Construction And Sequencing Of Normalized Full-length CDNA Library And Analysis Of Bioinformatics In Common Carp (Cyprinus Carpio)

The common carp (Cyprinus carpio) is a widespread freshwater fish of eutrophic waters inlakes and large rivers in Europe and Asia. To provide reference sequences for transcriptomeanalysis and assist in differentiating duplicated gene copies, a full-length cDNA library ofcommon carp (Cyprinus carpio) was constructed via Cap-trapper method. mRNAs from eighttissues of an adult female common carp, including blood, gill, ovary, liver, head kidney, kidney,heart, and brain, were used for library construction. Total number of colony-forming units (CFU)of the primary full-length cDNA library was calculated to be6.4×106. After normalization withgenomic DNA, the redundancy of the normalized cDNA library was reduced by87folds. CFUof the normalized cDNA library was2.0×105, which was enough to cover all the common carpgenes and their potential duplicates. The average insert size was estimated to be1.0kb with arecombination of about100%.RandomLy selected43008cDNA clones were sequenced from5’ end by use of M13F (-20)primer, in order to further characterize the constructed cDNA library.29779EST sequences wereobtained from sequenced. After removed, these EST sequences were assembled into15116unigenes. Genes GC concent analysis demonstrated that expressed gene of common carp had aGC concent of45.53％with highly approaching zebra fish. By use of the BLASTX analysis,8437unique sequences were campared to Gene Ontology (GO) databases. Gene Ontology (GO)analysis indicated that the constructed cDNA library exhibited well gene diversity in biologicalfunction.13,12and22of groups were assigned to biological process, molecular function, andcellular component GO categories, respectively.Some genes involved in hypoxia of Rab1a were identified in this libary, named by Rab1a-1、Rab1a-2and Rab1a-3, repectively．The structure of Rab1a gene and protein were analyzed bysoftware. Subsequently, Rab1a were analyzed by qPCR under hypoxia.