Molecular Mechanism Of Regulation Of Natural Rubber Biosynthesis By 14-3-3 Proteins In Laticifer Of Hevea Brasiliensis

Natural rubber(cis-1,4-polyisoprene)is the main economic product synthesised via MVA pathway in highly specialized laticifer,a cellular network created by anastomosis of latex cells which is embedded in phloem tissues,and is widely used industrially.Although some genes controlling natural rubber biosynthesis have been cloned and characterized,little is known about the regulatory mechanism of natural rubber biosynthesis in laticifer of Hevea brasiliensis at molecular and biochemical levels.The interaction partners of 14-3-3s include proteins involved in signal transduction,metabolism,chromatin function,ion transport and vesicle trafficking.In plants,The main function of 14-3-3s are in the regulation of abiotic and biotic stress response,primary metabolism,response to light,hormone pathways,growth and cell division,and other 'housekeeping' functions.Although the functions in secondary metablism have been conjectured since the discovery of plant 14-3-3 proteins,it is only in the past two to three years that the number of clear examples has increased substantially.Our previous studies have suggested that HbGF14 have a role in the pathway of natural rubber biosynthesis,but molecular mechanism of regulation of natural rubber biosynthesis by 14-3-3 proteins in laticifer of Hevea brasiliensis is still unknown.To obtain the information of the molecular and regulational mechanism of 14-3-3 proteins in the pathway of natural rubber biosynthesis,a total of ten novel 14-3-3 genes(HbGF14a-j)from laticifer were identified in the Hevea brasiliensis genome.Phylogenetic analysis demonstrated that the presence of two divergent classes of HbGF14s,according to the structures of exon-intron and sequence of amino acid,as same as their homologues from rice and Arabidopsis.Tissue-specific expression analysis of the HbGF14s demonstrated diverse spatiotemporal expression patterns,while their expression profiles under JA and ET treataments indicated that HbGF14s were response to hormone signaling,similar to their plant homologues.In addition,the fluorescence signal in the onion epidermal cells released by HbGF14s::GFP fusion was detected in nucleus and cytoplasm.For screening the target proteins,a library was built using the cDNA which synthesized by reverse transcription of mRNA extracted from laticifers.81 interaction partners of the seven 14-3-3s(HbGF14a,HbGF14b,HbGF14c,HbGF14e,HbGF14f,HbGF14g and HbGF14h),which are involved in plant signal transduction,metabolism,development,and other cellular processes,were identified in laticifers.To clarify the regulational mechanism of natural rubber biosynthesis,our study focused on the target proteins of HbGF14s,including RZF,SRPP,MYB,MYC,cis-prenyltransferase and glutaredoxin.Furtherly,the interaction of HbGF 14a and HbRZF was demonstrated by assay of yeast two-hybrid,BiFC and Pull-Down.The results indicated that the RKVTCP motif of HbRZF and a-helice 7-8 of HbGF 14a is the recognition motifs.Transient dual-LUC assay in Nicotiana benthamiana demonstrated that target gene transcription was repressed by the binding of HbRZF with pHbHRT,which was interfered by HbGF14a.How HbGF14c regulate natural rubber biosynthesis through interacting with HbSRPP need furtherly study,which was also demonstrated by assay of yeast two-hybrid,BiFC and Pull-Down.