Screening AtGRP7Interacted Proteins From Arabidopsis Thaliana Using Yeast Two-hybrid Technology

Under the continuing impact of earth rotation, organisms show circadian rhythm with a period of24hours to adapt to the change of earth. Biological rhythm is essential for the organisms to adapt the environment and enhance survival possibility. About ten percent of plant genes are regulated by biological clock. The regulators involving in circadian rhythm include central oscillator and slave oscillator. The slave oscillators are under the control of oscillator gene and further regulate the expression of downstream target genes. AtGRP7is a kind of RNA binding protein with rich glycine in Arabidopsis. AtGRP7expression was effected by central oscillator and under a feedback regulation by its own protein, AtGRP7was considered to be a slave oscillator gene. Previous studies suggested that AtGRP7involved in cold response and flowering period regulation. AtGRP7also took aprt in plant immune response. However, the molecular mechanism of AtGRP7and which proteins interact with AtGRP7are so far less known.In order to screen the proteins interacted with AtGRP7, in this study, a bait vector of yeast two-hybrid for AtGRP7was first constructed and transformation in Y2H gold indicated that AtGRP7did not show autoactivation and toxicity in host. Library titering test showed that the titer of Arabidopsis cDNA library used in this was1.45×107cfu/ml. After hybridization between Y2Hgold strain of bait vector and Y187strain of cDNA library and auxotroph screening, colonies grown on Quadruple Dropout medium (QDO) were collected. The plasmids were isolated from these colonies and subsequently co-transformed with AtGRP7bait vector into Y2Hgold strain, five clones were finally identified to contain AtGRP7interacted proteins. Sequencing and Blantx analyses showed that, one of these interacted proteins encodes ribosomal protein, one encodes carbohydrate combining protein, one encodes xylulokinase, the other two genes were not functionally characterized. These genes may be associated with immune response or other processes, which requires further characterization. However, this work paves a way to reveal the molecular mechanism and lays a foundation of further identification of AtGRP7function.