Third-Generation Sequencing and Analysis of Four Complete Pig Liver Esterase Gene Sequences in Clones Identified by Screening BAC Library.
OBJECTIVE
Pig liver carboxylesterase (PLE) gene sequences in GenBank are incomplete, which has led to difficulties in studying the genetic structure and regulation mechanisms of gene expression of PLE family genes. The aim of this study was to obtain and analysis of complete gene sequences of PLE family by screening from a Rongchang pig BAC library and third-generation PacBio gene sequencing.


METHODS
After a number of existing incomplete PLE isoform gene sequences were analysed, primers were designed based on conserved regions in PLE exons, and the whole pig genome used as a template for Polymerase chain reaction (PCR) amplification. Specific primers were then selected based on the PCR amplification results. A three-step PCR screening method was used to identify PLE-positive clones by screening a Rongchang pig BAC library and PacBio third-generation sequencing was performed. BLAST comparisons and other bioinformatics methods were applied for sequence analysis.


RESULTS
Five PLE-positive BAC clones, designated BAC-10, BAC-70, BAC-75, BAC-119 and BAC-206, were identified. Sequence analysis yielded the complete sequences of four PLE genes, PLE1, PLE-B9, PLE-C4, and PLE-G2. Complete PLE gene sequences were defined as those containing regulatory sequences, exons, and introns. It was found that, not only did the PLE exon sequences of the four genes show a high degree of homology, but also that the intron sequences were highly similar. Additionally, the regulatory region of the genes contained two 720bps reverse complement sequences that may have an important function in the regulation of PLE gene expression.


CONCLUSIONS
This is the first report to confirm the complete sequences of four PLE genes. In addition, the study demonstrates that each PLE isoform is encoded by a single gene and that the various genes exhibit a high degree of sequence homology, suggesting that the PLE family evolved from a single ancestral gene. Obtaining the complete sequences of these PLE genes provides the necessary foundation for investigation of the genetic structure, function, and regulatory mechanisms of the PLE gene family.
State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, Hubei, China.|Key Laboratory of Development of Veterinary Diagnostic Products of Ministry of Agricultural, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, Hubei, China.
Qiongqiong
Q
Zhou
State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agriculture University, Beijing, China.
Wenjuan
W
Sun
State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, Hubei, China.|Key Laboratory of Development of Veterinary Diagnostic Products of Ministry of Agricultural, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, Hubei, China.
Xiyan
X
Liu
State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, Hubei, China.|Key Laboratory of Development of Veterinary Diagnostic Products of Ministry of Agricultural, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, Hubei, China.
Xiliang
X
Wang
State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, Hubei, China.|Key Laboratory of Development of Veterinary Diagnostic Products of Ministry of Agricultural, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, Hubei, China.
Yuncai
Y
Xiao
State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, Hubei, China.|Key Laboratory of Development of Veterinary Diagnostic Products of Ministry of Agricultural, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, Hubei, China.
Dingren
D
Bi
State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agriculture University, Beijing, China.
Jingdong
J
Yin
State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, Hubei, China.|Key Laboratory of Development of Veterinary Diagnostic Products of Ministry of Agricultural, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, Hubei, China.
Deshi
D
Shi
D000818:Animals; D043182:Carboxylesterase / Q000235:genetics* / Q000302:isolation & purification; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics; D003001:Cloning, Molecular; D005091:Exons / Q000235:genetics; D059014:High-Throughput Nucleotide Sequencing; D007438:Introns / Q000235:genetics; D008099:Liver / Q000201:enzymology*; D058977:Molecular Sequence Annotation; D005810:Multigene Family; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics; D017422:Sequence Analysis, DNA; D034421:Sus scrofa / Q000235:genetics*
D043182:Carboxylesterase
Eur J Biochem. 1998 Oct 1;257(1):142-8
9799112
Arch Biochem Biophys. 1980 Aug;203(1):214-26
7406498
Drug Metab Lett. 2008 Dec;2(4):275-9
19356105
Lipids Health Dis. 2015 Jun 30;14:64
26121977
Angew Chem Int Ed Engl. 2001 Aug 3;40(15):2851-2853
29712000
J Biotechnol. 2008 Feb 1;133(3):301-10
18078679
Angew Chem Int Ed Engl. 2007;46(44):8492-4
17902087
J Biol Chem. 1961 Dec;236:3240-5
13859431
Eur J Biochem. 1979 Apr;95(3):519-25
446478
Bioorg Med Chem. 2009 Dec 1;17(23):7878-83
19884014
Nature. 2015 Nov 26;527(7579):508-11
26560029
Science. 2009 Jan 2;323(5910):133-8
19023044
Biochem Biophys Res Commun. 2010 Jan 8;391(2):1280-4
20018173
Chembiochem. 2001 Aug 3;2(7-8):576-82
11828491
J Mol Model. 2011 Jun;17(6):1493-506
20862595
Protein Eng. 2003 Dec;16(12):1139-45
14983097