Screening Of The Proteins Interacting With The Aluminum Tolerance Related Transcriptional Factor STOP1in Arabidopsis Using Yeast Two-hybrid System

Plants develop lots of tolerant mechanisms to adapt to various environmentstresses, such as drought, salinity, and soil acidity. Molecular characterization of suchsystems is critical to breed high productivity crop plants under stress environment.About40%of world arable land is categorized as acid soil and aluminum toxicity inacid soils is an important abiotic stress factor that reduces crop yield.STOP1is a C2H2-type zinc-finger protein belonging to a family of transcriptionfactors. The stop1mutant is sensitive to Al3+, but not other metal ions. STOP1cancontrol the transcription of a number of downstream genes. In order to find out themechanism of transcriptional control by STOP1and to illustrate the proteins whichdirectly binding to STOP1, a yeast two-hybrid system screening using STOP1as abait was performed to screen the Arabidopsis cDNA library. One positive clone wasobtained and sequenced. Using the BLAST on tair website, it was confirmed to be thefragments cDNA of NaKR1. In the previous study, the phloem transport and the metalbinding properties of the NaKR1protein are presented. NaKR1is specificallyexpressed in the phloem, encoding a heavy metal coordinating protein. To investigatethe interaction between NaKR1and STOP1and the role of NaKR1in the aluminumtolerance in Arabidopsis, we conducted the experiment as below:1. We found a protein NaKR1that can interact with STOP1through yeast twohybrid screening. NaKR1encodes a soluble protein of319amino acids, with a heavymetal-associated domain of59amino acids at the C-terminal region end.2. We tested the interaction between the full length STOP1,STOP1N terminal,and NaKR1in yeast and found that the interaction between STOP1N terminal and NaKR1is stronger.3. We confirmed the interaction between the two proteins in human293T cellusing pull down assay.4. We constructed the yellow fluorescent protein (YFP) fused expression vectorsof STOP1and NaKR1and transformed into Arabidopsis protoplast. Results indicatedthat both proteins are localized in the nucleus.5. We analyzed the relative expression level of NaKR1in Arabidopsis rootsunder the Al treatment by quantitative real-time PCR. Results showed that with25μM aluminum treatment up to36h, the NaKR1respond to25μM aluminumtreatment. The expression level increased constantly.6. We cloned NaKR1into the expression vector pEGAD-3XHA-LUC. By themethod of agro bacterium-mediated genetic transformation, the vectors wastransferred into the wild Arabidopsis Colombia-4. Analyzing the phenotype of thistransgenic plant under aluminum stress, the root length was longer than the wild type.