Functions And Molecular Regulatory Mechanisms Of OsTIPs In Rice

In the whole growth of the plant,flowering is an important indicator of the growth of plants from vegetative into reproductive,which is determined by endogenous and environmental signals.Arabidopsis thaliana is a typical low temperature and long daylight plant.Photoperiod pathway,vernalization,autonomous,gibberellin pathway,FRIGIDA(FRI)pathway and RNA polymerase ? associated factor 1(PAF 1)complex pathway to control the flowering time of Arabidopsis thaliana.Rice(Oryza sativa)is a short-day crop,and short daylight promotes flowering.Rice flowering(heading date)affects rice yield,quality and regional adaptability,which is a very important agronomic trait.At present,Heading date 1(Hd1)and Early heading date 1(Ehd1)as key factors have been found to control the heading date of rice in molecular mechanisms.These pathways regulate rice flowering by affecting the expression of the florigen genes heading date 3a(Hd3a)and Rice Flowering Locus T 1(RFT1).florigen genes is synthesized in leaves,and it transports through the phloem to the growth point to regulat rice flowering.However,the molecular mechanism of florigen transport in rice is unclear.In Arabidopsis,FT-interacting protein 1(FTIP1)has been shown to regulate the flowering of Arabidopsis thaliana by controlling the florigen Flowering Locus T(FT)interaction with it and then control the flowering process of Arabidopsis thaliana.However,whether the homologous genes of FTIP1 in rice are involved in the transport of florigen has not been reported.In this study,the function of FTIP1 homologous genes in rice are studied by reversed genetics.The main findings are as follows:1.OsTIPs from rice is phylogenetically conserved to FTIP1.We take use of bioinformatics to find the homologous genes of Arabidopsis FTIP1 in rice and named it OsTIPs gene family,including OsTIP1,OsTIP5,OsTIP6,and OsTIP7.Sequence alignment and evolution analysis show that OsTIPs and FTIP1 are highly conserved.They encode proteins all have the typical C2 domains and transmembrane domains.However,whether OsTIPs has the similar function to FTIP1 is unclear whether or not to it take part in the transport of florigen proteins.This project mainly studies the function of OsTIPl and OsTIP6.2.OsTIPl and OsTIP6 in rice can restore the late flower phenotype of ftipl-2 mutant in Arabidopsis thaliana.In order to study whether OsTIPl,OsTIP6 and FTIP1 in Arabidopsis thaliana have similar functions,we transfer the overexpression vector into the ftipl-2 mutant in Arabidopsis thaliana and observe the flowering time of the transgenic lines.The results show that OsTIP1 and OsTIP6 could restore the late flower phenotype of ftip1-2 mutant,indicating that OsTIPl and OsTIP6 have the similar function to FTIP1 in Arabidopsis thaliana.3.The overexpression of OsTIPl and OsTIP6 promot flowering in Arabidopsis thaliana.In order to study whether OsTIPl and OsTIP6are involved in the regulation of flowering time,we creat transgenic materials of overexpressing OsTIPl and OsTIP6 in Arabidopsis thaliana.They show early flowering phenotype.Compared with wild type(WT),the number of rosette leaves of overexpressing transgenic lines is significantly reduced,and the days of bud emerging and the days of the first flower opening time are significantly ahead of time.The results of molecular level show that the expression of CO and SOC1 is up-regulated,which promots the flowering of Arabidopsis thaliana.These results indicate that overexpression of OsTIPl and OsTIP6 are involved in the regulation of flowering in Arabidopsis thaliana,which indicates that OsTIPl and OsTIP6 may be involved in the regulation of flowering in rice.4.Expression pattern and subcellular localization of OsTIP1andOsTIP6 Results of real-timequantitative RT-PCR and GUS staining assay showsthatOsTIP1 and OsTIP6 are expressed in different tissues and organs,and the expression level reach its peak in ASA;A peak expression of them at the tillering period is observed.In addition,OsTIP1 and OsTIP6 expression has circadian rhythm changes,The peak of the expressionof OsTIP1 in Nipponbare is at 5 am and reaches the peak in SN9816 at 8 pm-2 am;the expression of OsTIP6 peak at 5 am in Nipponbare,and it peak at 11 pm in SN9816.At the same time,by using confocal laser microscopy we observe the subcellular localization of 35S:GFP-OsTIPl and 35S:GFP-OsTIP6 transgenic plants.The results showed that OsTIP1 and OsTIP6 are localized on the cell membrane.5.At the same time,we use the ordered T-DNA insertion mutant to find that the ostip6 mutants show late flowering,and the expression level of the gene is significantly decreased by RNA level detection.At the same time the mutant plays a role in inhibiting the expression of the florigen gene,OsMADS14,Ehd1 and other genes.oftip6 also affected some yield traits(1000-grain weight,spike weight,secondary branch number,etc.),Which further demonstrates that the gene is associated with flowering of rice.In ostip1 the T-DNA insertion position is not good,so no phenotypic differences are found.