Cloning And Characterization Of Ethylene Signaling Related Genes In Hevea Brasiliensis

Hevea brasiliensis is the main source of natural rubber.Biosynthesis and drainage of latex is one of wounding responses of rubber tree.Treatment with exogenous ethylene(ET)can stimulate the drainage of laxtex and improve the production of rubber.However,the molecular mechanism underlying the ET stimulation of latex drainage is so far less known.In this study,the genes of ET signaling pathway are cloned and characterizied,so as to reveal the the role of ET in latex biosynthesis and drainage.First,the ET responsive genes were identified by digital gene expression(DGE).Additionally,the homologous genes of key components of ET signaling pathway were in siliso cloned and characterized.Furthermore,the interacted proteins of the key genes and downstreaming genes were also identified.Following results were obtained:(1)DGE analysis revealed that 2216 genes showed differentiated expression after ethylene treatment in leaves,which including 1018 up-regulated gens and 1198 down-regulated genes.At 3h after ethylene treatment,a total of 10629 differentially expressed genes were observed,in which 3697 genes were up-regulated and 6932 genes were down-regulated.Between the 1h and 3h ET-treated samples,there were 9465 differentially expressed genes,including 3984 up-regulated genes and 5481 down-regulated genes.Gene function classification assay showed that the most differentially expressioned genes involved in metabolic pathways,biosynthesis of secondary metabolites,and plant hormone signal transductionDGE results also indicated that fewer genes showed differential expression in latex than that in leaves after ET treatment.Only 656 genes differentially express in latex at 1h after ethylene treatment,including 56 up-regulated gens and 600 down-regulated genes.At 3h after ethylene treatment,only 1047 genes showed differential expression,in which 180 genes were up-regulated and 867 genes were down-regulated.Between the 1h and 3h ethylene treated latex samples,there were 175 differentially expressed genes,including 102 up-regulated genes and 73 down-regulated genes.(2)Ein3(ethylene insensitive 3)is key transcriptional factor in plant ET signaling pathway.Four genes of EIN3 genes family were cloned in Hevea brasiliensis,termed as HbEIN3,HbEIN3-1,HbEIL3-1 and HbEIL3-2.Bioinformatics analysis showed that these genes are similar to other plants EIN3/EIL1,have a domain with 1-52 acidic amino acids and DNA bingding domain such as BD ?,BD ?,BD ?,BD ?,and BD ?,and a proline domain.Subcellular localization showed that HbEIN3 located in the nucleus.Additionally,HbEIN3 has autoactivation,suggesting that HbEINs are transcriptional factors.Yeast one hybrid analysis showed that HbEIN3s interacted with DRE and G-box cis-elements.The promoters of HbEBF2 and HbAOS,that contained these two elements,can interact with HbEIN3s,indicating that HbEBF2 and HbAOS might be the target genes of HbEINs.Additionally,Western-Blot showed that HbEIN3 protein exists in C-whey of rubber latex.(3)Stabilization of Ein3 is regulated by F-box protein EBF(Ein3 binding factor)through ubiquitin degradation pathway.Through homologous cloning method,2 EBF genes of Hevea brasiliensis were cloned,which were termed as HbEBFl and HbEBF2.HbEBF1 encoded a 655 amino acids protein and HbEBF2 encoded a 672 amino acids protein.Both proteins contain F-box domain and many LRR(leucine rich repeat)domains.HbEBF1 and HbEBF2 did not show autoactivation,but could interact with HbEIN3s,suggesting that they might regulate the stabilization of HbEIN3s at posttranscriptional level.(4)ERFs(ethylene responsive factors)are a group of transcriptional factors in downstream of ethylene signaling pathway.In this work,two ERF genes of Hevea brasiliensis,termed as HbERFl and HbERF2,were cloned.Both of them have one AP domain and encode 385 amino acids and 258 amino acids proteins,respectively.Both HbERF1 and HbERF2 showed autoactivation.HbERP1 was located in the nucleus.Western-Blot revealed that HbERFl protein existed in the C-whey of rubber latex.(5)The interaction between the cis-elements(DRE and G-box)and HbEINs observed enable us to predict the potential target genes of HbEIN3s using bioinformation analysis.After global scanning the DRE and G-box containing genes,7600 genes promoters containing DRE and 2800 genes promoters containing G-box were revealed.These gens includea seriel of genes involved in latex biosynthesis and drainage,including 5 tonoplast intrinsic proteins(TIP),sugar transporter(ST),rubber elongation factor(REF),hydroxymethylglutaryl-CoA reductase(HMGR),and small rubber particle protein(SRPP),suggesting that HbEIN3s might control the latex biosynthesis and drainage through regulating the expression of these genes.The results obtained in this work lay a foundation for investigation of ethylene signaling pathway in rubber tree and molecular mechanism of latex biosynthesis and drainage.