Construction And Partial ESTs Analysis Of A Normalized Full-length CDNA Library Of Myrmeleon Sagax

Medicinal insects Myrmeleon sagax's larvae known as ant-lion, is Neuroptera Myrmeleontidae insects. Since ancient times, ant-lion was used to treat a variety of incurable diseases on traditional medicine. Its dry powder can be used for treatment of urinary tract stones, vasculitis, hypertension, otitis media and thrombosis and other chronic diseases. Ant-lion body may contain a variety of antimicrobial proteins and some unknown proteins, and these proteins may have medicinal function and special use.In this study, the mRNA was isolated from the ant-lions whole insect which induced by E. coli before and after induction of 3hr,6hr and 12hr periods. The normalized full-length cDNA library was constructed with pDNR-LIB as a carrier using SMART (Switching Mechanism At 5 'end of RNA Transcript) technique combined with duplex-specific nuclease enzyme normalization. Library titer was calculated by plate counting and insert fragments were evaluated by selected colony PCR. The results showed that the titer of the original library was 7.5 x 105 cfu/ml, while the amplified library was 1.95×1011cfu/ml, the library average insert length is 1200 bps.192 clones were randomly selected from the library for the 5 'end EST sequencing and obtained 190 valid sequences with average read length of 1340bps. The recombination rate of library is 98.96% and the sequences information has been submitted to GenBank, accession number:HO059798-HO059987. Using ESTpiper software platforms for processing and assembling EST sequences and 174 unique sequences (Singlets) and 5 contigs were gained. In 179 unigenes, the averaged length of sequences are mainly in the 650bps-850bps with an average length of 683bp.The library non-redundant rate is 97.26% and meet large-scale EST sequencing requirements.Unigenes were annotated and classified of GO team by Blast2go and DNAstar softwares, the results indicated that a variety of potential medicinal function related genes were found in ant-lion:1) insect immune-ralated genes, including defensin-1 precursor, defensin-C and attacin-E; 2) cardiovascular-related genes, such as angiotensin receptor-related protein and the enzymes kallikrein; 3) Digestion-related enzymes, such as chymotrypsin, serine protease, elastase, protease and trypsin, etc.; addition, there are a variety of energy metabolism and other related proteins. These enzymes or proteins can be expressed by a large-scale genetic engineering, for medicine, food, livestock breeding, bio-pesticides and other fields.