Using GST-qUBAs Bait Protein To Enrich And Study Rice Ubiquitination Proteins Under Drought Stress

Rice is one of the most important food crop, and it occupies an irreplaceable important role in national economic development. In recent years, water scarcity and frequent seasonal droughts have become important factors affecting rice yield. So it is significant to study the regulation mechanism of the responses under drought stress in rice and to improve rice drought resistance. Recent studies have shown that ubiquitination in plant play a very important role in the regulation of drought stress. Moreover, some E3ligases and ubiquitinated proteins had been successfully identified as key regulatory factors. Using proteomics to study the ubiquitination regulatory network is helpful to understand regulatory mechanism of responsing drought stress and to identify the function of more drought tolerance genes in rice.The efficiency of identification of ubiquitinated protein and E3ligases directly from the total protein is relatively low, because the uniquitinated proteins are unstable in vivo. When we investigate the ubiquitinated protein using proteomics technology, the first step is to enrich ubiquitinated proteins from the total proteins of rice leaves. In this study we used GST-qUBAs protein as bait to enrich ubiquitinated protein, which fused quadruple ubiquitin-associated domains with GST and6xHis tags. And a total of134ubiquitinated proteins were successfully identified using GST-qUBAs as bait protein after two-step affinity purification process. Compared with the mothod of directly identifying ubiquitinated protein from crude protein sample, the identification efficiency in this study was increased more than three times. Moreover, all the identified ubiquitinated protein contained at least one ubiquitinated modified site. Based on the above results, it meant that this approach could high-efficiency enrich and identify ubiquitinated protein in rice.Then a total of58special ubiquitinated proteins and35special E3ligases were successfully identified from protein samples of rice leaves, which were involved in the response of drought stress in rice. And most of them were novel protein taking part in ubiquitin and proteasome system (UPS). Meanwhile, some of them were reported as ubiquitinated proteins including OsCP12and OsNek6, some of them wereE3ligases including OsPUB15and OsCTRl, which were involed in responses of drought stress in rice. Then this research further discovered the proteins possibily interacted with the identified special proteins, and preliminarily established a regulatory network of responses of drought stress in rice.. Although there are lots of small isolated networks and the linkages of them are not clear in this preliminary networks, further research and more protein-protein interactions will be constantly discovered. The primary network in this study could be further improved, which is an important part of our laboratory future research.