Construction Of CDNA Library And Analysis Of Expressed Sequence Tag In Haploid 10 Of Laminaria Japonica

Laminaria is one of the most important economic marine macroalga, both farming area and production of Laminaria of China rank first in the world. Laminaria can be used as food, chemical material and cardiovascular drugs, so it plays an irreplaceable role in the national economy and people's lives. Laminaria, with tremendous biomass, contributes greatly in the primary productivity of the subtidal area in warm temperate zone of the Pacific and Atlantic Ocean, and constructs high bio-diversity and productivity ecosystem with its living marine environment. Heterogenesis, parthenogenesis and apogamy of Laminaria make it a better model organism for algology and genetics researches.Rich polysaccharides and phenolic compounds, numerous chromosomes (2n=44), and a huge genome(434Mb~720Mb) of Laminaria, bring many problems for the study of molecular genetics of Laminaria. Researches for Laminaria gene and new molecular marker are extremely slow; as of today, the quantity of Laminaria Expressed Sequence Tags published is only 3496. And there are no reports about SSR markers that can be directly applied to the genetic characteristics study.This experiment has constructed the cDNA library for Haploid 10 of Laminaria japonica, and then carry out EST sequencing and bioinformatics analysis. The results are listed here:(1) Extract total RNA respectively by guanidine isothiocyanate, Trizol and Total RNA Extraction Kits, then carry out UV detection and gel electrophoresis. The results show that all of the three methods could get integrated and high-quality total RNA of Laminaria japonica gametophyte, and guanidine isothiocyanate method is the best one.(2) Isolate mRNA from total RNA using magnetic particles, and synthesize double-stranded cDNA with cDNA synthesis kit (Stratagene), then collect segments over 500bp with QIAquick gel extraction kit (QIAGEN), followed by connecting to carrier PBluescriptⅡSK(+) and transfecting DH10B E.coli. Finally the cDNA library is constructed, with 1.31×106 clones and 87% recombination rate.(3) Based on cDNA library, large-scale sequencing puts out 1478 ESTs. 805 valid sequences of more than 100bp after wiping off carrier and low-quality sequences are got. These ESTs stitch to 76 contigs and 321 singlets. 119 of the contigs and singlets match after Blastx and NCBI non-redundant protein database comparation, which accout for 30% of all; 52 of the contigs and singlets match after Blastn and NCBI non-redundant nucleic acid database comparation, which is 13.1%. 42 sequences can be searched from both of these two databases. 58 of the contigs and singlets match when compared with dbEST of Laminaria, which is 14.6%. Gene Ontology of the 119 contigs and singlets show that 109 attribute to cellular component, molecular function, biological processs categories, while the function of the remaining 10 contigs and singlets are unkown.The ESTs gained here offer direct information for Laminaria genomics, and lay good foundation for Laminaria biological research and application.