Analysis Of The Functional Gene Rpl11 Related To Mammary Gland Development Of Dairy Goat

With the developed of the research areas of life sciences,more and more new genes and functions of genes were discovered.It would be the main task to study the function of these genes in the next few years.An optimized procedure of RACE was adopted to amplify Rpl11 gene sequence in Long-SAGE tags of dairy goat mammary gland.SMARTTM RACE c DNA Ampliication Kit was used to m RNA of dairy goat mammary gland tissue reverse transcribe to c DNA by RT-PCR.3? and 5? c DNA fragments of Rpl11 gene were extended by 3? RACE and 5? RACE respectively.Products of PCR were T/A-cloned and transformed to E.coli.Positive colonies were sequenced.Total length Rpl11 gene was obtained by sequence contrasting and splicing.Complete Rpl11 sequence of dairy goat mammary gland were successfully identified by applying this high-throughput procedure of Rpl11.Bioinformatics analysis for sequence,Rpl11 gene encoding 246 amino acids,amino acid sequence homology by NCBI BLAST tool analysis,the highest homology is Bos(Rpl11,m RNA homology 95%),followed by Sus(Rpl11,m RNA homology 93%),again for the Hs(Rpl11,m RNA homology of 91%).The sh RNA expression vector targeting Rpl11 gene was designed and constructed.Dairy goat mammary epithelial cells were transfected with p IRES2-EGFP fusion protein expression vector and sh RNA expression vector transiently and the cells without sh RNA-transfection and with non-specific sh RNA transfection were used as negetaive controls.Inhibitory effect of RNAi was detected real-time fluorescence quantificational RT–PCR tranfected before and after.Effective inhibition of Rpl11 gene expression by plasmid-based RNAi provides analternative for dairy goat mammary epithelial cells.With breast development hormonal Esr,Pgr gene expression changes.The result show that after transiently transfeced 48 h compared with blank controls,transfected Rpl11 m RNA expression was significantly inhibited(p<0.05).Negetaive controls and blank controls were not significant(p>0.05),stable transfected 30 days,transfected Rpl11 m RNA expression was significantly lower than the negative control group(p<0.05),negetaive control and blank control is not significant(p>0.05).In this study,the gene of Rpl11 was cloned and eukaryotic expression vector p IRES2-EGFP-Rpl11 was constructed.Liposome techinigue was used to transfect dairy goat mammary epithelial cell.By G418 selecting a stable transfected cell line was gained.The stable transfected cells were screened by G418 with 500?g/m L.The expression changes of Rpl11 Esr Pgr gene were analyzed by RT-PCR.The results show that transiently transfeced 48 h compared with blank controls,transfected Rpl11 m RNA expression was significantly inhibited(p<0.05).Negetaive controls and blank controls were not significant(p>0.05).The results show that steadly transfeced 30 d compared with blank controls,transfected Pgr ?Esr m RNA expression was significantly increased(p<0.05).Negetaive controls and blank controls were not significant(p>0.05).