Study Of Interacting Protein Of The PAC Protein:Essential Protein Of Chloroplast Development In Arabidopsis Thaliana

Photosynthesis is one of the most important chemical reactions in the biosphere.Chloroplast is the place where photosynthesis takes place,so the research of chloroplast development should be one of the highest priorities for improving the efficiency of photosynthesis.Arabidopsis variegation mutants have played a prominent role in the study of chloroplast genes' function.Variegated2?var2?and pale cress?pac?are the two important variegation mutants related to the chloroplast development.In addition,VAR2 and PAC are their mutant genes,respectively.Meanwhile,VAR2 encodes the Fts H metalloproteinase,and PAC encodes an unknown chloroplast protein.Also,it is important to note that loss of PAC leads to a seedling lethal phenotype,which brought great inconvenience for the study of PAC function.To elucidate the mechanism of how PAC is involved in regulating chloroplast development,our laboratory has carried out yeast two hybrid screens for PAC interacting proteins.Consequently,we found that VAR2 could interacte with PAC.In this study we used different methods to verify the interaction between VAR2 and PAC.The main results obtained are as follows:1.We first constructed a prokaryotic expression vector p ET28a-VAR2_714-1995,and then transformed it into E.coli expressive strain BL21?DE3?.His-tag fused VAR2238-665 existed as inclusion body,and the recombinant protein was purified by Nickel affinity chromatography under denatured condition.Consequently,the target protein we obtained with high purity.The recombinant VAR2238-665 was used as antigen to immune rabbit to prepare polyclonal antibody.Prepared VAR2238-665 polyclonal antibody could be used to detect VAR2238-665 with high purity and specificity through western blotting detection.2.We constructed VAR2 overexpression lines in Arabidopsis to analyze the VAR2 level in vivo.The results of western blotting indicated that there are dramatic differences of VAR2 content in WT,var2-4 and VAR2 OE lines.So the prepared VAR2238-665 polyclonal antibody can meet the requirement of further studying interacting proteins,such as Co-IP,GST Pull-down and so on.3.By constructing Bi FC vectors and an Arabidopsis leaf protoplast transient expression system,we confirmed that the interaction of VAR2 and PAC.Moreover,they are all localized in the chloroplast.4.Taking advantage of the yeast two-hybrid system,we constructed a series of vectors to identify the specific VAR2 domain interacting with PAC protein.The PAC gene?except c TP?is connected to the BD vector,and different VAR2 domains are connected with AD vector.In the end,we found that the amino acid region 476-665 of VAR2 was confirmed to interact with PAC through the yeast two-hybrid.Taken together,this work laid the foundation for the future research on the verification of VAR2 and PAC interaction.What's more,it also facilitates future study of how PAC exerts its role in chloroplast development.