| In the present study, a transcriptome library and six DGE libraries at different development stages of pearl sac from Pinctada martensi were constructed, sequenced and assembled by RNA-high-throughput sequencing approach with bioinformatics analysis. And then, fluorescent real-time quantitative PCR was employed to examine if the expression patterns of some immunology and biominerization relavent genes at six developmental stages of pearl sac was as corrected as sequenced.Approximately 39,400,004-bp pair end (PE) raw reads from pearl sac of Pinctada martensii was generated by using Illumina/Hiseq-2000 RNA-seq deep sequencing analysis, and the total number of nucleotides was 3,546,000,360 nt. The GC content, Q20 and unknown bases were 42.94%, 94.65% and 0.01%, respectively. Repetitive, low-complexity, and low-quality reads were filtered out prior to assembly of sequence reads for non-redundant consensus. Transcriptome library analysis generated 102,762 Unigenes, of which there are 46,387 functional transcripts with complete or various length encoding regions being identified, and most of them may code proteins with more than 200 animo acid. Approximately 23,081 transcripts may be involved in 219 metabolic or signalling pathways. Blasted with the Nr, Swissprot, GO and COG databases, 36989, 30593, 7700 and 10414 transcripts were annotated, respectively. Additionally,the fuctions of 447 transcripts to be left unkwon. There are 453 Unigenes being associated with immunity and 103 Unigenes with biomineralization.Six DGE libraries of pearl sac which sampled separately at the 5th, 10th, 15th, 20th, 30th and 60th day after neclei-insert-operation were sequenced and analysized. Approximately 5 million raw tags were generated for each of the six DGE libraries. After filtering the low quality tags, the total number of clean tags in each library ranged from 2.5 to 3.4 million, with a mean value of 94.91%. The clean tags of six DGE libraries which can be aligned with that of transcriptome library was only about 26.32%, which means some highly expressed genes did not occur in transcriptome library. Compared the DGE library at the 60th day with other developemt stages of pearl sac, there were 2192, 1902, 2108, 1475 and 1368 genes expressed differentially. Especially, at the 15th day, the number of differencially expressed genes increased apparently. Of the 153 immun-erelevant genes, 30 genes expressed at deferent development of pearl sac with significant difference (P<0.05), they code lysozyme, Alkaline phosphatase, Acidic phosphatase, Toll-like receptor and lectin and so on, respectively. Of the 103 biomineralization-relevant genes, 13 genes expressed at deferent development of pearl sac with significant difference (P<0.05), they code dermatopontin, tyrosinase-like protein, perlwapin, Calmodulin like protein and so on.One immune-relevant gene and one biomineralizati-on-relevant gene which expressed significantly at different stage were choosed to investigate their acture expression levels by quantitative realtime PCR (qRT-PCR). The results show that the expression of lysozyme was at a low level before 30 days of pearl sac development, at the 30th day the expressed level significantly increased, and then decreased at the 60th day, recovering to its original state. Dermatopontin gene did not express differencially before 20 days of pearl sac development, however, its expression level increased significantly at the 30th day, and kept its high expression to the 60th day. The results of qRT-PCR were accord well with those obtained by DGE expression profiling.Our research enriched the information on cDNA database and provided further inferrence to investigate the molecular immune and mineralization mechanism of pearl oyster Pintada martensii. Meanwhile, the discovery and expression study of genes will facilitate genetic breeding of pearl oysters and high quality pearl production. |
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