Molecular Cloning And Functional Analysis Of Key Genes PsIPPI And PsCPT Associated With Cis-1,4-polyisoprene Biosynthysis In Periploca Sepium

Natural rubber is a key strategic material that is vital to the people's livelihood,the national economy and security.Its primary component is one kind of isoprenoids,known as cis-1,4-polyisoprene.The source of natural rubber is almost exclusive produced from Hevea brasiliensis Muell.Arg,whose strict growing conditions limit its cultivation to specific tropical environments.The problem of insufficient capacity of natural rubber production become more and more urgent that lead to the search for alternative sources in China.In the present research,a shrub named Periploca sepium Bunge was selected as the target for development.The key enzymes and their encoding genes associated with cis-1,4-polyisoprene biosynthesis were characterized using transcriptome sequencing and bioinformatics analysis;The key gene functions were identified to elucidate their roles in cis-1,4-polyisoprene biosynthesis by over-expression in P.sepium.The results are as follows:1.Based on the extraction rate and the extractive molecular weight distribution,the polyisoprene extraction method has been optimized by comparing the pretreatment and the extraction methods.The result showed that using chloroform as organic solvent in water bath by Soxhlet extraction was more efficiency,and using chloroform assisted with sonication,a quantity of small and big molecular of polyisoprene could be more fully dissolved and extracted.2.Three kinds of shrubs or herbages that produce latex,Cynanchum chinense R.Br.,Apocynum venetum L.and P.sepium,which are widely distributed in the arid and the semi-arid areas of Northwest,Northeast and North of China,were used as materials for polyisoprene extranction and identification.The result showed that only P.sepium including the component of cis-1,4-polyisoprene and was considered promising enough as an alternative natural rubber source.Its molecular weight distribution was analized using gel permeation chromatography(GPC)and its molecular structure was determined by nuclear magnetic resonance(NMR).3.The total RNA were extracted form the leaves,stem and bark of P.sepium for transcriptome sequencing and bioinformatics analysis.The key enzymes and their encoding genes associated with cis-1,4-polyisoprene biosynthesis were characterized,including isopentenyl diphosphate isomerase(PsIPPI),geranyl diphosphate synthase(PsGPPS),farnesyl diphosphate synthase(PsFPPS),geranylgeranyl diphosphatesynthase(PsGGPPS),and cis-prenyltransferase(PsCPT).4.Two key genes,PsIPPI and PsCPT that associated with cis-1,4-polyisoprene biosynthesis were selected from transcriptome sequencing.Their full length cDNA were amplified by PCR and were individually or simultaneously constructed into 3 kinds of overexpression vectors,for identifying their functions in cis-1,4-polyisoprene biosynthesis in P.sepium.5.Using Agrobacterium-mediated transformation,the 3 kinds of overexpression vectors were transferred into P.sepium.The result showed that compare with wild type control,PsIPPI-overexpressing transgenic plantlets had no significant difference in the total contents of cis-1,4-polyisoprenes,while PsCPT-overexpressing transgenic plantlets had about 5-fold increase,PsIPPI and PsCPT-co-overexpressing transgenic plantlets were significantly greater in cis-1,4-polyisoprene biosynthesis,especially in the high molar mass region.The research will play a promoting role on the development of P.sepium as an alternative source for the production of natural rubber,and completely solve the bottleneck in the shortage of natural rubber existing in China.