Functional Exploration Of Mg-Chelatase Ⅰ Subunit And Thioredoxin F From Pea Chloroplast

Mg-chelatase is the first enzyme in chlorophyll synthesize pathway (Mg2+ branch), it plays an essential role in chlorophyll synthesizing pathway to catalyze the reaction of Mg2+ insertion into protoporphyrinⅨ. As we know Mg-chelatase consists of three different subunits:I subunit (CHLI)、D subunit (CHLD)、and H subunit (CHLH), these subunits form a enzyme complex to accomplish the reaction and some of them may involve in other metabolism pathway. Moreover, some cofactors also regulate the acitiviry of Mg-chelatase. GUN4 protein is a well-known cofactor of Mg-chelatase and can interact with H subunit and bind the protoporphyrinⅨin order to enhance the activity. Recently, a Japanese group have found that I subunit is a target protein of thioredoxin-f (TRX-f), which can reduce I subunit and affect the ATPase activity of I subunit.In order to further study of the thioredoxin-depend regulation of Mg-chelatase, firstly, we isolated the full length chll cDNA sequence by using the method of PCR screenλgt11 pea leaf full-length cDNA library and 5’RACE assay. These cDNA is 1452 bp with a 1266 bp of open reading frame encoding 422 amino acids, whose theoretical molecular weight is about 45.98 KD. Then, we expressed and purified the recombinant proteins CHLI and TRX-f, after analyzing in vitro activities of ATPase and Mg-chelatase, we found that the recombinant TRX-f can reduced the CHLI and activate the ATPase of CHLI and Mg-chelatase activities in vitro.For further analyzing the regulation of CHLI by TRX-f, we performed the yeast two hybrid assay to determine the interaction between TRX-f and the subunits of Mg-chelatase and GUN4. The result shows that TRX-f only interacts with CHLI in yeast. Finally, we utilize VIGS (virus induced gene silencing) technology to study the function of CHLI and Trx-f in Pisum sativum plant. As a result, the leaves, stem, calyces and pod turn yellow in VIGS of CHLI pea plant. Meantime, the activity of Mg-chelatase, the contents of photosynthesis relative pigments decrease strongly as well as the expression level of Trx-f gene in RNA level in in VIGS of CHLI pea plant. In contrast, no significant phenotype is observed in the VIGS of TRX-f pea plant, and the activity of Mg-chelatase, the contents of photosynthesis relative pigments, and the expression level of chlI gene were not changed in the VIGS-Trx-f pea plants although the expression of the Trx-f gene was suppressed. All of these experimental results will provide good understand of the function of CHLI and Trx-f.