Gracilaria/Gracilariopsis lemaneiformis, an economically important agarophyte, has been under cultivation on a large scale in China and has been appreciated as a food and feed for culturing marine animals and formerly as a binding matural in the preparation of lime for painting walls. The most important use of Gracilaria, however, is the production of agar (Tseng, 2001). Besides its economic importance, G. lemaneiformis is also an ideal material for a variety of genetical studies owning the following traits (Zhang et al. 2004). Firstly, it can complete its life cycle under laboratory conditions. Secondly, pigmentation and morphological mutants are easy to be generated by physical or chemical mutagenesis. Thirdly, tetraspores and carpospores can be obtained at anytime under proper culture conditions.

The species possesses three isomorphic life-history phases, gametophyte, carposporophyte and tetrasporophyte. Phase differentiation is associated with numerous important physiological changes and many genes are developmentally regulated and expressed in a phase relative manner. Since 1976, investigations have been conducted on differential detection of proteins to find phase relative genes in algae (Hoxmark, 1976; Hushovd et al. 1982). Due to the drawbacks of applicable methods at that time, there were no satisfactory results. With the development of molecular biology, large-scale gene expression studies have been undertaken to understand the molecular mechanisms of phase differentiation in algae, such as Liu et al. (1994) and Sun et al. (2002). Despite these advances, the molecular mechanisms of phase differentiation in algae are still poorly understood and many phase relative genes remain to be discovered. Suppression subtractive hybridization (SSH) was developed by Diatchenko et al. (1996) and turned out to be a successful tool for rapid screening of differentially expressed genes (Wei et al. 2001; Caturla et al. 2002; Lee et al. 2002; Osherov et al. 2002; Cramer and Lawrence, 2004). Under this background, SSH was performed between RNA isolated from tetrasporophytes and female gametophytes of G. lemaneiformis. The findings in this report enhance our understandings of the phase differentiation process in Gracilaria.

In this study, 14 cDNAs were isolated and sequence-identified. Virtual Northern blot was used to study the different expression profiles. Clone SSH4 had high homology with other reported Rab GTPases, whose expression was a little bit higher (0.3 fold, p < 0.05) in female gametophytes compared to tetrasporophytes. This increase might enhance vesicle-mediated transports. It was observed that the expression of Rab5a was up-regulated during root development in rice Oryza sative L., maybe because endocytosis was responsible for the uptake of essential nutrients from the external environment (Wang et al. 2002).

Clone SSH 302 was homologous to thiamin pyrophosphokinase (TPK) (EC 2.7.6.2). TPK catalyzes the pyrophosphorylation of thiamin with adenosine 5'-triphosphate to form thiamin pyrophosphate (TPP). TPP is a necessary compound associated with glycolysis and tricarboxylic acid cycle. Thus, the differential expression of the TPK gene during phase differentiation in G. lemaneiformis could cause energy difference between the two phases.

Clone SSH407 had homology to GMP synthase. By densitometric analysis of blots normalized to GAPDH, the expression of SSH407 increased nearly 3 fold in tetrasporophytes as compared to female gametophytes. Clone SSH424 was homologous to RP42 which was developmentally regulated and may be involved in proliferative functions (Mas et al. 2000). Clone SSH528 was homologous to aspartate aminotransferase which was reported to be correlated with the content of certain amino acids, such as the content of cysteine, asparagine, glutamine and aspartic acid (O'Farrell et al. 1997). As a result of the differential expressions of the above metabolism involved genes, the amount of some nucleic acids and amino acids synthesized may be different between the two developmental stages. Consequently, the levels of metabolism are probably different between them. This is consistent with the fact that they are different in some physiological characters such as growth rate, etc. (Kain and Destombe, 1995; Zhang and Van der Meer, 1988). Therefore, the genes identified in this study may be utilized as novel molecular markers in future genetic breeding of G. lemaneiformis.

Among the genes of unknown functions, clone SSH391 did not hybridize with either of the samples, maybe because its expression level was too low to be detected. Two cDNAs are false positive, expressed at the same level between the two samples. Of particular interest is clone SSH466 which appears to be expressed solely in tetrasporophytes and is likely to be a tetrasporophyte-specific gene. Clone SSH486 showed significantly differential expression and increased 4.2 fold in tetrasporophytes compared to female gametophytes. It will be of great interest to study the specific role of clone SSH486 during phase differentiation in Gracilaria. Future studies will be focused on cloning the full-length cDNA sequences of these genes and using in situ hybridization and gene disruption techniques, etc. to elucidate their functions. The identification and characterization of all these differentially expressed genes will provide important clues to the probable metabolic pathways and/or structural features unique to the maturation of red algae

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