Cloning And Analysis Of SA Biosynthetic Genes ICS And PAL In Barley

Salicylic acid (SA) is an important plant hormones, it involves in a variety ofphysiological activities of plants. Isochorismate synthase gene (ICS) and Phenylalanineammonia lyase gene (PAL) are key genes in the SA biosynthesis pathway, where ICS convertsChorismate into Isochorismate and PAL catalizes Phenylalanine into trans-Cinnamic acid.However, biosynthesis and function of SA is largely unknown in Triticeae species. In thisstudy, we identified one HvICS and eleven HvPAL homologues according to genomic data ofbarley ‘morex’, cloned one HvICS and seven HvPAL from barley ‘Tamalpais’, characterizedtheir tissuse-specific transcription using real-time quantitative PCR (RT-qPCR). All eighttarget genes have been pursued for overexpression, gene silencing and GFP fusion in barleyor Brachypodium. Further study of these transgenic plants will enable us to understand thebiosynthesis and functin of SA in Triticeae. Main results are presented below.(1) Annotation and cloning of HvICS and HvPAL: Using ICS and PAL genes fromArabidopsis and rice as references, we identified one barley HvICS and eleven HvPAL genesin ‘Morex’ genomic sequence. In comparison, barley HvPAL and Arabidopsis AtPAL share ca.49%~56%similarity in their coding regions, and barley HvPAL genes have more than80%similarity between each other. Phylogenetic analysis revealled that all11barley PAL geneswere assigned to four clusters. According to their relationship, HvICS and seven HvPAL geneswere targed for further research.(2) Tissue-specific transcription of HvICS and HvPAL: Transcription of eight targetsgenes were examined in flag leaf, peduncle and young spike of ‘Tamalpais’ and ‘GoldenPromise’ using real-time quantatitive PCR(RT-qPCR). In general, HvICS was homogenousacross selected tissues, but HvPAL genes were more variable between each other and amongdifferent tissues. In detail, HvPAL_AK248841and HvPAL_AK249266were highly expressedin all tested tissues; HvPAL_AK253101transcripts were abundant in peduncle and young spike, ca.500times more than those in leaf tissue; expression of other four HvPAL genes waslow in all selected tissues.(3) Construction of plant expression vectors: HvICS and7HvPAL genes were clonedfrom barley ‘Tamalpais’. Each target gene was used to construct three types of plantexpression vectors, including overexpression, RNAi silencing, and fused green fluorescentprotein (GFP).(4) Plant genetic transformation: We developed an Agrobacterium-mediatedtransformation systme on barley ‘Golden Promise’. The transformation efficiency was ca.5%.Overexpression and RNAi silencing were performed on barley. Five to twenty independent T0plants were obtained for each target construct. GFP-based fusion expression were performedon Brachypodium; independent transgenic T0plants were generated for all target genes. GFP-based fusion was designed to reveal cellular localiation of target gene product.(5) Prokaryotic expression of target proteins: HvICS and HvPAL were expressed in E.coli strain Transetta (DE3). Expected proteins were visulized on Coomassie-stained PAGEgels. Unfortunately, all target proteins were in inclusion body; protein refolding is required torecover soluable proteins for each target.