Syti Molecular Characterization Analysis And Mirna Regulation Of Silk Protein

Synaptotagmins(syt) is a family of membrane transporter proteins that are present in the nervous system;it contains two calcium-binding domains:C2A and C2B.It induced synaptic vesicles and exocytosis and the release of neurotransmitters through the control Ca²⁺ influx.In this paper,some molecular characteristics of syt I affecting the occurrence of RNA editing and the organization of C2 domain are expatiated on.RNA editing is a major mechanism of regulating biological function in organism. In this article,we unexpectedly discover that there are much lower GC3 and GC-content and higher Gibbs free energy on editing exons than other exons in the Drosophila syt I Uranscripts,which was further,confirmed statistically by others 47 experimentally-validated samples.Sequence alignment,Ks and Ka/Ks assays suggest that rapidly ascending purifying selection occur in editing exons which constrains nucleotide divergency.The presence of specific molecular characteristics such as lower GC-content in editing exons imply an unexpected requirement and are likely to direct RNA editing occurrence.Thus,relations between molecular characteristics of DNA,RNA editing and purifying selection might be present.The GC content mutation anasysis of syt I further indicate that some changes in the characterization of DNA molecules,such as increasing or reducing GC content,can significantly affect the editing efficiency.This is perhaps related to the molecular activity.Another molecular characteristic of syt I is that it includes conserved C2 domain.In order to further study its biological role in evolution,we have identified 104,44,44 and 142 genes containing 154,65,63 and 218 C2 domains in Arabidopsis,Caenorhabditis, Drosophila and Homo,respectively.Phylogenetic analysis indicates that the C2 superfamily is composed a number of conserved subfamilies or clades and no C2 domain clades simultaneously containing animal and plant are detected,indicating independent origins of C2 domain genes.Conservation of the exon-intron structures within individual clades lends further support for the evolutionary relationships deduced from amino acid sequence similarity.The uneven distribution of intron phases implies specific evolution selection.We observe intron loss,not only from the 3'-region,but also from other regions of the coding sequence,with implications for the molecular mechanisms of intron deletion.MicroRNAs(miRNAs) are an extensive class of small noncoding RNAs with important roles in the regulation of gene expression.No study has been preformed on identifying miRNAs and their targets in Bombyx mori(silkworm) although silk is one of the most important fabric and staples of international trade around the world.In this study,we identify 41 miRNAs which belong to 37 miRNA families and 12 miRNAs of them are selected at random to be experimentally confirmed.Some characteristics such as position-specific nucleotide distribution;evolutionary conservation and clustered organization have been described.Next,we also identify a total of 129 potential miRNA targets in silkworm genome.GO molecular function analysis indicates a majority of these targets holding binding;enzyme and transcription regulator activity,suggesting that miRNAs take part in a variety of critical biological processes,such as development,cell proliferation,differentiation, apoptosis and metabolic regularity.We also find that at least 4 miRNAs (miR-33/-190/-276/-7) target fibroin,suggesting that miRNAs play an important role in silk development.Next,the 3'UTR of the silk fibroin was cloned and mutated on the two miRNA binding sites.We constructed four expression vectors using these segments linked to the back of report gene(GUS).And we also constructed four defferent miRNA-expression vectors based on their sequence and mutation. Agrobacterium-mediated transient assays for GUS staining,PR-PCR and GUS activity indicated that 33-miR and 7-miR can regulate the expression of fibrin and that such regulation was based on the complementary combination between miRNA and target sites.This study also found that synthetic miRNA can play a role on the regulation of target gene expression.In addition,compared with the normally used cell transformation or stable transgenic measure,leaves transient expression system is a highly efficient and easy way which can be used with a variety of miRNA regulation in the flora and fauna.