Open Access Anim Biosci Vol. 34, No. 5:922-930 May 2021 https://doi.org/10.5713/ajas.20.0342 pISSN 2765-0189 eISSN 2765-0235 Integrated analysis of transcriptomic and proteomic analyses reveals different metabolic patterns in the livers of Tibetan and Yorkshire pigs Mengqi Duan1,a, Zhenmei Wang1,a, Xinying Guo1, Kejun Wang2, Siyuan Liu1, Bo Zhang3,*, and Peng Shang1,* * Corresponding Authors: Objective: Tibetan pigs, predominantly originating from the Tibetan Plateau, have been Bo Zhang Tel: +86-010-62734852, subjected to long-term natural selection in an extreme environment. To characterize the Fax: +86-010-62734852, metabolic adaptations to hypoxic conditions, transcriptomic and proteomic expression E-mail: [email protected] patterns in the livers of Tibetan and Yorkshire pigs were compared. Peng Shang Tel: +86-0894-5822924, Methods: RNA and protein were extracted from liver tissue of Tibetan and Yorkshire pigs Fax: +86-0894-5822924, (n = 3, each). Differentially expressed genes and proteins were subjected to gene ontology E-mail: [email protected] and Kyoto encyclopedia of genes and genomes functional enrichment analyses. 1 College of Animal Science, Tibet Agriculture Results: In the RNA-Seq and isobaric tags for relative and absolute quantitation analyses, a and Animal Husbandry University, Linzhi, total of 18,791 genes and 3,390 proteins were detected and compared. Of these, 273 and Xizang 86000, China 257 differentially expressed genes and proteins were identified. Evidence from functional 2 College of Animal Sciences and Veterinary Medicine, Henan Agricultural University, enrichment analysis showed that many genes were involved in metabolic processes. The Zhengzhou, Henan 450046, China combined transcriptomic and proteomic analyses revealed that small molecular biosynthesis, 3 National Engineering Laboratory for Animal metabolic processes, and organic hydroxyl compound metabolic processes were the major Breeding/Beijing Key Laboratory for Animal processes operating differently in the two breeds. The important genes include retinol Genetic Improvement, China Agricultural University, Beijing 100193, China dehydrogenase 16, adenine phosphoribosyltransferase, prenylcysteine oxidase 1, sorbin and SH3 domain containing 2, ENSSSCG00000036224, perilipin 2, ladinin 1, kynurenine a These authors contributed equally to this aminotransferase 1, and dimethylarginine dimethylaminohydrolase 1. work. Conclusion: The findings of this study provide novel insight into the high-altitude metabolic ORCID adaptation of Tibetan pigs. Mengqi Duan https://orcid.org/0000-0002-9792-129X Zhenmei Wang Keywords: Hypoxia; Metabolic; Proteome; Tibetan Pig; Transcriptome https://orcid.org/0000-0002-0696-3276 Xinying Guo https://orcid.org/0000-0003-1405-4483 Kejun Wang https://orcid.org/0000-0003-4337-980X Siyuan Liu INTRODUCTION https://orcid.org/0000-0003-0418-5762 Bo Zhang https://orcid.org/0000-0002-7331-1550 Tibetan pigs, mainly originating from the Tibetan Plateau, have endured long-term natural Peng Shang selection in an extreme environment, such as hypoxia, low temperature, and high solar https://orcid.org/0000-0001-5427-7986 radiation [1,2]. Adaptation to the extreme environment is complex and reflected in several Submitted May 16, 2020; Revised Jun 23, 2020; aspects, including cardiopulmonary function, fat metabolism, immunity, blood pressure, and Accepted Sept 13, 2020 coat color [3-5]. Some studies have been conducted on hypoxia adaptation, physiological features, meat quality, and animal origin [6-8] of Tibetan pigs. Liver metabolic processes are also an important adaptive feature, as they play a key role in catalytic decomposition, biosynthesis, and energy delivery. Characterization of mRNA and protein expression in the liver tissues of Tibetan pigs remains to be performed. Recently, high-throughput sequenc- ing of DNA, RNA, and proteins, has been widely used to study physiological processes and traits [9,10] in pigs. In the present study, we combined proteomic and transcriptomic analyses to characterize liver function in Tibetan and Yorkshire pigs, to better under- Copyright © 2021 by Animal Bioscience This is an open-access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, 922 and reproduction in any medium, provided the original work is properly cited. www.animbiosci.org Duan et al (2021) Anim Biosci 34:922-930 stand the high-altitude adaptations of Tibetan pigs. according to the manufacturer’s instructions (Thermo Fisher Scientific, Waltham, MA, USA). The cDNA synthesis was MATERIALS AND METHODS performed with 1 μg of total RNA, following the protocol accompanying the FastQuant RT Kit (Tiangen Biotech (Bei- Ethics statement and sample collection jing) Co., LTD, Beijing, China). The qPCR amplification was The procedures for animal care were approved by the Ani- performed as described previously [1]. Relative expression is mal Welfare Committee of the Tibet Agriculture and Animal presented as mean±standard error of the mean. Differences Husbandry College, and all experiments were conducted in were tested for statistical significance using the Student’s t- accordance with approved relevant guidelines and regula- test. A p-value <0.05 was considered as the threshold for tions. Tibetan and Yorkshire pigs were raised under identical statistical significance (* p<0.05; ** p<0.01). conditions with same diet at the farm of the Tibet Agricul- ture and Animal Husbandry College, located in Linzhi city Protein extraction and labeling (Tibet, 2,900 m, above mean sea level). All pigs were housed Liver tissue protein was extracted using a mammalian tissue in standard conditions with natural, uncontrolled room tem- total protein extraction kit (AP0601-50, Beijing Bangfei perature and light. Complete formula meal feed was fed Biotechnology Co., Ltd., Beijing, China). Quantification three times per day and pigs had ad libitum access to water. was investigated using a protein quantification kit (Dingguo At the age of 6 months, three Tibetan pigs and three York- Changsheng, Beijing, China). Protein peptides from each shire pigs were randomly selected to slaughter and sample. group were labeled using the 8-plex isobaric tags for rela- Liver tissues from each individual were collected, immedi- tive and absolute quantitation (iTRAQ) reagents multiplex ately frozen in liquid nitrogen, and stored at –80°C until use. kit (ABI, Foster City, CA, USA). For each sample, approxi- mately 200 μg protein was precipitated with 10 volumes of RNA isolation and library construction acetone at –20°C overnight. After centrifugation, the pro- The total RNA from liver tissues was extracted following the tein pellet was dissolved in 60 μL of dissolution buffer. The phenol-chloroform method. RNA concentration and integ- iTRAQ labeling reagents were added to the peptide samples rity were determined using an Agilent 2100 Bioanalyzer and and reacted at room temperature for 1 h, before investiga- the Agilent RNA 6000 Nano Kit (Agilent Technologies, Palo tion of the labeling and extraction efficiency. Samples were Alto, CA, USA). RNA-seq libraries were constructed using pooled, vacuum-dried, and dissolved in solution. The re- the NEBNext Ultra RNA Library Prep Kit for Illumina (# constituted peptides were analyzed with a Q-Exactive mass E7530L, NEB; San Diego, CA, USA) according to the manu- spectrometer (Thermo Fisher Scientific, USA), coupled with facturer’s instructions. Paired-end 150 bp reads were generated a nano high-performance liquid chromatography system using the HiSeq Xten platform. (Thermo Fisher Scientific, USA). The detailed steps for en- zymolysis and iTRAQ labeling have been described previously Data analysis of RNA-Seq [5,9]. Data analysis included quality control of raw reads, filtering, alignment, assembly, expression count, and annotation. Con- Retrieval of database and protein data analysis taminated reads were filtered using a Perl script in-house. Data analysis was performed using Proteome Discover v2.1 Clean reads were mapped to the reference genome (Sus scrofa (Thermo Fisher Scientific, USA) software. Peptide identifi- genome v11.1, downloaded from ENSEMBL web server) cation was performed using the SEQUEST search engine, using HISAT2 v2.1.0 [11]. Reads for each gene in each sample using the Uniprot database (Sus scrofa 49003 entries, 20190102). were counted by HTSeq v0.6.0 (https://github.com/simon- Proteins were identified according to the following criteria: anders/htseq). Fragments per kilobase per million mapped mass error was set to 15 ppm for precursor ions and 0.02 Da reads were then calculated to estimate the expression level of for fragment ions. Trypsin was used as a cleavage enzyme, each gene. Deseq (http://bioconductor.org/packages/release/ and two missing cleavages were allowed. The false discovery bioc/html/DESeq.html) was used to identify the differentially rate was adjusted to 0.01. Differentially expressed proteins expressed genes (DEGs), with the criteria of fold change >2 (DEPs) were identified as having a fold change >1.2 and p- and adjusted p-value <0.05 [12]. value <0.05. Expression level validation by quantitative-polymerase Functional enrichment analysis chain reaction Gene ontology (GO) and Kyoto encyclopedia of genes and Differentially expressed mRNAs were selected for validation genomes (KEGG) functional enrichment analyses were per- by quantitative-polymerase chain reaction (qPCR). Total formed, using the