The Design And Construction Of The Rice Protein Chip And Its Initial Identification

Rice (Oryza sativa) is an important food crop, the basal genetic material for the study of crop product, transgenic crop, Heterosis; insect-resistant plant and other stress resistance ways, and a good model for the fundamental research. The rice protein kinases play an important role in the rice cellular transduetion, and also concern in many development and proliferation process in rice. Studying theses kinases, especially their phosphorylation process and screening their substrate, which can help us know the plant proliferation mechanism and important to the development agriculture.This experiment design a new method, which is based on Pooling system to construct the rice protein chip in high-throughput way. Firstly, Based on Pooling system, ten clone of the rice eDNA library were incubated in every well of the 96-well plate, after induced express protein, extracted the protein with lysozyme, and the total number of extracted rice library protein is 60 plates, which are made up of 6000 protein sample and include 60000 clones. After that, pooled the protein sample 10 times, and got the Pooling protein samples for 15 plates, which have 1200 protein samples. Secondly, test the general expression level of the mixed protein sample with a quick, direct, facility way-Dot blotting way. Moreover, expressed rice 57 GST fusion protein and 4 MBP fusion protein were all put into one 96-well plate, eventually, spot all these protein sample in the same protein array. Thirdly, test the quality of the rice protein array with Anti-β-gal and Anti-GST hybrid, and screen the substrates of the rice protein kinase-PI-D2 with this rice protein array. The results were summarized as follows:1. Based on Pooling system, we expressed about for 60000 rice root cDNA clone. protein.2. Lysosyme can extract many proteins at the same time, which is suit to extract much protein for construction rice protein library.3. With Dot blotting method, test the express level of mixed protein based on Pooling system, results indicated the quality of the protein samples is good and can be used made into rice protein array.4. Spotted a protein array, which include 7271 protein samples, and test its quantity with Anti-β-gal and Anti-GST, results indicated the quality of rice protein is good. 5. With this rice protein array, Screened the PI-D2 rice protein kinase substrates and got a number of potential substrate, which suggest that this rice protein array can be used into screening a lots substrates of protein kinase.This rice protein array are constructed based on Pooling system, which used shorten work time, less work quantity, saving work cost and reagent quantity .The rice protein array can be used in many biological way, and make the test process systematization and miniaturization, lessen the test time and improved the sensitivity and veracity. For instance, study of the interaction between the protein and protein, protein and DNA, protein and lipid, protein and drug and so on. The screening substrate of kinase protein with protein array is a representative method among them, which can be used to screen substrates in a systematization way, and let people understand the biological mechanism of protein kinase and the signal introduction pathway in the rice cellular perfectly, all this is important for the many biological study in future.