| Dunaliella, a single-celled green algae classified to Chlorophyceae of Chlorophyta, distributes throughout salt lakes and sea over the world. Dunaliella inhabits itself in the environment rich in Na+ and Cl-. Although wild types of Dunaliella screened from different environments presents distinct salt stress tolerance and optimal salt concentration, all of them are capable of living in the environment of a wide range of NaCl. Dunaliella. salina, has the greatest tolerance to salt stress, and can live in saturated salt solution. Accordingly, Dunaliella. salina is usually named as "Salt algae".Currently the study on the mechanism of plant's tolerance to salt stress via molecular genetics is getting popular, there is still a long way, however, to decipher the secret due to the complexity of the factors influencing the salt stress in advanced plants. Dunaliella.salina, a single-celled eukaryotic organism, has a relatively simple mechanism of saturation regulation, therefore it is an ideal model organism to be used to study the mechanism of salt stress tolerance. In this paper, Dunaliella salina was used as the research material, and its ability to regulate the osmotic pressure, as well as the interaction of saturation regulation and ultramicro-structure of Dunaliella. salina have been fully examined. Furthermore, its enolase gene, related to salt stress toleration, has been cloned and identified for its function.Under Transmission Electron Microscope and Scanning Electron Microscope, Dunaliella salina had thin and asymmetric outer membrane, the flagellum of thetypical "9+2" structure, a mass of endoplasmic reticulum, mitochondria of various shapes, "cup"-shaped chloroplast, with the eyespot located in the plasm. Two big amyloplastids were observed in DunaUella.salina for the first time. Its nucleus was located in the concave of chloroplast and nucleolus occurs for a short period. Under high osmotic shock, the size of the cells obviously shrink, so did the chloroplasts, mitochondria and Golgi bodies, etc. The membranes of these organelles partially melt with that of endoplasmic reticulum, which makes its surface area increase. By using Inverted Microscope, it was observed that Dunaliella salina of different growth stages after the high osmotic shocks can live in the medium with NaCl concentration between 0.1M and 5.0M, but its growth status and period showed differently. The optimal concentration for the growth of Dunaliella salina was 0.5-1.5M, and this organism could stand a variety range of osmotic shock.Enolase gene, the anti-adversity gene of D. Salina, was cloned by modified degenerate PCR technique. The traditional homogene cloning method was modified on two aspects: first, homo-primers was designed at the sites with minimum degeneracy instead of maximum homology, Secondly, some "N" bases were replaced with inosine in degenerate codes, resulting hi the degeneracy reduced from over 1000 times to below 100 times. A pair of degenerate primers were designed as 5' - GCI YTN CAR GAR TTY AGA T-3' /5' - CAT IAC NCC CCA NCC-3', and a 630bp-long cDNA fragment of D. Salina was obtained, which had high similarity to the enolases from other species. A full-length enolase cDNA of 1734bp from D. Salina (GeneBank accession No. 245549) was acquired by 5' RACE and 3' RACE.The sequence of Enolase gene from D. Salina (DsENO gene) Was analyzed by using bioinformadcs. BLAST search was used to characterize the sequence feature. The nucleic acid sequence of DsENO gene had high similarity to that of other species. The DNA sequence had a complete ORF (open reading frame), which coded a protein of 479 amino acid residues. The protein sequence of enolase which contained the conserved domain, was homologue to enolase of other organisms. It showed 83% ideaity and 89% similarity compared to the enolase in Chlamydomonas reinhardtii. The physical and chemical property of enolase and the predicted three dimensional strucure revealed that DsENO protein was well matched to identified enolase. Multiple sequence alignments and syste...
|
PDF Full Text Request