Enzymatic Properties And Gene Expression Of Key Sucrose Metabolizing Enzymes In Leaves Of Hevea Brasiliensis

Natural rubber(NR)is an important raw material whose only commercial source is rubber tree(Hevea brasiliensis).Rubber biosynthesis occurs in laticifers and needs sucrose to be the sole initial precursor molecule.Sucrose metabolism and regulation in laticifers have a key effect on latex regeneration and rubber production.Sucrose is the major form of photoassimilate in Hevea leaves and is transferred from source leaves to laticifers by sucrose transporter.It has been found that leaf phenology and developmental stage affected latex production directly.However,it is seldom reported about sucrose metabolism and regulation in leaves of rubber tree.In this study,expression patterns of genes involved in sucrose metabolism were investigated systematically during leaf development,including HbNINl,HbNIN2,HbSuss,HbSPSs,HbVINs and HbCWIs which have been previously cloned by our lab.In the meanwhile,soluble sugar and starch contents and enzyme activity of NIN,VIN,CWI,SPS and Sus(synthesis and cleavage direction)were investigated.The main results are as follows:1.Methods of sucrose metabolism enzyme activities assay using crude enzyme had been established.2.Kinetics including optimal pH,temperature,Km and stability of NIN?VIN?CWI?SPS and Sus had been investigated.The results showed that CWI may play an important role in sucrose cleavage.3.HPLC-ELSD chromatographic conditions have been optimized.The analysis was carried out with isocratic elution of acetonitrile:water(78:22,v/v).The drift tube temperature of the ELSD system were set to 82? and nitrogen flow rate was 2.0 standard liter per minute(SLM).The method was validated for accuracy and precision.4.Using the optimized HPLC-ELSD methods,the contents of soluble sugar in leaves were assayed which are 10 times more than that of starch.The results demonstrate that sucrose is the main reserve photoassimilate in Hevea leaves.5.Appropriate candidate reference genes applying for analyzing the expression pattern of interested gene during leaf development had been screened by Normfinder and Genorm,which was YLS8.6.Expression patterns of HbNIN,HbVIN,HbCWI,HbSPS and HbSus in different developmental stage of leaves had been analyzed.Expression patterns of genes except HbSPS correlated with their enzyme activities.This paper analyzed the dynamic characteristics of the sucrose metabolism enzyme and key genes during Hevea leaf development in the biochemical and molecular level for the first time.The results helps to deeply understand the sucrose metabolic pathways and regulation in developing Hevea leaves and lay the foundation to further reveal the molecular interaction mechanism between the 'source' leaf and the 'sink' laticifer which is of significance in latex regeneration.