Transcriptome Analysis Of In Vitro Shoot Organogenesis And Identification Of Related Genes In Ramie (Boehmeria Nivea L. Gaud)

In vitro shoot organogenesis, one of the most common pathways leading to in vitro plant regeneration, is widely used in biotechnology and the fundamental study of plant biology. Although previous studies have constructed a complex regulatory network model for Arabidopsis in vitro shoot organogenesis, no related study has been reported in ramie. We found there were distinct shoot regeneration ablities of two ramie cultivars, Huazhu No.5 and Dazhuhuangbaima. Therefore, Huazhu No.5, with high shoot regeneration ability, was used for RNA collection and transcriptome analysis of different development stages. We further selected auxin and cytokinin signaling related genes for qRT-PCR validation in Huazhu No.5 and Dazhuhuangbaima. Moreover, there was no study about auxin signaling related genes in ramie, although they have been reported to play an important role in shoot organogenesis. We also identified and examined the phylogenetic evolution and expression pattern of auxin signaling related genes in ramie according to trancriptome information. Main results were as follows:1. Transcriptome analysis of in vitro shoot organogenesis in ramieTo generate more complete observations of transcriptome content and dynamics during ramie in vitro organogenesis, we constructed a reference transcriptome library and ten digital gene expression(DGE) libraries using illumina sequencing. Approximately 111.34 million clean reads were obtained for transcriptome and the DGE libraries generated between 13.5 and 18.8 million clean reads. De novo assembly produced 43,222 unigenes and a total of 5,760 differentially expressed genes(DEGs) were filtered. Searching against the Kyoto Encyclopedia of Genes and Genomes Pathway database, 26 auxin related and 11 cytokinin related DEGs were selected for qRT-PCR validation of two ramie cultivars, which had high(Huazhu No.5) or extremely low(Dazhuhuangbaima) shoot regeneration abilities. The expression of 8 genes showed high correlations(>0.8) in two cultivars. The results revealed differing regulation patterns of auxin and cytokinin in different genotypes.2. Indentification of auxin signaling related genes in ramieIn this study, we used Arabidopsis and mulberry sequences as query to search against ramie transcriptome database and the searched sequences were analyzed for full-length coding sequence. In total, we obtained 16 BnIAA, 14 BnARF, 16 BnLBD, 71 BnSAUR, 4 BnAUX/LAX and 6 BnPIN genes, on which evolutionary analysis and expression profiling were conducted. Phylogenetic analysis revealed the Urticales-specific expansion of SAUR genes. Expression analysis showed these genes were actively expressed in major ramie tissues and 16 BnIAA, 6 BnARF, 16 BnLBD, 12 BnSAUR, 1 BnAUX/LAX and 4 BnPIN genes were differentially expressed after IAA treatment. Under drought(PEG induced) and high temperature(40 °C) stresses, 12 BnIAA, 10 BnARF, 16 BnLBD and 14 BnSAUR genes were differentially expressed at least in one stress, which could be considered as stress-responsive genes.