Cloning and Analyzing Chlorophyllase Genes of Glycine max

• Main Authors: Shen, Lee-Yen, 沈里彥
• Other Authors: Ling-ling Yang
• Format: Others
• Language: zh-TW
• Published: 2004
• Online Access: http://ndltd.ncl.edu.tw/handle/55052435190773183040

博士 === 臺北醫學大學 === 藥學系 === 92 === Soybean (Glycine max (L.) Merrill), containing highly amount of isoflavonoids protein and lipid, is an important food source. It can also be used as industrial and medicinal raw material. This project will use soybean as material 1) to clone the Chlase genes, 2) to study their expression and location of the enzymes, 3) and to analyze the phylogeny of our new genes along with the already published Chlase genes. It is expected that the obtained results will clarify the tissue specific, transportation and spatial distribution of the Chlase genes. From this, one can deduce what type of Chlase evolves first by reconstruct the phylogenic tree. We isolated and characterized three Chlorophyllase (Chlase) genes, GmCLH1, GmCLH2, and GmCLH3, from Glycine max var. Shi-Shi. The cDNAs of GmCLH2 and GmCLH1 and 3 encode 316 and 326 amino acids, respectively. GmCLH1 has no intron, and its cDNA sequence is 99.3% similar to that of GmCLH1. GmCLH1 and GmCLH3 have only 48% identity to GmCLH2 and 46% to 48% identity to the other three reported soybean Chlases. Constitutive expression of GmCLH1+3 and GmCLH2 was detected in young and senescent cotyledons and leaves, supporting the hypothesis that Chlase is active during chlorophyll turnover. GmCLH1+3 genes are tissue specific in pods and closely associated with pod yellowing (or maturation). Removing 51 N-terminal residues (a putative plastid transit peptide) in GmCLH1 significantly increased chlorophyllide production, whereas a 35-residue N-terminal deletion in GmCLH2 did not affect activity. These data, combined with predictions of a transmembrane domain for the Chlases, suggest that GmCLH1 is targeted to chloroplasts and located at the inner membrane of chloroplasts, whereas GmCLH2 is targeted to organelles other than chloroplasts. Phylogenetic analysis of the known Chlase sequences revealed two Chlase lineages, I and II, co-existing in angiosperms and that their divergence predated the split of monocots and dicots. The Chlase I lineage is likely a chloroplast protein and specific to fruit ripening, whereas Chlase II lineage is probably localized in the cytosol and mainly responsible for Chl turnover. Our findings further contribute to the research on promoting production in soybean specifically and all plants, in general.