Pseudomonas Fluorescens Flagellin Effect On Cell Transcriptome Analysis Pine Cloning And Expression Of Silver Pine Pinus Thunbergii Synthase Gene

Illumina solexa sequencing was performed in this study to characterize the transcriptome of Pinus thunbergii and identify candidate genes that respond to treatment of flagellin secreted by Pseudomonas fluorescens GcM5-1A associated with pine wood nematode (PWN). A total of29,475isoforms were obtained from flagellin treated and untreated P. thunbergii callus cells and were assigned GO terms based on their involvement in biological processes, cellular components and/or molecular functions. Comparison of expression profiles of the flagellin treated and untreated samples showed1,779significantly differentially expressed isoforms. After GO and KEGG enrichment analyses of significantly differentially expressed isoforms,286enriched GO isoforms and11enriched pathways were obtained. Expression of some genes that play an important role in disease resistance was found to be up-regulated in pine cells challenged with flagellin. These include chitinase, a-amylase subtilisin inhibitor, lipoxygenase1, CoA ligase, peroxidase, sucrose synthase, pinosylvin synthase, phenylalanine ammonia lyase, WRKY-1, PR5and PR10. In this study, we have provided the genetic architecture of the P. thunbergii transcriptome, and identified candidate genes potentially regulated by flagellin, which will facilitate further understanding of the roles of flagellin in pine wilt disease(PWD).In order to study the resistant mechanism of Japanese black pine against PWN, the cDNA coding pinosylvin synthase was amplified by RT-PCR based on the transcriptics data collected from P. thunbergii. The cDNA was cloned into pET-15b to construct expression vector pET-15b-PLS, and then pET-15b-PLS was transformed into E. coli BL21(DE3). Recombinant pinosylvin synthase was expressed abundantly in engineering bacteria after IPTG induction, and purified by affinity chromatography on a nickel column. The study will provide a substantial foundation for further elucidating the functions of pinosylvin synthase and culturing of PWD resistant pines.