Screening For Protein Interacting With ALP1

Study of protein-protein interactions is one of current research focus. Understanding the interaction mechanisms among proteins will help to analyze protein function and solve many problems related to life. The yeast two-hybrid technology is widely used in screening for and identification of interact proteins, as well as constructing protein interaction networks to illustrate the law of life. It is an important tool for system biology.There are a lot of DNA-J proteins in the plant, J protein and HSP70 could form a molecular chaperone involved in the response to stress. ALP1 is a J protein, it was confirmed that ALP1 loss-of-function mutant is sensitive to salt, ABA and JA in seedling. The research about ALP1 will help us understand the mechanism of plant stress resistance. In order to further reveal the ALP1 cell signal transduction pathways, it is first to explore the interaction mechanism of ALP1.The work of this thesis is based on the prior research work in our laboratory. First, the potential interacting proteins of the J protein ALP1 in Arabidopsis thaliana were predicted by bioinformatics method. Then parts of these potential proteins were screened by MATCHMAKER GAL4 Two-Hybrid System 3. The results show that all of the tested protein did not interact with ALP1. At the same time, a yeast bait expression vector pGBKT7-ALP1 and the cDNA library of Arabidopsis were successfully constructed. The library transformation efficiency was 1.2×107transformants/3μgpGADT7-Rec, library titer was 2.46×109pfu/mL. Polymorphism and Recombination rate met the yeast two-hybrid library requirements.This will be used to screen for the interacting protein of ALP1 and construct the interaction map in the future.