CDNA Library Construction Of Planarian (Dujesia Japonica) And The Phylogenetic Analysis,Expression Of DjGon Gene

Dujesia japonica is the typical in the phylum Platyhelminthes and shares with vertebrates keys traits such as bilateral,symmetry,three germ layers-ectoderm,mesoderm and endoderm, and dorsoventral and anteroposterior polarities.Due to these characters and its remarkable regeneration,it is the model animal to study the origin and evolution of the vertebrates, embryogenesis,chromosomes,stem cells and regeneration.In order to separate and identify the developmental and regenerative gene from Dujesia japonica,the total RNA as template are constructed the cDNA library,according to SMART^TM cDNA Library Construction Kit and Advantage 2 PCR Kit.The unamplified cDNA library contained 1.22×10⁵ independent clones in capability.The recombination rate was more than 98%.The average inserted cDNA fragments were about 900 bp,with the majority ranging from 500 to 1500 bp.From the library,we randomly selected some DNA fragments to sequence and blast in NCBI database on line.The results showed that it contained 208 rRNA genes,148 encoding genes and 78 genes in the chromosome.It is undoubted that our result established a solid molecular basis for the depth study of development and regeneration in planarian.Planarian DjGon encoding 249 amino acids were identified from the cDNA library of the Dujesia japonica.Structural analysis suggested that it was a member of ADAMTS protein family,with the conserved domain GON.The deduced planarian protein has more than 30% identity with some mammals.And the molecular analysis shows that the planarian gene may be represent the precursor gene that existed prior to the origin of ADAMTS-9 and-20.The paper used the molecular blot to study the expression pattern of this DjGon gene in Dujesia japonica.The southern blot revealed that this gene has two copies in Dujesia japonica. The whole mount in situ hybridization blot suggested that DjGon gene expression is restricted mainly to the gut and dermo-muscular lac in the planarian larvae.And the signal in gut is stronger than that in dermo-muscular lac.