| The genus Platanus,a member of Platanaceae family,perennial tall deciduous trees are widely planted for greening in temperate and subtropical cities,highways and factories in the entire world.In China,titled as the "King of the Street Trees",they are extensively planted and cultivated in northwest,northeast and southeast China.However,with the development of automobile industry in China,large quantities of Pb2+ discharged by automobiles have become one of the main sources of the urban environmental pollutant.Therefore,as the main greening specie,Platanus are facing severe lead stress,which leads declined chlorophyll content,hinder of plant respiration and photosynthesis,internal oxidative stress,cell membrane lipid peroxidation,oxidative damage of protein,pigments,enzymes,nucleic acid,etc.Lead stress also causes leaf chlorosis,growth retardation,and even death of the plants.At present,most researches stay on the analysis and assessment of heavy metal contents in polluted regions and very few focuses on harm of lead pollution to Platanus,and its damage mechanism has not been clear.Taking Platanus acerifolia seedlings as materials,lead nitrate soaked(XLM-12-40)seedlings as treated sets,distilled water soaked seedlings(XLM-0)as the controlled sets,using new generation Solexa/Illumina transcriptomic technology,this research built up a more comprehensive P.acerifolia transcriptome database,discovered differentially expressed gene between various treatments,identified the critical genes affecting lead stress by using bioinformatics analysis,constructed sRNA library,appraised the known and new miRNA,determined response of specific miRNA and their target genes lead stress,initially clarified the molecular mechanisms of gene expression under lead stress conditions and miRNA change regulation.The main research results are as follows:1.Completed sequencing and assembling P.acerifolia transcriptome in pre and post lead treatment.Through transcriptome sequencing of the seedling leaves,nearly seventy million raw reads sequences were obtained from each sample.By using bioinformatics software analysis and genome assembly,140,989(Control sets),140,989(Treatament sets)and 115,066(control sets and Treatmentsets mixed together,All-unigene)transcriptomic sequences were gained respectively;66,875 CDS gene sequences were also obtained by applying the Blast and ESTscan software analysis.2.Made functional annotation analysis on P.acerifolia All-unigene.115,066 All-Unigenes were made homological alignment in NCBI Nr,and Swiss-Prot databases,63,750 and 37,142 of its sequences presented,to a certain extent,homology with other organism;18,084 sequences were with functions based on COG classification;GO classifications showed 41,539 sequences were in 25,16 and 14 category respectively based on biological processes,molecular function and cellular localization;34,556 sequences were associated with in 128 pathways based on KEGG analysis.3.Selection of Differentially expressed unigene.The expression level of the established P.acerifolia transcriptome sequences were calculated,and corrected by multiple hypothesis detection.Under the criterions of absolute value of differentiation less than 4,P-Value less than 0.01and FDR less than 0.001,16,246 differentially expressed unigenes were obtained.4.Accomplished selection and analysis on P.acerifolia genes response to lead stress.Differentially expressed unigene were analyzed by GO function classification,and KEGG metabolic pathways enrichment analysis.Further BLAST comparison and related metabolic pathways analysis were carried out,and 356 candidate unigenes associated with lead stress responses were obtained.These unigene functions involved in chlorophyll biosynthesis,photosynthesis-antenna protein,photosynthesis,respiration,carbon fixation metabolic process and brassinosteroids synthesis.Through comprehensive analysis,it concluded that under lead stress condition,the expressions of CHLH?CRD1?CHLG?CAO are hindered,which affects growth and development of P.acerifolia,MAPK?CDPK?GSH?MT may strengthen its ability in resisting lead stress.5.Established small RNA libraries of P.acerifolia samples in pre and post lead treatment.A total number of 13.8 million small RNA were obtained,small RNAs in range of 21 to 24 nt accounted for 80%and the proportion of the total of consensus sequence accounted for more than 80%of the total number of small RNA.6.Carried out the prediction and analysis on P.acerifolia miRNA response to lead stress.Mapping sRNA sequences into P.cerifolia transcriptome,all types of sRNA sequences and quantities of each type sRNA sequences were comprehensively and systematically compared.165 known miRNA were identified,185 new miRNA were discovered,in which 105 related to lead stress.640 target genes associated with 75 miRNA were predicted,and functionally annotated through Nr,Nt,Swissport database.Results showed that the functions of miRNA target genes associated with lead stress included the oxidative stress response,transcription factors,hormone signal transduction,protein reversible phosphorylation,self feedback,poison effluent and ubiquitin,which are functions closely related to adversity stress.The target genes involved in the growth and other physiological metabolic processes of P.acerifolia,directly or indirectly participated in its resistance to lead stress.In summary,using a new generation of sequencing technology(Solexa/Illumina sequencing),we constructed the first P.acerifolia transcriptome library,made selection of differentially expressed unigenes,obtained 356 key genes related to responding lead stress,built up P.acerifolia sRNA libraries,identified known miRNA,predicted and discoveried new miRNAs,and miRNA related to lead stress,predicted the corresponding miRNA target genes.Under the condition of lead stress,the molecular mechanism in strengthening resistance to lead stress of P.acerifolia was studied from different levels and angles.Meanwhile,this research provides a technology and information platform for thoroughly understanding the molecular and regulation mechanisms of growth and development of Platanus. |
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