CDNA-AFLP Analysis Of Inducible Gene Expression In Zinc Hyperaccumulator Sedum Alfredii Hance Under Zinc Induction

Phytoremediation is defined as the use of plants to decontaminate and/or remove pollutants from the environment.It has emerged as an alternative technique for removing toxic metals from soil and offers the benefits of being in situ,cost effective and environmentally sustainable plants with high metal uptake and accumulation capacity have been termed as hyperaccumulator species.Sedum Alfredii Hance(S.Alfredii)was found in an old Pb/Zn mined area,Southern China and was originally identified as a new Zn/Cd co-hyperaccumulating plant,it was probably mutants from the non-hyperaccumulating ecotypes of S.Alfredii after a considerably long period(more than one thousand years)of inhabitation the highly heavy metal polluted environments.It has an extraordinary ability to tolerate and accumulate high concentration of environmental Zn and Cd,and with the characteristics of large biomass,fast growth speed,asexual propagation and perennial. Therefore,it is an ideal plant for studying mechanisms which are responsible for hyperaccumulation and phytoremediation practice.In the present study,we focused on generating dendrogram by using RAPD methods,and the analysis of the band sequences that only observed in HE as compared with other species of Sedum;otherwise, cDNA-amplified fragment length polymorphism(cDNA-AFLP)analysis was employed to identify the genes of Sedum alfredii H.that exhibit a modulated expression under Zn treatment applied in hydroponics culture;in addition,Zn and Cd concentrations in plants, reduced glutathione and Hydrogen Peroxide,total Sulphur were detected,and the role were analyzed in hyperaccumulations.The major results were summarized as follows:1.Sedum alfredii Hance has been identified as a new Zn/Cd co-hyperaccumulating plant species from heavy metal polluted area native to China.However,little information is available for the genetic differences among this ecotype and its relatives.In this paper, genetic variation and relationship among several species of Sedum including hyperaccumulating ecotype(HE)and the non-hyperaccumulating ecotype(NHE)of S. alfredii H,well as Sedum including Sedum Makinoi Maxim(SMM),Sedum emarginatum Migo(SEM),Sedum Sarmentoum Bunge(SSB),and Sedum Bulbiferum Makino:(SBM),was determined by random amplified polymorphic DNA(RAPD) analysis.Phytogenetic analysis suggested that they can be divided into two groups and the non-hyperaccumulating ecotype(NHE)of Sedum alfredii H is the nearest relatives of the hyperaccumulating ecotype(HE).However,genetics variation of HE and NHE is notable,which indicated that long term pollution of soil exerted great influenceon the genetic diversity and plant evolution.2.Hyperaccumulation of heavy metals in plants is closely related to metal detoxification in plant cells to metal toxicity,however the mechanisms is not fully understood yet.In this study,the possible roles of superoxide(H₂O₂)in zinc and cadmium hyperaccumulation of Sedum alfredii Hance were analyzed.The results showed that Zn/Cd concentration in the shoots of Sedum afredii Hance increased linearly within 15 days,after then leveled off.Concentration of total GSH,total S,as well as H₂O₂ in the shoot also increased within 15 days correspondingly,and then decreased with further increasing time of metal treatment.Shoot total S and glutathione(GSH)were higher for Cd than for Zn treatment.However,reverse trends in shoot H₂O₂ concentration were noted,i.e.much higher H₁O₂ concentration was observed in Zn-treated plant shoot than in Cd-treated plant shoot.Increasing external S supply could increase Zn and Cd accumulation in Sedum alfredii H.These results suggested that S containing compounds coundn't be the main chelator of Zn or Cd in the hyperaccumulating ecotype of Sedum alfredii Hance.GSH may play an important role in the detoxification of dissociated Zn and Cd,and H₂O₂ may be a kind of signal molecule in correlation with Zn and/or Cd hyperaccumulation.3.A pair of degenerate primers were designed using the plant actin sequences already available,actin genes from the hyperaccumulator Sedum alfredii H.were PCR-amplified, cloned,and sequenced.Then,both 5'-and 3'-rapid amplification of cDNA ends(RACE) were performed.The actin gene of full-length is 1418bp,and putative reading frame from 19bp to 1149bp(together 113 lbp).4.cDNA-amplified fragment length polymorphism(cDNA-AFLP)analysis was employed to identify genes that exhibited an indusibleexpression under Zn treatment in Sedum alfredii H.grown in hydroponic culture,plants were treated for 3h,8h,24h and 1 weeks with 500μM Zn and untreated plants were used as controls.The results showed that there existed over 4000 expression bands appeared at different treatment times.Among over 100 transcript-derived fragments(TDFs)87 TDPs cloned as Zn responsive.. According to the analysis with genomic database,about were identified 47 TDFswere identified t are obviously homologous to the genes that have known or putative function, 6 TDFs were homologous to uncharacterized genes while 34 TDFs did not show significant matches to any genes in the Genodatabase.Real-time PCR analysis showed that the expression profiles of 16 in 23 TDFs which were randomly selected from 87 TDFs had high similarity to those with cDNA-AFLP.Thirty eight genes of known function TDFs coud be devided into different functional groups including transcriptional factors,expression regulators,and stress responding and transport facilitation genes,as well as genes involved in cellular metabolism and organization and the photosynthetic process.These results,suggest that a multitude of processes are implicated in Zn hyperaccumulating process in Sedum alfredii H..There have been 49 responsive genes identified in the HE of Sedum alfredii H composing of,34 known genes with blast and 15 unknown or putative genes,which accounted for 56.3%of total 87 genes,. implying Zn hyperaccumulation is associated with many related to the functions of many unkonown genes.According to BLAST analysis of the functions for the known 38 genes, zinc hyper-accumulation seemed to be controlled by different pathways including signal transduction,stress response,transcriptal factors,and expression regulation.These results indicate that the process of Zn hyperaccumulation could be regulated by a complex system of signal induction and physi-biochemical metablism.