ABSTRACT
OBJECTIVE:To develop a detection LC-MS/MS method for global DNA methylation in medicinal plants. METH-ODS:Genomic DNA was isolated using plant DNA extraction kit,and then hydrolyzed by 88% formic acid at 140 ℃. After dried with nitrogen,extracted DNA was dissolved again with mobile phase. LC separation was performed on HILIC column with mobile phase consisted of 7 mmol/L ammonium formate-acetonitrile (gradient elution) at flow rate of 0.3 ml/min. The analysis was con-ducted by tandem MS with positive ion electrospray ionization in multiple reaction monitoring(MRM)mode. The ratio of genomic DNA methylation in 10 commonly used medicinal plants was calculated. RESULTS:The linear ranges of Cyt and 5mC were 1-500 ng/ml(r=0.999 5)and 0.2-100 ng/ml(r=0.999 6). The relative standard deviations(RSDs)of accuracy were 1.12% and 3.68%(n=6). The RSDs of intra-day precision were 2.36% and 4.02% for Cyt and 5mC,respectively (n=5). The RSDs of inter-day precision were 1.04% and 3.54% for Cyt and 5mC,respectively (n=3). The RSDs of repeatability test were 1.53% and 3.27%for Cyt and 5mC,respectively(n=6). The recoveries of Cyt and 5mC were 98.7%-102.1% and 91.2%-103.5%. The percentages of global DNA methylation in 10 medicinal plants were ranged from 17.63% to 25.18%. CONCLUSIONS:LC-MS/MS method is simple,rapid,sensitive and precise,and can be used for the detection of global DNA methylation in medicinal plants.
ABSTRACT
Objective An investigation of new cultivars of Amomum villosum Lour.(AVL) with high yield and good quality was carried out,thus to supply evidences for the identification of AVL cultivars in accordance with the morphological features of their flowers and fruits.Methods An investigation of AVL species from the genuine producing areas of Yangchun city of Guangdong province was performed.The morphological features of flowers and fruits of two cultivars(Changguo and Yuanguo) as well as one breeding type(Chunxuan type) were examined.Results Specific and significant features were screened out in different cultivars of AVL.Conclusion There exit specific features in flowers and fruits of different cultivars of AVL from Yangchun.
ABSTRACT
Objective To establish a DNA extraction method for DNA barcoding analysis of Chinese medicinal materials.Methods Seven different DNA extraction methods were used to extract DNA from 6 medicinal recalcitrant plants which are rich in secondary metabolites.Results CTAB method 3 was fast,simple,universal and effective,by which a high DNA concentration and qualified ratio were obtained as compared with the other methods.The DNA extracted by this method could provide good results for DNA barcoding analysis.The main improved steps of this methods were as follows:①adoption of 3 %CTAB rather than 2 %CTAB in the exaction;②adding 1 %polyvinylpyrrolidone(PVP) and 0.2 %?-mercaptoethnoal in extraction solution to remove secondary metabolites and to prevent DNA degradation;③centrifuge at 10000 r/min for 15 min to remove protein and impurity.Conclusion CTAB method 3 is a proper method of DNA extraction for DNA barcoding analysis of Chinese medicinal materials.
ABSTRACT
Objective To establish a molecular identification method for three cultivars of Amomum villosum Lour.(AVL),thus to provide scientific evidence for the identification,selection and breeding of AVL.Methods The fragments of 26S rDNA D1-D3 region and matK gene of three cultivars of AVL and Amomum longiligulare T.L.Wu were amplified by polymerase chain reaction(PCR) and with corresponding primers,and then their sequences were analyzed,and phylogenetic tree was constructed based on the sequences.Results We obtained 739 bp in 26S rDNA D1-D3 sequence.Differences in 4 basic sites of 739 bp were shown between AVL and Amomum longiligulare T.L.Wu.The two cultivars of AVL,Changguo and Yuanguo,had the same sequence,but there was a difference in one basic site of Changguo and Yuanguo from Chunxuan.The phylogenetic tree based on 26S rDNA D1-D3 sequence revealed the difference between Chunxuan and the other two cultivars of AVL.We also obtained 824 bp in matK gene sequence.The three cultivars of AVL showed the consistent sequence,but there was a difference in one basic site of three cultivars of AVL from Amomum longiligulare T.L.Wu.Conclusion We can identify the three cultivars of AVL through the sequence differences at the molecular level,and Chunxuan has a closer genetic relationship with Amomum longiligulare T.L.Wu.