Identify The Substrates Interacting With DX16 Through SELEX

Alternative splicing of pre-mRNA, which happens in high frequency and generally exists in genome, is the main reason that increases the diversity of proteins and multiplicity of gene expression. Recent research shows that some Ser and Arg amino acid rich proteins (SR protein) play an essential role in alternative splicing. SR protein is a kind of splicing factors that help recognition splicing site correctly and promote mature of splicesome,they are essential to constitutive splicing and have pivotal contribution to alternative splicing.Dxl6 gene, a novel gene encoding SR protein, has been cloned in 2000. According to its amino acid and nuclear sequence, Dxl6 is considered as the candidate gene of splicing factors. In situ hybridization shows that Dxl6 expresses mainly in nervous system in later embryo stage and is thought to associate with nervous development. The 2-nucleotide deletion mutation induced by EMS results in lethal to drosophila in later embryo stage or first instar-larvae. The insertion mutation (inserted in intron region) caused by P element results in lethal to drosophila in later pupae, although there is 5 percent escaper of P element k00230 insertion, these escapers have abnormity in bristles, wings, legs, eyes and the like.The aim of the research is to study the role that Dxl6 plays in fly development in central nervous system in embryo as well as after embryo stage, SELEX is used to find the substrates interacting with Dxl6 during splicing procedure.First of all, clone the gene fragments including the two RRM regions into prokaryotic expression vector, and finally the protein is expressed in E.coli in a soluble condition and purified successfully. It provides a good tool for the next experiment.Dxl6N and a random RNA library are hatched together in a certain condition, after six rounds of SELEX selections, the sequences that interact with Dxl6 are gotten. With the help of biological information analysis, the consensus sequence of candidate genes and their common motif and second structure are acquired. RT-PCR analysis the mRNA expression level of the target genes in Dxl6 mutation fly lines and wild type flies. The results lay a basis for studying splicing in vitro and more regulation mechanism of Dxl6 in fly development.