title: Cloning of ubiquitin-activating enzyme and ubiquitin-conjugating enzyme genes from Gracilaria lemaneiformis and their activity under heat shock.
abstract: To study the response of Gracilaria lemaneiformis to heat stress, two key enzymes - ubiquitin-activating enzyme (E1) and ubiquitin-conjugating enzyme (E2) - of the Ubiquitin/26S proteasome pathway (UPP) were studied in three strains of G. lemaneiformis-wild type, heat-tolerant cultivar 981 and heat-tolerant cultivar 07-2. The full length DNA sequence of E1 contained only one exon. The open reading frame (ORF) sequence was 981 nucleotides encoding 326 amino acids, which contained conserved ATP binding sites (LYDRQIRLWGLE, ELAKNVLLAGV, LKEMN, VVCAI) and the ubiquitin-activating domains (VVCAI…LMTEAC, VFLDLGDEYSYQ, AIVGGMWGRE). The gene sequence of E2 contained four exons and three introns. The sum of the four exons gave an open reading frame sequence of 444 nucleotides encoding 147 amino acids, which contained a conserved ubiquitin-activating domain (GSICLDIL), ubiquitin-conjugating domains (RIYHPNIN, KVLLSICSLL, DDPLV) and ubiquitin-ligase (E3) recognition sites (KRI, YPF, WSP). Real-time-PCR analysis of transcription levels of E1 and E2 under heat shock conditions (28°C and 32°C) showed that in wild type, transcriptions of E1 and E2 were up-regulated at 28°C, while at 32°C, transcriptions of the two enzymes were below the normal level. In cultivar 981 and cultivar 07-2 of G. lemaneiformis, the transcription levels of the two enzymes were up-regulated at 32°C, and transcription level of cultivar 07-2 was even higher than that of cultivar 981. These results suggest that the UPP plays an important role in high temperature resistance of G. lemaneiformis and the bioactivity of UPP is directly related to the heat-resistant ability of G. lemaneiformis.
authors.affiliation: Key Laboratory of Marine Genetics and Breeding, Ministry of Education, Ocean University of China, Qingdao 266003, Shandong, China.
authors.forename: Guang-Qi
authors.initials: GQ
authors.lastname: Li
authors.affiliation: Key Laboratory of Marine Genetics and Breeding, Ministry of Education, Ocean University of China, Qingdao 266003, Shandong, China. Electronic address: xnzang@ouc.edu.cn.
authors.forename: Xiao-Nan
authors.initials: XN
authors.lastname: Zang
authors.affiliation: Key Laboratory of Marine Genetics and Breeding, Ministry of Education, Ocean University of China, Qingdao 266003, Shandong, China.
authors.forename: Xue-Cheng
authors.initials: XC
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Marine Genetics and Breeding, Ministry of Education, Ocean University of China, Qingdao 266003, Shandong, China.
authors.forename: Ning
authors.initials: N
authors.lastname: Lu
authors.affiliation: Key Laboratory of Marine Genetics and Breeding, Ministry of Education, Ocean University of China, Qingdao 266003, Shandong, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Ding
authors.affiliation: Key Laboratory of Marine Genetics and Breeding, Ministry of Education, Ocean University of China, Qingdao 266003, Shandong, China.
authors.forename: Le
authors.initials: L
authors.lastname: Gong
authors.affiliation: Key Laboratory of Marine Genetics and Breeding, Ministry of Education, Ocean University of China, Qingdao 266003, Shandong, China.
authors.forename: Wen-Chao
authors.initials: WC
authors.lastname: Chen
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D017343:Genes, Plant; D044724:Gracilaria / Q000201:enzymology / Q000235:genetics*; D018869:Heat-Shock Response; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D014158:Transcription, Genetic; D044764:Ubiquitin-Activating Enzymes / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
chemical_list: D010940:Plant Proteins; D000255:Adenosine Triphosphate; D044763:Ubiquitin-Conjugating Enzymes; D044764:Ubiquitin-Activating Enzymes
keywords: Gene cloning; Gracilaria lemaneiformis; Heat shock; Transcription analysis; Ubiquitin-activating enzyme; Ubiquitin-conjugating enzyme