Expressed sequence tag analysis and cloning of trehalose-6-phosphate synthase gene from marine alga Laminaria japonica (Phaeophyta)
doi: 10.1007/s13131-012-0260-6
Expressed sequence tag analysis and cloning of trehalose-6-phosphate synthase gene from marine alga Laminaria japonica (Phaeophyta)
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摘要: A high quality cDNA library was constructed from the brown alga Laminaria japonica, with the titer of 1.2×105 pfu/ml. The average insert size of the cDNA library is about 1.6 kb. From the cDNA library, 591 cDNA clones were randomly selected and sequenced. As a result, 574 EST (expressed sequence tag) sequences were generated. All of the 574 ESTs were submitted to the dbEST database section of GenBank with the accession numbers from CX942625 to CX943198. The cDNA library was screened with a α-32p labeled 453 bp TPS gene probe, which is a partial sequence yielded from Porphyra yezoensis. Four positive cDNA clones were screened and the sequencing data showed that these four cDNA clones covered majority of L. japonica TPS cDNA sequence. After PCR amplification, sequencing and assembling, the entire ORF (open reading frame) sequence of the TPS gene was obtained, which was named LjTPS. LjTPS encodes a protein containing 908 amino acids with a calculated molecular mass of 101 674 Daltons. The LjTPS gene was successfully expressed in E. coli and rice. The LjTPS gene has potential application both in plant breeding to stress tolerance and in deciphering the TPS gene function and mechanism to stress tolerance.Abstract: A high quality cDNA library was constructed from the brown alga Laminaria japonica, with the titer of 1.2×105 pfu/ml. The average insert size of the cDNA library is about 1.6 kb. From the cDNA library, 591 cDNA clones were randomly selected and sequenced. As a result, 574 EST (expressed sequence tag) sequences were generated. All of the 574 ESTs were submitted to the dbEST database section of GenBank with the accession numbers from CX942625 to CX943198. The cDNA library was screened with a α-32p labeled 453 bp TPS gene probe, which is a partial sequence yielded from Porphyra yezoensis. Four positive cDNA clones were screened and the sequencing data showed that these four cDNA clones covered majority of L. japonica TPS cDNA sequence. After PCR amplification, sequencing and assembling, the entire ORF (open reading frame) sequence of the TPS gene was obtained, which was named LjTPS. LjTPS encodes a protein containing 908 amino acids with a calculated molecular mass of 101 674 Daltons. The LjTPS gene was successfully expressed in E. coli and rice. The LjTPS gene has potential application both in plant breeding to stress tolerance and in deciphering the TPS gene function and mechanism to stress tolerance.
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