Cloning,expression, and characterization of Aureobasidium melanogenum lipase in Pichia pastoris

cDNA of Aureobasidium melanogenum lipase comprises 1254 bp encoding 417 amino acids, whereas genomic DNA of lipase comprises 1311 bp with one intron (57 bp). The lipase gene contains a putative signal peptide encoding 26 amino acids. The A. melanogenum lipase gene was successfully expressed in Pichia pastoris. Recombinant lipase in an inducible expression system showed the highest lipase activity of 3.8 U/mL after six days of 2% v/v methanol induction. The molecular mass of purified recombinant lipase was estimated as 39 kDa using SDS-PAGE. Optimal lipase activity was observed at 35–37 °C and pH 7.0 using p-nitrophenyl laurate as the substrate. Lipase activity was enhanced by Mg²⁺, Mn²⁺, Li⁺, Ca²⁺, Ni²⁺, CHAPS, DTT, and EDTA and inhibited by Hg²⁺, Ag⁺, SDS, Tween 20, and Triton X-100. The addition of 10% v/v acetone, DMSO, p-xylene, and octanol increased lipase activity, whereas that of propanol and butanol strongly inhibited it.     

Genetic Diversity and Molecular Markers of Cupped Oysters (Genera Crassostrea, Saccostrea, and Striostrea) in Thailand Revealed by Randomly Amplified Polymorphic DNA Analysis

Genetic diversity and species-diagnostic markers of 5 oysters in Thailand, Crassostrea belcheri (Sowerby, 1871), Crassostrea iredalei (Faustino, 1932), Saccostrea cucullata (Born, 1778), Saccostrea forskali (Gmelin, 1791), and Striostrea (Parastriostrea) mytiloides (Lamarck, 1819), were investigated by randomly amplified polymorphic DNA (RAPD) analysis. In a total, 135, 127, and 108 genotypes were observed from primers OPA09, OPB01, and OPB08 (Operon Technologies Inc., kits A and B), and 131 and 122 genotypes from primers UBC210 and UBC220 (University of British Columbia), respectively. Two hundred fifty-four reproducible and polymorphic fragments (200–2500 bp in length) were generated across the 5 investigated species. The average number of bands per primer varied between 12.4 and 32.2. The percentage of polymorphic bands within Crassostrea (53.23%–77.67%) was lower than that within Saccostrea and Striostrea oysters (86.21%–99.36%). Nine, species-specific markers were found in C. belcheri, 4 in C. iredalei, and 2 in S. cucullata. The mean of a ratio between the number of genotypes generated by each primer and the number of investigated specimens of C. belcheri (0.58) was lower than that of the remaining species (0.90–1.00). Genetic distances between pairs of oyster samples were between 0.105 and 0.811. A neighbor-joining tree indicated distant relationships between Crassostrea and Saccostrea oysters, but closer relationships were observed between the latter and Striostrea mytiloides. Received June 6, 2000; accepted September 12, 2000     

Molecular Genetic Identification Tools for Three Commercially Cultured Oysters (<Emphasis Type="Italic">Crassostrea belcheri</Emphasis>, <Emphasis Type="Italic">Crassostrea iredalei</Emphasis>, and <Emphasis Type="Italic">Saccostrea cucullata</Emphasis>) in Thailand

Abstract Molecular genetic keys for identification of 3 commercially cultured oysters (Crassostrea belcheri, Crassostrea iredalei, and Saccostrea cucullata) in Thailand were developed based on restriction analysis of 18S ribosomal DNA and cytochrome oxidase subunit I (COI). Digestion of the amplified 18S rDNA with Hinf I unambiguously differentiated Crassostrea oysters from Saccostrea oysters and Striostrea (Parastriostrea) mytiloides. In addition, species-specific restriction fragment length polymorphism patterns of C. belcheri, C. iredalei, and S. cucullata were consistently observed when the gel-eluted COI was digested with Mbo I and Dde I. Thirty composite haplotypes were observed across all individuals. Species-specific composite haplotypes were found in C. belcheri (AAAA and AAAB), C. iredalei (AABC and AABU), and S. cucullata (BBCD and BBCE), respectively. The most common composite haplotype of COI in C. belcheri (AAAA), C. iredalei (AABC), and S. cucullata (BBCD) was amplified, cloned, and sequenced. Detection of C. belcheri and C. iredalei based on polymerase chain reaction was further developed using more specific primers (HCO2198 and R372) followed by digestion of a 372-bp product with Mbo I.     

Genetic relationships and species authentication of Boesenbergia (Zingiberaceae) in Thailand based on AFLP and SSCP analyses

Genetic relationships of 15 Boesenbergia species were examined using AFLP analysis. A total of 893 fragments were generated from six primer combinations; of these, 99.78% were polymorphic. The mean genetic distance between pairs of taxa ranged from 0.435 to 0.935. The neighbor-joining tree resolved investigated species into two separate lineages (corresponding to species possessing compact or elongated inflorescences) and suggests a rapid radiation in Boesenbergia. In addition, SSCP patterns of the partial psbA–trnH spacer were not overlapping between different species and therefore can be used for authentication of Boesenbergia species. Morphologically similar Boesenbergia longiflora and Boesenbergia sp., which showed three SNPs based on psbA–trnH polymorphism were successfully differentiated by SSCP. Moreover, Boesenbergia curtisii displayed intraspecific ecomorphological variation but exhibited the same SSCP pattern. Therefore, AFLP and SSCP are rapid and cost-effective methods for analysis of genetic similarity and species identification in Boesenbergia.     

Genetic Diversity and Geographic Differentiation of the Giant Tiger Shrimp (Penaeus monodon) in Thailand Analyzed by Mitochondrial COI Sequences

Genetic diversity and geographic differentiation of the giant tiger shrimp, Penaeus monodon, in Thai waters (Satun, Trang, Phangnga, and Ranong in the Andaman Sea and Chumphon and Trat in the Gulf of Thailand) were examined by COI polymorphism (N = 128). We observed 28 COI mitotypes across all investigated individuals. The sequence divergence between pairs of mitotypes was 0.00–20.76%. A neighbor-joining tree clearly indicated lineage separation of Thai P. monodon and large nucleotide divergence between interlineage mitotypes but limited divergence between intralineage mitotypes. High genetic diversity was found (mean sequence divergence = 6.604%, haplotype diversity = 0.716–0.927, π = 2.936–8.532%). F-statistics (F _ST) and an analysis of molecular variance (AMOVA) indicated that the gene pool of Thai P. monodon was not homogeneous but genetically differentiated intraspecifically (P < 0.05). Six samples of P. monodon could be allocated into three different genetic populations: Trat (A), Chumphon (B), and the Andaman samples Satun, Trang, Phangnga, and Ranong (C). Contradictory results regarding patterns of geographic differentiation previously reported by various molecular approaches were clarified by this study.     

Genetic Differentiation of the Stingless Bee Tetragonula pagdeni in Thailand Using SSCP Analysis of a Large Subunit of Mitochondrial Ribosomal DNA

Genetic diversity and population differentiation of the stingless bee Tetragonula pagdeni (Schwarz) was assessed using single-strand conformational polymorphism (SSCP) analysis of a large subunit of the ribosomal RNA gene (16S rRNA). High levels of genetic variation among individuals within each population (North, Northeast, Central, Prachuap Khiri Khan, Chumphon, and Peninsular Thailand) of T. pagdeni were observed. Analysis of molecular variance indicated significant genetic differentiation among the six geographic populations (Φ (PT)?=?0.28, P?相似文献