Gene Expression Profile Analysis Of Gossypium Hirsutum Induced By Insect Pest And Functional Characterization Of Farnesyl Diphosphate Synthase

It will induce a series of genes specific expression in plant when the plant wasattacked by insect pest. Some of these genes involved in regulating the release of plantvolatile organic compounds.This volatile organic compounds would help the plant take theindirect defense action by attracting the natural enemies of the pest.So screeing andidentification the key genes which regulate the release of the herbivore induced plantvolatile will help us further understand the molecular mechanism of plants resistance toherbivore.First we analyzed the gene expression profiles of the Gossypium hirsutum when itwas attacked by Helicoverpa armigera by the data mining of the gene chip.Based on this,we isolated and functionally characterized farnesyl diphosphate synthase in the Gossypiumhirsutum which plays a important role in the terpene synthesis metabolic pathways. Theresults are as follows:The results showed that a total of3635genes were differently expressed after induced6h,2213of them were down-regulated,1422were up-regulated; a total of2339genes weredifferently expressed after induced12h,1155were down-regulated,1184were up-regulated; a total of3616genes were differently expressed after induced24h,2367down-regulated,1249up-regulated; a total of5042genes were differently expressed afterinduced48h,2621were down-regulated,2421were up-regulated. Through comparingwith the data in NCBI, we found out131pathways that the differentially expressed genesinvolved in after induced6h;99pathways after induced12h;115pathways after induced24h;111pathways after induced48h. Bioinformatics analysis showed that the metabolismof differertially expressed gene which was induced by insect pest involve terpenoidssynthesis and metabolism, energy metabolism, carbon metabolism. To prove the results ofgene chip profiling,we chosed allene oxide cyclase,Lipoxygenase genes,acc synthase andother6genes from differertially expressed genes for real-time quantitative polymerasechain reaction.The results showed that the trend of gene expression in Q-PCR is similar tochip pro-filing.We used the GC-MS technology to comparison the changes of three kinds ofterpenoids in cotton when it attacked by insect pest. Compared with the healthy plant,wedetected E-β-farnesene from herbivore-induced plant volatiles and there was a significantincrease in releasing the limonene and alpha pinene.We screened and identified a gene from large of ESTs obtained from microarrayexperiments which was related to terpenoids synthesis and metabolism. The gene wasamplified by smart race strategy. The open reading frame of FPS is1029bp in length,FPSencodes a protein comprised342amino acid. Residues with acaculated molecular mass of39.4ku and an isoelectric point of5.06. Multiple sequence alignment relealed that the deduced amino acid sequence has71%identity with rice and80%with arabidopsis andchili. To further investigate the temporal expression of FPS in different organizations in G.hirsutum induced by larvae of Helicoverpa armigera, the gene transcript was examined byreal-time quantitative PCR method. It was found that FPS transcript level was significantlyincreased at the young leaves.We built the fusion expression vector FPS-Peasy E1and getthe fusion protein which was Purificated.