Screening And Preliminary Analysis Of Differentially Expressed Genes In The Latex Of Super-high-yielding Rubber Tree Compared With Its Average Counterparts

Natural rubber(cis-1,4-polyisoprene) is an important raw material of great industrial and defence importance.Due to the limitations of rubber cultivation areas and climate conditions,Chinese natural rubber industry relies largely on imports.Therefore, a reliable way to improve this condition is to substantially increase per unit rubber yield. In Yunnan rubber plantations of China,several rubber trees were found to be extraordinarily high-yielding,and termed super-high-yielding(SHY) rubber trees. Among these SHY trees,a clone PR107 tree had a record of 127 kg dry rubber yield per year,5-10 times larger than that of an average high-yielding rubber tree,making it a precious rubber tree for the purpose of unraveling high-yielding mechanisms.In this paper,a silver-staining cDNA-AFLP technique suitable profiling of latex transcriptome was established and then applied to identify genes that were differentially expressed in the latex ofPR107 SHY rubber tree and its average counterparts.The major results we obtained were as follows:1.Considering the research conditions of our laboratory and utilizing the pulic available Hevea gene sequences,we conducted improvement and optimization on the classical cDNA-AFLP protocols,including the selection of suitable combinations of restrition enzymes,optimization of PCR reaction parameters,and reduction of the background of silver staining,and finally a silver staining cDNA-AFLP protocol was set up suitable for latex transcriptome analysis.This protocol has high sensitivity,good repeatability,and after the usage of two restriction enzyme pairs,it could cover more than 90%of latex transcriptome of rubber tree in theory.2.Using our silver staining cDNA-AFLP protocol,we made comparative latex transcriptome anlysis between the SHY tree and its average counterparts.A comprehensive screening including all the 384 selective primer combinations of retriction enzyme pairs,TaqI/MseI and ApoI/MseI,were performed and 453 unique differentially expressed transcript-derived fragments(TDFs) were obtained and further verified by semiquantitaive RT-PCR,among which 247 were functionally annotated by blastX aginast publicly available non-reduntant protein database.These TDFs functioned in cell metabolism,transcription and translation,defense and stress response,protein degradation and cell signal transduction and so on,but surprisingly,only two of them were directly involved in rubber biosynthesis.Preliminary analysis indicates that the highly active laticiferous metabolism and the ability of timely and effectively alleviating stress are probably the main reason for its high yield.3.The expression levels of most known rubber-biosynthesis directly-involved genes correlated neither with the yields of SHY tree and its average counterparts,nor with those of different rubber clones,further suggesting that rubber biosynthesis did not account for the high-yielding mechanisms underlying SHY tree.