Analyzing Differential Sex Gene Of Tilapia Using A Siliver Staining Based DD-PCR Protocol

1.Analyzing Differential Sex Gene of Tilapia Using a Silver Staining BasedDDRT-PCR Protocol.To develop mRNA differential display polymerase chain reaction (DD-PCR) method with silver staining , isolate and clone sexal differential genes in Tilapias. Total RNA were extracted from the gonads of two types of Tilapias.The cDNA copies of differential expressed mRNA species were amplified by DD-PCR using an anchored primer 5' dTuN, combined with two of arbitrary' primers and one long primer, respectively. The DNA bands on gel were displayed by silver stain method. After running on 10% polyacrylamide gels, the differential displayed DNA bands were visually recovered from the gel and reamplified by PCR. Test by agrose gel ,we get four differential bands. The reamplified products were cloned into pUCm-T vector, and send to biotech compare for sequence analyzing. Speculate the structure and fuction of the differential gene by comparing its sequence with multiple public bioinformation data. Four gene were found in this research , two of them were come from the O.reochromis male, two of them were come from the O.niloticus male, these genes were considerated as new genes after many types of analyses2.compare of SRY sequence between different type TilapiasThe Sex-determining region Y (SRY) is a strong candidate for the mammalian testis determining factor. In this article using the primers which specially amplify the 600bp fragments of human SRY gene ,we studied O.reochromis aureus and O.niloticus . The results showed that both males and females all have three band in these tilapias . the amplified product which are about 600bp were cloned into pUCm-T vector and were sepuenceed . these sequence of Tilapias were analyzed by clustalW method . the result showed there was no difference between male and female, no differences were between these two species. .