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Molecular Cloning, Characterization and Evolutionary Analysis of Leptin

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Molecular Cloning, Characterization and Evolutionary Analysis of Leptin Open Life Sci. 2017; 12: 406–417 Research Article Hai-feng Tian, Qiao-mu Hu, Yan Meng, Han-bing Xiao* Molecular cloning, characterization and evolutionary analysis of leptin gene in Chinese giant salamander, Andrias davidianus https://doi.org/10.1515/biol-2017-0048 Received March 30, 2017; accepted May 15, 2017 1 Introduction Abstract: Leptin is an important hormone possessing Leptin, the protein product of the obese (ob or Lep) gene, diverse physiological roles in mammals and teleosts. was first cloned in ob/ob mice [1], and then was identified However, it has been characterized only in a few amphibian in human and other mammals (reviewed in [2]). Leptins species, and its evolutions are still under debate. Here, also were identified in non-mammalians, including birds the full length of the leptin (Adlep) cDNA of Chinese giant [3-5], reptiles [2], amphibians [6-8], and teleosts, the salamander (Andrias davidianus), an early diverging later generally possess duplicated leptins [8-15]. Leptin amphibian species, is characterized and according to the has been found to be responsible for the regulation of results of the primary sequence analysis, tertiary structure body weight and energy homeostasis [16,17], and it also reconstruction and phylogenetic analysis is confirmed is involved in regulating appetite, reproduction [18], the to be an ortholog of mammalian leptin. An intron was immune system [19], bone formation [20], angiogenesis identified between the coding exons of A. davidianus [21], and stress response [22,23]. leptin, which indicated that the leptin is present in the The primary amino acid sequences of leptins show low salamander genome and contains a conserved gene conservation among vertebrates. The identity of the leptin structure in vertebrates. Adlep is widely distributed but protein in Xenopus to that of pufferfish, human, and tiger expression levels vary among different tissues, with salamander (Ambystoma tigrinum) is 13%, 35%, and 60%, highest expression levels in the muscle. Additionally, the respectively [6,7,9]. Although, positive selections of leptins leptin receptor and other genes were mapped to three are revealed in several mammal lineages, for example, known leptin signaling pathways, suggesting that the pikas (Ochotona curzoniae), Cetacea and Pinnipedia, and leptin signaling pathways are present in A. davidianus. heterothermic bats [24-26], the conserved gene structure Phylogenetic topology of leptins are consistent with (three exons separated by two introns) and secondary the generally accepted evolutionary relationships of and tertiary structures of leptins were found from teleosts vertebrates, and multiple leptin members found in teleosts to mammals [2,3,6,11,27,28]. Phylogeny reconstruction seem to be obtained through a Cluopeocephala-specific of vertebrate leptins showed that most vertebrates form gene duplication event. Our results will lay a foundation distinct clades with topology consistent with the generally for further investigations into the physiological roles of accepted vertebrate topology except that the relationships leptin in A. davidianus. among teleosts remain inconsistent [2,11,29]. Leptin is mainly expressed in adipose tissue, Keywords: Leptin (Lep); tissue expression; evolution; stomach and liver in mammals [2], however, expression leptin signaling pathways; Andrias davidianus of leptins in non-mammalians is variable. For example, leptin mRNA is widely distributed in brain, pituitary and heart tissue in frogs [6], different individuals show different tissue distributions in tiger salamander [7], and *Corresponding author: Han-bing Xiao, No. 8, 1st Wudayuan Road, different tissue expressions among taxa in teleosts, such Donghu Hi-Tech Development Zone, Wuhan 430223, China, E-mail: as mainly expressed in liver in most fish species [29], [email protected] weakly and transiently expressed in adipose of rainbow Hai-feng Tian, Qiao-mu Hu, Yan Meng, Yangtze River Fisheries trout (Oncorhynchus mykiss) [30], and widely expressed in Research Institute, Chinese Academy of Fishery Sciences, Wuhan 430223, P. R. China tilapia [31]. Interestingly, leptins seem to exert pleiotropic Open Access. © 2017 Hai-feng Tian et al., published by De Gruyter Open. This work is licensed under the Creative Commons Attribution- NonCommercial-NoDerivs 4.0 License. Molecular cloning, characterization and evolutionary analysis of leptin gene in Chinese giant salamander 407 effects in both mammals and non-mammalians 2.2 Cloning the full-length cDNA and [2,3,6,29,32-34]. For example, leptins possess roles in food intronic DNA of Adlep intake and energy metabolism in mammals [35,36], growth and reproduction in birds [3], growth and development Total RNA of 12 tissues were isolated using TRIZOL of the hind limb in X. laevis [6], and food intake and (Takara, Dalian, China) based on the manufacturer’s reproduction in teleosts [37,38]. Generally, more diverse protocol, and then tissues were treated with DNase I physiological roles have been characterized in teleosts (THERMO SCIENTIFIC) to remove genomic DNA and in contrast to those found in mammals [39]. Hence, the used as templates for the following experiments. The evolution of multiple functional leptins becomes an first stand of cDNA was obtained by using SuperScript III interesting question. However, due to the inconsistency transcriptase (Invitrogene, Carlsbad, CA, USA) following of the phylogenetic analysis of leptins, especially for the manufacturer’s instructions. One annotated leptin those teleostean homologs [2,29,40,41], identifying more gene sequence (m.112490) was found in the transcriptome leptins and characterizing their physiological roles in of A. davidianus in our previous work [50]. Here, the non-mammals, especially in amphibians, would help to internal region of leptin in A. davidianus was obtained by address this question. To our knowledge, leptin had only using one pair of primers designed according to the partial been characterized in two amphibian species, in which sequence of leptin in A. davidianus mentioned above they are involved in regulating food intake, growth rate, (Table 1). Then, based on the obtained sequence, primers and bone and lung development [6,7,42,43]. were designed (Table 1) and the 5’- and 3’- terminus were The Chinese giant salamander (A. davidianus) obtained by using 5’- and 3’- RACE System (Clontech, belongs to the family Cryptobranchidae, which is thought Palo Alto, CA, USA) according to the manufacturer’s to be a primitive group within the Caudata based on recommendations. PCR amplification products were molecular and fossil evidence [44-46]. Moreover, a examined by electrophoresis using 1% agarose gel with rapidly growing industry to farm this species has been the DL2000 marker (Takara, Dalian, China). The purified developed throughout China during past two decades sequence fragments were cloned into a pMD-18 T vector and [47], which is mainly based on controlled artificial sequenced. All obtained sequence data were assembled propagation [48] and ecological breeding methods [49]. using the LasergeneSeqMan program (DNAStar, Madison, Noticeably, significant weight differences (5- to 15- fold) WI, USA). Prediction of Open Reading Frames (ORF) was have been found in one- and two-year-old artificial carried out using the EditSeq program (DNASTAR). The propagated populations (unpublished data), which led sequence of the signal peptide was predicted using the us to identify and characterize leptin in A. davidianus. program SignalP 4.1 (http://www.cbs.dtu.dk/services/ Therefore, the objectives of the present study were: (1) to SignalP/) [51]. The domains were predicted on the Pfam clone and analyze the characteristics of leptin genes in A. server (http://pfam.xfam.org/). davidianus; (2) to examine the tissue distribution of the For the quantitative real-time PCR (qPCR) analysis, genes in A. davidianus; and (3) to investigate the evolution total RNA from 12 different tissues were extracted, and of the leptin gene in vertebrates. their first-strand cDNAs were synthesized as mentioned above. The qPCR was performed on a BIO-RAD Connect™ CFX using Power SYBR® Green PCR Master Mix. The 2 Materials and Methods specific primers for qPCR are listed in Table 1. The reaction contained: 10 µL of 2× PCR Master Mix, 1 µL of forward 2.1 Sample collection primer, 1 µL of reverse primer, 1 µL of cDNA template, and 7 µL of H2O. The PCR protocols were as follows: initial The individuals of A. davidianus used in this study were denaturation at 95°C for 2 min, 40 cycles of 95°C for 10 cultured in Yangtze River Fisheries Research Institute, s, 55°C for 20 s, 72°C for 20 s. The melting curves were Chinese Academy of Fisheries Science. Three one-year- analyzed at 65–95°C after 40 cycles. Each PCR analysis old male salamanders were anesthetized according to was performed in triplicate. Relative expression analysis the standards of the Chinese Council on Animal Care. is normalized against the β-actin. The values were Organ tissues, including kidney, spleen, lung, stomach, expressed as mean ± standard error of the mean (SEM). skin, muscle, intestine, gonad, heart, liver, brain, and The expression difference was analyzed by one-way pituitary gland, were collected and preserved in RNA Lock ANOVA with Tukey adjustment using GraphPad Prism Stabilizer Reagent (E.Z.N.A.® RNA-Lock Reagent; Omega Software (GraphPad Software Inc., San Diego, CA, USA). Bio-Tek Inc.) for RNA extraction. Significance was set at P < 0.05. 408 H. Tian, et al. In order to obtain the genomic sequence of the protein modeling program MODELLER9v14 [52-54]. leptin in A. davidianus, genomic DNA of muscle was After alignment, a bundle of 20 models from random isolated using the Blood & Cell Culture DNA Kit (QIAGEN, generation of the starting structure was calculated, and Hilden, Germany) according to the manufacturer’s the best one was selected according to the lowest molpdf recommendations. Primers for PCR amplification of the and DOPE score implemented within Modeller [52-54]. leptin gene intron are listed in Table 1. The obtained The selected best model was further subjected to loop PCR products were sequenced and then assembled by refinement in Modeller.
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