Identification Of Novel Genes Involved In Ovarian Follicular Development In Shaoxing Duck

The silver staining differential display method was employed to identify the novel genes involved in ovarian follicular development in Shaoxing ducks. 5' RACE was used to extend cDNA fragment. The expression patterns of discovered ESTs were investigated to elucidate their possible biological functions on hierarchical follicular development in Shaoxing ducks.1. Differential display of genes expressed in hierarchical follicles in Shaoxing ducks3 expressed sequence tags (ESTs) were identified by silver staining differential display. The analysis results in National Center for Biotechnology (NCBI) GenBank/BLAST database revealed that 1 of them, named SXDF0201 (271bp), was a novel EST, and it was then registered in GenBank (GenBank Account: CB072629). EST_SXDF0201 was found to be highly expressed in smaller hierarchical follicles (p<0.01), such as Fw, F5 follicles by Semi-quantitative RT-PCR. Another EST (SXDF0202) was expressed stronger in Fw follicle (p<0.05), although no significant difference shown among Fl, F3, F5 follicles.2. The extension of EST_SXDF0201 by 5' RACE544bp EST_SXDF0201 was extended by 5' RACE. The BLAST research further confirmed that the EST_SXDF0201 was a novel EST in the follicles of Shaoxing ducks.3. The temporal and spatial expression patterns of identified ESTsI. The expression patterns of ESTs in granulose and theca layers of ovarian follicles In this series of experiment, granulose layers and theca layers were separated from hierarchical follicles to study the expression patterns of identified ESTs. The result showed that granulose layers was expressed higher ESTJSXDF0201 than theca layers in almost all hierarchical follicles, but no differences were observed among hierarchical follicles hi the same layer; The expression of EST_SXDF0202 ingranulose layers was increased along with follicular maturation (p<0.01) from Fw to F3 follicles, but decreased dramatically to the lowest in pre-ovulatory F1 follicles (p<0.01). In Theca layers, the highest expression of EST_SXDF0202 was found in Fw follicles (p<0.01).II. The expression of ESTs in different tissuesTotal RNA was extracted from hypothalamus, pituitary, muscle, liver, and fat tissue of Shaoxing ducks at laying peak for the study of EST tissue-specific expression by RT-PCR, with beta-actin as an internal standard. The result revealed that neither EST_SXDF0201 nor EST_SXDF0202 was ovary-specific, but expressed in all of investigated tissues. High expression level of EST_SXDF0201 was found in hypothalamus and pituitary whereas muscle and fat tissues had lower expression level.III. The expression patterns of ESTs in different stages of ovary developmentThe semi-quantitative RT-PCR was applied to investigate the expression pattern of those ESTs in developmental stages of Shaoxing duck ovary at 30, 60 and 90 days of age. The result showed that EST_SXDF0201 was significantly higher expressed in D30 than D60 (p<0.05) and D90 (p=0.015), but the expression of EST_SXDF0202 was almost consistent throughout the ovarian development.