Indian Journal of Animal Research

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Research Article

Indian Journal of Animal Research, volume 54 issue 6 (june 2020) : 679-684

Molecular cloning, characterization and tissue specificity of the expression in the TAC1 genes from Henan Huai goat (Capra hircus)

Zhilong Tian1,3, Yuqin Wang1,2, Huibin Shi1, Zhibo Wu1, Xiaohui Zhang1, Jianping Wang1, Yuanxiao Li1, Fang Yang1, Yumei Liu1, Mingxing Chu3
1College of Animal Science and Technology, Henan University of Science and Technology, Luoyang-471 003, China.
2Mutton Sheep Breeding Engineering Technology Research Center of Henan Province, Luoyang-471 003, China.
3Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing-100 193, China.
Cite article:- Tian Zhilong, Wang Yuqin, Shi Huibin, Wu Zhibo, Zhang Xiaohui, Wang Jianping, Li Yuanxiao, Yang Fang, Liu Yumei, Chu Mingxing (2019). Molecular cloning, characterization and tissue specificity of the expression in the TAC1 genes from Henan Huai goat (Capra hircus) . Indian Journal of Animal Research. 54(6): 679-684. doi: 10.18805/ijar.B-1025.

ABSTRACT

To further to understand the structure and function of the TAC1 gene, we cloned the full-length cDNAs of the TAC1 genes from goat by rapid amplification of cDNA ends-PCR and the qRT-PCR was used to analyze the TAC1 mRNA expression patterns of goat various tissues. The full-length cDNA of goat TAC1 was 1176 bp, with a 339 bp open reading frame encoding 112 amino acids. The amino acid sequence analysis revealed that goat TAC1 gene encoded a water-drain protein and its relative molecular weight and isoelectric point was 13,012.86 Da and 6.29 respectively. Alignment and phylogenetic analyses revealed that their amino acid sequences were highly similar to those of other vertebrates. TAC1 expression of the goat of the brain, cerebellum, medulla oblongata, heart, liver, spleen, lung, kidney, uterus, ovaries. These results serve as a foundation for further study on the Capra hircus TAC1 gene.

INTRODUCTION

Litter size plays an important role in the Livestock economy (Gupta et al., 2016). The litter size in goat is a complex trait that is influenced by many factors, such as genetic background (Maitra et al., 2016), nutritional level (Mahfuz et al., 2018) and feeding management. The genetic background principally includes the number of ovulation, fertilization efficiency and estrus (Mahfuz et al., 2018; Muayad et al., 2019; Rahman et al., 2017). Among them, the estrus is particularly important, which can affect the number of lambs per year in the goat.
        
Over the past 10 years, several upstream neuropheno types have been implicated in stimulatory and/or inhibitory regulation of GnRH secretion. A model had proposed in which Kiss1 neurons in the arcuate nucleus (ARC), called as KNDy neurons, release kisspeptin (a potent GnRH secretagogue) in a pulsatile manner to drive GnRH pulses under the coordinated auto-synaptic action of its co-transmitters, the tachykinin neurokinin B (NKB, stimulatory) and dynorphin (inhibitory) (Fergani and Navarro 2017). many studies support this model; however, additional regulatory mechanisms (upstream of KNDy neurons) and alternative pathways of GnRH secretion (kisspeptin independent) exist but remain ill-defined. In this aspect, attention to other members of the tachykinin family, namely substance P (SP) and neurokinin A (NKA). Early studies documented a robust stimulatory action of LH release by SP in rats, rabbits and humans (Traczyk et al., 1992) and recent electrophysiological studies have described potent depolarizing effects of SP and NKA on ARC Kiss1 neurons in the mouse (de Roux et al., 2003) indicating that LH stimulation by these tachykinins involves, in part, a kisspeptin-dependent mechanism. Mammalian tachykinins (TKs) are a family of biologically active and structurally related peptides derived from three different genes: TAC1 encodes for substance P (SP) and neurokinin A (NKA), TAC3 encodes for neurokinin B (NKB), and TAC4 encodes for hemokinin-1 (HK-1) (Satake 2016). Tachykinins exert most of their actions by interacting with specific G protein-coupled membrane receptors: neurokinin 1 receptor (NK1R), NK2R and NK3R encoded by the TACR1, TACR2 and TACR3 genes, respectively (Satake 2016). Substance P and HK-1 bind preferentially to the NK1 receptor, NKA to the NK2 receptor and NKB to the NK3 receptor. Many experimental findings point of a possible role of tachykinins as modulators of the secretion of pituitary and gonadal hormones, acting as paracrine factors (Navarro et al., 2015).
        
However, the gene and cDNA sequences of the Capra hircus TAC1 gene have not been published in GenBank. The precise physiological function and expression pattern of the TAC1 gene in the goat brain to remain unclear. In the current study, we cloned the cDNA of the goat TAC1 gene, predicted the corresponding protein sequence and then performed phylogenetic and structural analyses. The cDNA sequences and characteristics will enrich the comprehension of the Capra hircus genome. Using quantitative real-time PCR (qRT-PCR), we analyzed the expression profile of TAC1 mRNA in different types of goat tissue and sought to identify the possible relationship between the TAC1 gene and goat reproduction. The data obtained and the results of this analysis will aid in comprehending the function of the goat TAC1 gene.

MATERIALS AND METHODS

All experimental procedures involving goat followed the policies and guidelines on the Henan University of Science and Technology Animal Care and Use Committee.
 
Goat samples collection and tissue preparation
 
Eight months old Henan Huai goat was obtained from a local livestock farm in Luo-Yang, Henan Province. Different tissues, including the brain, cerebellum, medulla oblongata, heart, liver, spleen, lung, kidney, uterus, ovary, oviduct as well as the gluteal muscle and mesangial fat were immediately collected and frozen in liquid nitrogen until RNA isolation.
 
RNA isolation and cDNA synthesis
 
Total RNA was extracted from the Henan Huai goat brain using RNAiso Plus Reagent (Takara, Japan) according to the manufacturer’s instructions. The quality of the total RNA was checked by electrophoresis on a 1.2% agarose gel. The final RNA concentration was determined using a protein and nucleic acid spectrophotometer (Eppendorf, Germany) at 260 and 280 nm wavelengths. Then, total RNA was reverse transcribed for cDNA using a PrimeScript® RT Reagent Kit (Takara, Japan) according to the manufacturer’s instructions. The cDNA was used as the template to amplify TAC1 and was stored at -20°C.
 
Amplification and cloning of full-length TAC1 cDNA
 
A pair of forward (F1) and reverse (R1) primers (Table 1) was designed with the Primer Premier 5 program using Capra hircus TAC1 (XM_005678955.3) as the reference gene sequence. The 5’ /3’ RACE first-strand cDNA was synthesized using the SMARTer RACE 5’ /3’ Kit (Takara, Dalian, China) according to the manufacturer’s protocol.
 

Table 1: Primers used for amplifying cDNA and RACE of the TAC1 gene in goat.


 
5' RACE was performed by touchdown PCR and it employed the goat TAC1 specific primers GSP5 and the universal primers UPM (Table 1). The PCR program is divided into 4 stages, stage 1: 94°C, 5 min; stage 2: 94°C, 30 s, 72°C, 2 min, 5 cycles; stage 3: 94°C, 30 s, 70-60°C touchdown PCR, 30 s, 72°C, 2 min, every 5 cycles down 2°C, total 30 cycles; 72°C, 10 min; finally 4°C to terminate the reaction. Then the 32 RACE components and procedure was identical to those of 52 RACE, except that the GSP3 primer was used (Table 1). All PCR products, including the internal fragment, 52 RACE and 32 RACE, were detected by agarose gel electrophoresis and the PCR products were recovered using an Agarose Gel DNA Purification Kit (Tiangen, Beijing, China). The products were cloned into the pMDl8-T vector (Takara, Dalian, China) and sent to Sangon Biotech Co, Ltd. (Shanghai, China) for nucleotide sequence.
 
Bioinformatic analyses of TAC1
 
Sequence analysis of the Henan Huai White goat TAC1 gene was performed using the BLAST program at NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The ORF was predicted using the ORF Finder program at NCBI (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). Protein prediction and analysis were performed using the Conserved Domain Architecture Retrieval Tool within the BLAST program at NCBI (http://www.ncbi. nlm.nih.gov/BLAST) and the DNAman program (Lynnon LLC, USA). The molecular weight and pI were calculated by the Compute pI/Mw program (http://us.expasy.org/tools/pi_tool.html). The signal peptide was predicted using the SignalP4.0 program (http://www.cbs.dtu.dk/services/SignalP/) (Petersen et al., 2011). The PSort II program (http://psort.hgc.jp/) was used to predict protein sorting signals and the intracellular localization. The secondary structure of the deduced amino acid sequence was predicted by the SOPMA program(http://npsa-pbil.ibcp.fr/) (Combet et al., 2000). The SWISS-MODEL program (http://www.expasy.org/swissmod/SWISSMODEL.html) was used to model the 3D protein structure(Biasini et al., 2014). The phylogenetic tree constructed from the alignment was generated with the neighbor-joining method using Molecular Evolutionary Genetic Analysis (MEGA) software version 7 (http://www.megasoftware.net/), followed by phylogeny tests with 1000 bootstrap replicates (Kumar et al., 2016).
 
TAC1 mRNA expression in various tissues
 
The first strand cDNA was synthesized from 1 μg of total RNA that was extracted with TRIzol Reagent. Results were quantified using NanoDrop 2000 software. The Q-F and Q-R primers (Table 1) were designed (Sangon Biotech Co, Ltd.) using the Primer Premier 5 program. The relative mRNA levels were normalized to that of GAPDH. The 20 μL reaction volume contained 10 μL of 2×SYBR Premix Exa °C Taq (Takara), 0.8 μL of each primer, 2 μL of cDNA and sterile water to a volume 20 μL. The PCR program was performed at 95°C for 5 min, followed by 40 cycles at 95°C for 5 s and 60°C for 1 min. Thereafter, a dissociation program was carried out at 95°C for 15 s, 60°C for 1 min and 95°C for 15 s. All samples were amplified in triplicate and the mean was used for further analysis. The stability of the target genes was evaluated by the 2-DDCt method (Schmittgen and Livak 2008).
 
Statistical analyses
 
For the comparison of relative TAC1 mRNA expression levels, the data were analyzed using multiple analysis of variance (ANOVA) comparisons. Means were analyzed with Duncan’s Multiple Range test and are presented as the means ± standard errors (SEs), with a significant difference level of P<0.05 and a highly significant difference level of P<0.01. All statistical analyses were performed using Statistics Analysis System (SAS) software version 9.2 (SAS Institute Inc, Cary, NC, USA).

RESULTS AND DISCUSSION

cDNA cloning and sequence analysis of the TAC1 gene
 
The 1176 bp TAC1 sequence was obtained by cloning and splicing using a cDNA from the brain of the goat as the template. It consisted of a 339 bp CDS and a 549 bp 3’  terminal UTR and a 288 bp 5’ terminal UTR. A BLAST search of the NCBI’s nucleotide sequence database revealed that the TAC1 fragment (GenBank: MG757439) of goat origin was highly similar (99%) to the predicted TAC1 gene sequence of the goat (XM_005678955.3) (Fig 1A). The goat TAC1 nucleotide and deduced amino acid sequences are shown in (Fig 1B). In addition, the ORF Finder program identified the ORF between nucleotides 289-627, encoding a protein of 112 amino acids.
 

Fig 1: (A) BLAST search of NCBI’s nucleotide sequence database showing a distribution of BLAST hits on the queried sequence (all BLAST hits are not shown).


 
 Analysis of the amino acid sequence of TAC1
 
The deduced amino acid sequence had a molecular weight of 13012.86 Da and a pI of 6.29. TAC1 had possessed a signal peptide, as determined by the Signal P4.0 program (Fig 2A, Table 2). In addition, the C-, S-, Y-and D-scores were more than 0.5, indicating that TAC1 had a signal peptide and could be secret outside of cells. Prediction of its subcellular localization showed that TAC1 protein sequence belonged to secret protein (Fig 2B); it plays a biological role mainly in extracellular 55.6 % and nucleus 33.3%, mitochondrial 11.1%. The secondary structure of the protein was predicted to consist mainly of α-helixes, β-folds and random coils (Fig 2C). The ProtScale programs at ExPASy calculated the Hydropathicity profiles of goat TAC1 (Fig 2D). The ordinate represented the Hydropathicity score of the protein; a high score was indicative of an overall high Hydropathicity, while a low score was indicative of an overall low Hydropathicity. The abscissa represented the position of the amino acids. As shown in Fig 2D, the first 20 amino acids of TAC1 were Hydropathicity, while the remaining amino acids were hydrophobic. The amount of hydrophobic amino acids is larger than that of hydrophilic amino acids. The tertiary structures of TAC1 were predicted using SWISS-MODEL. The results showed that the protein comprises a single peptide and the structure of the closest similarity is mammalian tachykinin peptide, Neuropeptide K (Fig 2E).
 

Table 2: Prediction of a signal peptide sequence in porcine TAC1.


 

Fig 2: (A) Prediction of the signal peptide of TACI (C-Score: Cleavage site score, S-score: Signal peptide score, Y-Score: Combined cleavage site score).


 
Characteristics of the deduced protein and phylogenetic analysis of TAC1
 
The deduced amino acid sequence of TAC1 from the Henan Huai goat was compared to that from eight other animals using the MEGA7.0 program. The coding sequence and amino acid sequences accession number of the Henan Huai goat TAC1 gene is shown in Table 3. The phylogenetic tree was constructed from the deduced Henan Huai goat TAC1 and the TAC1 sequences in other mammals using the neighbor-joining method of the MEGA7.0 program (Fig 3). The results showed that TAC1 from the Capra hircus TAC1 clustered with other mammals. This means that the Capra hircus TAC1 gene is conserved in mammals, the highest homology was with ovis aries and the lowest homology was with sapiens and papio.
 

Table 3: Homology of TAC1 nucleotide and amino acid sequences between Capra hircus and other species.


 

Fig 3: Phylogenetic tree and alignment of TAC1 amino acid sequences from the goat and other species. The tree was constructed using the neighbor joining method in the MEGA 7.0 program.


 
Expression of TAC1 mRNA in different tissues
 
The TAC1 mRNA level was normalized against that of GAPDH. qPCR was used to analyze the TAC1 mRNA level in different organs, including the brain, cerebellum, medulla oblongata, heart, liver, spleen, lung, kidney, uterus, ovaries, tubal and gluteal muscle from a goat. The results showed that the TAC1 mRNA expression in the lung, kidney, ovary, liver higher than the uterus, tubal and gluteal muscle (P<0.05) (Fig 4).
 

Fig 4: Relative mRNA expression levels of TAC1 in various tissues from Henan huai goat.


        
Seasonal reproduction is the adaptive behavior of mammals to environmental changes. The photoperiod is the main environmental factor that affects this activity. Reproduction of Seasonal in goat is controlled by a neuroendocrine axis comprised of the hypothalamus-pituitary-gonads. In recent years, many research has shown that there is a neuropeptide in sheep pituitary, can promote prolactin release. TAC1 encodes tachykinins (Page 2005), bioactive peptides including substance P and neurokinin A (NKA), which have been shown to be capable of regulating PRL release in vivo (Debeljuk and Lasaga 2006). In the past research, SP has largely been associated with processes unrelated to reproductive function, such as pain perception and inflammatory activity in the brain (Felipe et al., 1998) as well as with psychiatric disorders (Ebner and Singewald 2006). But now studies reported substance P and NKA in the ovine PT and suggested that these peptides could act as PRL secretagogues (Hu et al., 2014). Previous studies in rodents showed that substance P and NKA can act as potent regulators of pituitary hormone secretion. Simavli found that SP acts through Kiss1 neurons to stimulate GnRH release and is involved in the reproduction of female mice (Simavli et al., 2015).
        
Mus musculus TAC1 localizes on mouse chromosome 6 and primarily expressed in the central nervous system (Ebner and Singewald 2006). However, there is no study on the function of TAC1 in the goat and its complete sequence remains unavailable. We report the cDNA sequence of TAC1 from the Henan Huai goat. It localizes on goat chromosome 4, possesses 7 exons and contains a 339 bp coding sequence (CDS) that encodes 112 amino acids, and a 549 bp 32 terminal UTR, 288 bp 52 terminal UTR. The deduced amino acid sequence of TAC1 was highly homologous (92-99%) with that of other mammalian species. The phylogenetic tree analysis revealed that the Henan Huai goat TAC1 amino acid sequence had a close genetic relationship of Ovis aries. goat TAC1 had possessed a signal peptide, similar to tac1 from other animals. It belongs to the extracellular signaling molecules, obvious transmembrane domain.
        
The qPCR results showed that the Henan Huai goat TAC1 mRNA expression levels were similar in most of the tissues. However, compared to its expression in the other tissues, TAC1 mRNA was more highly expressed in the kidney and lung, liver, ovary of the goat. It is known that SP can induce tumor cell proliferation, angiogenesis, and migration via NK1R and that the SP/NK1R complex is an integral part of the cancer cell itself, as well as its tumor microenvironment (Rosso et al., 2012). Thus in the cholangiocarcinoma (CCA) research, found that SP secreted by CCA promotes CCA growth via autocrine pathway. As we all know, In the ovary of mammals, tachykinins have been shown to be present in nerve fibers, blood vessels and in granulosa, luteal and interstitial cells. This suggests that TAC1 may play a role in goat reproduction.

CONCLUSION

In this study, we cloned the full-length TAC1 cDNA sequences of goat brain, determined their cDNA and amino acid sequences and performed structural and functional analyses using bioinformatics and phylogenetics and assessed their relative mRNA and protein expression levels in various goat tissues. Phylogenetic analysis shows that mammalian TAC1 is conserved. The result from qRT-PCR and indicated that transcription of TAC1 might be related to the reproduction. Our findings would be helpful for the further comprehension of the functions of the goat TAC1 genes. Detailed studies should be performed in the future to clarify the functions of goat TAC1.

CONFLICT OF INTEREST

The authors declare that there are no conflicts of interest.

ACKNOWLEDGMENT

This research was supported by the National Natural Science Foundation of China (31472059).

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