RESUMO
Growth rate of the Kuruma prawn, Marsupenaeus japonicus is an important economic trait, with larger animals commanding higher market prices. To identify gene markers associated with growth, a genetic map of a full-sib F(2) intercross family of M. japonicus has previously been generated and quantitative trait loci (QTL) influencing weight, total length, and carapace length were identified. In this study, amplified fragment length polymorphism (AFLP) markers associated with the major QTL region, contributing 16% to phenotypic variation, were characterized. Flanking sequence has been obtained and allelic variants responsible for segregation patterns of these markers have been identified. The genomic sequence surrounding the AFLP band 7.21a, residing under the QTL peak, contains a gene sequence homologous to the elongation of very long chain fatty acids-like (ELOVL) protein family. A full-length mRNA (ELOVL-MJ) encoding this protein was isolated from M. japonicus, representing both the first ELOVL gene in crustacea and the first candidate gene identified via QTL studies in crustacea.
Assuntos
Tamanho Corporal/genética , Proteínas de Membrana/genética , Penaeidae/genética , Sequência de Aminoácidos , Análise do Polimorfismo de Comprimento de Fragmentos Amplificados/veterinária , Animais , Sequência de Bases , Mapeamento Cromossômico/veterinária , Primers do DNA/química , DNA Complementar/química , Genótipo , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Penaeidae/crescimento & desenvolvimento , Reação em Cadeia da Polimerase/veterinária , Polimorfismo de Nucleotídeo Único/genética , Locos de Características Quantitativas , Reprodutibilidade dos Testes , Alinhamento de Sequência/veterináriaRESUMO
Temperature-regulated expression of recombinant proteins in the tac promoter (Ptac) system was investigated. Expression levels of fungal xylanase and cellulase from N. patriciarum in E. coli strains containing the natural lacI gene under the control of the Ptac markedly increased with increasing cultivation temperature in the absence of a chemical inducer. The specific activities (units per milligram protein of crude enzyme) of the fungal xylanase and cellulase produced from recombinant E. coli strain pop2136 grown at 42 degrees C were about 4.5 times higher than those of the cells grown at 23 degrees C and were even slightly higher when compared with cells grown in the presence of the inducer isopropyl beta-D-thiogalactopyranoside. The xylanase expression level in the temperature-regulated Ptac system was about 35% of total cellular protein. However, this system can not be applied to E. coli strains containing lacIq, which confers over production of the lac repressor, for high-level expression of recombinant proteins. In comparison with the lambda PL system, the Ptac-based xylanase plasmid in E. coli pop2136 gave a considerably higher specific activity of the xylanase than did the best lambda PL-based construct using the same thermal induction procedure. The high-level expression of the xylanase using the temperature-regulated Ptac system was also obtained in 10-litre fermentation studies using a fed-batch process. These results unambiguously demonstrated that the temperature-modulated Ptac system can be used for overproduction of some non-toxic recombinant proteins.
Assuntos
Escherichia coli/genética , Xilosidases/biossíntese , Xilosidases/genética , Sequência de Bases , Celulase/biossíntese , Celulase/genética , Primers do DNA/genética , DNA Complementar/genética , Escherichia coli/metabolismo , Fungos/enzimologia , Fungos/genética , Expressão Gênica , Óperon Lac , Dados de Sequência Molecular , Plasmídeos/genética , Regiões Promotoras Genéticas , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Temperatura , Xilano Endo-1,3-beta-XilosidaseRESUMO
A Neocallimastix patriciarum xylanase cDNA with the core coding sequence essentially identical to xynA was isolated and modified for high-level expression in Escherichia coli. The xylanase cDNA was truncated into individual catalytic domains, which were modified at the N-terminus. These modified xylanases were synthesised as non-fusion proteins under the control of the tac promoter. High-level expression was obtained with the modified domain II construct, accounting for approx. 25% of total cellular protein. However, with the same vector and expression cassette, expression levels of constructs containing domain I or domains I and II fused in tandem were very low. RNA analysis revealed that the striking difference in expression levels of these three constructs was not due to transcription efficiency, but was mainly related to transcript stability. Further analysis of the domain II construct revealed that the high-level expression of the domain II xylanase was largely attributed to the presence of a favourable N-terminal coding sequence, as mutation at the N-terminus of the domain II dramatically reduced the expression level. The modified domain II xylanase produced in E. coli had a specific activity of 1229 U mg-1 protein at pH 7 and 50 degrees C without purification. The availability of a recombinant fungal xylanase with high specific activity and in high yield offers a potentially attractive source of xylanase for industrial applications.
