Screening And Function Analysis Of Flowering Related Genes In Common Wild Rice (Oryza Rufipogon Griff.)

Flowering is one of the important characters of transition from vegetative to reproductive stage forseed reproduction and environment adaptation. The common wild rice (Oryza rufipogon Griff. AA)widely distributed in China, Southeast Asia and South Asia is the most closely related ancestral speciesto Asian cultivated rice (O. sativa L.) and has abundant genetic diversity. Three O. rufipogon cDNAlibraries, two vegetative stages (CWRT-V1and CWRT-V2) and one flowering stage (CWRT-F2) weresequenced using Solexa Illumina deep sequencing. After comparison among three libraries, we selectdifferently expressed genes (DGEs) and construct plant expression vectors to transform Arabidopsisthalina for function identification. The results not only provide a large number of information aboutflowering related genes, but also lay a foundation for the research of molecular mechanism of floweringdiversity, flowering process and early flowering mutant.Analysis of high throughput sequencing resultsThree cDNA libraries were constructed with the materials of common wild rice in Gaozhou,Guangdong Province. CWRT-V1come from vegetable stage for single-flowering sub-group, CWRT-V2come from vegetable stage for double flowering sub-group and CWRT-F2come from reproductivestage for double flowering sub-group, respectively. A total of7046714，7231932and7155366highquality reads were obtained. After mapping to the reference genome,27405，27333，28979unique geneswere found respectively.Identification and verification of differentially expressed genesWe compared the gene expression level between vegetable stage of and reproductive stage ofCWRT-V2, and found that2808genes were significantly up-regulated, while1496unigenes weresignificantly down-regulated. Meanwhile, between vegetable stage of CWRT-V1and reproductive stageof CWRT-F2,1740genes were significantly up-regulated, while982unigenes were significantlydown-regulated. For confirming the gene expression data,12genes were randomly chosen for qRT-PCRanalysis in CWRT-V1and CWRT-F2libraries and10genes were randomly chosen in CWRT-V2andCWRT-F2libraries. It was in consistent with that in the sequencing result.Screen of differentially expressed genes related to early floweringAfter comparison of DGEs among the three libraries, we acquired375genes related to earlyflowering in which the majority is putative expressed protein and transcription factors. GO termannotation reflect these genes were involved in cell development, signal transduction and response tostimulus. Some known flower development genes were also found, such as OsMADS6，17，5，58，1，34，26，OsLFL1，FON1. All of these showed that the high throughput sequencing technology could reallyhelp discovering the genes which controlling flowering in common wild riceFunction verification of the candidate genes10plant expression vectors were constructed and made them highly expressed in Arabidopsis thaliana. Identify early flowering related traits of transgenic Arabidopsis thaliana. By analyzing theKEGG pathways and did Go term annotation, it was found that these genes involved in MYBtranscription factors, MADS transcription factors and expressed protein. The rosette leaves havesignificant difference between transgenic plants and the wild type, such as LOC_Os05g51900.1，LOC_Os11g36230.1and LOC_Os02g46030.1.