Identification Of Key Enzymes Of DMNT/TMTT Pathyway In Lima Bean And Rice

The irregular acyclic homoterpenes, (E)-4,8-dimethylnona-1,3,7-triene (DMNT) and (E,E)-4,8,12-trimethyltrideca-1,3,7, 11-tetraene (TMTT), are the most widespread volatiles produced by angiosperms with emissions from flowers and from vegetative tissues upon herbivore feeding. Special attention has been placed on the role of homoterpenes in attracting parasitoids and predators of herbivores and has sparked interest in engineering homoterpene formation to improve biological pest control.In order to elucidate these valuable attractants in genetic engineered crop, we sequenced the transcriptome and Digital Gene Expression Profiling of one Phaseolus species lima bean using four different lima bean samples (Alamethicin treatment leaf sample, Tetranychus cinnabarinus damage leaf sample and two healthy leaf samples).The results showed that the transcriptome of lima bean contains seven putative terpene synthase (TPS) genes, of which three are predicted to be functional. We isolated and functionally characterized these TPS enzymes. The results showed that PITPS3 and PITPS4 were multifunctional, producing linalool and (E)-nerolidol precursors of DMNT. In addition, PITPS4 protein could produce (E,E)-geranyllinalool precursors of TMTT also. PITPS5 could account for the terpene volatiles of (E)-β-caryophyllene and (E)-a-humulene.Accoding to boinformation anysis, we predict the last committed step in the production of DMNT and TMTT are performed by a cytochrome P450 enzyme, PIP450.We isolated the PIP450 genes from lima bean. the TPS contained an open reading frame encoding 528 amino acid residues. The amplified PIP450 gene was cloned into PYES2 vector. The constructed recombinant plasmid,name PYES2-PIP450, was transformed in to Saccharomyces cerevisiae according to the LiAc/PEG method.The PIP450 expressed in yeast was able to oxidative degradation (E)-nerolidol and (E,E)-geranyllilool to DMNT and TMTT,indicating that it had activity.In addition, we conduct a comparison the transcriptome of lima bean with the genomes of six other legume species, including the common bean, Medicago, lotus, soybean, chickpea, and pigeonpea, revealed 15 and four orthologous groups with signatures of positive selection among the two Phaseolus species and among the seven legume species, respectively. Characterization of these positively selected genes revealed that these genes are mostly involved in thylakoids, photosynthesis and metabolism.The headspace volatiles from one indica rice cultivar mudgo with different treatments were collected and analyzed with GC-MS. DMNT in volatile emissions was observed for rice plants with three different treatments (small brown planthopper, ALA and (E)-nerolidol). Then, we performed transcriptome analysis on mudgo leaves derived from small brown planthopper 96h treatment and healthy control. Transcriptome analysis identified 21 rice genes whose expression were potentially related to DMNT biosynthesis including two terpene synthase genes and 19 P450 genes.