Cloning and characterization of a female gametophyte- specific gene in Gracilaria lemaneiformis (Gracilariales, Rhodophyte)

1 The CAS/Shandong Provincial, Key Laboratory of Coastal Environmental Processes, Yantai Institute of Costal Zone Research, Chinese Academy of Sciences (CAS), Yantai 264003, China. 2 Institute for Life Sciences, Qingdao University of Science and Technology (QUST), Qingdao 266042, China. 3 College of Marine Life Sciences, Ocean University of China, Qingdao 266002, China. 4 Graduate University of the Chinese Academy of Sciences (CAS), Beijing 100049, China.


INTRODUCTION
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).Gracilaria, along with Porphyra, Laminaria and Undaria has a bulk production by farming in China.It is not only an economically important algal species, but also a good material for genetical studies (Chen et al., 2009).Thus, it is important to pursue basic studies on G. lemaneiformis.
tetrasporophytes.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 (Guzman-Uriostegui 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.Researchers have been engaged in the study of the mechanisms of phase formation since 1976 (Ren and Zhang, 2008).However, no satisfactory results were obtained due to the limitation of applicable methods in the past.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 be able to yield clear bands in Gracilaria, were used to distinguish the phases and sexes of G. lemaneiformis (Sun et al., 2003).Until now, several phase-specific and sex-specific genes have been identified.Eight unique cDNAs for the sporophyte and seven specific for the gametophyte, including elongation factor alpha and lipoxygenase encoding genes have been isolated from Porphyra purpurea (Liu et al., 1996).A heat-shock protein encoding gene, which might be involved in the differentiation of female gametophyte, has been identified from Griffithsia japonica (Lee et al., 1998).An ubiquitin gene of G. lemaneiformis during phase formation is identified and characterized (Ren et al., 2009).GlRab11, the first functional Rab-like protein identified in G. lemaneiformis was isolated and the cDNA full-length of GlRab11 was obtained (Ren et al., 2008).cDNA subtracted hybridization was employed to study Porphyra purpurea phase-specific genes (Liu et al., 1994) and suppression subtractive hybridization (SSH) was deve-loped by Diatchenko et al. (1996), which turned out to be a successful tool for rapid screening of differentially expressed genes (Shim and Dunkle, 2002;De la Vega et al., 2007).SSH had been applied to study differential expression of genes in developmental stages (Brun et al., 2003;Zhu et al., 2003;Singh et al., 2007) and under stress conditions (Bahn et al., 2001;Caturla et al., 2002).Sun et al. (2002) reported an analysis of 180 ESTs of the G. lemaneiformis tetrasporophyte cDNA library.Suppression subtractive hybridization (SSH) was employed between RNA extracted from female gametophyte and tetrasporophyte.Fourteen cDNAs were identified, among which SSH466 was a putative tetrasporophyte-specific gene (Ren et al., 2006).
In this study, we constructed SSH libraries between female and male gametophyte of G. lemaneiformis and isolated the cDNA full-length of GMF-01, which is a female gametophyte-specific Gene.

Algae materials and cultivation
G. lemaneiformis used in this study were collected from Zhanshan Bay (Qingdao, China).The healthy and mature fronds were used.Tetrasporophytes with released tetraspores were separated from female and male gametophytes under the microscope.Then one female gametophytes and one male gametophytes developed from tetraspores were picked out.The separated algae materials were brushed and rinsed in sterilized seawater until they were completely divorced from epiphytes.The materials were cultivated in Provasoli medium (Provasoli, 1966) under a light intensity of 50 µmol photon m −2 s −1 with a 12:12 (L: D) cycle at 15±1°C.The thallis were used for RNA extraction.

SSH library construction
To isolate sex-relative genes, suppression subtractive hybridization (SSH) was performed between RNA isolated from male and female Chen et al. 2591 gametophytes of G. lemaneiformis.Total RNA was extracted from each sample with RNeasy Plant Mini Kit (Qiagen, China), reversetranscribed and amplified using a SMART PCR cDNA Synthesis Kit (BD Biosciences Clontech, USA).Both forward (female gametophyte as tester and male gametophyte as driver) and backward (male gametophyte as tester and female gametophyte as driver) SSH were performed using a PCR-select cDNA subtraction kit (BD Biosciences Clontech) according to the manufacturer's instructions.
In order to confirm differential expressions of the clones, cDNA dotblots were performed.Based on the results of cDNA dot-blots, clones that expressed differentially between female and male gametophyte were sequenced and aligned with the BLAST algorithms at the National Center for Biotechnology Information (NCBI).The sequences were also analysed with ContigExpress (Vector NTI Suite 6.0) to find contigs.

Screening of the SSH cDNA libraries
A total of 768 clones were randomly selected from the SSH libraries (384 clones from each of the two SSH libraries) and screened by macro-array dot-blots.Both forward and backward subtracted radioactively labeled cDNA populations were then used as individual probes for identical blots (Figure 1).411 clones were found to be positive clones.When analysing the sequencing results with ContigExpress (Vector NTI Suite 6.0), 136 contigs were found and one female gametophyte-specific sequence (480 bp) showed significantly differential expression between the two sexes.It was designated as GMF-01 and was chosen to clone its full-length cDNA sequence.We are still analyzing the rest partial sequences and they will be published later.The partial sequence of GMF-01 is as follows: GCAGACTTCTACTATTCAGTATAGATGGGTTGAAGAA TATAGGGCCACGTTTACCACCAAAGTGGAGATCGGA GAAATTATTCGGACGCAGGACATCATCAACTCGCCT GACTTTAACATGGGACAATCCGTTTCATTCGATGGAG TCGAGTGGAGTCCTCCAGTCAGCGACCGGAAGCCG CCGAACATTGGGGTAGCATACAAGGTTGACACGAAC GCTCTGCATCCAGTTCTACTCGCTTCTTATACCATGA

Cloning of the full-length cDNA and sequence analysis of GMF-01
Based on the partial sequence obtained from the SSH library, two primers B5 and B3 were designed to amplify the 5' and 3' cDNA ends of GMF-01.1019 bp and 548 bp were amplified in the 5' and 3' SMART RACE reactions respectively.The full length of GMF-01 cDNA had 1357 nucleotides.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.Searches from the public sequence databases using NCBI BLASTx showed that there was no significant match with GMF-01.The predicted protein had a calculated molecular weight of 36.7 kDa and a theoretical pI of 7.92.The instability index was computed to be 43.61 which classified the protein as unstable.The GRAVY (Grand average of hydropathicity) of this protein was -0.131, which indicated that the protein was hydrophilic.Results of prediction showed 45.05% of its secondary structure is random coil (Figure 2).Sub-cellular location prediction results with ProtComp, TMHMM (Figure 3) and PSORT all indicated that it's probably an extracellular protein.The cDNA and amino acid sequences are indicated in Figures 2 and 3.

DISCUSSION
As an agarophyte, components of G. lemaneiformis cells are extremely complicated.In the construction of 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).
Thus, SMART strategy was taken before SSH library construction was carried out.That is the key point of the successfully construction of SSH library.We identified 3 practical sequences that are differentially expressed between female gametophytes and male gametophytes.One putative female gametophyte-specific gene GMF-01 was selected to isolate the full-length of cDNA for further analysis.
In this study, we identified a female gametophytespecific gene of G. lemaneiformis.The protein encoded by GMF-01 may be a extracellular protein.It was not known yet whether GMF-01 was red-algae specific.
In the SSH library, we found some special gene function of GMF-01.The construction of additional transgenic clones in which GMF-01 is knocked out should allow a better assignment of its function.Based on the SSH libraries constructed in this study, more differentially expressed genes could be found.The differentially expressed genes obtained in this study are closely related with gametogenesis of G. lemaneiformis.Studies on these genes may play important roles in understanding sex determination mechanisms and will provide clues for red algal evolution pathways.

Figure 1 .
Figure 1.Dot blots of cDNA in subtractive.Two identical membranes blotted with PCR amplified cDNA sequences from subtracted libraries were probed with the forward subtracted probes (A) and the backward subtracted probes (B).