Analysis Of Expressed Sequence Tags From Halophyte Atriplex Canescens And Functional Characterization Of AcEBP1 Under Abiotic Stress

With the continuous expansion of urban land and increasing population,the food supply will be insufficient.More seriously,the abiotic stress,such as drought,salinity,extreme temperature,light intensity,deficiency or toxic levels of nutrients and disease,are major limiatation factors causing loss of crop production and food crisis.How to increase crops abiotic stress in the world is now in great need.Clone and function character of some stress relative genes are generally the initial step for improving resistance of abiotics stress.With twenty years' development of molecular and biology,genomics and bioinformatics,scientists have made important progress in the mechanisms of signal transduction pathways under abiotic stress,and obtained many stress relative genes as well.However,most researches are focus on herbaceous plant and its response pathway of increasing the stress resistance by regulating the functional protein.The mechanism of inhibiting plant growth after abiotic stress stimulation is rarely reported.As halophyte Atriplex canescens exhibit its endurable traits for drought,salinity and other abiotic stress,investigation of abiotic stress genes in this woody plant would provide new insights for both basic exploration and applied sciences.In this research,three major objects were investigated as below:1.Little information is available on gene expression profiling in abiotic stress of A.canescens.Using similar research method in functional genomics,a full-length cDNA library was generated from A.canescens exposed to 400 mM NaCl abiotic stress,and 343 high-quality ESTs were obtained after PCR and sequencing and deposited in the dbEST GenBank under accession numbers JZ535802 to JZ536144.In an evaluation of annotated 343 EST sequences,190 unigenes from 197 assembled unigenes were identified with known functions.Based on Arabidopsis MIPS functional category and GO classifications,72 genes sharing similarities with genes related to the defense response.According to reported abiotic stress genes information,23 genes were identified as NaCl responsive genes,and 17 genes were up-regulated under salt stress identified by qRT-PCR.These 17 genes provided a rich genetic resource for further research in Arabidopsis.Additionally,among these 343 unigenes sequences,22 simple sequence repeats(SSRs)were also identified contributing to the study of A.canescens resources.What's more,Six canidiate genes have been indentified by overexpressing in Arabidopsis or yeast under abiotic stress and increased the abiotic stress resistance of Arabidopsis and yeast.2.Simple,efficient,and economical recombinant plant binary expression vectors for functional research of large-scale candidate genes in Arabidopsis and promoting crop improvement by genetically biotechnology is in great demand.In this study,using the pCAMBIA1301,pCAMBIA3300,pCAMBIA3301 and pCHF3 vectors,we constructed series of plant expression vectors(total 12)carrying different promoters(CaMV35S and rd29A),different marker genes(hyg?bar?eGFP and GUS)and same multiple cloning sites(MCS: SacI,Kpn I,SmaI,BamHI,XbaI,Sal I,and Pst I).These recombinant binary vectors could be utilized for overexpressing,inducedexpressing,subcellular localization and promoter activity analysis.Our results demonstrated these vectors could be successfully transferred into Arabidopsis and tabaco with functional marker elements,which indicated this system was a convenient and economic vector system for transformants screening and subcellular localization.Besides,the optimization of basta resistance screening also makes the selection of transgenic plants more simple,convenient and efficient.Therefore,the recombination vectors provided a relatively high-efficiency and reliable platform for researchers in vector construction and exhibit its great application and potential in plants.3.From previous work on the EST sequencing,An ErbB-3-binding protein gene AcEBP1,also known as proliferation-associated 2G4 gene(PA2G4s)belonging to the M24 superfamily,was obtained from the A.canescens.Subcellular localization imaging showed the fusion protein AcEBP1-eGFP was located in the nucleus of epidermal cellsin Nicotiana benthamiana.The AcEBP1 gene expression levels were up-regulated under salt,osmoticstress,and hormones treatment as revealed by qRT-PCR.Overexpression of AcEBP1 in Arabidopsis could negative regulate stress responsive genes and PR genes but without any obvious phenotypes under normal conditions.However,AcEBP1 could inhibit root cell growth and exhibit sensitive phenotypes under stress NaCl,osmotic stress,ABA and low temperature.Unexpected,the AcEBP1 overexpressing increased resistance to drought stress.Together,these results demonstrate that AcEBP1 may negatively affect cell growth and other abiotic stress response genes,which resulted in the sensitive phenotypes under most of stress conditions.As a negative regulator under drought,AcEBP1 overexpressing Arabidopisis might keep less energy and water consumption,which increased water retention rate and enhanced drought resistance.Overall,current plant abiotic stress research is mainly focusing on the herbaceous plants,and the investigation of abiotic stress resistance mechanism is still missing.In this research,we isolated and characterized series novel resistance related genes by cDNA libarary construction and biomatics analysis of woody plant A.canescens,which provided much more candidate genes and information for genetics breeding of crops.Meanwhile,we constructed series of plant binary expressing vectors,which provided high-efficiency and ecnomic vector resource for researchers in functional character in plants.What's more,several experiments were performed to functionally characterize proliferation associated protein AcEBP1 under abiotic stress,which may play important role in inhibiting plant growth under abiotic stress.All the research not only contribute the understanding of abiotic stress response in woody plants,but also provide much more novel condidate genes for plant genetics engineering,which is important and potential.AcEBP1 from elite halophyte A.canescens could also provide new insights into plant growth under abiotic stresses.