Study On Interaction Between Ankyrin ANK6 And SIG5 In Arabidopsis Thaliana

Ankyrin (ANK) proteins contain a variety of ankyrin domains which consist of ankyrin repeats motifs existed widely in organisms from bacterial, fungi to mammals. ANK domains mediate protein-protein interactions to achieve complex biological functions. In Arabidopsis, there spread at 108 ANK genes in the genome. Our previous work proved that one of the ANK genes, ANK6, played essential role in plant double fertilization. Using ANK6 as "bait" protein, we screened the Arabidopsis cDNA library and screened out a number of interaction protein candidates. SIG5 is one of them. Our goal is to confirm the interaction relation between SIG5 and ANK6, and the results are shown below:Firstly, by putting SIG5 and ANK6 constructs into AH109 strains, we confirmed ANK6 interacted with SIG5 protein in yeast two-hybrid system. We contructed six SIG5 deletion mutants and pair with ANK6 separately in yeast two-hybrid system. The results showed that ANK6 interacted with N-terminal of SIG5 protein.To further confirm the interaction between ANK6 and SIG5 protein, we expressed soluble recombinant proteins-ANK6-GST and SIG5-His6 in bacterial BL21 cells. Then we confirmed ANK6 proteins indeed interacted with SIG5 proteins by pull-down experiments and western-blotting.The mutant plants of SIG5 and ANK6 showed similar phenotype of homozygous lethal. We built the histochemical localization vectors and obtained transgenic plants by floral dip method. The different tissues and organs of transgenic plants were stained with GUS staining solution. The results shown that the transgenic plants of SIG5 gene expressed space-specific, mainly expressed in the pollens, leavies and ovules.In summary, our study mainly on the interaction between Ankyrin ANK6 and SIG5 in Arabidopsis thaliana. Using the methods of yeast two-hybrid and pull-down to identify and verify that the ANK6 interacted with SIG5 protein. The mutants of SIG5 and ANK6 gene had a very similar phenotypes of homozygous lethal and also very similar in tissue-specific expression. From the study in the three aspects of ANK6 genes layed the foundation about the growth and development of physiological functions in Arabidopsis thaliana.