| Amur tiger (Panthera tigris altaica) located in north-east Asia, it is the largest extant volume feline subspecies, one of the most endangered species in the world. For a long time, due to the destruction of the living environment, habitat loss, poaching poaching affect human behavior, resulting in a sharp decline in the number of wild Siberian tiger population dangerous endangered, therefore, all aspects of the Amur tiger genetic resources protection is necessary. cDNA library is one of the basic means of functional genomics research, its advantage is reflected in genetic resources conservation, aspects of cloning and screening of new genes for endangered species of wild genetic resources was of far-reaching significance. In this paper, experimental materials taken from 2.5 years in captivity Amur tiger heart tissue, based on modern molecular biology and bioinformatics methods to construct the Siberian tiger heart tissue cDNA libraries, while the library and the quality of the resulting ESTs are analyzed, the main results are as follows:1、SMART method successfully constructed Amur tiger heart tissue cDNA library. Heart total RNA after reverse transcription synthesis of ss-cDNA, LD-PCR synthesized ds-cDNA, the resulting double-stranded cDNA by proteinase K digestion and Sfil I digested, over Chroma SPIN-400 column purification, yielding large cDNA fragment with λTriplEx2 carrier connection, packaging, namely acquire Amur tiger heart tissue cDNA library. The resulting recombinant cDNA library was 93.47%, titer and titer amplified when unamplified library were 2.69 × 106 pfu/ml and 4.96 × 109pfu/ml, described amplified library has a large storage capacity and still has a high quality. Library insert an average length of 795bp.2、160 randomly selected clones were sequenced, sequencing results by GENE BANK after the initial post-treatment, etc. BLAST alignments, obtained 129 of the original sequence, to remove low-quality 108 valid sequences. Mainly in sequence length between 750～1000bp, sequencing success rate of 80.6%.3、 The resulting sequence GO analysis, classification of proteins and pathways.Maximum cell component sequence ratio 19.7%, the proportion of molecules having a sequence of functions catalytically active function of maximum 47.9%, the proportion of the sequence of biological processes play a role in the metabolic process a maximum of 59.2%, protein hydrolase classification sequence ratio a maximum of 15.5%. Sequence analysis of the metabolic pathways involved in the integration of the proportion of protein sequence signal path up to 7%.4、The resulting effective analysis of the relationship between gene sequences obtained genetic relationships. Binding GO annotation, metabolic pathway analysis and reading relevant literature, and screened Amur tiger metabolism, heart development-related gene functional analysis center for Amur tiger survival improve disease prevention and reproductive capacity of the foundation. |
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