The Construction And Analysis Of Gonad Suppression Subtractive Hybridization Library Of Monopterus Albus

Rice field eel (Monopterus albus) was used as experimental material in this study. Applying histological section technology, we had made the paraffin section of the ovary, testis, and inner sex gonad. We selected ovarian tissue and testis tissue in stage IV as material. Two subtractive libraries were successfully constructed. Positive clones from the libraries were screened and sequenced. By preliminarily analyzing the bioinformatics of these expressed sequence tags (ESTs), we tried to get some differentially expressed genes from the gonad of rice field eel in this stage,expecting to accumulate basic data for the study of its sex reversal mechanism. The main results were as follows:1. We adopted supression subtractive hybridization (SSH) technique, using rice field eel ovarian in stage IV as tester and testis in stage IV as driver to construct forward subtractive library; using rice field eel testis in stage IV as tester and ovarian in stage IV as driver to construct reverse subtractive library. Blue-white spot screening results showed that recombination rate in forward library and reverse library were 83.3% and 83.1% respectively. PCR analysis showed that the length of the insert gene fragments were mainly distributed between 150-1000 bp, with the average length of about 400 bp.2. Positive clones were selected to be sequenced. After removing vector sequences and sequences with length shorter than 100 bp,473 valid sequences were obtained, representing 34 unigenes. The ESTs were analyzed with BLAST by comparing sequences in NCBI non-redundant nucleotide databases (Nt) and non-redundant protein database (Nr).52.9% of the ESTs showed high identity with the existing nucleic acid sequences, and 32.4% of ESTs showed high identity with the existing protein.3.34 of the unigens in the libraries were annotated into functional categories. According to the cellular processes in the GO classification, the ESTs could be divided into three categories:cellular component, molecular function and biological process. In the COG classification, according to participate in the metabolic process is divided into the following five categories:inorganicion transport and metabolism, energy production and conversion, translation, carbohydrate transport and metabolism, replication, recombination and repair.4. After bioinformatics analysis of the sequenced clones, we screened some genes, including cytochrome c oxidase subunit Ⅱ, Mitochondrial Innermembrane protein Mitofilin, DNA topoisomerase, type Ⅱ A subunit B and ribosomal protein S5 domain 2-type fold.