ABSTRACT
Genome shuffling for improving the activity of alkaline pectinase in Bacillus subtilis FS105 and its molecular mechanism were investigated. The fused strain B. subtilis FS105 with the highest activity of alkaline pectinase was obtained after two rounds of genome shuffling. The activity of alkaline pectinase in B. subtilis FS105 was 499 U/ml, which was improved by 1.6 times compared to that in original strain. To elucidate its molecular mechanism, rpsL gene sequences from original and fused strains were cloned and aligned, and the space structure of their coding proteins were also analyzed and compared. The alignment of the rpsL gene sequences indicated that three bases G, G and C were respectively replaced by A, A and G in the positions 52, 408 and 409 after genome shuffling. This resulted in the substitution of two amino acid residues in ribosomal protein S12: D18N and P137A, and therefore improving the biosynthesis of alkaline pectinase. This study lays a foundation for improving the activity of alkaline pectinase by genome shuffling and understanding its molecular mechanism.
Subject(s)
Bacillus subtilis/enzymology , Bacillus subtilis/genetics , DNA Shuffling/methods , Genes, Bacterial/genetics , Polygalacturonase/genetics , Polygalacturonase/metabolism , Amino Acid Sequence , Bacillus subtilis/isolation & purification , Base Sequence , DNA, Bacterial , Models, Molecular , Mutagenesis , Pectins/metabolism , Protein Conformation , Protoplasts , Ribosomal Proteins/chemistry , Ribosomal Proteins/genetics , Sequence AlignmentABSTRACT
Potato can be used as a source of modified starches for culinary and industrial processes, but its allelic diversity and tetraploid genome make the identification of novel alleles a challenge, and breeding such alleles into elite lines is a slow and difficult process. An efficient and reliable strategy has been developed for the rapid introduction and identification of new alleles in elite potato breeding lines, based on the ethylmethanesulphonate mutagenesis of dihaploid seeds. Using the granule-bound starch synthase I gene (waxy) as a model, a series of point mutations that potentially affect gene expression or enzyme function was identified. The most promising loss-of-function allele (waxy(E1100)) carried a mutation in the 5'-splice donor site of intron 1 that caused mis-splicing and protein truncation. This was used to establish elite breeding lineages lacking granule-bound starch synthase I protein activity and producing high-amylopectin starch. This is the first report of rapid and efficient mutation analysis in potato, a genetically complex and vegetatively propagated crop.
Subject(s)
Plants, Genetically Modified/metabolism , Solanum tuberosum/metabolism , Starch Synthase/metabolism , Starch/biosynthesis , Alleles , Amino Acid Sequence , Amylopectin/genetics , Amylopectin/metabolism , Base Sequence , DNA Shuffling/methods , Introns/genetics , Models, Genetic , Plants, Genetically Modified/genetics , Point Mutation , Reverse Transcriptase Polymerase Chain Reaction , Sequence Alignment , Solanum tuberosum/genetics , Starch Synthase/geneticsABSTRACT
AIM: To directed promote the antithrombotic activity of hirudin in vitro with DNA family shuffling method. METHODS: PCR products of HV1, HV2 and HV3 genes of hirudin were combined and digested by DNase I. Then random fragments about 50 bp were purified and reassembled to the same size of hirudin gene by two amplifications of PCR with or without primers. The shuffled hirudin genes were inserted into phagemid pCANTAB5E vector and the hirudin mutants were displayed on the surface of bacteriophage M13. Mutants with increased specific activity of antithrombin were screened by affinity panning with decreased amounts of thrombin. RESULTS: After shuffling and selection, a clone (HV2-N47K) with high antithrombotic activity was obtained. CONCLUSION: Hirudin variants with improved properties could be hopefully aquired by DNA family shuffling and affinity panning.