| Amur tiger is the strongest felid of five existing tiger subspecies which is the first class national protected animal, one of the world’s most endangered species and flagship species in temperate forest ecosystems. For a long time, due to the serious destruction of Amur tiger habitat environment, food shortages and human factors such as disorderly catching, leading to a sharp drop in the number of wild Amur tigers. Therefore, it has a very important scientific value and signification to the conservation of genetic resources and the research related to molecular biology in a timely manner.This experiment with captive Amur tiger placenta tissue for research materials, using modern molecular biological methods to construct Amur tiger placental cDNA library, and identificate the cDNA library quality. The results are as follows:This experiment takes 2.5 years old Amur tiger liver tissue as research materials, using of one-step Trizol extraction to get total RNA of placenta. Synthesis first chain with reverse transcription, then double-stranded cDNA is synthesized by LD-PCR, through proteinase K digestion and Sfi I enzyme after chromatography column to isolate the cDNA larger pieces connected with carrier lambda TriplEx2, finally packed and connection, obtained the Amur tiger placenta cDNA library. And finally identified the quality of the library. The original library titer is 2.68× 106pfu/ml and recombination rate is 97.2%, the amplified library titer reaches to 4.8x 109pfu/ml and recombination rate is 94.6%.200positive clones from random selection were examed by PCR, obtained the library insert fragments ranging in length between 0.5 and 3.0Kb, and the average length of the fragment is 0.78Kb.By selecting 200 clones randomly, using the BLAST of GENE BANK to compare and analysis, we obtained 138 original sequences, removed poor quality and short (< 200 bp) sequences, finally obtained the 105valid sequences of ESTs. Most of the sequences length is larger than 300 bp, mainly between 600 and 900 bp, the success rate of sequencing achieved to 89.3%. These sequences with GO analysis are divided into three categories, namely biological process, molecular function, cellular component.GO classification results,80 sequences,71 were comments,10 kinds of molecular function was recorded 107 times,12 and biological processes involved in the six kinds of cell components. Category GO gene was found with different molecular functions, they can also participate in different biological process, which reflects the complexity of the Siberian tiger life processes.80 genes were classified proteins and metabolic pathway analysis, with 24 proteins and 33 signaling pathways. And important proteins and metabolic pathways were analyzed.By constructing gene-related liver tissue interaction network diagram, and use GO annotations, KEGG metabolic pathway analysis and retrieval tools such as InterPro and find the relevant literature, screening related genes in liver tissue, such as SOD1, CTNNB1, LIF2 etc., the function of these genes were studied, and to improve the biological point of Amur tigers metabolism of awareness, and enhance their physical and Siberian tiger improve their quality of life. |
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