Sreening Of Gonad Development-related Genes And Cloning And Expression Analysis Of Cf-phb2in Chlamys Farreri

The gonadal development is one of the important fields in development biology,which is regulated by many genes. PHB (Prohibitin) proteins participate in severalbiological processes, including negative regulation to transcriptional activity of sometranscription factors and to the cell cycle as well as suppression of tumor andapoptosis. In this study I detected10Tags of the differentially expresses profilebetween the testis and ovary at proliferative stage in Chlamys farreri bysemi-quantitative RT-PCR; cloned a full-length cDNA of C. farreri phb2by RACEtechnique, and analysed its spatial expression in tissues by semi-quatitaitive RT-PCRas well as expression pattern of the Cf-phb2in C. farreri testis by Real-Time PCR andin situ hybridization. The main results as follows:By semi-quatitative RT-PCR detection of10selected tags,3tags were highlyexpression in ovary tags and4tags were highly expression in testis.The coincidencerate was40%. Furhermore, one testis highly expression tag>F0BWI0G01A0M8X wasselected to clone its sequence and study its expression characteristics. The full-lengthcDNA sequence was2,122bp and contained an882bp open reading frame (ORF)that encoding a deduced protein of293amino acids, and was finally judged to bephb2of C. farreri (Cf-phb2) by sequence alignment and homology analysis.Semi-quantitative RT-PCR detected that the Cf-phb2expressed in all detected C.farreri organs, and the expression was highest in testis. Furthermore, the Cf-phb2mRNA was detected in the testes of4developmental stages (resting stage, proliferative stage, growing stage and mature stage) during the reproductive cycle inC. farreri using quantitative real-time PCR technique, and the expression of Cf-phb2increased with the testis development, which was the lowest in that of resting stageand the highest in that of mature stage which was about15-fold higher than that ofproliferative stage. Moreover, positive signals of the Cf-phb2were observed in alltypes of germ cells in the testis, including spermatogonia, spermatocytes, spermatidsand spermatozoa by in situ hybridization technique. Intensity of the positive signalsincreased with spermatogenesis except the very faint expression in spermatozoa,suggesting the Cf-phb2participats in the spermatogenesis in C. farreri, and may playa potential role in cell proliferation suppression of spermatogenesis in C. farreri testis.