Study Of Transcriptomics And Proteomics On Uptake And Tolerance Mechanism Of Copper In Elsholtzia Splendens And Its CDNA Library Construction

Elsholtzia splendens is an annual herbaceous plant,belonging to the family Labiatae,which can endure elevated copper concentration and accumulate high level copper content.In China,it has been identified and accepted as Chinese native Cu-tolerant and accumulating plant,in the last few years,more research works have been conducted on the phytoremediation of copper contamination soil and its mechanism in copper tolerance and accumulation.In this study, hydroponics was used to investigate the transcriptomic changes of E.splendens in response to Cu-excess(100μmol/L) and Cu-deficiency(0μmol/L) stresses by cDNA-AFLP and Real-time quantity PCR technique,to explore the proteomic changes of E.splendens in response to Cu-excess (100μmol/L) and Cu-deficiency(0μmol/L) stresses by SDS-PAGE,2D-PAGE,MALDI-TOF MS and LTQ-ESI-MS/MS technique.The mechanisms of Cu uptake,accumulation,tolerance and detoxification in E.splendens were elucidated at the transcriptome and proteome levels for the first time.In addition,the fully length cDNA libraries of E.splendens treated with 100μmol/L and 0μmol/L Cu were constructed,which provided costful resources for functional genome research such as clone sequencing and gene screening and so on.The main results are listed as follows:1.Study of transcriptomics in E.splendens upon Cu-excess and Cu-deficiency stressesIn the transcriptomic study,cDNA-AFLP analysis was used to investigate the gene expression profile in E.splendens root tips(about 3～5cm) at four time-points(0h,3h,6h and 24h) during the Cu-excess and Cu-deficiency stresses.Sixty-six transcript derived fragments(TDFs) shown differential expression pattern were obtained.62 unique genes were found by Blast analysis,which were composed of 43 function-known genes,6 function-unknown genes and 13 novel genes.Five TDFs were selected random and the expression levels were assessed using real-time quantitative reverse transcription PCR(qRT-PCR) and the results were consistent with that of cDNA-AFLP. Among the 62 unique genes,12 genes were induced by Cu-excess stress,one gene was induced by Cu-deficiency stress,and other 49 genes were induced by both Cu-excess and Cu-deficiency stress. Of the 42 function-known genes,two genes are involved in redox homeostasis,six for signal transduction,three for protein metabolism,one for energy metabolism,nine for stress resistance, three for metallic tolerance and detoxification,four for cell wall metabolism,two for secondary metabolism,and includes one eukaryotic translation initiation factor,two transcription factors,nine ribosomal genes and one predominantly leaf-expressed protein gene.These Cu-induced genes indicate that Cu toxicity affected different physiological and biochemical pathways of the plant.2.Study of proteomics technique of E.splendensThe results of comparison on three extraction methods(TCA/acetone method;homogenization buffer method;phenol extraction method) of E.spIendens total protein showed that the E. splendens root and leaf protein extracted using phenol extraction method were adapted for two dimensional electrophoresis.E.splendens root and leaf proteins were Separated by two dimensional electrophoresis using IPG strips with different pH,and the maps of 2-DE using IPG strip with pH5-8 have the best results with high resolutions and much more protein spots.3.Study of proteomics in E.splendens upon Cu-excess stressThe present study investigated the Cu accumulation and proteome changes in E.splendens tissues upon Cu stress.After 6 days of treatment with 100μM Cu,Copper contents were increased with the 100μM Cu treatment in both leaves and roots,the concentration in the roots being 92-fold increased compared to controls,reaching 5170.5 mg/kg dry weight,but only 3-fold increased in the leaves.SDS-PAGE analysis indicated that the proteins changed more intensively in roots than that in leaves proteins upon Cu treatment.2-DE and image analyses found that 45 protein spots, including 16 up- and 29 down-regulated protein spots were significantly changed in roots,but only 6 protein spots with equal up- and down-regulated protein spots in leaves.Gene ontology analyses showed that these root proteins have many molecular functions and are involved in many biological processes,which indicates that complicated physiological and biochemical changes were involved in the mechanism of Cu tolerance and detoxification in E.splendens.The identified E.splendens root proteins were classified into several functional categories including signal transduction,redox homeostasis,regulation of transcription and translation,energy metabolism,cell wall metabolism, cell cytoskeleton rearrangement,transport and stress resistance.The roles of these different expressed proteins gained insight into the molecular mechanisms of E.splendens Cu tolerance and detoxification.4.Study of proteomies in E.splendens upon Cu-deficiency stressThe present study investigated the Cu accumulation and proteome changes in E.splendens tissues upon Cu-deficiency stress.After 6 days of treatment with 0μM Cu,the decrease of copper contents was 25.98 percent in roots and 15.55 percent in leaves.SDS-PAGE analysis showed that the intensity of some root and leaf protein bands was slightly increased.To further investigate the changes of protein profiles during Cu-deficiency stress,2-DE analysis of the total proteins in E. splendens roots and leaves were carried out.Quantitative image analysis revealed a total of 79 and 14 protein spots that changed their intensities significantly by more than 2.0-fold in roots and leaves respectively.There were 49 up-regulated spots and 30 down-regulated spots in roots,9 up-regulated spots and 5 down-regulated spots in leaves.Gene ontology analyses showed that these root proteins have many molecular functions and are involved in many biological processes,which indicates that complicated physiologiCal and biochemical changes were involved in the uptake process and response to Cu-deficiency stress.The identified E.splendens root proteins under Cu-deficient condition are closely associated with starch metabolism,signal transduction,energy metabolism,redox homeostasis,regulation of transcription and translation,energy metabolism, protein modification,degradation and transport,disease resistance,cell wall metabolism and cell cytoskeleton rearrangement.The differently expressed proteins in E.splendens leaves were manly involved in ion transport across the tonoplast,energy metabolism and carbon assimilation.In addition,Rubisco might undergo enhanced degradation under Cu stress.The results of these proteomic data indicated that E.splendens changed the biochemical reactions and metabolic pathways to enhance copper uptake,reutilization and other adaptive changes.All together,the proteomic analysis of the differentially displayed proteins in E.splendens roots and leaves upon Cu-excess and Cu-deficiency stresses indicated that E.splendens root organs played an important role in Cu uptake and tolerance,which might act as a Cu-sink.E.splendens adaptation to Cu-excess and Cu-deficiency stresses can synthesize some proteins involving in the same processes such as redox homeostasis,signal transduction,transcription and translation, energy metabolism,cell wall metabolism,cell cytoskeleton rearrangement and ion transport.But, the roles of these proteins between Cu-excess and Cu-deficiency stresses might be different.5.Construction of cDNA library from E.splendens roots challenged by Cu-exeess and Cu-defieieney stressesIn this study,root tips of E.splendens treated with 0 and 100μmol/L Cu for 3h,6h and 24h were used for construction of cDNA library.Two nonnormalized full-length cDNA library were constructed based on SMART technique.The titer of unamplified Cu-deficient library is 1.22E+08pfu/mL.Individual clones of the library are 6.41E+07.The titer of amplified Cu-deficient library is 3.96E+12 pfu/mL.The percentage of recombinant clones is 98%.82%of the inserted cDNA is long over 500bp.The titer of unamplified Cu-excessive library is 3.44E+07pfu/mL. Individual clones of the library are 1.81E+07.The titer of amplified Cu-excessive library is 4.86E+12pfu/mL.The percentage of recombinant clones is 99%.79%of the inserted cDNA is long over 500bp.