RESUMEN
The genetic diversity of 10 Machilus thunbergii populations in eastern China was analyzed using inter-simple sequence repeat markers. The populations showed high genetic diversity, with an overall population genetic diversity of 0.2343. Genetic diversity varied largely among populations, and populations with the highest genetic diversity were mainly from the eastern and western parts of the natural distribution area. Small populations, lack of effective gene flow, and fragmentation of habitats have led to greater genetic differentiation among populations, with 41.18% of genetic variation existing among populations. Unweighted pair-group method with arithmetic mean cluster analysis indicated that populations distributed between latitudes 25° and 31°N were clustered together and should be prioritized for in situ conservation. Northern, eastern, and southern populations were located in peripheral areas of the distribution range and were clustered separately. Collection of distinctive germplasm from peripheral populations should be promoted and ex situ conservation of elite germplasm should be implemented.
Asunto(s)
Especies en Peligro de Extinción , Variación Genética , Genética de Población , Lauraceae/genética , China , Análisis por Conglomerados , Conservación de los Recursos Naturales , ADN de Plantas/genética , Ecosistema , Flujo Génico , Sitios Genéticos , Repeticiones de Microsatélite , Filogenia , Reacción en Cadena de la Polimerasa , Árboles/genéticaRESUMEN
An anchor-chain molecular system was constructed for controlled orientation and high activity in enzyme immobilization. A streptavidin recognition peptide (streptag) coding sequence was fused to the 3' end of the phoA gene, which codes for E. coli alkaline phosphatase (EAP). Both the wild-type (WT) and the Asp-101 --> Ser (D1O1S) mutant were modified with the streptag sequence with or without the insertion of a flexible linker peptide [-(Gly-Ser)(5)-] coding sequence. The fused genes were cloned into the vector pASK75 and expressed in the periplasm of the host cell Escherichia coli SM547. The proteins were released by osmotic shock and purified by ion-exchange chromatography. Enzyme activities of all proteins were measured spectrophotometrically with rho-nitrophenyl phosphate as the substrate. Specific activities of D101S-streptag and D101S-linker-streptag enzymes were increased 25- or 34-fold over the WT, respectively. These fusion proteins were then immobilized on microtiter plates through streptag-streptavidin binding reaction. After immobilization, the D101S-linker-streptag enzyme displayed the highest residual activity and the ratio of enzyme activities of the linker to nonlinker enzymes was 8.4. These results show that the addition of a linker peptide provides a spacer so as to minimize steric hindrance between the enzyme and streptavidin. The method provides a solution for controlled enzyme immobilization with high recover activity, which is especially important in construction of biosensors, biochips, or other biodevices.