Construction And Analysis Of Subtractive Library About The Goldfish Wen-related Genes

Goldfish belonged to Cypriniformes, Cyprinidae, Carassius in taxonomy and was Chinese unique aquarium fish which originated from red carp at the Jin dynasty period and had experienced many generations of hybrid. Although people could obtain goldfish with high ornamental value, the breeding methods were still traditional selection and hybridization with low efficiency and randomness. Wen-class goldfish was popular for the ornamental value of the wen among all kinds of goldfish varieties, currently study had discussed the biochemical composition and structure of the wen in some goldfish species, but the functional gene of goldfish and their mechanism of action remained unknown.In this study suppression subtractive hybridization (SSH) strategy was employed to construct two libraries that represented genes related to wen. Wen tissue of1-year oranda goldfish was treated as the tester, epithelial tissue on the head of40-days oranda fry and red carp were treated as the driver. After hybridization the housekeeping gene a-tubilin was used to detect the efficiency of libraries, and a-tubilin mRNA abundance was decreased by up to210-fold in the subtracted libraries compared with the unsubtracted samples, indicating that the libraries was successful. A total of243ESTs were identified by nest PCR and dot-blot screen and sequenced. Sequences were checked for homologies in the GenBank nr database by using the Blast search, only matched with E-values smaller than10e-5were retained as significant. By Blast analysis163sequences of the libraries were identified as homologues of genes encoding proteins possessing known functions(E-values＜10-5),35sequences were identified as unknown genes (hypothetical proteins), and45sequences yielded no BlastHits or had poor similarity (E-values≥10-5).The sequences were divided into several potential categories according to GO functional annotations, as a result the most highly represented classes were binding, catalytic activity, and transporter activity.Uniprot database was used to analyse function, structure, mechanism and tissue specificity of related genes and we selected a set of genes that could be involved in wen formation process of oranda goldfish. To further confirm and analysis wen related genes, analysis of tissue distribution and expression levels of putative genes were performed. Skin、wen、liver、spleen、gut、kidney、heart、muscle and gill of adult oranda were extracted to investigate tissue distribution of putative genes by semi-quantitative RT-PCR. As a result, five genes that showed tissue specificity in the process of wen developed were identified in the two SSH libraries. Expression levels of these five genes were achieved by real-time quantitative PCR, the tester of two libraries(1-year oranda) was compared with2-months oranda (wen-like would just begin to form) and4-months oranda (wen was just formed), these genes all presented different expression patterns:the gene expression quantities of RhoV, K18and PTX3had a significant increase during2-months oranda and4-months oranda, Fat2changed more during4-months oranda and1-year oranda, MAM2kept stable relatively during the whole process of wen growth.