Screening The Interaction Protein With AtCYSb Using Yeast Two-hybrid System

The yeast two-hybrid system is an effeetive method for the study of Protein-Protein, Protein-RNA, Protein-Small molecules interaction in yeast in vivo in recent years. From the establishment of this technology, it has been widely used in various fields.Cysteine proteinase inhibitor exists in various sorts and varieties creature, it take part in the activity of proteinase, interreaction of signaling receptor and the responder of adversity. At present, cysteine proteinase inhibitor in cowpea tomato poyato soyabean and rice has been isolated and extracted, and their functions have been studied furtherly. AtCYSb is one of cysteine proteinase inhibitor in Arabidopsis thaliana, some studies show that low temperature, drought, high salt, and oxidative stress can induce the expression of AtCYSb in Arabidopsis thaliana and overexpression of the gene also can improve the ability of Arabidopsis to resist the low temperature, drought, high salt and oxidative stress. But until now, it is still not clear that how the resilience mechanism is performed. So we plan to use "AtCYSb" as bait, using the yeast two-hybrid system, finding out the interacting protein, and to explore the resilience mechanism furtherly.In this study, the AtCYSb gene was cloned in vector pGBKT7, and we designate the combinant plamid as pGBKT7-AtCYSb. After the combinant plamid pGBKT7-AtCYSb was transformed to yeast Y2HGold, we transformed Arabidopsis cDNA library plasmids to yeast Y2HGold containing pGBKT7-AtCYSb. Positive clones were obtained by the selection of autotrophic phenotype, and then we extacted the yeast total DNA, amplified the total DNA with specific primers by PCR and digestion detected. According to the PCR and digestion results, we removed the same fragment. We transformed pGADT7-Rec-cDNA plasmids to yeast Y2HGold containing pGBKT7-AtCYSb for preliminary co-transformation test. After sequencing and blast in NCBI, we get a Ca(2+)-dependent nuclease(AtCaN2) containing 226 amino acids, and the similarity is 100%. The full-length cDNA of AtCaN2 was cloned in pGADT7 AD vector. We again transformed pGADT7 AD-AtCaN2 plasmid to yeast Y2HGold containing pGBKT7-AtCYSb for co-transformation test. The results show that both AtCaN2 (partial) and AtCaN2 (full-length) can interact with AtCYSb protein, activate the expression of reporter gene.We use pull-down and agarose gel electrophoresis to study the interaction between AtCYSb and AtCaN2 furtherly. The pull-down result shows that it has interaction between AtCYSb and AtCaN2; the agarose gel electrophoresis result shows that there is a role of relationship between AtCYSb and AtCAN, and AtCYSb inhibited the activity of AtCaN2 when uas A. DNA as a substrate.The detection of nuclease AtCaN2 experiment shows that:the nuclease could degrade both double-stranded DNA and single-stranded RNA and the nuclease activity of the His-AtCaN2 fusion protein was highest at 37 ℃, required a neutral or weakly-alkaline environment, and was stimulated by Ca2+ and Mg2+, but inhibited by Zn2+ and high concentration of Mn2+.