| The rate of ovulation is determined by, among other things, the availability of small follicles that can be recruited into the follicular hierarchy. A decrease in the rate of lay with, for instance, aging has been attributed to both an increase in the rate of atresia and a decrease in the number of small, growing follicles that provide the pool from which follicles are selected into the final growth phase (the preovulatory hierarchy). Previous studies on follicles of human and mammal ian have shown that the growth and differ entiation of follicles is a complex physiological and biochemical process regulated by many factors, and regulated by not only endocrine hormone of reproductive axis, but also many of the paracrine and autocrine factors secreted by the local ovarian. For research on avian ovarian follicle development, some differentially expressed genes have already been detected in different development stages, but to complex organism is concerned, it is only one small part. Until now, there is still no reasonable hypothesis to illustrate the mechanism of avian hierarchal follicle establishment. Through the screening and comparing of the differentially expressed genes in duck follicles of different development stages, we can furtherly understand the effects of these genes on the process of follicular growth and differentiation, the molecular biology mechanism of the poultry sexual maturity, the start of laying and maintenance of a high yield performance. This study will provide a theoretical foundation for finally revealing the mechanism of avian hierarchal follicle establishment. A better understanding of differentially expressed genes during follicle selection and differentiation should lead to the development of management practices or genetic manipulations (molecular), which could enhance the rate of laying at times during the production cycles when egg production is suboptimal. In this experiment, we used the follicles of Shaoxing duck as the experimental material, and the DSN mediated normalization subtractive hybridization method to construct two forward-and reverse-substracted cDNA libraries of follicular development-related genes, thus we can find the new genes, and explore the molecular mechanism of avian hierarchal follicle establishment. The results are as follows:The first forward-and reverse-substracted cDNA libraries between Fl and PF were constructed by SSH technique. In the forward-subs tracted cDNA library, the follicles of F1and PF were used as tester and driver, respectively. Conversely, the reverse library was constructed. Results showed that the qualities total RNA and reverse transcription of cDNA were very good, and the products of SSH were well enr iched. In the forward library, a total of88differentially expressed genes were obtained. After analysis by BLAST, the results showed that60genes have homo logy and the other28genes have no homology from NCBI. Further analysis showed that some important functional genes were isolated in the forward library, including the LHR, ECFR, SLC family, NADH1β, PRKG2and AKAP2, etc. In the reverse-substracted cDNA library, a total of72differentially expressed genes were obtained. After analysis by BLAST, the results showed that51genes have homology and the other21genes have no homology from NCBI. The genes were isolated, including RBMII, p32and U2AF subunit of splicing factor SF2, tensin, EIF3I, α-tubulin, ACTG2, CD58, MCM5, RCC2, Flotillin-2and some other genes encoding enzymes and their homologies, such as FG3ATPase family gene3-like2, AK3L2and TAOK3.The second forward-and reverse-substracted cDNA libraries between Fl and F6were constructed by SSH technique. In the forward-substracted cDNA library, the follicles of Fl and F6were used as tester and driver, respectively. Conversely, the reverse library was constructed. Resuts showed that the qualities of total RNA and reverse transcription of cDNA were very good, and the products of SSH were well enriched. In the forward library, a total of79differentially expressed genes were obtained. After analysis by BLAST, the results showed that67genes have homology and the other12genes have no homology from NCBI. Further analysis showed that some important functional genes were isolated in the library, including EGFR, ADAMTS-1, MAGUK family, SLC family, lymphocyte antigen86-like, IL17RA, and death domain-containing tumor necrosis factor receptor superfami ly member23. Some genes encoding channel or transporter proteins were isolated, such as sodium/myo-inositol cotransporter-like, calcium/calmodulin-dependent serine protein kinase, SLC9A8, potassium inwardly-rectifying channel, subfamily J, member2. SLC2A1and PCTP, etc. In addition, RNF14, TTC17, PHF21A, PLEKHB2, ADD3, RGS12, SVEP1, HDAC4, STOM, SMARCE1, SETD4, CEBPG, STRADA, ZC3H14, RHOBTB1, ALDH1A2, EXOC2and GAPVD genes were also isolated in this forward library. In the reverse-substracted cDNA library, a total of37differentially expressed genes were obtained. After analysis by BLAST, the results showed that29genes have homology and the other8genes have no homology from NCBI. In this library RBMII, α-tubulin1c, CD58, EIF3I and MCM5genes were isolated. |
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