Construction of a Full-Length Enriched cDNA Library and Preliminary Analysis of Expressed Sequence Tags from Bengal Tiger Panthera tigris tigris

International Journal of Molecular Sciences

Changqing Liu⁴  Dan Liu³  Yu Guo¹  Taofeng Lu⁴  Xiangchen Li⁴  Minghai Zhang²  Jianzhang Ma²  Yuehui Ma⁴ 

[1] Department of Bioscience, Bengbu Medical College, Bengbu 233000, China; E-Mail:;College of Wildlife Resource, Northeast Forestry University, Harbin 150028, China; E-Mails:;The Northeast Tiger Wooden Land of Heilongjiang, Harbin 150028, China; E-Mail:;Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, China; E-Mails:

关键词: Bengal tiger;    Panthera tigris tigris;    fibroblast cell line;    SMART cDNA library;    expressed sequence tags;   

DOI  :  10.3390/ijms140611072

来源: mdpi

【 摘 要 】

In this study, a full-length enriched cDNA library was successfully constructed from Bengal tiger, Panthera tigris tigris, the most well-known wild Animal. Total RNA was extracted from cultured Bengal tiger fibroblasts in vitro. The titers of primary and amplified libraries were 1.28 × 10⁶ pfu/mL and 1.56 × 10⁹ pfu/mL respectively. The percentage of recombinants from unamplified library was 90.2% and average length of exogenous inserts was 0.98 kb. A total of 212 individual ESTs with sizes ranging from 356 to 1108 bps were then analyzed. The BLASTX score revealed that 48.1% of the sequences were classified as a strong match, 45.3% as nominal and 6.6% as a weak match. Among the ESTs with known putative function, 26.4% ESTs were found to be related to all kinds of metabolisms, 19.3% ESTs to information storage and processing, 11.3% ESTs to posttranslational modification, protein turnover, chaperones, 11.3% ESTs to transport, 9.9% ESTs to signal transducer/cell communication, 9.0% ESTs to structure protein, 3.8% ESTs to cell cycle, and only 6.6% ESTs classified as novel genes. By EST sequencing, a full-length gene coding ferritin was identified and characterized. The recombinant plasmid pET32a-TAT-Ferritin was constructed, coded for the TAT-Ferritin fusion protein with two 6× His-tags in N and C-terminal. After BCA assay, the concentration of soluble Trx-TAT-Ferritin recombinant protein was 2.32 ± 0.12 mg/mL. These results demonstrated that the reliability and representativeness of the cDNA library attained to the requirements of a standard cDNA library. This library provided a useful platform for the functional genome and transcriptome research of Bengal tigers.

【 授权许可】

CC BY   
© 2013 by the authors; licensee MDPI, Basel, Switzerland