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Development of EST-SSR Markers from Laminaria and Cloning and Comparative Studies of Seaweed Trehalose-6-phosphate Synthase Genes

Author: WangGuoLiang
Tutor: LiuTao;WengManLi
School: Ocean University of China
Course: Marine biology
Keywords: Laminaria EST-SSR trehalose-6-phosphate synthase genes gene cloning comparative genetics
CLC: S968.421
Type: Master's thesis
Year: 2010
Downloads: 84
Quote: 1
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Laminaria is one of the most important seaweeds, which is the earliest cultivatedand and high-yielding algae in china. Its farming area and yield-production rank first of seaweed in the world. The main producing area of Laminaria is concentrated in northwest Pacific and Atlantic, especially in China and Japan.Laminaria can be widely used as food, marine drugs, chemical materials and agricultural fertilizers, so it plays an important role in our aquaculture industries. In addition, with huge biomass, Laminaria contributes a large proportion of the primary productivity of the subtidal area, and improves the culture environment and prevent red tide correspondingly, therefore utilization and sustainable development research of Laminaria should be given more and more attention. Heterogenesis, parthenogenesis and apogamy in the life history of Laminaria make it a favourable model organism for genetics and algology, both academically and practically. After introduced to our country, the genetic breeding, propagation and cultivation techniques of Laminaria have made great progress. Compared with cultivation of seaweed, the researches in molecular biological levele are delayed relatively. Rich in polysaccharides and phenolic compounds, numerous chromosomes, and a huge genome, makes the progress in researches for Laminaria genes and new molecular markers more slowly. Nowadays, there are not enough recearches for molecular markers for Laminaria. Through the sequence analysis of 4099 ESTs from Laminaria (971 ESTs were generated from our two constructed cDNA libraries and 3128 ESTs were downloaded from NCBI databases) with SSRIT software (www.gramene.org/db/searches/ssrtool), we found 254 SSRs in 201 ESTs, from which 63 SSR primer-pairs were designed and then tested in optimized SSR reaction conditions using 12 Laminara DNA samples as templates. Results showed that 23 SSR primer-pairs gave good amplification patterns on most (more than 80%) of the testing samples and can be used as SSR markers. The developed 23 SSR markers were used in genetic diversity analysis of 12 selected seaweed species, which belong to red, brown and green algae respectively; the obtained result is basically similar to that from traditional classifications. The developed EST-SSR markers showed a good practicability. They will be useful in molecular biological research in Laminaria, especially for functional gene research.Trehalose is a non-reducing disaccharide sugar, composed of two glucose units, and can be found in many organisms. It could serve as a protective agent against heat, and saline-alkali stresses. And this character makes trehalose synthase gene as the target gene for plant gene transformation to improve its drought-salinity tolerance and adverse resistance. In this study, the trehalose-6-phosphate synthase gene (TPS) was isolated by homologous cloning method from Laminaria japonica and other 9 seaweed species. The obtained ORF (open reading frame) contained 2727bp. Sequences of the obtained TPS genes from seaweeds were deposited in GenBank. All of the ten seaweed TPS genes encoded peptides of 908 amino acids and the sequences were highly conservative both in nucleotide composition (>94.4%) and in amino acid composition (>96.6%). Homology alignment for seaweed TPS and the TPS proteins from bacteria, yeast and higher plants indicated that, unlike the TPS genes from some other plants, there was no intron existed in all of the ten isolated seaweed TPS genes. And the most closely related sequences to seaweed TPS gene were those from higher plants (OsTPS and AtTPS5, about 40% similarity), whereas the most distant sequence was the one from bacteria (EcOtsAB, about 20% similarity). Based on the living environment, LyTPS and other seaweed TPS genes are considered as good candidate gene resources for genetic improvement of the drought- and salt-resitance in crops. If the seaweed TPS genes are transformed into crops, the transgenic plants are expected to increase salt/ drought resistance. That will be useful in crop improvement and with bright application furture.

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CLC: > Agricultural Sciences > Aquaculture, fisheries > Aquaculture technology > A variety of seafood, animal and plant breeding > Algae farming > Brown algae > Kelp
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