| 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 placenta 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 (?)dentmea the quality or the (?)brary. Tne original morary liter is 2.79×10(?)pru/ ml and recombination rate is 98.2%, the amplified library titer reaches to 5×109pfu/ ml and recombination rate is 95.3%.300 positive 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 0.89Kb.By selecting 300 clones randomly, using the BLAST of GENE BANK to compare and analysis, we obtained 208 original sequences, removed poor quality and short (<200 bp) sequences, finally obtained the 158 valid 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 90.2%. These sequences with GO analysis are divided into three categories, namely biological process, molecular function, cellular component.In the GO classification results, the number of concerning the molecular function is 12, and associated with cellular component is 19, and related to the biological process annotation is 25, respectively accounted for 21.5%,33.9% and 44.6%. Ranked first in cellular component is cell part, followed by cell. Molecular function is ranked first in all kinds of DNA, RNA, proteins, such as ion binding activity. Cellular process has the largest proportion in biological process; The second one is the metabolic process. GO classification find one gene also has different molecular function,it also can participate in various biological processes, which reflects the complexity of life.The 95 sequences protein structure are analysied by InterPro retrieval. The results of analysis is that 56 Unigene get annotations in the InterPro database involving 181 ORF. With KEGG retrieval, the 95 sequences get systematic analysis of gene function. Obtained 32 annotations and 23 pathways relate to metabolic progress.Using of GO annotation, KEGG pathway analysis and retrieval tools such as InterPro and the relevant literature to filter out the placenta and fetal growth-related genes, such as PLAC8, PEG 10, IGF-Ⅱ, etc. Studing of the function from these genes can enhance the awareness of the Amur tiger pregnance from biological view, and improved the survival of fetal Amur tiger and their quality of life. |
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