title: Cloning and characterization of indole synthase (INS) and a putative tryptophan synthase α-subunit (TSA) genes from Polygonum tinctorium.
abstract: Two cDNAs for indole-3-glycerol phosphate lyase homolog were cloned from Polygonum tinctorium. One encoded cytosolic indole synthase possibly in indigoid synthesis, whereas the other encoded a putative tryptophan synthase α-subunit. Indigo is an old natural blue dye produced by plants such as Polygonum tinctorium. Key step in plant indigoid biosynthesis is production of indole by indole-3-glycerol phosphate lyase (IGL). Two tryptophan synthase α-subunit (TSA) homologs, PtIGL-short and -long, were isolated by RACE PCR from P. tinctorium. The genome of the plant contained two genes coding for IGL. The short and the long forms, respectively, encoded 273 and 316 amino acid residue-long proteins. The short form complemented E. coli ΔtnaA ΔtrpA mutant on tryptophan-depleted agar plate signifying production of free indole, and thus was named indole synthase gene (PtINS). The long form, either intact or without the transit peptide sequence, did not complement the mutant and was tentatively named PtTSA. PtTSA was delivered into chloroplast as predicted by 42-residue-long targeting sequence, whereas PtINS was localized in cytosol. Genomic structure analysis suggested that a TSA duplicate acquired splicing sites during the course of evolution toward PtINS so that the targeting sequence-containing pre-mRNA segment was deleted as an intron. PtINS had about two to fivefolds higher transcript level than that of PtTSA, and treatment of 2,1,3-benzothiadiazole caused the relative transcript level of PtINS over PtTSA was significantly enhanced in the plant. The results indicate participation of PtINS in indigoid production.
authors: Department of Agricultural Biotechnology and Research Institute of Agriculture and Life Sciences, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul, 08826, Republic of Korea.
authors: Zhehao
authors: Z
authors: Jin
authors: Department of Agricultural Biotechnology and Research Institute of Agriculture and Life Sciences, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul, 08826, Republic of Korea.
authors: Jin-Hee
authors: JH
authors: Kim
authors: Department of Crop Science, Chungnam National University, 99 Daehak-ro, Yuseong-gu, Daejeon, 34134, Republic of Korea.
authors: Sang Un
authors: SU
authors: Park
authors: Department of Agricultural Biotechnology and Research Institute of Agriculture and Life Sciences, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul, 08826, Republic of Korea. soounkim@snu.ac.kr.|College of Horticulture and Gardening, Yangtze University, Jingzhou, 434023, Hubei, China. soounkim@snu.ac.kr.
authors: Soo-Un
authors: SU
authors: Kim
mesh_terms: D020121:5' Untranslated Regions / Q000235:genetics; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli; D018506:Gene Expression Regulation, Plant / Q000187:drug effects; D017343:Genes, Plant; D005816:Genetic Complementation Test; D049452:Green Fluorescent Proteins / Q000378:metabolism; D007211:Indoles / Q000737:chemistry / Q000378:metabolism*; D009928:Organ Specificity / Q000187:drug effects / Q000235:genetics; D010802:Phylogeny; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D028502:Polygonum / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics*; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013347:Subcellular Fractions / Q000378:metabolism; D013830:Thiadiazoles / Q000494:pharmacology; D014367:Tryptophan Synthase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
chemical_list: D020121:5' Untranslated Regions; D007211:Indoles; D010940:Plant Proteins; D021122:Protein Subunits; D012333:RNA, Messenger; D013830:Thiadiazoles; D049452:Green Fluorescent Proteins; C015700:benzo-1,2,3-thiadiazole; C030374:indole; D014367:Tryptophan Synthase
keywords: Complementation assay; Homologous recombination; Indigoids; Indole synthase; Indole-3-glycerol phosphate lyase; Polygonum tinctorium; Tryptophan synthase α-subunit