Functional Exploration Of A Mg Chelatase D Subunit Interacting Protein P43

In plant, chlorophylls are the most abundant tetrapyrrole molecules, and are essential for photosynthesis. Magnesium chelatase (Mg chelatase) is the first and key enzyme in chlorophyll biosynthesis branch. It catalyzes the insertion of Mg2+into protoporphyrin Ⅸ. Mg chelatase is a heterologous complex composed of I, D and H (CHLI, CHLD and CHLH) subunits.Subunits of Mg chelatase were reported to be involved in other different pathways, and interact with other proteins. For example, H subunit is an anti-sigma factor for SigE, and interacts with Mg-protoporphyrin Ⅸ methyltransferase.This study is mainly focused on P43, a glycosylated accessory protein of pea chloroplast DNA polymerase, which is hypothesized to interact with CHLD. The work includes two parts below.On one hand, yeast two-hybrid system, GST-Pull down assay, BiFC, CoIP technologies were used to verify the interaction between P43and CHLD both in vivo and in vitro. The interaction between P43and CHLD may suggest some biological relationship between chlorophyll biosynthesis and DNA replication.On the other hand, taking advantage of VIGS, yeast two-hybrid system and Western blotting, function of P43was explored. The results revealed that homologous proteins of P43can be found in Nicotiana benthamiana and Arabidopsis thalina. By screening a yeast two hybrid cDNA library using P43as a bait, two putative positive clones were obtained. Analysis of VIGS plants of CHLI and CHLD indicated that the expression of P43in VIGS-CHLI (CHLD) plants showed no difference to VIGS-GFP plant, which suggested that reduction of CHLD in pea did not cause an obvious altered expression of P43although they physically interact with each other. RNA binding assay suggested that P43may interact with RNA and has RNase activity.