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Abstract
In this study, forward and reverse suppression subtractive hybridization (SSH) libraries were constructed between female and male gametophyte of Gracilaria lemaneiformis to isolate genes differentially expressed between gametophytes. Dot-blots were performed to screen 384 colonies randomly selected from each subtracted libraries. Partial gene (designated as GMF-01) which is female gametophyte-specific was selected to isolate its cDNA full length sequences and to be characterized. Then we got its cDNA full length sequences using SMART-RACE technic. Sequence analysis showed that the open reading frame (ORF) of GMF-01 is 1002 bp long with a GC content of 47.7%, encoding 333 amino acids. GMF-01 does not have significant match in the databases when examined on NCBI website. Amino acid sequence analysis showed that the theoretical pI of this translated protein is 7.92, while the instability index is computed to be 43.61, which has classified the protein as unstable. Results of prediction showed 45.05% of its secondary structure has random coil and sub-cellular location prediction indicated that it is probably a extracellular protein.
Highlights
Gracilaria lemaneiformis is a commercially important agarophyte that can be used to produce agar, a major ingredient of dairy products, surgical jellies, ointments, cosmetics and healthcare products (Tseng, 2001)
In the construction of suppression subtractive hybridization (SSH) library, RNA isolated from G. lemaneiformis was too difficult to purify enough due to the polysaccharides
The SMART approach is a PCR-based amplification system that allows the creation of cDNA from a very small amount of total RNA (Cramer and Lawrence, 2004; Shary and Guha-Mukherjee, 2004; Pavan, 2011)
Summary
Gracilaria lemaneiformis is a commercially important agarophyte that can be used to produce agar, a major ingredient of dairy products, surgical jellies, ointments, cosmetics and healthcare products (Tseng, 2001). Even though the phases and sexes of Gracilaria look identical before sexual maturation, there are physical differences between them (Kain and Destombe, 1995), such as growth rate in phases of G. lemaneiformis (Zhang and van der Meer, 1988), levels of polyamines in sexes and phases of G. cornea (GuzmanUriostegui et al, 2002), and lipid composition among different developmental stages of Gracilaria verrucosa (Khotimchenko, 2006). Owing to their particular life history, differentiation of phase and sex in red algae has already attracted researchers' attention. With the development of molecular biology, great progress has been made recently, such as that made by Ye et al (2006). 6 ISSR primers, which had proved previously to
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