Arch. Anim. Breed., 61, 59–70, 2018 https://doi.org/10.5194/aab-61-59-2018 Open Access © Author(s) 2018. This work is distributed under the Creative Commons Attribution 4.0 License. Archives Animal Breeding Novel alternative splicing variants of ACOX1 and their differential expression patterns in goats Xian-Feng Wu1,*, Yuan Liu1,*, Cheng-Fang Gao1, Xin-Zhu Chen1, Xiao-Pei Zhang1, and Wen-Yang Li1 1Institute of Animal Husbandry and Veterinary Medicine, Fujian Academy of Agricultural Sciences, Fuzhou, Fujian 350013, PR China *These authors contributed equally to this work. Correspondence: Wen-Yang Li ([email protected]) Received: 6 August 2017 – Revised: 29 October 2017 – Accepted: 14 November 2017 – Published: 24 January 2018 Abstract. As the first and rate-limiting enzyme of the peroxisomal β-oxidation pathway, acyl-coenzyme A ox- idase 1 (ACOX1), which is regulated by peroxisome proliferator-activated alfa (PPARα), is vital for fatty acid oxidation and deposition, especially in the lipid metabolism of very long-chain fatty acids. Alternative splicing events of ACOX1 have been detected in rodents, Nile tilapia, zebra fish and humans but not in goats. Herein, we identified a novel splice variant of the ACOX1 gene, which was designated as ACOX1-SV1, in addition to the complete transcript, ACOX1, in goats. The length of the ACOX1-SV1 coding sequence was 1983 bp, which presented a novel exon 2 variation owing to alternative 50-splice site selection in exon 2 and partial in- tron 1, compared to that in ACOX1. The protein sequence analysis indicated that ACOX1-SV1 was conserved across different species. Reverse-transcription quantitative real-time polymerase chain reaction (RT-qPCR) anal- ysis showed that these two isoforms were expressed spatially and differently in different tissue types. ACOX1 and ACOX1-SV1 were expressed at high levels in liver, spleen, brain and adipose tissue in kid goats, and they were abundantly expressed in the fat, liver and spleen of adults. Interestingly, whether in kids or in adults, in fat, the mRNA level of ACOX1 was considerably higher than that of ACOX1-SV1. In contrast, in the liver, the expression of ACOX1-SV1 was considerably higher than that of ACOX1. This differential expression patterns showed the existence of a tissue-dependent splice regulation. These novel findings for ACOX1 should provide new insights for further studies on the function of ACOX1 and its variants that should aid in the breeding of goats with improved meat quality. 1 Introduction The peroxisomal fatty acid β-oxidation pathway, which is an essential metabolic pathway that operates in all eu- Goat meat is expected to assume greater importance in the karyotic cells (Kunau et al., 1995; Shi et al., 2016), is the future owing to the rapidly growing world population and a main metabolic destination of very long-chain fatty acids steady increase in the consumption of this meat (Henchion (VLCFAs). This pathway plays a key role in the infants of et al., 2014); moreover, meat with better qualities and im- mammals, wherein lipids in the breast milk become the ma- proved flavor is desirable and demanded by consumers. The jor source of energy (Ehara et al., 2015). Importantly, this Fuqing goat breed is one of the finest local breeds found on pathway is regulated directly by the peroxisome proliferator- the east coast of China and provides high-quality meat that activated receptor alfa (PPARα), which is a master transcrip- is tender and has only a slight odor. Nonetheless, research on tional regulator of the fatty acid metabolism (Poirier et al., goat breeding for improving the quality of meat has lagged 2006). behind that on pork and beef. Data on candidate genes that Acyl-coenzyme A oxidase 1 (ACOX1) is the first and the affect the performance and quality of meat have largely been rate-limiting enzyme of the peroxisomal β-oxidation path- lacking, warranting detailed studies in this direction. way. It is mainly activated in peroxisome proliferators (a Published by Copernicus Publications on behalf of the Leibniz Institute for Farm Animal Biology. 60 X.-F. Wu et al.: Novel alternative splicing variants of ACOX1 and their differential expression patterns in goats kind of organelle in the liver) and oxidizes long and medium tee of the Fujian Academy of Agricultural Sciences (FAAS), straight-chain fatty acid substrates (Poirier et al., 2006; Wang Fujian province, China. The care and use of experimental an- et al., 2014). A deficiency of this enzyme leads to severe mi- imals fully complied with local animal welfare laws, guide- crovesicular steatohepatitis in mice (Fan et al., 1998; Yu et lines and policies. al., 2003). The lack of both ACOX1 and PPARα causes the absence of spontaneous hepatic peroxisome proliferation in 2.1 Animals and tissue collection aged mice (Hashimoto et al., 1999; Tanaka et al., 2017), high- lighting the close connection between ACOX1 and PPARα. In Seven Fuqing goats (four adult wethers and three female humans, the absence of ACOX1 is associated with pseudo- kids (lactation period)) were fed and slaughtered in the goat neonatal adrenoleukodystrophy (P-NALD), which is a disor- breeding farm of FAAS. Because the development of gonads der of the peroxisomes (El Hajj et al., 2012). The ACOX1 in goats does not begin during the lactation period, the ex- gene holds great promise as a potential target for improv- periments would not be influenced by gender. Nine types of ing meat performance in pig and cattle. For instance, an A/C tissue were collected for the study: heart, liver, spleen, lung, polymorphism in intron 9 was found to be significantly as- kidney, leg muscle, the longissimus muscle, perirenal fat and sociated with fat deposition in pig (Zuo et al., 2007), and the brain. All the samples were snap-frozen in liquid nitrogen ◦ other one (A1865C) in exon 13 was significantly related to and subsequently stored at −80 C. back fat thickness and marbling score in cattle, as evidenced from ultrasound imaging (Jiao et al., 2011). These observa- 2.2 Total RNA isolation, first-strand cDNA synthesis and tions point to the critical role of ACOX1 in the fatty acid cDNA pool construction metabolism and suggest its potential functions in affecting Total RNA was isolated from the collected tissue samples us- the development and deposition of fat. ing an RNA extraction kit and RNase-free DNase I (TaKaRa, Alternative splicing (AS), which exists in almost all kinds Dalian, China). The genomic DNA was removed by treat- of mammals (Kelemen et al., 2013), is a fundamental molec- ment with RNase-free DNase I, and the quality of RNA ular event that results in the production of different mature was determined using a NanoDrop 1000 spectrophotome- transcripts from the same primary RNA sequence (Sammeth ter (Thermo Fisher Scientific Inc., Wilmington, DE) and et al., 2008). These transcripts contribute to diverse biolog- 1 % agarose gel electrophoresis (Li et al., 2013). Reverse- ical processes, such as cell differentiation (Kianianmomeni transcription polymerase chain reaction (RT-PCR) was per- et al., 2014; Cotter et al., 2015), disease development (So- formed to synthesize complementary DNA (cDNA) using hail et al., 2014) and the adipocyte metabolism (Fiszbein et a PrimeScript™ RT reagent Kit (TaKaRa), according to the al., 2017). Previous studies reported that the splicing vari- manufacturer’s recommended procedure (S. H. Zhang et al., ants of PPARG (PPARG1 and PPARG2) could affect the 2016). lipogenesis of mammary tissue via the upregulated expres- sions of key lipogenic gene networks (Shi et al., 2014). The different transcripts of the SFRS18 gene (SFRS18_V1 and 2.3 Identification of ACOX1 alternative splice variants SFRS18_V2) were revealed to correlate with intramuscular The primers were designed, based on the predicted goat fat content in pigs (Wang et al., 2009). Thus, AS may af- ACOX1 mRNA sequence (XM_013972412.1), using Primer fect meat quality by regulating the expressions of key genes Premier 5 software (Premier BioSoft, Palo Alto, CA, USA); within the myocyte and adipocyte metabolism, especially the the sequences of the primers are shown in Table 1. A pair intramuscular lipocyte generation. Splicing variants of the of primers (P1) was designed for the identification of dif- ACOX1 gene were detected in Nile tilapia (He et al., 2014), ferent splice transcripts in the goat ACOX1 gene (Table 1). mice (Vluggens et al., 2010) and humans (Oaxaca-Castillo The polymerase chain reactions (PCRs) were performed as et al., 2007); however, they have not been reported in goats. described in our previous study (X. Y. Zhang et al., 2015). These studies all lend credence to the notion that the ACOX1 gene plays fundamental roles in meat quality. Therefore, this 2.4 Clone sequencing and validation of ACOX1 study aims to explore potential splicing transcripts of the goat transcript variants ACOX1 gene and the tissue expression pattern in kid and adult goats, which would be of benefit for further study on The PCR products were separated by electrophoresis on the function of the ACOX1 gene and breeding programs in agarose gel and were cloned into a pGEM-T easy vec- goats. tor (Promega, Shanghai, China) after purification. The re- combinant vector thus obtained was then transferred to Es- cherichia coli DH5α competent cells (TaKaRa) and se- 2 Materials and methods quenced (X. Y. Zhang et al., 2016). Experimental animals and procedures used in this study were approved by the International Animal Care and Use Commit- Arch. Anim. Breed., 61, 59–70, 2018 www.arch-anim-breed.net/61/59/2018/