As an important basic metabolic pathway, glycolysis is ubiquitous in all kinds of organisms. But the genes/enzymes involved in this pathway and the compartmentation of the pathway in some algae of important evolutionary status remains unknown or controversial. In the present work, firstly, triosephosphate isomerase (TIM), one of the important enzymes of the pathway, was identified in two trophic modes of euglenoids, and phylogenetic analysis was carried out combined with sequences from other organisms including chlorophytes, rhodophytes and kinetoplastids. Secondly, the compartmentation of the pathway was investigated in Chlamydomonas, and furthermore phylogenetic analyses of each classic glycolytic enzyme were conducted to trace the evolutionary history of the pathway in this species. The results and conclusions are as follows: 1) By 3’-RACE and 5’-RACE, two types of cDNA sequences were obtained in phototrophic Euglena gracilis and Euglena intermedia, and saprotrophic Astasia longa. Meanwhile tim gene sequences of two chlorophytes and rhodophytes were acquired by genome DNA-PCR and database searches. Sequence analyses and presequence prediction indicated that the shorter one encodes a cytosol TIM (cTIM) and the longer one encodes a chloroplast TIM (cpTIM) or a plastid TIM (pTIM). The typical presequence of the putative A. longa pTIM and the high sequence similarity between A. longa pTIM and E. gracilis cpTIM imply that A. longa pTIM is targeted to plastids, and therefore that A. longa plastids still have function(s) involving in TIM (e.g. probably glycolysis and fatty acid synthesis). Sequence alignment and phylogenetic analyses of TIM indicated I) euglenoid and rhodophyte TIMs share a unique 2-aa insertion within their loop-4 areas; II) most important, euglenoid TIMs neither group with TIMs of kinetoplastids, which share the nearest common ancestor with euglenoids, nor are closely related to TIMs of III chlorophytes, which are considered to offer euglenoids chloroplasts through secondary endosymbiosis, but group with TIMs of rhodophytes. These suggest that tim lateral gene transfer might have occurred between euglenoids and rhodophytes after the divergence of euglenoids with kinetoplastids. 2) By using the methods of bioinformatics and molecular biology experiments, glycolyticsis-associated genes/enzymes were identified and analyzed for their sub-cellular targeting and expression level based on Chlamydomonas reinhardtii genomic and transcriptomic databases. The results indicated that different from the majority of eukaryotes, Chlamydomonas cytosol does not possess a complete glycolytic pathway. Although the last three steps of the pathway occur in cytosol, the forward seven steps take place in the chloroplast. Molecular phylogenetic analyses indicated all the enzymes involved in the forward six steps in Chlamydomonas chloroplast and the last two steps in Chlamydomonas cytosol derived from the original cytosolic genes, and chloroplast FBAs involved in the fourth step were most probably from the duplication of the endogenous cytosolic gene at the very early stage; chloroplast PGK involved in the seventh step originated from cyanobacterial endosymbionts by lateral gene transfer. These suggest the lack of glycolytic enzymes in Chlamydomonas cytosol was not a primary character but due to a secondary lost. The occurrence of the forward seven glycolytic steps in chloroplast should result from the original cytosolic enzymes by relocalization to the chloroplast either directly or after gene duplication, or derive from cyanobacterial endosymbionts by lateral gene transfer.
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