Construction Of Yeast Two-hybrid CDNA Library And Screening Of The Proteins Interacting With ARC1 From The Stigma Of Ornamental Kale

Self-incompatibility(SI) allows the pistil to distinguish between self and non-self pollens of the same species and reject self or self-related pollens.Many flowering plants have evolved this physiological strategy to prevent inbreeding and maintain genetic diversity in a population. The stigma,in which the self-incompatibility reaction takes place,can inhibit the growth and development of self-pollen through signaling cascade response in papillar cell in Brassica.Arm repeat containing 1(ARC1),a stigmatic U-box protein with E3 ubiquitin ligase activity, functions as a positive mediator of SI signaling.However,little is known about substrates of the ARC1 in the rejection of incompatible pollen during the SI response.Ornamental kale(Brassica oleracea var.acephala) homozygous for S_13-b haplotype was used in this study.The total RNA was isolated from stigma,and the mRNA was purified by affinity chromatography.The cDNA library from the stigma,which applied to yeast two-hybrid screening,was constructed.The total size of the library transformed into Escherichia coli was 2.5×10⁵ clones,and the total size of the amplified library was 4×10⁸ clones.The percentage of vectors with inserts was above 96%.The insert size by PCR from the colony and plasmids from library with Myr-specific primers was 0.4-3 kb,and the average insert size was about 800 bp in length.The full-length of ARC1 was cloned into pSos vector.And then,it was confirmed that the pSos-ARC1 plasmid was suitable for detecting protein-protein interactions in the CytoTrap system.On the base of CytoTrap cytoplasmic yeast two-hybrid system,we used ARC1 as bait to screen in the cDNA library from the stigma of ornamental kale,and obtained two proteins which could interact with ARC1.One of the two proteins was composed of 71 amino acids,whose homologous protein is Nucellin of Arabidopsis thaliana with 79%identity. Nucellin is a new aspartic protease,which may play a role in progressive degeneration of the barley nucellar cells after ovule fertilization.The other protein consists of 288 amino acids, whose homologous protein is DnaJ of Arabidopsis thaliana with 88%identity.DnaJ proteins are a class of chaperone proteins,which function as part of the Hsp70 chaperone machine,and play important role in protein translation,folding,unfolding,translocation,and degradation.These results provided important clues to further investigate the function of ARC1 and mechanism of the SI signal pathway.