Study On Function Of Phragmoplastin-interacting Protein 1 (PhrIP1) Of Arabidopsis Thaliana During The Formation Of Cell Plate In Plant

During anaphase in plant cells, Golgi-derived vesicles travel along thephragmoplast microtubules toward and reach the equatorial region of the cell. Theygradually align into the cylinder-like or barrel-like phragmoplast. Phragmoplast-in-helical arrays wrap around and squeeze vesicles into the dumbbell-shaped vesicle-tubule-vesicle (VTV) structures on the equatorial plane. The VTVs fuse with each otherand consolidate into the membrane-like structure cell plate. Therefore, membrane fusionduring cell plate formation is a continuous process. As the cell plate grows centrifugally,membrane fusion events occur exclusively at the growing margin of the outwardlyexpanding cell plate until reach and fuse with the parent cell wall into the new cell walldividing the parent into two daughter cells. The formation and expansion of cell plateare required for cytokinesis in plant cells and closely related to plant cytokinetic cycle,organelle differentiation and morphologic architecture.A plant dynamin-like protein—Phrogmoplastin in phroGmoplast plays a pivot rolein early membrane fusion event during cell plate formation. Phragmoplastin-interactingproteinl (PhrIP1) isolated from Arabidopsis AD-cDNA library has been studied andindicated that this protein interacted with phrogmoplastin and co-localized with thephragmoplastin at the cell plate during cytokinesis. PhrIP1 contains a lysine rich region,two RNA-binding domains and three CCHC-type zinc-fingers and it may be a novelRNA binding protein.We presume that PhrIP1 may mediate the expression of post-transcription of thetarget mRNA whose protein plays a key role in the formation of cell plate or cytokinesisby PhrIP1 interacting with target-mRNA during interphase; During anaphase, PhrIP1may interact with the protein from target mRNA or other proteins to co-mediate orfacilitate the initiation of phramoplastin complex which triggers an early vesicles fusionevent at the formation and expanding cell plate. To study the function of PhrIP1 at theformation of cell plate, the key results from this research are summarized as below.1, PhrIP1 interacting with other proteins by yeast-two hybrid systemUsing PhrIP1 as bait, 46 genes were isolated from AD-cDNA library ofArabidopsis thaliana by yeast two hybrid screening. Four genes of them were expressedrespectively in E.coli ER2566 and purified proteins could be interacted with PhrIP1 in a protein-protein binding assay in vitro. Ran2 small GTP binding protein by 33^# cloneencoding is determined as the target protein to study by the analysis of protein blast.2, PhrIP1 interacting with RNA by affinity adsorptionThe total RNA was isolated from seedling of Arabidopsis thaliana and bound withGST-PhrIP1 beads. Then, the beads were washed out free RNA for several times. Thespecific bound RNAs were extracted from GST-PhrIP1 beads and measured OD₂₆₀ byspectrophotometer and analyzed with RT-PCR using five pairs of primers. The cDNA ofthe specific mRNA bound to GST-PhrIP1 was cloned into the pTYB2 plasmid andcontrolled under T7-promoter. The mRNA probe was transcribed with [α-³²p]rUTPlabel in vitro and incubated with GST-PhrIP1 fusion protein by UV-cross linking andRNA electrophoretic mobility shift assays (REMSA). The results showed that thePhrIP1 could specifically bind to Ran2-mRNA.3, Ran2 GTPase activity, its expression and subceullar localization in plantAnalysis of gene structure showed that Ran2 was consisted of five exons and fourintrons, and its full-length cDNA encodes 24 kD protein with 221 amino acids. Ran2protein expressed and purified from E. coli was determined activity of GTPase by(NH₄)₂MoO₄-FeSO₄ assay and the results showed that it had intrinsic activity of GTPase.The Ran2 was all expressed in root, stem, flower and leaf of Arabidopsis by RT-PCRanalysis. The results of immunofluorescence localization of Ran2 showed that Ran2 wasinvolved in each phase during plant mitosis, and mainly localized on the nucleusmembrane at interphase, and dispersed in the cytoplasm in patches from preprophase toprometaphase, and on the equatorial plane (cell plate) and the spindle at anaphase, andrelocated on the newly forming daughter nucleus membranes at telophase.4, Cloning and homologous analysis of Ran2 gene from different plantsThe total RNAs were isolated from Allium cepa, Allium sativum and Brassicanapus and the full-length cDNAs of Ran2 was generated by RT-PCR with primers ofAtRan2. After sequencing, Ran2 genes from different plants were compared each otherby DNAMAN. The result showed that Ran2 homologous with nucleotide sequences wasrespectively 99.70 %, 100 %, 89.94 % and with amino acid sequences was respectively99.10 %, 100 %, 96.38 % between Arabidopsis thaliana and Allium cepa, Alliumsativum, Brassica napus by BLASTn and BLASTp, except lack of Asp (D) C-terminal in Allium sativum. These results demonstrate that Ran2 is high conserved in plantevolution.5, Analysis of PhrIP1 mutant and construction of PhrIP1, Ran2 overexpression vector and RNAi vector and GFP vector of PhrIP1After observation of phenotypes and analysis of PhrIP1 mutant and wildArabidopsis thaliana by RT-PCR and western blot, the results showed that no obviousdifference was observed in phenotypes and RT-PCR products of PhrIP1 were positivebetween PhrIP1 mutant and wild-type in this study. PhrIP1 was not detected indissolvable protein of seedling of Arabidopsis by western blot. These results suggestthat PhrIP1 is an indissolvable associated membrane protein and no loss of function inPhrlP1 mutant (T-DNA), and its mutant may not exist in purified homologous. Tofurther study the function of PhrIP1 and Ran2 genes in cell plate formation in plant, theplasmids of pBI-PhrIP1, pBI-Ran2 for over-expression in plant and RNAi recombinantplasmids Hellsgate2-PhrIP1 and Hellsgate2-Ran2 for gene silence at mRNA level andGFP fusion plasmid pMON-GFP-PhrIP1for localization in living cells were constructed,inheritably transferred into Arabidopsis to gain their T₀ seeds. Now the phenotypes andfunctions of T₀ are being studied.