ABSTRACT
Objective To establish an optimum DNA extraction method for Chinese traditional medical herbs in order to meet necessary for DNA barcoding research.Methods Four Chinese traditional herbs, Glycyrrhiza uralensis Fisch, Phellodendron Chinensis Cortex, Cistanche tubulosa Wight and Cistanche deserticola Ma were chosen as the experimental materials, the DNA was extracted by 6 different kinds of DNA extraction method, including the improved method of high-salt combined low-pH,the improved method of SDS,CTAB method,PVP method,PlantZol Kit and Ezup Kit, the quality of DNA was investigated by ultraviolet spectrophotometry,agarose gel electrophoresis and PCR amplification by using specific primers of ITS2 and psbA-trnH. ResuIts The quality of the DNA was better than other four kinds of methods by the improved method of high-salt combined low-pH and Ezup Kit, the value of OD260/OD280 was between 1.7 ~1.9,the yield of DNA was the highest by the PlantZol kit , followed by the improved method of high-salt combined low-pH( P <0.05 ) , but the purity of DNA was poor by the PlantZol kit.The DNA electrophoresis tests showed that the DNA integrity of Glycyrrhiza uralensis Fisch and Cistanche tubulosa Wight were better with the improved method of high-salt combined low-pH, the improved SDS method, the CTAB method and the PlantZol kit.The DNA of Phellodendron Chinensis Cortex and Cistanche deserticola Ma were extracted by the six methods appeared diffuse status in the lanes.But only the improved method of high-salt combined low-pH could make the PCR amplification of the success rate 100% by using specific primers of ITS2 and trnH-psbA.ConcIusion The DNA extraction method of high-salt combined low-pH can be used to establish the Chinese DNA barcoding which has the advantages of lower cost, simpler procedure and less time.
ABSTRACT
Objective To construct in the extracellular matrix metalloproteinase inducer (EMMPRIN)glycosylation single point mutation plasmid,and to explore its relationship with tumor cell proliferation.Methods PCR point mutantion technology was used to construct the mutantion plasimid of EMMPRIN glycosylation single point.After successful mutation, the function of mutantion plasmids were detected. Western blotting was used to detect the expression of EMMPRIN protein,immunofluorescence method was used to detemine the morphological changes of the cells,and MTT assay was performed to detect the relationship between mutantion pasmid and tumor cell proliferation.Results Confirmed by restriction enzyme digestion and sequencing,the 44th,the 152th,and the 186th Asn were successfully mutated to Gln in the sequence of EMMPRIN;EMMPRIN/GFP (N44Q), EMMPRIN/GFP(N152Q),and EMMPRIN/ GFP (N186Q)glycosylation single point mutation plasmids were constructed.Compared with wild-type,thel morphology of the cells was significantly changed,the core division of mutant-type cells was significantly reduced, the number of filopodia was reduced. The results of MTT assay showed that the survival rate of the cells in wild-type group were significantly increased compared with control group (P<0.05 );the survival rates of the cells in EMMPRIN (N44Q) group, EMMPRIN (N152Q) group and EMMPRIN(N186Q)were significantly decreased compared with wild-type group(P<0.05).Conclusion Mutant-type EMMPRIN can inhibit the proliferation of tumor cells;with the duration increasing, the inhibitory effect is weakened.There is a correlation between EMMPRIN glycosylation and proliferation of tumor cells.