Molecular Cloning And Functional Analysis Of Flowering Related RING Protein 1 (FRRP1) In Rice

Rice [Oryza sativa) is one of the most important cereal crops which provide the major food supply for a half of world’s population. Flowering time is a key trait that decides the cultivating areas and seasons of rice. Therefore, it is especially important to clarify the mechanisms that control the flowering time and to excavate the vital genes for controlling flowering time, which will speed up the screen of new rice variety with good quality and high yield.In this study, we cloned and characterized a gene which controls flowering time in rice, and named it as Flowering Related RING Protein 1 (FRRP1). The full-length mRNA of FRRPl is 2873 nt with an ORF of 2535 nt, encoding an 844-amino acid protein around 96.5 kDa and a calculated pI of 4.90. Functional domain prediction revealed that FRRP1 contains a single C3HC4-type RING domain at its carboxyl terminal region. This protein is highly conserved in many dicotyledonous and monocotyledonous plants including rice. FRRP1 was a homolog of AtHUB2 in Arabidopsis, which is an E3 ubiquitin ligase with a RING domain controlling flowering time by monoubiquitinating histone H2B and regulating the methylation of histone H3. Enhanced expression of FRRP1 in Arabidopsis mutant hub2 could rescue its early-flowering phenotype, FRRP1 was also found to interact with AtH2B and AtUBCl (ubiquitin conjugating enzyme), respectively. These results indicate that FRRP1 contains the similar function of regulating flowering time by AtHUB2.By transformation of the RNAi vector pTCK303-FRRPl into the callus of rice Nipponbare with Agrobacterium-mediated transformation technique, we created FRRP1 RNAi transgenic lines. We got 13 transgenic hygromycin resistant lines at To generation. Then we carried out our experiments in three positive lines of T3 generation. First we confirmed FRRP1 RNAi was stably inherited and passed to the T3 generation by PCR, qRT-PCR and GUS staining. Compared to wild-type, the FRRP1 knockdown plants flowered earlier by 23 d,25 d and 26 d in the three lines respectively, which is significant in Rice. Furthermore, FRRP1 knockdown transgenic rice had a compound changes on an array of agronomic traits, including plant height, panicle length and grain length, etc. The expressions of some key genes associated with the flowering time and other agronomic traits were also significantly different between transgenic rice and wild-type, like Hd3a, RFT1, Ghd7, etc.Further researches showed FRRP1-F1 had the ability of self-ubiquitination in vitro, and the result indicated that FRRP1 could function as an E3 ligase. Interaction between FRRP1 and OsH2B was detected by using yeast two-hybrid assays, and the level of histone H2B monoubiquitination was significantly reduced in the FRRP1 knockdown transgenic plants comparing to wild-type, showing FRRP1 monoubiquitinate histone H2B. In conclusion, FRRP1 may control flowering time in rice by regulating key genes associated with the flowering time with histone H2B monoubiquitination.This study not only created the stable genetic rice new materials with early flowering, but also did the researches in the functions of FRRP1. It is instructive to clarify the relation between the early flowering and monoubiquitination of Histone H2B.