucose 4-epimerase gene family, designated BrUGE1 to BrUGE5, have been cloned and characterized. Quantitative PCR shows that the BrUGE1and BrUGE4 mRNA are most abundant among other BrUGE genes, accounting for more than 55% of total BrUGE transcripts in most of the tissues examined. All genes showed organ specific expression pattern, two of which (BrUGE1 and 4) actively responded after Pectobacterium carotovorum subsp. carotovorum infection, while four genes (BrUGE-1, -3, -4 and -5)were shown to respond considerably against salt, drought and abscisic acid (ABA) treatments. To better understand the function of the UGE gene, we constructed a recombinant pART vector carrying the BrUGE1 gene under the control of the CaMV 35S promoter and nos terminator and transformed using Agrobacterium tumefaciens. We then investigated BrUGE1 overexpressing rice lines at the physiological and molecular levels under biotic and abiotic stress conditions. Bioassay of T3 progeny lines of the transgenic plants in Yoshida solution containing 120 mM Nacl for 2 weeks, confirmed that the BrUGE1 enhances salt tolerance to transgenic rice plants. Also T3 progeny lines of the transgenic plants, when exposed to infection caused by Xanthomonas oryzae pv oryzae, showed tolerance to bacterial blight. These results showed that BrUGE1 can be used as potential genetic resource for engineering Brassica with multiple stress resistance.

Secondary plant metabolites undergo several modification reactions, including glycosylation and physiological functions. Glycosylation, which is mediated by UDP-glycosyltransferase (UGT), plays a role in the storage of secondary metabolites and in defending plants against stress. In this study, a UDP-glucosyltransferase cDNA was isolated from Brassica rapa hereinafter referred to as BrUGT. It has a full-length cDNA of 1,236 bp that contains a single open reading frame of 834 bp which encodes a polypeptide of 277 amino acid residues with a calculated mass of 31.19 kDa. BLASTX analysis hits a catalytic domain of glycos_transf_1 super family (c112012) that belongs to the glycosyltransferases group 1 with tetratricopeptide (TPR) regions. UGT gene expression analysis showed high mRNA transcripts in pistil, followed by petal, seed and calyx of flower in Brassica rapa. Furthermore, we constructed a recombinant pFLCIII vector carrying the BrUGT gene under the control of ubiquitin promoter and NOS terminator and transformed into rice using Agrobacterium tumefaciens. The UGT overexpressing rice lines were then characterized at the physiological and molecular levels. To further understand the biological function of BrUGT, transcriptional profiling of the gene in transgenic rice lines under cold, salt, PEG, H2O2, ABA and drought stress condition is underway.

UDP-glucose 4-epimerase catalyzes the reversible conversion of UDP-glucose to UDP-galactose. The gene, named BrUGE1, isolated from a Chinese cabbage composes of a total length of 1,328 bp that contains a single open reading frame (ORF) of 1,056 bp which encodes a polypeptide of 351 amino acid residues with a calculated mass of 39.0 kDa. Expression analysis showed that BrUGE1 is tissue specific and highly expressed in stem of rice plant. Interestingly, BrUGE1 mRNA was highly accumulated by drought stress with significantly higher amount of soluble sugar. Morphological evaluation showed an increase in yield and yield components compared to the wild type. Moreover, a better growth performance on galactose as well as higher UGE1 expression was observed in transgenic rice lines than in wild type. In the Ubi-1::BrUGE1 lines, the increase of UGE1 expression was apparently sufficient to overcome the toxic effects of galactose. Taken together, the Ubi-1::BrGUE1 rice lines increased yield probably by increasing the rate of filled grains. The enhanced drought tolerance may be due to the induction of soluble sugar which may act as osmolyte to compensate dehydration during drought stress.

UDP-glucose 4-epimerase (UGE) catalyzes the reversible conversion of UDP-glucose to UDP-galactose. The gene, named BrUGE1, isolated from a Chinese cabbage had a total length of 1,328 bp that contains a single open reading frame (ORF) of 1,056 bp which encodes a polypeptide of 351 amino acid residues with a calculated mass of 39.0 kDa. Sequence analysis of BrUGE1 protein has the characteristic of an active site tetrad and NAD-binding motif (typically TGXXGXXG) of the extended short chain dehydrogenase/ reductase (SRD) superfamily. Expression analysis showed that BrUGE1 is tissue specific and highly expressed in stem of rice plant. Interestingly, BrUGE1 mRNA was highly accumulated by drought stress with significantly higher amount of soluble sugar. Morphological evaluation showed an increase in yield by 27%. Panicle length, number of productive tillers/hill, and filled spikelets were significantly increased by 17~20% compared to the wild type Gopum. Moreover, the growth of the wild type Gopum seedlings on galactose was increasingly inhibited with a decrease in UDP-glc epimerase 1 expression compared to the transgenic rice lines. In the Ubi-1::BrUGE1 lines, the increase of UDP-glc epimerase 1 expression was apparently sufficient to overcome the toxic effects of galactose. Taken together, the Ubi-1::BrGUE1 rice lines increased yield probably by increasing the rate of filled grains. The enhanced drought tolerance may be due to the induction of soluble sugar which may act as osmolyte to compensate dehydration during drought stress.

Limonoid는 항바이러스 및 항균제로써의 치료적인 목적으로 널리 연구되고 있는 성분으로 감귤에서 풍부하게 존재한다. 그러나 성분 자체의 쓴맛으로 인하여 기호성이 저하되므로 이를 해결하는 노력이 필요하며 제품 개발이 요구되고 있는 실정이다. 이러한 쓴맛을 제거할 수 있는 훌륭한 효소로써 LUGT가 주목되고 있으며, 이에 효소의 특성화 연구를 위하여 분리 및 정제를 시도하였다. Plasmid vector인 pET30a(+)을 사용하여 대장균에서

Limonoid UDP-glucosyltransferase (LUGT)는 리모노이드에 포도당을 붙여줌으로써 궁극적으로 감귤에서 발생하는 limonoid bitterness를 제거해 주는 효소이다. 본 연구에서는 10종의 제주산 감귤로부터 LUGT유전자를 PCR 클로닝하고 그 염기서열을 비교했다. 실험에 사용한 모든 종에서 카르복실기 말단에 식물 당전달 효소에서 발견되는 전형적인 아미노산 서열인 p1ant secondary product glyco