Physiological And Biochemical Identification Of Six Dunaliella Species And Analysis Of Expressed Sequence Tags(ests) From High-quality CDNA Library Of Dunaliella Salina

The unicellular, halotolerant, green alga, Dunaliella salina (Dunaliellaceae, Dunaliella) has no cell wall, but has the unique ability to adapt and grow in a wide range of salt conditions from about 0.05 to 5.5 M. Dunaliella salina is rich in various valuable bioactive substances, such asβ-carotene, while can also produce large amounts of bio-oil under salt-stress conditions. Dunaliella salina is the best model organism to study molecular mechanisms of salt-tolerance since that it is the most salt-tolerant photoautotrophic eukaryote and also has simple cell structure.Currently, there are few research reports on indoor training system, microscopic morphology and cell growth curve of various Dunaliella species, which makes Dunaliella's large-scale biotechnology application and basic molecular research relatively difficult. In the present study, we collected six Dunaliella species: Dunaliella salina, Dunaliella parva, Dunaliella primolecta, Dunaliella bardawil, Dunaliella tertiolecta and Dunaliella sp., established and improved culture conditions of the above six Dunaliella species to achieve the success of indoor cultivation. Based on the successful cultivation, microscopic morphology structure of six Dunaliella species was observed by inverted microscope, showing that all of six species are unicellular green algae, have round, oval or spindle shape, contain a large cup-like chloroplast, give birth to two long flagellum on the top, as well as that different species have different cell size and Dunaliella salina has the largest cell volume. Then, three of the six Dunaliella species: Dunaliella parva, Dunaliella primolecta and Dunaliella Salina were selected for measuring their growth curve under 0.75M, 1.5M, 3M and 4M salt conditions respectively. The results of growth curve showed that following the salt concentration, the growth state of three species cell weakened, and through growth of 10 days and 50 days, three species went into the logarithmic phase and plateau phase respectively. Comparison of growth curve of three species under 1.5M conditions indicated that parva grow best, primolecta second and salina worst.At present, the D. salina's genome sequencing program has not been completed, and there are also a limited number of D. salina's nucleotide sequences and ESTs in NCBI's Genbank database. Therefore, a representative high-quality full-length cDNA library need to be built for large-scale expressed sequence tags analysis in order to fully carry out the D. salina's functionl genomic research and understand the basic molecular mechanisms of it's salinity-tolerance. In the present study, a high quality cDNA library was constructed from D. salina cells adapted to 1.5M NaCl by using Clontech's SMART? technology and its SMART? cDNA Library Construction Kit. Detection results of library quality show that well-constructed cDNA library from D. salina reaches standards of high-quality full-length cDNA library. Aiming for obtain some interesting information about D. salina's functional genes and salt-tolerance mechanism, 487 ESTs were successfully identified for generating a small expressed sequence tags (ESTs) database. Putative functions were assigned to 236 ESTs (48.5%) after comparison with protein databases by Blast of NCBI, and an additional 59 ESTs (12.1%) just had significant similarity to known ESTs of D.salina while had no homology with any known genes or proteins. However, up to 192 ESTs(39%) had no similarity to any known sequences, and may represent a large number of novel genes in D. salina. For those D. salina ESTs for which functional assignments could be made, the largest functional categories included protein synthesis and rate (43.6%), energy metabolism and photosynthesis (21.2%), and primary metabolism (5.5%), while a small parts of ESTs may be related to resistance of D. salina cell. Within the protein synthesis and rate category, the vast majority of ESTs encoded ribosomal proteins indicating that D.salina invests substantial resources in the production and maintenance of protein synthesis. Comparison results between D.salina ESTs(487) and known ESTs, gene or protein of Chlamydomonas reinhardtii and Volvox carteri f. nagariensis respectively indicated that 245 ESTs had significant similarity to known sequences of both species, which reveals that others of 487 ESTs may be specificly related to salt-resistance of D. salina cell. The full-length predictive analysis of these ESTs by using ORF Finder of NCBI led to discovery of complete encoding sequence of some unknown genes.