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

文献类型: 外文期刊

第一作者: Liu, Changqing

作者: Liu, Changqing;Lu, Taofeng;Li, Xiangchen;Ma, Yuehui;Guan, Weijun;Liu, Changqing;Guo, Yu;Liu, Dan;Zhang, Minghai;Ma, Jianzhang

作者机构:

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

期刊名称:INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES ( 影响因子:5.923; 五年影响因子:6.132 )

ISSN: 1422-0067

年卷期: 2013 年 14 卷 6 期

页码:

收录情况: SCI

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 x 10(6) pfu/mL and 1.56 x 10(9) 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 6x 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.

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