Identification And Alternative Splice Events Analysis Of Genes Related To Adventitious Root Formation

Adventitious root(AR)formation,which is controlled by endogenous and environmental factors,is indispensable for vegetative asexual propagation.Nowadays,the studies on AR formation are mostly focused on the transcriptional level of genes,while those at other levels are relatively weak.In this study,apple rootstocks ‘T337' were used as test material,i TRAQ and SMRT sequencing techniques were used to explore the expression profiles of proteins and the complex alternative splicing,which was of great significance to enrich the molecular mechanism of AR formation.The main results were as follows:1 The proteome database of ‘T337' apple rootstocks in AR induction period was established,the differences of morphology and hormone levels changed,differentially expressed proteins and biological process were identified.(1)Exogenous IBA treatment induced AR formation while rooting rate and root length increased significantly.The contents of auxin(IAA),abscisic acid(ABA)and brassinolide(BR)increased in the early stage and then decreased,while gibberellin(GA),zeatin(ZR)and methyl jasmonate(JA)were opposite.(2)To explore the underlying molecular mechanism,an i TRAQ-based proteomic technique was employed to identify the expression profiles of proteins at a key period of AR induction(3 days after IBA treatment).In total,3,355 differentially expressed proteins(DEPs)were identified.Further analysis showed that DEPs were mainly enriched at phytohormone metabolism and signaling,carbohydrate metabolism and energy production,ubiquitin-proteasome system mediated protein homeostasis,reactive oxygen and nitric oxide signaling,and cell wall remodeling biological processes.(3)RT-q PCR analysis was used to confirm the transcriptional levels of nine genes involved in phytohormone metabolism and signaling.Finally,a putative work model involved in the IBA-induced AR formation was proposed.2 The database of full-length transcriptome in the process of AR formation of 'T337' apple rootstocks was established,the gene structure annotation of reference genome 'Golden delicious' was updated,the alternative splice events during AR formation and differentially expressed alternative splice events were also identified.(1)We obtained 71637 transcript isoforms involving of 23,672 genes with the single molecule real time sequencing(SMRT)technology.Among these,12,234 were known transcripts,52,334 were novel transcripts of known genes and 7,069 were novel transcripts of novel genes.Totally,we identified 5,358 novel genes.(2)About 20 % of the differentially expressed genes were not differentially expressed after updating the reference annotation,novel differentially expressed genes were then identified,indicating that our SMRT data can import the reference annotation.(3)We detected a total of 27,005 alternative splice(AS)events from 8,026 genes,including skipping exons(SE),retained intron(RI),alternative 5' splice-site(A5),alternative 3' splice-site(A3),alternative first exon(AF),alternative last exon(AL),mutually exclusive exons(MX),and the majority of AS events were RI(48.87 %).The result of GO enrichment analysis indicated potential AR formation related GO terms including root development,such as root morphogenesis,root meristem growth,root cell differentiation,and auxin transport and signal transduction,were significantly enriched.Additionally,KEGG pathways,including carbon metabolism,biosynthesis of amino acids,RNA transport,RNA degradation and so on,were significantly enriched.(4)482,435 and 338 differentially alternative spliced events,involved in 384,354 and 271 genes,were identified during AR induction period,initiation period and expression period,respectively.The result of GO enrichment analysis indicated that AS may participate in AR formation by regulate the proportion of transcript isoforms involved in the auxin-activated signaling,root development,lateral root formation,meristem initiation,shoot system morphogenesis pathways.(5)According to the result of GO enrichment,we identified 26 differentially alternative spliced genes that were related to AR formation,which including 35 alternative events.The exact gene function of these transcript isoforms if worthy of further study.