Comparative Proteomics Anaylsis Of Pogostemon Cablin Leaves After Continuous Cropping

Pogostemon cablin(Blanco)Benth.(Lamiaceae)is a traditional Chinese medicine,which is cultivated extensively in the Philippies,Malaysia,India,China and other Southeast Asian countries.P.cablin has been widely used in pharmaceuticals,food and cosmetics,because the essential oil of P.cablin(knows as patchouli oil)has antifungal properties,which gives rise to an increasing demand.However,P.cablin has suffered from very serious consecutive monoculture problems,which leaded to severe diseases,significantly reduced biomass and quality of overground part(leaves),or low harvest rate.Therefore,it is necessary to understand the leaves growth and development by examining protein expression after continuous cropping,especially in the transformation period from fast to slow growth.In this paper,the dynamic accumulation mode of patchoulol during the development of P.cablin after continuous cropping(TR)and normal growth(CK)P.cablin were tested.The two-dimensional electrophoresis(2-DE)conditions suitable for P.cablin leaves were explored.The resistance response proteins of P.cablin after continuous cropping were separated and identified by proteomic techniques.The main results are summarized as follows:(1)The essential oil contents in the two different cropping treatments showed growing trends from 90 to 210 days after cultivation(DAC)and reached their maximum levels at 210 DAC.The essential oil content in the TR group patchouli significantly lower than the CK group patchouli from 150 to 210 DAC.(2)The patchoulol contents of TR group patchouli oil and CK group patchouli oil have the similar accumulation trends,showed growing trends from 90 to 210 DAC and reached their maximum levels at 210 DAC.The patchoulol contents in the TR group patchouli oil significantly lower than the CK group patchouli oil,during its different growth periods.(3)Sample preparation and isoelectric focusing conditions were established and optimized to get better 2-DE profiles of total proteins in the P.cablin leaves.The results showed that the clearly 2-DE profile of the total leaf protein was been attained using TCA-acetone method better than BPP method,and isoelectric focusing on the 100 000 Vhrs was better than 110 000 Vhrs,meanwhile,the rate of success identification of the leaf protein was 90%.The method was suitable for patchouli leaves for two-dimensional electrophoresis.(4)To investigate the changes of the protein profiles between the two monoculture treatment in different temporal(90,150,and 210 DAC),2-DE was applied to display and compare all extractable P.cablin leaves proteins.Quantitative image analysis using Image Master 2D Platinum 5.0 revealed that 183 proteins were exhibiting more than 2.0 fold differential expression.The differential expression proteins(DEPs)were excised from the gels,digested with trypsin,and the peptides were analyzed using MALDI-TOF/TOF MS coupled with database searches.In total,119 proteins were successfully identified by MS/MS analysis,with 64 up-regulated protein spots and 55 down-regulated ones compared to CK,among them,large number of DEPs related to Post-translational modification,protein turnover,and chaperones and Carbohydrate transport and metabolism mainly located on Chloroplast and Cytoplasm.Those DEPs have carrier catalytic activity,binding function activity,transporter activity,structural molecule activity and antioxidant activity,mainly involved in cellular physiological process,metabolic process,and response to stimulus.Continuous cropping leaded to change in the expression of proteins that related to energy,sugar and amino acid metabolism in P.cablin leaves,which suggested that these proteins were involved in continuous cropping obstacle response activities.