title: Construction of a full-length enriched cDNA library and preliminary analysis of expressed sequence tags from Bengal Tiger Panthera tigris tigris.
abstract: 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.
authors.affiliation: Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, China. wjguan86@hotmail.com.
authors.forename: Changqing
authors.initials: C
authors.lastname: Liu
authors.forename: Dan
authors.initials: D
authors.lastname: Liu
authors.forename: Yu
authors.initials: Y
authors.lastname: Guo
authors.forename: Taofeng
authors.initials: T
authors.lastname: Lu
authors.forename: Xiangchen
authors.initials: X
authors.lastname: Li
authors.forename: Minghai
authors.initials: M
authors.lastname: Zhang
authors.forename: Jianzhang
authors.initials: J
authors.lastname: Ma
authors.forename: Yuehui
authors.initials: Y
authors.lastname: Ma
authors.forename: Weijun
authors.initials: W
authors.lastname: Guan
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D048430:Cell Shape; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D020224:Expressed Sequence Tags / Q000378:metabolism*; D005260:Female; D005293:Ferritins / Q000737:chemistry / Q000235:genetics; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D015723:Gene Library; D007194:India; D007621:Karyotyping; D008297:Male; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012313:RNA / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D017422:Sequence Analysis, DNA; D046010:Tigers / Q000235:genetics*
chemical_list: D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D012313:RNA; D005293:Ferritins