Preliminary Identification Of The Function Of OsRhoGAP2Gene As A Rice Rho Gtpase Activating Protein

The existing research results show that RhoGAP/RopGAP play an important role in Theprocess of pollen development. In this paper, Yeast Two Hybrid System was used to screenout an interacting protein of OsRacD from young panicles which named OsRhoGAP2. Inorder to identify the functions of the gene OsRhoGAP2in rice fertility control and lay thefoundation for studing the rice Rho signal mechanism，a series of studies has conducted forthis gene.Using bioinformatics analysis, the genetic structure, physicochemical characteristics,secondary structure of protein, strcture of transmembrane and protein subcellular location ofOsRhoGAP2had been predicted and analyzed.In this study, The OsRhoGAP2gene wasfused with green fluorescent protein (eGFP) and was recombined with a strong promoterCaMV35S to obtain a binary expression vector pCAMBIA1302-OsRhoGAP2-eGFP. Themethod of agrobacterium infection was used to guide the recombinant gene into onionepidermal cells, so that by detecting the distribution of green fluorescent proteins in the cells,the subcellular localization of OsRhoGAP2was determined. Results showed thatOsRhoGAP2-eGFP fusion protein was mainly localized in the cell membrane,By comparison of the promoter sequences of OsRhoGAP2with promoter cis-elementdatabase, results showed that OsRhoGAP2gene may be regulated byexogenous hormone regulation and abiotic stress,such as high salt and drought. Usingrealtime fluorescence quantitative PCR method to detect the relationship betweenOsRhoGAP2with exogenous hormone and abiotic stress，The results showed thatexpression of the gene had a different degree of increase in the regulation of the abovefactors.By the treatments of exogenous hormone such as IAA、6-BA、GA、SA，expressionof the gene had improved over2folds. The transcriptional expression of OsRhoGAP2enhanced progressively under the treatments of IAA; Under the treatments of6-BA, thetranscription of OsRhoGAP2increased before3h to3.7folds but decreased thereafter;The transcription of OsRhoGAP2was increased after6h under the treatments of GA and SA,respectively; The transcription of OsRhoGAP2were increased within2folds under thetreatments of MeJA and ABA, respectively. The expression peak were appeared in3h aftersalt stress and drought stress,up to3.5and2.4folds, respectively. These results showed that， OsRhoGAP2may play an important role in different signal pathways.By rice genome databases blast and sequence alignments, the1912bp-length promoter ofOsRhoGAP2is obtained, and the cis-elements were predicted and analyzed. In order to laythe foundation for studing the mechanism for regulating gene of OsRhoGAP2,Six promoterdeletion fragments of this gene promoter was further cloned by PCR，and fused with thebinary expression vector pCAMBIA1391containing GUS tag.In order to identify the function of OsRhoGAP2gene, the binary plant expression vectorpCAMBIA1302-OsRhoGAP2-eGFP had been constructed and transformed into rice callus byAgrobacterium-mediated method. after infection, screening and re-differentiation, nineteenpositive transgenic rice were obtained. RT-PCR detection showed the expression level of thegene OsRhoGAP2were higher than that of control, suggest that the gene over-expressed intransgenic rice. In the meantime, using Gateway cloning technology, RNA interferencevectors pANDA-OsRhoGAP2had been constructed in this study. At present, RNAi genetictransformation of Rice and screening of transgenic rice were ongoing.In this study, a series of experiments had been done for analysising the functiong,genetic component and genes expression and regulation of OsRhoGAP2,laying a foundationfor study the functiong of this gene in development of rice and the relationship betweenOsRacD and OsRhoGAP2.