Construction Of CDNA Library For Siberian Tiger’s Spleen And Bioinformatic Analysis Of ESTs

cDNA library is a collection of cells or tissue specific period information of all mRNA. Construction of Siberian tiger cDNA library is not only a representation of technological advancement, but an important process in Siberian tiger conservation.This study is divided into two main parts:1.Construction of Siberian tiger’s spleen cDNA libraryThe total RNA was extracted from Siberian tiger’s spleen using Trizol one-step method. The ultraviolet spectrophotometer estimated the concentration of total RNA was 0.58μg/μl, OD260/OD280 was 1.97, testifying that purity and concentration all reach the requirements. After formaldehyde deformation electrophoresis identify the integrity and quality of total RNA, the total RNA are integrated and not be degraded cause the lines are clear in 28S and 18S, and the brightness ratio is about 2:1 according the electrophoresis image. The cDNA library was constructed with SMARTTM cDNA library construction kit. Total RNA forming a double-stranded cDNA by reverse transcription, the ds cDNA were then digested by SfiI and fractionated by CHROMA SPIN-400 columns. Collected the cDNA fragments longer than 400bp and then ligated to λTripIEx2 vector. Then X,-phage packaging reaction and library amplification were performed. Quality of the original and the amplified cDNA libraries was strictly checked by using conventional titer determination. The results showed that titer of the primary library was 2.98 ×106 pfu/ml and the recombination rate was 94.6%. And the titer is 9.70×109 pfu/ml in the amplified library.23 plaques were randomly picked up to detect the average length of intercalary sections in the library. The length of insert sections ranging between 500bp-1200bp. All of these results show that the established cDNA library of Siberian tiger spleen accord with the quality requirements, which laid a solid foundation for the later analysis.2. Bioinformatics analysis of EST sequencesRandomly picked 300 clones and sequenced, there were 273 of the original sequence obtained. After initial screened, there were 239 sequence identified as valid EST sequences. Then BLAST the sequences in GenBank and there were 80 functional annotation genes. Then the annotated genes were taken analysis of PANTHER, GOrilla, PPINet and KEGG resepectively. PANTHER is a kind of analysis towards genes and protein, including a step of classify towards 80 annotated genes and interpreted protein by using 5 methods, The results show in a histogram and a table resepectively. GOrilla is an analysis of GO terms and genes enrichment. The results showed that the genes enrichment effect occurs in energy reserve metabolic process, and the key genes were IQGAPI, CALMland FASN. PPINet analysis is defined as the protein- protein interaction network and exhibit the relationship in visualized Cytoscape graph as a network, then selected the central genes, CALMl and CALR. Analysis the signal pathway with KEGG, and the results showed that CALM1 plays a essential role in the Calcium signaling pathway as main sensor and CALR participates the immune response as binding protein, as well as maintenance of intracellular calcium homeostasis. All of these biological information not only preliminary interpretation towards the genetic information of the Siberian tiger spleen, but also provides the basic data for the research of the Siberian tiger genome.