Functional Analysis Ofubiquitin-Binding Zinc Finger Protein ZFP319 From Rice(Oryza Sativa L.)

Rice (Oryza sativa L.) is one of the major food crops inChina and rice growth and productivity are largely affected by abiotic stresses such as salt, drought and cold.Expecially the drought stress, its harm for agricultural productionof rice and other corps is more and more serious. The function of AN1-type zinc finger proteins is very important for the plant to response and control abiotic stress response, however, its mechanismremainpoorlyunderstood. In our laboratory, we cloned a special member of rice AN1 zinc finger family proteins, ZFP319, which containsa specific zinc finger architecture harboring an AN1 and C2H2 zinc finger motifs (AN1/C2H2). We analyzed the molecular propertiesand function of ZFP319, and preliminaryexplored it’s probably mechanismfor rice responsiveness to abiotic stresses. The following are my major research results:1. ZFP319 contains an AN1 and a C2H2 zinc finger motifs. Through the analysis of interaction between the ZFP319 protein and ubiquitin protein (AIP1 and AIP2), we confirmed that ZFP319 could interact with ubiquitin proteins AIP1 and AIP2 in yeast cell. In addition, both AN1 and C2H2 zinc finger architecture influenced their anchoring strength and the AN1 truncated protein lost the interaction capability with AIP2. Theresult indicated thatan AN1 zinc finger motif is necessary for binding ubiquitin.2. ZFP319 localizes in the whole cytoplasm, indicating that ZFP319 may not function as a nucleus transcription factor while participate in the 26S- proteasome pathways via binding ubiquitins in plant.3. Through tissue expression analysisit was found that ZFP319 was constitutively expressed in root, stem, leaf, panicle of rice plant expecially in root, and markedly induced by salt, drought, osmotic stress and oxidative stress (H2O2).4. In Rice Mutant Database, we discovered the ZFP319 T-DNA insertion mutant. Using transgenic technology, we obtained the first generation of over-expressing rice lines of ZFP319. Thepositive mutant and transgenic rice lines have been confirmed by PCR.5. In yeast cells,ZFP319 and GAL4 BD domain fusion protein lacked the transcriptional activity. ZFP319 wan used as bait to screen the rice young panicle cDNA library, and finally we identified five different ZFP319 interacting proteins (ADP1-5). Through the analysis of NCBI protein BLAST tool and SMART protein domain, five proteins that could interact with ZFP319 were characterized as follows:ADP1 (Glucose:fructose 6-diphosphate amino transferase), ADP2 (putative RNase), ADP3 (Clathrin adaptor proteins),ADP4 (metal peptidase) and AIP5 (family protein Rad21/Rec8). Yeast in vivo analysis results showed that ZFP319 could interact with those proteins.On the basis of the above results we speculated that the function ofZFP319 may play a regulatory role in rice stress responses byparticipating the 26Subiquitin-proteasome pathway.