CDNA Library Construction,EST Analysis Of Nannochloropsis Oculata And Primarily Study On The Cloning Of Δ6 Desaturase Gene

Nannochloropsis oculata (Eustigmatophyceae), a marine eukaryotic unicellular alga, which is considered to be of high nutritional value owing to its large amounts of proteins, polyunsaturated fatty acids (especially EPA) and diverse valuable pigments.In respect that it grows well under conditions of mass culture, N. oculata has considerable potential for commercial exploitation. However, previous studies of this microalga focused on its taxonomy, culture and biochemistry and little has been known at the molecular level until now. Therefore, a cDNA library was constructed as follows:1)Algal culture.Nannochloropsis oculata was grown in 250 mL flasks containing 150 mL sterilized seawater enriched with f/2 medium. The cultivation was conducted at 20±1℃and 70μmol photon s-1m-2 white fluorescent light on 12h/12h light/dark rhythm.2)Construction of cDNA library.Cells were collected at the middle growth phase. Total RNA was extracted by the guanidine thiocyanate-phenol-chloroform extraction method. Total RNA were run on agarose gel to investigate the quality. cDNA synthesis was performed and cDNA was inserted into EcoR I sites of pBluescript. A cDNA library consisting of 2.5×105 clones was constructed and over 95% of the clones were recombinants determined through X-Gal/IPTG screening. The insert size was determined by PCR, a rough estimate was made for randomly chosen clones,which ranged from 500 to 1000 bp.3)EST analysisA total of 5812 clones were then sequenced, a collection of 5315 EST reads was obtained. Sequence lengths varied from 100 to 716 bp, with an average of 420bp. The trimmed reads were clustered and assembled, yielding 668 contigs. Each sequence from the non-redundant collection was translated into amino acid sequences and subjected to similarity search against nr database by means of the BLASTX algorithm. Similarity between a deduced amino acid sequence and a known sequence was judged to be significant when the E value was less than 1e-04. As a result, only 637sequences showed significant similarity to proteins registered in nr database, and the remainder showed only weak similarity or no similarity. Among the sequences that could be annotated, there are 9 NRSs refered to lipid metabolism, interestingly, Contig638 shows highly sequence similarity to cyclophilin, and ZGW0529 is highly similar (87% amino acid identity) to sequence ofα-dioxygenase. In addition, such as Ribosomal proteins and light harvesting complex components are comparatively abundant.4)Cloning ofΔ6 desaturase geneDegenerate PCR used to findΔ6 desaturase turned to a falure. This work set the molecular basis for further studies on Nannochloropsis oculata to better understand its biochemical, physiological and cellular processes.