The Construction Of CDNA And Cloning Of Sox-3from Ovomermis Sinensis

Ovomermis sinensis belongs to Nematouda, Trichosyfingida, Mermithida, Mermithidae, Hexamermithinae, one kind of species that parasite in insects. This species carry through the parasitic life, have a wide range of host and parasite rate is almost equal to host mortality, so it has the great biological control potential. Nowadays people usually spray chemical pesticides to combat agricultural pests, but also indirectly killed the field O. sinensis, resulting in the sharp drop in their numbers and facing with the danger of extinction; At present, The conditions of large scale cultivate O. sinensis in vitro were not mature. The purpose of this study is to save the species' genetic resources to facilitate the screening and studying of related genes by building the O. sinensis cDNA library. cDNA library construction principle is using mRNA of specific tissue and cells as a template, through reverse transcriptase reverse the template into cDNA, after connected the vector and transformed the recipient strain to form a recombinant DNA cloning group. The library that we constructed consists of all the mRNA of the tissue or cell, reflecting its stage-specific gene expression status. Because cDNA library is smaller than the genomic library, so it is facilitate to screen specific genes. Since the first cDNA library was constructed in1976, one after another, standardization of cDNA, chromosome region-specific cDNA library, subtractive cDNA library, yeast two-hybrid cDNA library, the SMART cDNA library construction method appeared. With the development of science and technology, library construction methods are simpler, higher success rate and the quality.In this study, we used Trizol reagent extracted total RNA from the O. sinensis, and using it as reverse transcription template to build cDNA by Creator TM SMARTTM kit that purchased from Clontech Company, then connected with T vector, after E. coli electroporation. coated the product on the surface of LB solid plates with appropriate antibiotics,37℃overnight culture and next day collected the clones, titer the concentration, The experimental datas showed that the cDNA library titer was1.16×109cfu/ml, the number of clones were6.0×106. The recombination rate was approximate to100%. The quality of sinensis cDNA library that we constructed accorded with standard, and could be used for subsequent gene screening and functional studies.Sox gene family highly conserved, widely expressed in birds, fish, reptiles and other animals, research found it plays an important role in sex determination, cell differentiation, nervous system development, early embryonic development, cartilage, and formation of organs. The gene families were researched more in vertebrate. In invertebrate,the gene only been reported in Caenorhabditis elegans and Drosophila. This study was designed to use the O. sinensis as experimental materials to investigate whether this species also exist Sox-3gene, through analysis of homologous conservative sequence, the experimental result indicated we obtained the fragment of Sox-3gene.