The Identification And Functional Analysis Of Citrate Synthase In Laticifer Of Hevea Brasiliensis

Natural rubber is an important industrial raw material and strategic material,mainly from Hevea brasiliensis.The biosynthesis of natural rubber is carried out in the specialized cell-Laticifer,and the sugar metabolism in laticifers provides it with energy and substances.Energy ATP is one of the limiting factors for rubber synthesis,and its supply efficiency directly affects the regeneration ability of latex.Tricarboxylic acid cycle(TCA)is the most effective way of oxidizing sugar or other substances for energy production.Citrate synthase(CS)as a key enzyme catalyzes the condensation of acetyl-Co A with oxaloacetic acid to form citrate acid(CA).In order to reveal the regulation mechanism of energy supply during the process of rubber synthesis,the genes of citrate synthase,encoding the first rate-limiting enzyme of TCA cycle,were isolated and analyzed by tapping and ethephon yield increasing experiments.At the same time,the changes of transcription level,protein level and citric acid content with experimental treatment were analyzed comprehensively,and the putative interaction proteins with HbCS4 or HbCS5 were screened.The main results follows:1 According to the nucleotide sequences of CS from Arabidopsis thaliana,five HbCS genes(HbCS1-5)were isolated from the Hevea brasiliensis genome database.The physiochemical properties of HbCSs protein and their subcellular localization were evaluated with softwares.HbCS1 and HbCS3 were predicted to function in peroxisome,and HbCS2,HbCS4 and HbCS5 in mitochondria.2 The results of tissue exprssion profiles showed that HbCS4 was a highly abundant gene in latex,and it was speculated to be a key member of citrate synthase in latex according to its tissue preference.3 The changes of HbCSs transcription level,protein level and citric acid content were comprehensively analyzed in virgin rubber trees,that were treated by a series of tapping experiments.The results showed that the ATP content in latex decreased rapidly with tapping,while the transcript expression of HbCS4 and CS enzyme activity increased significantly.The contents of citric acid and acetyl-Co A in mitochondria were decreased.It was inferred that HbCS4 play a key role in the energy supply of latex synthesis.4 Ethephon treatment is common measure used to increase the yield of tapped rubber trees.The transcription level,protein level and citric acid content of HbCSs were also analyzed after ethephon treatment.The results showed that except HbCS5,its expression increased by 2 times after ethephon treatment for 12 h,the transcript levels of other genes changed little,while the CS enzyme activity in latex and the citric acid content in mitochondria were much higher than that of tapping treatments.The difference between these two treatments suggested that ethephon and tapping treatments may influence CS with different regulation mechanism.5 The comparison of CS enzyme activity and citric acid content in mitochondrion of in two variety of Reyan 7-33-97 and PR107 were conducted.The results showed that the CS enzyme activity level was consistent with the their productivity,showing the close relation between the activity of CS enzyme and the yield of rubber tree.6 The homogenized cDNA expression library of latex was constructed in yeast,and the bait vectors of p GBKT7-HbCS4 and p GBKT7-HbCS5 were constructed.Two interacting proteins with HbCS4 and three with HbCS5 were screened by yeast two-hybrid experiments.