The Exploration Of The Promoter Activity Area Of Goat Agouti Gene

On the previous study we had gotten a goat Agouti gene sequence with the length of12478bp through joined JN651404with EF587236.2together, we also used softwaresto predict a candidate goat Agouti gene promoter region was6450-7100bp, and the7100bp was the transcription initiation site of goat Agouti gene. In order to test thisresult we start this study for further experiment.Using Primer Premier5.0software designed primers at2kb upstream transcriptioninitiation site area we got a DNA fragment with the length of2537bp, of which1283-1935bp was the predicted promoter area, in this area we had found a typicaltranscription factor binding sites the TATA box, and HSF, SYR, GATA-1, ADR1,Nkx-2transcription factor binding sites and so on, while1895-2533bp had a highersimilarity to human Agouti gene promoter area (the similarity is75%), we predictedGATA-X, GATA-1, GATA-3transcription factor binding sites and so on.According to the distribution of transcription factor binding sites and the principle ofprimer design rationality, the2537bp DNA sequence gotten by PCR as the longestfragment cloning and other fragments designed by using Primer Premier5.0software,we built luciferase report gene plasmid as the following: pGL3-Basic(1-2537),pGL3-Basic (462-1964), pGL3-Basic (462-1817), pGL3-Basic (819-1817),pGL3-Basic (819-1585), pGL3-Basic (819-1402), pGL3-Basic (819-1248),pGL3-Basic (1846-2537), pGL3-Basic (1869-1981), pGL3-Basic (2128-2291).Using GFP，pGL3-Control and pRL-TK plasmid to explore the best transfectionsystem, for the A375cells the liposomes and plasmid volume ratio is1:1,pGL3-Control and pRL-TK ratio is100:1, while the293T cells the liposomes andplasmid volume ratio is1:1, pGL3-Control and pRL-TK ratio is800:1.According to the best transfection system, dual luciferase report gene transienttransfected A375cells and293T cells using plasmid constructed, and the resultanalysised by using SPSS17.0software, the result showed that the A375cells hadhigher transfection rate than293T cells, the result also showed that the constructedplasmids containing missing pieces had no significant difference compared withnegative control, verified that the the2537bp fragment at2kb upstream transcriptioninitiation site area of goat Agouti gene had no promoter activity area.In the subsequent study of goat Agouti promoter, we can continue to obtain goatAgouti5‘UTR sequences by designing primers, and analysis the different shears existing in this gene, ultimately determine the position of transcription initiation site,on the basis of which we can study the promoter activity area of goat Agouti gene.