Fungi play a significant role in biology-related domains, and with the molecular biology technology advancing, identification of fungi at molecular level and verification of genetic transformant has become the necessa...Fungi play a significant role in biology-related domains, and with the molecular biology technology advancing, identification of fungi at molecular level and verification of genetic transformant has become the necessary step of test. By far, however, there is no ideal high-throughput molecular identification method. In this paper, a high-throughput device was designed, and a novel PCR-mediated molecular identification method suitable for the device was developed. Through cloning of ccllulase encoding genes and regulatory genes on the genome of Trichoderma reesei, cloning of promoter of phesphoglycerate kinase gene 1 and xylanase encoding genes on the genome and expression vector of Saccharomyces cerevisiae, and ITS sequences cloning and RAPD analysis of T. reesei, S. cerevisiae, Penicil- lium oxalicum, Rhizopus stolonifer, Aspergillus niger, A. carbonarius and A. japonicas, the method turned out to be an effective one with wide application potential. The establishment of the method has worked out the bottleneck of high-throughput molecular identification.展开更多
1,3-propanediol (1,3-PD) is an important material for chemical industry,and there has been always much interest in the production of 1,3-PD using all possible routes. The genes encoding glyc-erol dehydratase (GDHt) fr...1,3-propanediol (1,3-PD) is an important material for chemical industry,and there has been always much interest in the production of 1,3-PD using all possible routes. The genes encoding glyc-erol dehydratase (GDHt) from Citrobacter freundii,Klebsiella pneumoniae and metagenome were cloned and expressed in E. coli. All glycerol dehy-dratases but the one from metagenome could be detected to show enzyme activities. In order to im-prove the enzymatic properties of GDHts,the genes encoding α and β-γ subunits were cloned,and the enzyme characteristics were evolved by rational de-sign based on their 3D structures which were con-structed by homology modeling. Six heteroenzymes were obtained by swapping the α subunit genes of these three different-source-derived GDHts. The pH,thermal stability and Vmax of some heteroenzymes were dramatically improved by 2―5 times compared with the wild one (GDHtKP). The GDHt cloned from metagenome,originally proved to be with no enzyme activity,was converted into active enzyme by swap-ping its subunits with other different GDHts. In addi-tion,the effect of subtle 3D structural changes on the properties of the enzyme was also observed.展开更多
基金Supported by Applied Fundamental Research Project of Guangxi Department of Science and Technology(14125008-2-13)
文摘Fungi play a significant role in biology-related domains, and with the molecular biology technology advancing, identification of fungi at molecular level and verification of genetic transformant has become the necessary step of test. By far, however, there is no ideal high-throughput molecular identification method. In this paper, a high-throughput device was designed, and a novel PCR-mediated molecular identification method suitable for the device was developed. Through cloning of ccllulase encoding genes and regulatory genes on the genome of Trichoderma reesei, cloning of promoter of phesphoglycerate kinase gene 1 and xylanase encoding genes on the genome and expression vector of Saccharomyces cerevisiae, and ITS sequences cloning and RAPD analysis of T. reesei, S. cerevisiae, Penicil- lium oxalicum, Rhizopus stolonifer, Aspergillus niger, A. carbonarius and A. japonicas, the method turned out to be an effective one with wide application potential. The establishment of the method has worked out the bottleneck of high-throughput molecular identification.
文摘1,3-propanediol (1,3-PD) is an important material for chemical industry,and there has been always much interest in the production of 1,3-PD using all possible routes. The genes encoding glyc-erol dehydratase (GDHt) from Citrobacter freundii,Klebsiella pneumoniae and metagenome were cloned and expressed in E. coli. All glycerol dehy-dratases but the one from metagenome could be detected to show enzyme activities. In order to im-prove the enzymatic properties of GDHts,the genes encoding α and β-γ subunits were cloned,and the enzyme characteristics were evolved by rational de-sign based on their 3D structures which were con-structed by homology modeling. Six heteroenzymes were obtained by swapping the α subunit genes of these three different-source-derived GDHts. The pH,thermal stability and Vmax of some heteroenzymes were dramatically improved by 2―5 times compared with the wild one (GDHtKP). The GDHt cloned from metagenome,originally proved to be with no enzyme activity,was converted into active enzyme by swap-ping its subunits with other different GDHts. In addi-tion,the effect of subtle 3D structural changes on the properties of the enzyme was also observed.
