Construction Of CDNA Library In The Amur Tiger Kidney And The Analysis Of ESTs

Amur tigers, mainly in Northeast of Asia, due to threating of poaching and habitat, such a sharp decline in Amur tiger population, are threatened with extinction. Protection of the Amur tiger has been known as one of the hot issues of concern in today’s world. cDNA library can store 100% messenger RNA of cells, be capable of specific expression of individual cells or tissues in the gene encoding and can also synthesis the protein during specific developmental stages. In summary, cDNA library has been widely used in various development periods and became an indispensable technology Endangered Species Resources Protection.This study we got high quality and purity of the Amur tiger kidney tissue RNA by Trizol extraction, while also successfully established a Amur tiger kidney cDNA library and analyzed ESTs fragments at the same time. The main results are as follows:1. In two and a half northeast of Amur tiger kidney tissue RNA material, using SMART technology through reverse transcription reaction (RT-PCR), RNA was reversed and transcribed into a strand cDNA (ss-cDNA).2 strand cDNA (ds-cDNA) was synthesized during long chain reaction (LD-PCR), after digesting by protease K and Sfill, the products were fractionated with CHROMA SPIN-400 column, the product obtained in vitro were then ligased with the vector XTripIEx2-Finally, the products were amplified in the host cell and the recombination rate were measured, we also assessed the quality of the library.2. We selected 200 bacteria colonies randomly to PCR assay, and obtained 187 positive clones. We delivered the clones to company for sequencing. After sequencing, ESTs sequence were performed clustering and splicing through bio-informatics, eventually we obtained 164 valid sequences. Using gene ontology database, we analyzed molecular function, biological process, protein fractionation, pathways and cell group of genes; we classified the genes into cell component, molecular function and biological process aspects through KEGG database and the proportion was 41.08%,23.23% and 35.69% respectively; we obtained 15 metabolic pathways by KEGG pathway, got 35 annotated protein by Interpro and selected related functional genes ultimately.