Screening And Verification Of The Proteins Interact With AT14A In Arabidopsis Thaliana

AT14A is a member protein from Arabidopsis thaliana which belongs to the DUF677superfamily. The function of this protein superfamily was little known. AT14A possesses a small region that has sequence similarities to integrins from animal. Therefore it was assumed to be integrin-like protein and perform some or all of the functions performed by animal integrins. Integrins has been shown to play an important role in diverse biological processes including cell-cell adhesion and cell-matrix adhesion, cell migration, cell proliferation cell differentiation. The existing research of AT14A protein shown that it was presented in the membranes, involved in the maintenance of cell shape, organization of the cytoskeleton of A. thaliana Suspension cultured cells, and AT14A may play a role in cell wall metabolism. Though some studies have been published on the physiological functions, however, seldom work has been carried out in molecular mechanism. In this paper, interaction proteins was screened and identified using AT14A intracellular domain as bait protein by Yeast Two Hybrid System, co-immunoprecipitation and GST-pull down. RT-PCR were used to evaluate AT14A interacting protein, exploring the molecular function of AT14A, which brought some new clues for studying the potential molecular mechanism of AT14A protein. Following results were reached:1. Two independent yeast two hybrid cDNA library screens resulted in134positive clones involved in37different interaction pairs.19and18potential interacting proteins were screened by AT14A-N and AT14A-C, respectively. Total10unknwon protein among above37potential interacting proteins,10of the proteins were screened in two or three clones. One protein was screened in two Yeast Two Hybrid.2.10proteins(ATSYP23, ATSNX1, CIP1, AtFH7, CSN4, Sec14p-like phosphatidylinositol transfer family protein, ATVDAC3, ATSYTA, EDM1, ARA4) which are screened by two independent Yeast Two Hybrid screens were selected and expressed in prokaryotic expression system. The interaction was analyzed by co-immunoprecipitation. Results suggested only AtFH7and EDM1interact with AT14A-N, AT14A-C obviously in vitro, respectively.3. The fragment encoding AT14A was inserted into pGEX-6p-1(with GST tag) form fusion expression vector and transformed into Escherichia coli, which was analyzed by pull-down and western blot. Results suggested no obviously interactions in vitro was detected in both of the protein pairs in this study. 4. To analysis the expression of AtFH7and EDM1, RT-PCR and Real-time PCR was performed in ecotypes Columbia, atl4a-1deletion mutant and AT14A overexpression A. thaliana plant. Results showed that AtFH.7and EDM1gene was induced by AT14A. In AT14A overexpression plant, both of the genes had significantly higher levels of mRNA than wild plant, but the expression level is in coincidence with the level of wild plant.In summary, we screened two proteins AtFH7and EDM1by Yeast Two Hibrid, and the interaction was analyzed successfully in vitro in this study. The discovery of interaction between AT14A and AtFH7, EDM1laid a foundation for further study of the molecular biology function.