Chanan Euaruksakul

Hideki Nakajima

Arunothai Rattanachata

Muhammad Y Hanna

Ahmad R T Nugraha

Mohamed Boutchich
email: mohamed.boutchich@sorbonne-universite.fr

Electronic and Thermoelectric Properties of Graphene on 4H-SiC (0001) Nanofacets Functionalized with F4-TCNQ

Keywords: epitaxial graphene, nanofacets, photoemission spectroscopy, work function, thermoelectric properties

Graphene grown on silicon carbide (SiC) substrate remains an interesting template for both fundamental studies and applications because it is monolithic to the substrate and does not require any transfer. The synthesized material comprises monolayer graphene on large terraces of several microns in both length and width, and step edges or nanofacets with multilayer graphene ribbons [1,2]. Owing to the tunability of electronic properties, graphene and 2D analogues have been considered to be potential candidates for thermoelectric applications [3][4][5][6]. However, in its 2D sheet configuration, graphene presents a thermal conductivity and a zero bandgap that are detrimental for the thermoelectric figure of merit (ZT) [7,8]. Interestingly, in a multilayer [9], under specific stacking configuration [10] or patterned into nanoribbons [11,12], owing to confinement effects, graphene exhibits a finite bandgap [13]. Moreover, these structures, when functionalized by heteroatoms, undergo substantial variation of their electronic and thermal properties. The doping level can be tailored to increase the electrical conductivity () and hence the PF. The ambipolar transport in graphene implies positive (holes) or negative (electrons) S coefficients adjusting the E F position by chemical doping or field-effect gating [14]. Therefore, E F can then be located anywhere in the electron dispersion spectra of graphene yielding to n-or p-type material. This feature allows unipolar carrier transport that prevents S being compensated for by the contribution of both carrier types. WF and barrier height at the interface of a heterostructure are modulated, and the carrier injection mechanism can be judiciously controlled. There have been numerous studies on the functionalization of monolayer graphene using various adsorbates, notably F4-TCNQ, an effective p-type dopant [15,16]. This molecule is a strong electron acceptor that compensates for the natural n-type doping of pristine graphene grown on SiC substrates. Much less work has focused on the electronic properties at the step edges or nanofacets. These nanostructures, which exhibit a structure different from the terraces, cannot be effectively probed using spatially averaged techniques [17,18]. Angle-resolved photoemission electron spectroscopy (ARPES) is a powerful tool that reveals the electron-dispersion spectrum as well as the overall impact of any adsorbate onto graphene [19]. However, it is not possible to isolate the local electronic variations with a typical micron size probe of ~ 100 m. On the other hand, PEEM provides localized information as well as the topography of the material.

In this work, we have investigated functionalized epitaxial graphene on SiC and probed its electronic properties at the step edges using PEEM mapping. We determined that the WF variation between the step edges and monolayer terraces is as high as 0.7 eV after doping. We calculated S and  within the range of the experimental E F and carrier density achieved and estimated the resulting PF for both monolayer terrace and the nanofacets. The results showed an optimum S ~ 100 μV/K and S ~125 μV/K for monolayer and bilayer graphene, respectively with a PF of ~ 10 -2 W/K 2 m. EXPERIMENTAL

Materials

The graphene was grown on n-type 4H-SiC (0001) substrates. The substrate was first heated to 1000 °C in an ultra-high vacuum (UHV) chamber and up to 1525 °C in an argon (Ar) atmosphere to favour the formation of a large and homogeneous graphene layer. The sample was then cooled from 1525 °C to room temperature. A homogenous topography showed several ~ m wide terraces on average, separated by step edges [1,3]. The F4 -TCNQ molecules with 97% purity were sourced from Sigma-Aldrich. The material was placed in a crucible in an in-situ effusion cell fitted to the ARPES and PEEM systems.

Instrumentations and measurements

The band structure of the sample was measured around the K-point of the graphene Brillouin zone (BZ) using a synchrotron radiation-based ARPES beamline 3.2Ua: PES of the Synchrotron Light Research Institute (SLRI). The spectra were averaged over a 100 m 2 probe diameter and acquired using a Scienta R4000 analyzer. The sample was placed in the 6-axis manipulator at room temperature at a photon energy of 40 eV with an energy resolution of ~ 130 meV [20]. PEEM was performed at the beamline 3.2Ub:PEEM with an Elmitec LEEM PEEM III [21]. The imaging electron energy analyzer was utilized for the WF mapping and micro-XPS (the description of WF mapping by PEEM is provided in the Supplementary Material). The in-situ LaB 6 electron gun was used as a source for low-energy electron microscopy (LEEM). PEEM and LEEM could be switched so that the analyses by both techniques could be carried out on the same microscopic area. The WF mapping in PEEM was measured at 110 eV photon energy.

Graphene on n-type 4H-SiC (0001) was degassed at 400 °C overnight and then left to cool to room temperature. After the degassing, the graphene layer was checked by LEEM, LEED and XPS for its structural integrity and cleanliness. F4-TCNQ was then evaporated from a low-temperature effusion cell (NTEZ -MBE Komponenten). The material was degassed at ~ 80 °C in a quartz crucible and for a few hours while the gate valve was closed. The temperature was then raised to 100 °C for the deposition, and when the gate valve opened, the pressure in the analysis chamber rose from ~ 10 -10 mbar to low 10 -9 mbar. The distance from the crucible lip to the sample is approximately 25 cm in the PEEM chamber and 10 cm in the ARPES chamber. In-situ LEEM observation during F4-TCNQ deposition and XPS result after the deposition are shown in supplementary Figures SI 1&2.

The near-edge X-ray absorption fine structure (NEXAFS) was measured at the ARPES chamber in the total electron yield mode, which monitors the drain current from the sample as a function of the photon energy over the nitrogen K edge. The energy resolution of the NEXAFS N K edge is 0.4 eV.

RESULTS AND DISCUSSION

Figure 1 shows the Raman spectrum of the as-deposited sample. For clarity, the SiC Raman signature has been subtracted to reveal the main features of graphene G ~ 1570 cm-1 and 2D ~ 2700 cm -1 . The D peak ~ 1350 cm -1 could be assigned to structural defects, such as Si clusters from broken Si-C bonds that are typically formed during the high-temperature graphitization. The LEEM image indicates that 76% of the area comprises monolayer graphene, whereas the bilayer and trilayer graphene occupy approximately 17% and 7% of the total area, respectively. These bilayer and trilayer graphene appear as narrow ribbons above the nanofacets all over the surface [23] of SiC substrate, where graphene grows thicker near kinks and defects [24,25]. Figure 3a was derived from the slope of the E(k) branches [15,26], Table I. The flat bands distributed at 0.5-0.7 and 1.3-1.5 eV in Fig. 3(c-d Gaussian fitting after background subtraction. The first peak located at 397.7 eV corresponds to the anionic species N -1 , and the second peak located at 399.6 eV is attributed to the neutral N 0 species.

The third peak appears at 401.5 eV and seems to be the satellite peak due to the shake-up process [30,31]. The anionic species contribute to the charge transfer through the CN bonds, whereas the fluorine atoms remain inactive. F1s spectra (supplementary Fig. SI1) present no difference independently of the deposition conditions. The XPS results agree with the literature and support the standing-upright orientation of F4-TCNQ molecules on the graphene substrate, a result that is also consistent with our NEXAFS results. Note that it is not possible using NEXAFS to resolve the nanofacets, but we believe (considering the tilted topography) that the molecules could be lying down, which is the most energetically stable position, as calculated by Tian et al. [32].

Furthermore, we observed the non-homogeneous modification of electronic structure for graphene functionalized with F4-TCNQ by WF mapping under PEEM, Fig. 5. Whereas the F4-TCNQ deposition in PEEM was made on a separate setup from the PES platform, the deposition parameters were identical. This ensured that F4-TCNQ molecules deposited in PEEM were in the same configuration and induced a similar effect on the electronic properties. Note that the absolute WF measurement in PEEM is tedious to perform directly due to both non-isochromaticity in the imaging XPS mode and the difficulty in determining the Fermi edge. To evaluate the absolute WF profile of pristine graphene (blue line) in Fig. 5c, the PEEM measurement before functionalization was offset by the absolute WF ~ 4.3 eV of the monolayer graphene measured in our previous work [1,3]. After F4-TCNQ deposition, we offset the PEEM data by the positive shift of 1.28 eV on monolayer graphene measured by PES (our WF shift measurement originated mostly from monolayer domains, which covered more than 76% of the surface, and the result also compares well with Chen et al. [28] and Coletti et al. [15] who reported shifts of WF for monolayer graphene on 6H-SiC(0001) after F4-TCNQ deposition of 1.3 eV and 1 eV, respectively). In the 25 μm field of view and before the onset of deposition (Fig. 5a), we observed a small WF difference between graphene on the terraces (monolayer) and nanofacets (bilayer and trilayer), which exhibit a ~ 0.1 eV larger WF than the monolayer areas. This observation has been reported on 6H-SiC(0001) by Hibino et al. [33] and on 6H-SiC(000 ) by Mathieu et al. [34,35]. We also noted smaller domains within monolayer graphene scattered all over the SiC terraces, which have slightly lower (less than 0.1 eV) WF. Figure 5b displays the mapping after 8-minute deposition of F4-TCNQ.

The WF profile dramatically changed, as shown by the line profiles in Fig. 5c. The WF of monolayer graphene on SiC terraces became ~ 0.7 eV larger than the graphene on top of nanofacets, as shown by the red line profile in Fig. 5c. The profile shows that electron transfer occurs strongly on the monolayer graphene on SiC terraces and, in contrast, there is a relatively much smaller effect induced by F4-TCNQ on bilayer and trilayer graphene domains on the nanofacets, where the WF shifts only by ~ 0.5-0.6 eV. This functionalization also makes the WF of the bilayer and trilayer graphene domains smaller than the monolayer graphene after F4-TCNQ doping. To provide a statistical representation of the work function changes under the entire field of view in Fig. 5a and5b, histograms of the WF data from individual pixels of the WF mapping before and after F4-TCNQ deposition are plotted together in Fig. 5d. Each histogram is fitted with Gaussian functions to determine the number of species distinguishable by a small difference in WF [34,36]. The main peaks before and after F4-TCNQ deposition (solid blue and red lines) represent the monolayer graphene on SiC terraces that covers most of the total surface area. The histogram shows the overall shifts over the entire area and a clear spreading of the work function over a wider range after doping by F4-TCNQ.

Shoulders are observed on both lower and higher energies of the main peak centred at 5.5 eV. This also shows up as a non-uniform colour for the monolayer graphene domains in the mapping and could be assigned to either the non-uniform layer of F4-TCNQ or interactions with the buffer layer at the substrate interface. Note that the large full width half maximum (FWHM) of the Gaussian peaks that represent the bilayer and trilayer graphene (at 0.53 eV lower than the main monolayer graphene) in the histogram are assigned to the artefacts inherent to the small electric field on the surface of the sample arising from the difference in WF itself [37].

To discuss the difference in charge transfers of F4-TCNQ that results in different shifts of WF on monolayer graphene on terraces, and bilayer and trilayer graphene on nanofacets, we compared our results with the literature. F4-TCNQ may adsorb on graphene with either standing or lying-down configurations depending on the types of supporting substrates [38,39] and the surface coverage. For F4-TCNQ on epitaxial monolayer graphene on SiC, the molecules lie down parallel to the surface at low coverage [40]. At high coverage, the configuration switches to standing upright with the cyano groups of F4-TCNQ bonding to the surface only on one side. This configuration change is evidenced by the deconvolution of N 1s spectra in XPS into N -1 and N 0 components [6,15] and by angulardependent NEXAFS in this work (which largely measures the monolayer graphene). To our knowledge, there is no report on the determination of the adsorption configurations of F4-TCNQ on pure bi-layer graphene on SiC. We could not clarify the orientation of the molecules on the narrow nanofacets, neither by NEXAFS nor by PEEM, due to the angle rotation constraints of our equipment.

However, DFT calculations by Tian et al. [32] have shown that the F4-TCNQ molecule lies down on free-standing bi-layer graphene [32], and this is likely to occur on the bilayer and trilayer graphene near the nanofacets. Although it is expected that lying down F4-TCNQ induces more charge transfer per molecule (they transfer electrons with the cyano groups on both sides instead of on one side), the adsorption footprint in this configuration is larger, and there could be repulsive forces between the molecules that makes the packing less dense compared to that of standing F4-TCNQ on monolayer graphene; hence, a lower WF shift is observed on the nanofacets.

Evaluation of thermoelectric properties

Functionalized graphene superlattices are thought to enhance the TE properties [41]. Considering the electronic properties measured on our graphene template functionalized with F4-TCNQ, we determined the TE properties of monolayer and bilayer graphene within the linear Boltzmann transport theory and constant relaxation time approximation (CRTA). In this framework, one can calculate S and σ assuming a phenomenological relaxation time constant τ 0 and electronic energy dispersion E sk , where s denotes the band index and k is the electron wave vector. The electronic structure of the functionalized graphene is calculated using Quantum ESPRESSO [42,43]. We carried out a geometrical optimization for the orientation of the molecule and found that the most stable configuration on both monolayer, bilayer and trilayer graphene was the F4-TCNQ molecule lying down above graphene. The expressions for S and  are [3][4][5][START_REF] Goldsmid | Introduction to Thermoelectricity[END_REF][START_REF] Liao | [END_REF][46] (

where e is the unit electric charge (defined to be positive), T is the average temperature of the material, N is the number of k in the unit cell, E F is the Fermi energy, f sk is the Fermi-Dirac distribution function and v sk is the component of the electronic group velocity for a specific direction at each k point. Note that the CRTA is valid for near-equilibrium transport involving elastic and isotropic scattering, which we assume in this work for simplicity. Figure 6 illustrates the model implemented.

The accuracy of the calculation thus mostly depends on the choice of τ 0 parameter, which can be measured experimentally or taken from the literature. Figure 7 presents the calculated results of S, σ

and PF (which is equal to S 2 σ) for the functionalized monolayer and bilayer graphene as a function of Fermi energy and the carrier concentration. For each thermoelectric quantity, there are two curves shown in Figure 7 scaled with E F and the carrier concentration, respectively for the experimental doping range achieved. The plots of σ and PF are scaled by τ 0 , where the value of this τ 0 parameter could vary depending on the sample, typically on the order of 0.5-10 ps as observed by pump probe techniques [47,48]. Note that from the formulas of S and σ, S does not depend on the relaxation time because τ 0 in the numerator will cancel with that in the denominator, as opposed to σ. In Figure 7, the maximum S values are close to 100 μV/K for functionalized monolayer graphene, whereas bilayer graphene exhibits a S ~125 μV/K that is higher than that of monolayer graphene owing to the bandgap opening. This order of magnitude is on par with experimental data [14,49] measured on flakes. We observe that upon appropriate doping within -0.5 to 0.5 eV or with a carrier concentration of the order of 10 12 cm -2 , which we attained experimentally (see Table I), the TE properties of graphene could be enhanced significantly. There exists an optimal doping to obtain the maximum PF in both p-and n-type graphene, where the p-type (n-type) corresponds to the negative (positive) E F or the positive (negative) carrier concentration. The presence of two maximum peaks in PF near E F = 0 is mainly due to the two peaks (negative and positive) S because the electrical conductivity monotonically increases with doping. However, the values of maximum PF strongly depend on the relaxation time τ 0 . Using Figure 7, and knowing τ 0 and the carrier concentration, one can estimate the thermoelectric properties of one's own functionalized graphene sample. For example, we can estimate by assuming the typical relaxation time within 0.5-10 ps, the maximum calculated PF in the order of 10 -2 W/K 2 m, which is in the same order of magnitude as the PF of bulk bismuth telluride [50], one of the most integrated TE materials available on the market. Functionalization or doping of graphene multilayer nanofacets exhibit enhanced thermoelectric performance. Practically, one would need to isolate the graphene patterns from the substrate to reduce heat losses. Substrate-graphene interaction always plays an important role in determining the electronic structure of graphene. In this work, we demonstrated that surface-transfer doping is also affected by different graphene thicknesses, particularly on the nanofacet structures of the substrate. This difference results in non-homogeneous charge transfers across the graphene layer. This inhomogeneity may either be detrimental to device performance or, on the other hand, be used as a bottom-up approach to control the energy level and create different graphene electronic structures suitable for TE applications. Here, we observed a direct change in the electronic properties and band structure of graphene upon in-situ functionalization by F4-TCNQ molecules. We have also mapped the WF across the sample and

Fig. 1 .Fig. 2 .

 12 Fig. 1. Raman spectrum of as-grown graphene. The presence of the G and 2D bands across the

  shows the electron dispersion E(k) for the pristine sample along the perpendicular to the ΓK graphene Brillouin zone, with a residual n-type doping with E F lying at ~ 0.43 eV above the Dirac point E D yielding a charge carrier concentration of n ~ 1.36 x 10 13 cm -2 . Figure3(b-d) represent the E(k) for the doped sample with an increasing number of molecules while the spectra are accumulated.

Fig. 3 .

 3 Fig. 3. ARPES measurement of graphene/4H-SiC (0001). (a) Pristine graphene, (b) after heating

Figure 4

 4 Figure4shows the near edge X-ray absorption fine structure (NEXAFS) of the nitrogen K edge on

Fig. 4 .

 4 Fig. 4. Angular-dependent N K edge NEXAFS spectra of F4-TCNQ on graphene measured for

Fig. 5 .

 5 Fig. 5. Work function mapping before (a) and after (b) F4-TCNQ deposition on graphene/SiC (25 μm field

Fig. 6 .

 6 Fig. 6. F4TCNQ on free-standing graphene configuration after relaxation. The molecule is in a lie-down position, which is the most energetically favourable configuration for both low and high coverage.

Fig. 7 .

 7 Fig. 7. Calculated Seebeck coefficient, electrical conductivity and power factor as a function of Fermi energy (upper horizontal axis) and carrier concentration (lower horizontal axis) for (a) monolayer and (b) bilayer graphene. Note that the electrical conductivity and power factor are scaled by the relaxation time. The range of Fermi energy in this figure covers a larger range of doping than the range of carrier concentration. The blue trace is scaled with E F , whereas the red one with the carrier concentration. CONCLUSION

Table I :

 I Dirac point and charge carrier concentration for each deposition time of F4 TCNQ.

	) correspond to the partially filled lowest-unoccupied molecular orbital
	(LUMO) and relaxed highest-occupied molecular orbital (HOMO) of F4-TCNQ resulting from the
	electron transfer [27,28], respectively.
		a	b	c	d
	Deposition time (min)	pristine	0	2	12
	F4-TCNQ (nm)	0	0.58	0.74	0.74
	Dirac point (eV)	0.43	0.22	0.13	0.2
	Carrier concentration (10 12 cm -2 )	13.6	3.55	1.24	2.94

observed a significant difference in the charge transfers from F4-TCNQ doping on the monolayer graphene on terraces as opposed to bilayer and trilayer graphene on nanofacets. Considering the electronic properties measured under the synchrotron beamline and a relaxation time range 0.5-10 ps, we obtained a maximum thermoelectric power factor PF in the order of 10 -2 W/K 2 m, comparable to commercial thermoelectric materials. Interestingly, we observed that the graphene on nanofacets located at the edges exhibits a PF nearly as high as the monolayer graphene on the terraces and a Seebeck coefficient 25 % larger. This improvement suggests that functionalization is an effective route to enhance the thermoelectric properties of nanostructured materials.

ACKNOWLEDGMENT

REFERENCES:


Sedigheh Mohammadzadeh

Fatemeh Moradian

Sakineh Yeganeh
email: skyeganeh@gmail.com

Bahram Falahatkar

Sylvain Milla

Design, production and purification of a novel recombinant gonadotropin-releasing hormone associated peptide as a spawning inducing agent for fish

Keywords: Decapeptide, GAP, Half-life, Proteolytic Stability, rGnRH

GnRH is a neuropeptide known to regulate reproduction in vertebrates. The purpose of this study was to design and produce recombinant gonadotropin-releasing hormone associated peptide (rGnRH/GAP) as an alternative of the previous GnRHs and native extracted hormone from tissue, to induce final maturation in fish. Decapeptide as well as GAP area sequences were compared between GnRH1, GnRH2, and mGnRH from Acipenser sp and Huso huso, respectively. Considering the conserved amino acids and the replacement of un-stable amino acids with those that were more stable against proteolytic digestion as well as had a longer halflife, the sequence was designed. The sequences of decapeptide and GAP region were synthesized and then cloned on pET28a expression vector and transformed into expression host Escherichia coli BL21(DE3). The supernatant of cultured recombinant bacteria was used for purification using TALON Metal affinity resin. The purity of the GnRH/GAP was confirmed by single 8 kDa band on SDS-PAGE and western blot. Bioinformatics studies were performed for evaluation of homology between GnRH protein sequences and prediction of 3D protein structure using Swiss Model. The result showed that the structure prediction of the recombinant GnRH decapeptide was relatively similar to decapeptide of GnRH2 from Beluga (Huso huso). The GAP structure was similar to GAP1 of Nile tilapia (Oreochromis niloticus) and sturgeon and GnRH2 of Chinese sturgeon (Acipenser sinensis). The mass analysis showed that the sequence was exactly the same as designated sequence. Biology activity of rGnRH/GAP was tested in mature goldfish (Carassius auratus) and results showed that rGnRH/GAP had a positive effect in final maturation. Indeed 17α, 20β-dihydroxy-4-pregnen-3-one (DHP) was increased 17 h and 24h after injection with rGnRH/GAP and spawning stemmed from that injection. These novel findings introduce the potential of utilizing rGnRH/GAP in aquaculture.

Introduction

Gonadotropin-releasing hormone (GnRH) belongs to conserved neurohormonal gene family that has 30 different isoforms, including 15 isoforms in vertebrata and 15 isoforms in non-vertebrates [START_REF] Roch | Evolution of GnRH: Diving deeper[END_REF]. GnRH is a decapeptide with 10 amino acids found in fish as well as other animals which are synthesized primarily on neurons in the brain [START_REF] Melamed | Hormones and their receptors in fish reproduction[END_REF]. Two forms of GnRH (mammalian GnRH and chicken GnRH-II) are found in fish as same as other vertebrates, but there are nine forms of GnRH in fish that are distinct from other vertebrates. The release of GnRH from hypothalamus regulates reproductive and sexual functions in vertebrates [START_REF] Bancroft | The endocrinology of sexual arousal[END_REF][START_REF] Parvathy | Role of serotonin in fish reproduction[END_REF].

Primary structure of GnRH protein precursor is composed of a signal peptide in the N-terminal region (approximately 28 amino acids), a section of ten amino acids (decapeptide), which is an active region of the peptide and is almost completely conserved in all fishes. Thereafter, there are three amino acids; Glycine, Lysine and Arginine, which are conserved in all species of fish and where the protease digestion occurs in the sequence of this peptide. Finally, in C-terminal, there is also a GnRH-associated peptide (GAP) that has about 60 amino acids, which is less conserved in fish than other regions of the peptide, and has different sequences and sizes [START_REF] Zmora | The GnRH system in the European sea bass (Dicentrarchus labrax)[END_REF][START_REF] Lethimonier | Evolutionary aspects of GnRHs, GnRH neuronal systems and GnRH receptors in teleost fish[END_REF]. Studies have shown that GAP has a role in the stability of GnRH structure and unlinked to the biological activity of the peptide [START_REF] Andersen | The salmon GnRH encoding gene in teleost fish[END_REF]. Nikolic et al [START_REF] Nikolics | A prolactin inhibiting factor within the precursor for human gonadotropin-releasing hormone[END_REF] reported that the inhibition of PRL basal secretion obtained by GAP was comparable to that reported for dopamine [START_REF] Pérez Sirkin | Conservation of Three-Dimensional Helix-Loop-Helix Structure through the Vertebrate Lineage Reopens the Cold Case of Gonadotropin-Releasing Hormone-Associated Peptide[END_REF]. It was shown that GAP was co-secreted with GnRH into the hypophyseal portal blood of ovariectomized sheep [START_REF] Clarke | GnRH-associated peptide (GAP) is co secreted with GnRH into the hypophyseal portal blood of ovariectomized sheep[END_REF]. In addition, some reports showed that GAP could stimulate gonadotropins release in rats in vitro and in vivo [START_REF] Nikolics | A prolactin inhibiting factor within the precursor for human gonadotropin-releasing hormone[END_REF][START_REF] Chandrashekar | Assessment of the effects of a synthetic gonadotropin-releasing hormone associated peptide on hormone release from the in situ and ectopic pituitaries in adult male rats[END_REF][START_REF] Yu | Effects of the gonadotropin-releasing hormone associated peptides (GAP) on the release of luteinizing hormone (LH), follicle stimulating hormone (FSH) and prolactin (PRL) in vivo[END_REF][START_REF] Milton | Total solid-phase synthesis and prolactin-inhibiting activity of the gonadotropin-releasing hormone precursor protein and the gonadotropin-releasing hormone associated peptide[END_REF][START_REF] Kerrigan | Regulation of gonadotropin subunit messenger ribonucleic acid expression in gonadotropin-releasing hormone (GnRH)-deficient female rats: effects of GnRH, galanin, GnRH-associated peptide, neuropeptide-Y, and thyrotropin-releasing hormone[END_REF][START_REF] Chavali | Helix-loop-helix motif in GnRH associated peptide is critical for negative regulation of prolactin secretion[END_REF]. The previous studies indicated that GAP could have a physiological significance in the regulation of pituitary function in mammals. However, there are also reports where these GAP effects were not observed, as for instance in human pituitary prolactinoma in vitro [START_REF] Ishibashi | Effect of GnRH-associated peptide on prolactin secretion from human lactotrope adenoma cells in culture[END_REF] or in sheep in vivo [START_REF] Thomas | Gonadotropinreleasing hormone associated peptide (GAP) and putative processed GAP peptides do not release luteinizing hormone or follicle-stimulating hormone or inhibit prolactin secretion in the sheep[END_REF]. To the best of our knowledge, a study by Planas et al. [START_REF] Planas | Effects of GnRH-associated peptide and its component peptides on prolactin secretion from the tilapia pituitary in vitro[END_REF], on the effects of human GAP on PRL release in tilapia, has been the only attempt so far to investigate the possible biological function of GAP in non-mammalian vertebrates.

One of the major goals of aquaculture practices is to artificially stimulate spawning via the manipulation of the hormonal axis. Different endocrine approaches have been considered to induce final sexual maturation in breeding fish [START_REF] Aizen | Biologically active recombinant carp LH as a spawning-inducing agent for carp[END_REF]. Pituitary extract, human chorionic GTH, hCG and different analogs of GnRH are used to induce spawning in finfish reproduction [START_REF] Aizen | Biologically active recombinant carp LH as a spawning-inducing agent for carp[END_REF][START_REF] Zohar | Endocrine manipulation of spawning in cultured fish: from hormones to genes[END_REF]. There are significant advantages to using GnRH as compared with gonadotropin-based preparations such as fish pituitary and synthetic gonadotropins. Synthetic GnRH eliminated the risk of the transmission of infectious diseases and allowed the possibility of applying exact doses of GnRH. Another important factor is the high degree of interspecies similarity between GnRH peptides allowing to be used for more than one fish species [START_REF] Gothilf | Clearance of different forms of GnRH from the circulation of the gilthead seabream, Sparus aurata, in relation to their degradation and bioactivities[END_REF]. Due to the structure of GnRH peptide, this hormone is exposed to a variety of endopeptidases in the body that reduced its halflife [START_REF] Gothilf | Clearance of different forms of GnRH from the circulation of the gilthead seabream, Sparus aurata, in relation to their degradation and bioactivities[END_REF]. This is a major challenge because GnRH peptide is digested by proteolytic enzymes shortly after the treatment [START_REF] Mylonas | Use of GnRHa-delivery systems for the control of reproduction in fish[END_REF][START_REF] Cleverly | Is the metalloid peptidase EC 3.4. 24.15 (EP24. 15), the enzyme that cleaves luteinizing hormone releasing hormone (LHRH), an activating enzyme?[END_REF]. The synthesized GnRHs are decapeptides that are designed to resist to enzymatic degradation in blood circulation. However, they had only a very short half-life period in fish (up to 23 minutes in gilthead seabream,Sparus aurata L) [START_REF] Gothilf | Clearance of different forms of GnRH from the circulation of the gilthead seabream, Sparus aurata, in relation to their degradation and bioactivities[END_REF][START_REF] Mylonas | Use of GnRHa-delivery systems for the control of reproduction in fish[END_REF][START_REF] Zarski | Effect of different commercial spawning agents and thermal regime on the effectiveness of pikeperch, Sander lucioperca (L.), reproduction under controlled conditions[END_REF]. Failure of singlestage injection protocols to stimulate final maturation in females is probably the result of short residence of GnRH in circulation. It is important to improve hormone formulations that have more resistance to enzymatic degradation with no requirement for further frequent use of hormone. The latter is a priority in development of spawning induction therapies.

There have been different strategies used for increasing the protein's half-life in blood in which one of them is changing the amino acid sequences [START_REF] Kontermann | Recombinant bispecific antibodies for cancer therapy[END_REF][START_REF] Szlachcic | Longer action means better drug: Tuning up protein therapeutics[END_REF][START_REF] Chekan | Structure of the lasso peptide Isopeptidase Identifies a topology for processing threaded substrates[END_REF]. Naturally, GnRH has a half-life of 2 to 4 minutes, which is the result of breaking the bound between amino acids Trp 3 -Ser 4 and Tyr 5 -Gly [START_REF] Cleverly | Is the metalloid peptidase EC 3.4. 24.15 (EP24. 15), the enzyme that cleaves luteinizing hormone releasing hormone (LHRH), an activating enzyme?[END_REF][START_REF] Moradi | The transport and efflux of glycosylated luteinizing hormone-releasing hormone analogs in Caco-2 cell model: contributions of glucose transporters and efflux systems[END_REF]. Typically, substitution of Gly at position 6 with a D-amino acid (D-Ala and D-Arg) in GnRH or with His and Lys increases the metabolic stability [START_REF] Moradi | The transport and efflux of glycosylated luteinizing hormone-releasing hormone analogs in Caco-2 cell model: contributions of glucose transporters and efflux systems[END_REF]. Studies have shown that GAP has a role in the stability of GnRH structure.

Recent advances in the construction of cDNA encoding target protein provided the potential for the production of recombinant peptides [START_REF] Levavi-Sivan | Fish recombinant gonadotropins[END_REF]. The recombinant hormones of fish have been produced in various systems such as yeast Pichia. pastoris, Drosophila S2, silk worm Bomyx mori and bacterial expression system and their biological activity has been evaluated in vitro and in vivo [START_REF] Aizen | Biologically active recombinant carp LH as a spawning-inducing agent for carp[END_REF][START_REF] Levavi-Sivan | Fish recombinant gonadotropins[END_REF][START_REF] Kamei | In vivo gonadotropic effects of recombinant Japanese eel follicle-stimulating hormone[END_REF][START_REF] Ohta | Follicle-stimulating hormone induces spermatogenesis mediated by androgen production in Japanese eel, Anguilla japonica[END_REF]. The production of rGnRH /GAP with acceptable biological activity in fish species can introduce an effective hormone therapy in aquaculture and it is used as specific homologous for the treatment of reproductive disorders in new species. Considering the positive effects mentioned in relation to rGnRH/GAP as an induction of final maturation in breeding fish.

In this study, the stable sequence of decapeptide GnRH was designed based on all native as well as synthetic and GAP sequences was added to decapaptide. After suitable expression of the peptide, purification was performed using affinity tag chromatography. Finally, biology activity of rGnRH/GAP was tested in its capacity of regulating the final maturation in goldfish.

Materials & methods

Bioinformatics studies

The gene sequences and amino acid sequences related to different fish pre-pro GnRH was acquired the GAP area that contributed to the sustainability of the pro-protein were compared among GnRH1 Amur sturgeon (Acipenser schernckii) (GenBank: AOW41585.1), GnRH1 Chinese sturgeon (Acipenser sinensis) (GenBank: AGK30598.1), GnRH2 Chinese sturgeon (GenBank: AGK30597.1), mGnRH Beluga (Huso huso) (GenBank: ABR18540.2), GnRH2 Beluga (GnBank:

EF534706.2). Considering the preserved amino acids in each of the species cited and the best displacement in amino acids in sequence that play a role increasing the half-life and the stability of the peptide against proteolytic digestion, the decapeptide sequence was selected. The relative halflife in the biological environment and the peptide stability were checked at ProtParam (https://web.expasy.org/protparam/) database.

The selected decapeptide (GnRH peptide) was translated to gene sequence using translate tools (www.expasy.org/tools/translate). The GAP region was added due to increase in sustainability of the pro-protein, as well as for ease of decapeptide purification due to the small size of the decapeptide consequently, was selected based on GnRH2 sequence of beluga.

Three-Dimensional (3D) structure of the protein was constructed by comparative modeling according to Swiss Model (http://www.swissmodel.expasy.org/spdbv/) corresponding to its amino acid sequence. Among all current theoretical approaches, comparative modeling is the only method that can reliably generate a 3D model of a protein from its amino acid sequence.

Gene synthesis and cloning

In order to facilitate cloning and increase the stability of the translated sequences, the start and stop codons as well as His-tag were added on the gene sequence and it was synthesized by Shainegene Company (Shanghai, China). The synthesized gene was cloned in an expression vector (pET28a + ). The designed protein contained 77 amino acids, so the expected molecular weight was around 8 kDa.

Transformation of recombinant vector

E. coli BL21 (DE3) became competent as a host for cloning and expression based on the protocol of Sambrook and Russell (2001). Cell suspension was dispensed into 500 µl aliquots stored at -80 °C. For transformation, 10 ng of recombinant vector (pET28a + /GnRH) was transferred into one tube of competent cells and tapping gently. It was incubated on ice for 30 min and heated at 42 °C for 30 seconds then quickly placed on ice. One ml of pre warmed LB medium was added to the vial, and it was placed in a shaking incubator and shaked at 37 °C for 1 h at 225 rpm. One hundred µl of grown recombinant bacteria were poured on a LB plate containing kanamycin antibiotic (50 µg/mL) and incubated at 37 °C overnight. After growth of bacterial colonies on the selective plate, a single colony was taken and transformation was confirmed using colony PCR with specific primer (table 1). The PCR were performed on a PTC-200 thermal cycler (BioRad, USA) by denaturation at 94 °C for 10 min, followed with 30 cycle of amplification at 94 °C for 1 min, 57 °C for 1 min and 72 °C for 90 s and an additional elongation at 72 °C for 10 min after the last cycle. The PCR products were checked by electrophoresis on 1.5 % agarose gel containing ethidium bromide

Gene expression and protein extraction

A single recombinant colony was cultured in 100 mL LB media containing kanamycin (50 µg/mL) at 37 °C overnight. Then 5 mL of starter culture were transferred into fresh LB media (2 L) containing kanamycin (50 µg/mL) and grown up to a cell density of OD 600 = 0.5-0.6. Cell density was determined by measuring the OD of culture at 600 nm with a spectrophotometer.

The culture was induced by adding IPTG to a final concentration of 1 mM and allowing further growth at 20 °C overnight. The induced cells were centrifuged at 5000 g for 10 min. The cell pellet was suspended in lysis buffer )100 mM Tris, pH 8.0, 100 mM Nacl, 10 mM Imidazole, 0.5 % (v/v) Triton x-100, ( and 1X EDTA-Free Protease Inhibitor Cocktail. The cell suspension was subjected to sonication for disruption (3 times for 30 s with 50% pulses), then it was centrifuged at 10000 g for 25 min in 4 °C, after which the supernatant was taken for protein production analysis using SDS-PAGE. The supernatants of induced and un-induced cells were taken on 15% SDS-PAGE. Proteins band were developed by Coomassie brilliant blue staining.

Purification of recombinant GnRH/GAP

The supernatant was purified using Batch/Gravity-Flow column purification with TALON metal affinity resin. The solubilized protein was loaded on Co 2+ -CMA, TALON resin (Clontech). 3 mL of resin suspension was transferred to a sterile column and washed with DD water then equilibrated with 20 bed volume of equilibration buffer containing; 50 mM Tris, 300 mM NaCl, 10 mM Imidazole and 0.05% Triton x-100 at pH 8.0. The solubilized sample (pH adjusted to 8)

was loaded on to the column and shacked at 4 °C for 1 h. After that the column was washed with 10 bed volumes with washing buffer that was the same as equilibration buffer. Finally, the Histagged protein was eluted with 5 bed volumes elution buffer (50 mM Tris, 150 mM Imidazole and 1 % (v/v) Triton x-100, pH 7.5) at a flow rate of 1 mL/min. The elute samples collected in 500 µl fractions. The majority of the His-tagged protein was recovered in the first one bed volume. The fractions were analyzed using 15% SDS-PAGE. The fractions containing rGnRH were dialyzed in sodium chloride serum (0.09%, pH 7.0) with slow stirrer at 4 °C for 18 h. The dialyzed sample was lyophilized and stored at -20 °C for further use.

Western blotting

Purified protein from SDS-PAGE was transferred to nitrocellulose membrane using transfer buffer (39 mM glycine, 48 mM Tris-base, 0.037% SDS, 20% methanol) by Bio-Rad Mini

Protean Tetra Cell System. The membrane was incubated in the blocking buffer of 3% BSA/phosphate-buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, pH7.3) for 2 h. The membrane was then incubated in a monoclonal anti-6xHis tag antibody (at 1:3000 dilution in PBST/BSA (PBS containing 1% Tween 20 and 2% BSA) by gentle shaking at 4 º C overnight. The membrane was washed with PBS/T three times and then incubated with HRPconjugated goat anti mouse IgG (Sigma Aldrich, USA) as secondary antibody diluted 1:5000 in PBST/BSA with gentle shaking for 1 hour for 1 h at 37 •C. The membrane was washed three times with PBS/T and detection was carried out using HRP staining solution. Chromogenic reaction was stopped by rinsing the membrane twice with distilled water.

Determination of protein concentration

Protein concentration was determined by Bradford's method (1976) with bovine serum albumin as a standard (0, 5.0, 10.0, 20.0, 30.0, 40.0, 50.0 and 60.0 µg/mL). The lyophilized peptide was dissolved in saline (0.09%, pH 7) and assayed with Bradford buffer and the absorbance of samples measured at 595 nm with spectrophotometer.

Mass spectroscopy analysis

The purified peptide was studied using MS/MS (MALDI-TOF/TOF) mass spectrometer to reveal amino acid sequence. Sample preparation for Mass spectroscopy was performed by Jean-Baptiste Vincourt in a scientific platform in Nancy, France. 500 ng of the lyophilized protein were suspended in SDS-loading buffer, and it was run on SDS-PAGE. Proteins band were developed by Coomassie brilliant blue staining. The corresponding band was excised and processed as follows for protein content identification:

Cysteines were reduced in 100 mM DTT, and they were prepared in 100 mM ammonium bicarbonate (BA), then they were alkylated in iodoacetamide, 100 mM in BA, each for 45 min. directly upon deposition. Sample acquisition in TOF and TOF/TOF modes were performed automatically using WARP-LC as a pilot software on an Auto flex speed MALDI mass spectrometer (both from Bruker). Peptide assignments, protein identification and scoring were managed on a Proteins cape server allowing a 50 ppm tolerance for mass measurements through interrogation of the whole swiss prot database of a local Mascot server. In addition, as the protein of interest was recombinant, its theoretical artificial sequence was used to assign non-naturally occurring peptides.

In vivo biological activity

Mature female goldfish (Carassius auratus) were obtained from Fish Hatchery Center (Sari, Mazandaran, Iran) and transferred to wet lab located at the Faculty of animal science, Sari, Iran.

The fish were acclimated to the experimental conditions into two 250 L tanks and fed with a commercial carp diet for two weeks. After 2 weeks of acclimation and gradual temperature increase, selected fish were randomly divided into two experimental groups (n = 6). Photoperiod for this indoor experiment was set at 12hL:12hD cycle and temperature was 25.2 ± 0.3 °C. To supply needed water, water was used after 24 h aeration. Air stone aeration was connected to a central air pump (Iran Pash, Urmia, Iran) in each aquarium.

The first treatment consisted in the injection of the recombinant GnRH (20 µg/kg body weight) dissolved in 0.9% NaCl and the second group of fish (control) received 0.9% NaCl at 1 ml/ kg of body weight. Blood samples were collected from 3 fish in each group before injection (0), 17 h after injection (ovulation time) and 24 h after injection for measurement of 17α, 20β-dihydroxy-4-pregnen-3-one (DHP). Spawning success was determined by the number of fish that spawned after the injection.

Enzyme-linked immune sorbent assay for 17α, 20β-dihydroxy-4-pregnen-3-one

DHP was determined by the enzyme-linked immunosorbent assay (ELISA) using a commercial kit (Cat. No: CK-E91529, Eastbiopharm, USA). The assay range is from 1 ng/mL to 400 ng/mL and the kit sensitivity was 0.52 ng/mL.

Statistical analysis

Statistical analyses were performed using the STATISTICA software (StatSoft Inc., Tulsa, OK, USA). Data are presented as a mean ± standard deviation of the mean (SD). Normality and homogeneity of variances were tested by the Kolmogorov-Smirnov and Bartlett methods, in order to comply with the prerequisites of analysis of variance (ANOVA). Within-group differences of DHP levels were analyzed by repeated-measures ANOVA, and between-group differences were analyzed using T-test at each sampling time.

Results

Bioinformatics study

GnRH/GAP protein sequences selected from GnRH1 Amur sturgeon, GnRH1 Chinese sturgeon, GnRH2 Chinese sturgeon, GnRH2 Beluga and mGnRH Beluga and they were aligned with multiple sequence alignment by Clustal Omega (www.EBI.ac.uk/service/tools). The Clustal Omega result showed that pre-pro-GnRH contained a signal peptide, the conserved decapeptide, the cleavage site and the GnRH associated peptide (Fig. 1). Three regions of GnRH including decapeptide, proteolytic processing site and GnRH-associated peptide (GAP) at C-terminal (60 amino acids) were selected according to the preserve amino acids, and resistance to proteolytic digestion and higher half-life and peptide stability and considered for its gene synthesis (Fig. 2).

The comparison of the signal peptide and GAP sequences of GnRH showed a very low conservation between different species of fish. The alignment of Beluga GnRH2 and GnRH2 of Chinese sturgeon was done. Resulting the GAP area selected from Beluga GnRH2 sequence with accession number in GeneBank, EF534706.2 that had up 99% sequence identity with GnRH2 of Chinese sturgeon (Fig. 3).

According to sequence analysis of designed peptide in Expasy/protparam, the estimated half-life was more than 10 h in E.coli, in vivo and up to 1 h in mammalian reticulocytes, in vitro. The instability index (II) was computed to be 10.92 that classified the peptide as stable. The instability index is a measure of proteins, used to determine whether it will be stable in a test tube. If the index is less than 40, then it is probably stable in the test tube. If it is greater than it is probably not stable.

Prediction of the three-Dimensional protein structure

Predicted tertiary protein structure of the rGnRH/GAP was obtained using spdbv in Swiss Model. 3D structure predicted in accordance with the decapeptide sequence designed and selected GAP sequence. Our result showed that the 3D structure prediction of rGnRH decapeptide was somewhat similar to GnRH2 from Beluga built with Swiss Model. The GnRHassociated peptide (GAP) structure was relatively similar to GAP1 of Nile tilapia and sturgeon as well as GnRH2 of Chinese sturgeon. They had a Helix-Loop-Helix structure (HLH) but the recombinant hormone had a large helix and a small helix with a short loop between them (Fig. 4).

GnRH/GAP gene sequence and synthesis

The peptide sequence of GnRH/GAP designed was translated to nucleic acids using translate tools in Expasy and after synthesis and checking up for correct gene sequence, it was then cloned at pET28a. After gene synthesis, the primary sequence was confirmed s (ShineGene, China).

Regarding GnRH sequences of various species of aquatic organism cited in databases and the ability to produce cDNA encoding of this hormone, it was possible to produce recombinant GnRH/GAP (rGnRH/GAP).

Transformation of recombinant vector and expression of the peptide

The Recombinant vector pET28a + which contains the gene of GnRH/GAP was transformed into E. coli BL21 (DE3). After the growth of the E. coli cells containing the recombinant vector a single colony took, transformation was confirmed using colony PCR and a fragment of 185 bp detected at the expected size (Fig. 5). The recombinant bacterial cultured and induced by IPTG then proteins in cells extract from both un-induced and induced cultures checked by SDS-PAGE.

The molecular weight of the expressed peptide was estimated at expected size of 8 kDa and it was detected in the induced culture (Fig. 6).

Purification of recombinant GnRH/GAP

The peptide was soluble in cytoplasm thus it was in cell lysate supernatant. The supernatant was purified using affinity chromatography. After purification the first fractions of elution loaded on a 15% SDS-PAGE and the purified recombinant peptide bands developed by Coomassie brilliant blue staining and a band of about 8 kDa (expected size) was detected according to protein molecular weight marker )Fig. 7).

Western blot analysis of recombinant GnRH/GAP

In Blotting analysis with anti-His-Tag antibodies, a band of 8 kDa developed in the membrane.

The six Histidine tag was designed at carboxyl terminal of recombinant peptide for affinity purification and the His-Tag sequence was not isolated from recombinant peptide (Fig. 8).

Protein concentration

The concentration of recombinant GnRH/GAP peptide determined after dialysis in sodium chloride serum and then lyophilized. The peptide powder dissolved in the same buffer and the concentration of the recombinant peptide was 3.5 mg/mL.

Mass spectrometry analysis

The results of the Mass spectroscopy on this peptide showed that the sequence of the recombinant peptide was exactly consistent with the designed sequence (Fig. 9, 10).

In vivo biological activity

The results showed that no significant difference of DHP level was observed between treatments before injection (P>0.05). A significant difference of DHP level between GnRH/GAP injected fish and control fish was observed 17 h and 24 h after the injection (P<0.01). DHP levels increased 17 h and subsequently decline at 24 h after injection in the fish group that received 20 µg/kg body weight of recombinant GnRH/GAP but no change was observed in DHP level 17 h after injection in the control fish (Fig. 11). All females injected with recombinant GnRH/GAP spawned, while none of the control fish spawned.

Discussion

GnRH is a decapeptide produced in the hypothalamus with a regulatory function in the reproductive system [START_REF] Kah | GnRH and GnRH receptors in metazoa: a historical, comparative, and evolutive perspective[END_REF][START_REF] Okubo | Structural and functional evolution of gonadotropin-releasing hormone in vertebrates[END_REF][START_REF] Gaillard | Characterization of Gonadotropin-Releasing Hormone (GnRH) Genes From Cartilaginous Fish: Evolutionary Perspectives[END_REF]. Different analogs of GnRH with different half-life exist [START_REF] Mylonas | Use of GnRHa-delivery systems for the control of reproduction in fish[END_REF]. These analogs are decapeptides and some amino acids are substituted at the native GnRH to increase half-life. Due to the structure of GnRH peptide, this hormone is exposed to a variety of endopeptidases in the body that reduced its half-life [START_REF] Gothilf | Clearance of different forms of GnRH from the circulation of the gilthead seabream, Sparus aurata, in relation to their degradation and bioactivities[END_REF]. Synthetic GnRH has only a half-life of 23 minutes in vivo because of degradation by enzymatic degradation [START_REF] Mylonas | Use of GnRHa-delivery systems for the control of reproduction in fish[END_REF][START_REF] Moreau | Luteinizing hormone-releasing hormone agonists in the treatment of prostate cancer: a review of their discovery, development, and place in therapy[END_REF]. In this study, the design of bioactive sites was based on interspecies conserved amino acids at position 2, 3, 4, 9 and 10 that remained unchanged. In contrast, the amino acids at positions 1, 5, 7 and 8 were varying among different species of fish [START_REF] Hildahl | Identification and gene expression analysis of three GnRH genes in female Atlantic cod during puberty provides insight into GnRH variant gene loss in fish[END_REF][START_REF] Yue | Molecular cloning of cDNA of gonadotropinreleasing hormones in the Chinese sturgeon (Acipenser sinensis) and the effect of 17βestradiol on gene expression[END_REF]. There were His or Tyr at position 5, Trp or Leu at position 7 and Arg or Tyr at position 8 in examined species. Thus, in order to increase the stability and half-life of the peptide His, Leu and Try were chosen at positions 5, 7 and 8, respectively. There was Glutamine at position 1 in most fishes but there was Glutamic acid instead of Glutamine in some fish species such as Russian sturgeon (Acipenser gueldenstaedti), (GenBank: AAB34379.1) and in other vertebrates and synthetic GnRH. Glu was selected rather than Gln because of differences in the stability index in favor of Glu. Amino acid at position 6 (Gly) was high conserved in different fish species but can be a target site for proteolytic digestion and thus potentially reduces the half-life so, we used serine instead of Gly [START_REF] Moradi | The transport and efflux of glycosylated luteinizing hormone-releasing hormone analogs in Caco-2 cell model: contributions of glucose transporters and efflux systems[END_REF][START_REF] Sealfon | Molecular mechanisms of ligand interaction with the gonadotropin-releasing hormone receptor[END_REF].

Typically, substitution of Gly at position 6 with a D-amino acid in GnRH or with His and Lys increases its metabolic stability [START_REF] Moradi | The transport and efflux of glycosylated luteinizing hormone-releasing hormone analogs in Caco-2 cell model: contributions of glucose transporters and efflux systems[END_REF]. In our design, serine increased more the stability than His and Lys. Substitution of Gly at position 6 with a D-amino acid (D-Ser) in LHRH increases its metabolic stability and the binding affinity to LHRH receptor [START_REF] Moradi | The transport and efflux of glycosylated luteinizing hormone-releasing hormone analogs in Caco-2 cell model: contributions of glucose transporters and efflux systems[END_REF]. Amino acids of Pro 9 and Gly 10 are conserved in all types of GnRH sequences. Since the presence of Gly at the end of Cterminal (position 10) even after proteolytic digestion of the precursor hormone was important for the biological function of the hormone so we did not make replace this the amino acid. Two of the most used analogues in the market are mGnRHa (pGlu-His-Trp-Ser-Tyr-DAla-Leu-Arg-Pro-Net) and sGnRH (pGlu-His-Trp-Ser-Tyr-DArg-Trp-Leu-Pro-Net). These analogues used D-Ala and D-Arg at position 6 (instead of Gly) to increase the stability and ethyl amide at position 10 instead of glycine to increase the binding of the receptor to the ligand [START_REF] Moradi | The transport and efflux of glycosylated luteinizing hormone-releasing hormone analogs in Caco-2 cell model: contributions of glucose transporters and efflux systems[END_REF][START_REF] Podhorec | Induction of final oocyte maturation in cyprididea fish by hypothalamic factors, a review[END_REF]. Despite the changes in the amino acid sequence in these analogues, the half-life of them was not so high [START_REF] Mylonas | Use of GnRHa-delivery systems for the control of reproduction in fish[END_REF]. After the decapeptide the proteolytic site is highly conserved and consist of three amino acids (Gly, Lys and Arg) where the protease digestion occurs in the sequence of this peptide and separates it from GAP area [START_REF] Hildahl | Identification and gene expression analysis of three GnRH genes in female Atlantic cod during puberty provides insight into GnRH variant gene loss in fish[END_REF]. The functional part of GnRH protein is decapeptide (bioactive site) that is processed from the precursor by removal of the signal peptide and cleavage at the dibasic amine acid to separate GAP region [START_REF] Zmora | The GnRH system in the European sea bass (Dicentrarchus labrax)[END_REF]. The signal peptide and GAP sequences showed a very low conservation between species [START_REF] Hildahl | Identification and gene expression analysis of three GnRH genes in female Atlantic cod during puberty provides insight into GnRH variant gene loss in fish[END_REF]. It is presumed that GAP domain involved in detecting the receptors for the precursor protein. Although still controversial, the function of the GAP is believed to be restricted to providing the correct secondary structure for precise processing of the GnRH precursor [START_REF] Zmora | The GnRH system in the European sea bass (Dicentrarchus labrax)[END_REF]. Several methods of peptide modification have been discovered to prolong its half-life and do not require repeated injection of the hormone. These techniques include chemical and genetic methods that increase the half-life of the polymers, such as polyethylene glycol (PEG) and add lipid to the peptide which reduces the rate of renal excretion [START_REF] Moradi | The transport and efflux of glycosylated luteinizing hormone-releasing hormone analogs in Caco-2 cell model: contributions of glucose transporters and efflux systems[END_REF]. It is also possible to change the amino acids in amino and carboxyl ends after chemical synthesis of peptide to increase binding to the cell surface receptors. In genetic engineering, methods with displacement of amino acids that are important in the stability of the structure can increase peptide stability against protease digestion [START_REF] Blomenrohr | Chimaeric gonadotropin-releasing hormone (GnRH) peptides with improved affinity for the catfish (Clarias gariepinus) GnRH receptor[END_REF]. The tertiary structure of GnRH in protein data bank (PDB) only for Gonadotropin-releasing hormone agonist (GnRH1, LHR.H) from homosapiens was recorded (Legrand, p. 2015, http://www.rcsb.org/structure/4D5M). Since the recombinant GnRH sequence with beluga GnRH2 that had up 99% sequence identity, the predicted tertiary structure of rGnRH was compared with the predicted 3-D structure of the peptide of this fish. There was Helix-Loop-Helix structure (HLH) in GAP of rGnRH/GAP, Nile tilapia and sturgeon as well as GnRH2 of Chinese sturgeon. There was a large helix and a small helix with a short loop between them in the recombinant hormone. In sturgeon there were two identical lengths of alpha helices with long loop between them but, in Nile Tilapia and Chinese sturgeon, there were one long and one short alpha helix with a long loop between them (9). Also, the HLH structure was predicted for GAP1 sequences of the vertebrate species analyzed. Some variations were observed in the length of the alpha helices, as well as in the length of the loop [START_REF] Pérez Sirkin | Conservation of Three-Dimensional Helix-Loop-Helix Structure through the Vertebrate Lineage Reopens the Cold Case of Gonadotropin-Releasing Hormone-Associated Peptide[END_REF]. Sirkin et al [START_REF] Pérez Sirkin | Conservation of Three-Dimensional Helix-Loop-Helix Structure through the Vertebrate Lineage Reopens the Cold Case of Gonadotropin-Releasing Hormone-Associated Peptide[END_REF] predicted 3D structure of GAP2, a Helix-loop-Helix structure, with long alpha helices, observed in some species like shark and other teleost. However, some other GAP2 structure showed one or very short or no alpha helix such as in a sauropsid (alligator), in a chondrostean (sturgeon) and in a teleost (arowana).

Comparison of the GAP sequences between GnRH2, mGnRH, GnRH1 and GnRH2 from fish showed the low conservation suggesting that GAP may participate in the folding and processing of GnRH prohormone and the regulation of gonadotropin secretion as suggested by) Bond [START_REF] Bond | Characterization of complementary DNA encoding the precursor for gonadotropin-releasing hormone and its associated peptide from a teleost fish[END_REF] and Seeburg [START_REF] Seeburg | Characterization of cDNA for precursor of human luteinizing hormone releasing hormone[END_REF]..

In this study, E. coli was used for expression system to produce the recombinant protein. Several cell types both prokaryotic (bacteria) and eukaryotic (animal, fungi, etc.) are used to produce recombinant proteins. Bacteria are a good host for production of recombinant peptides and are used for proteins that do not have post-translation changes such as GnRH. They also have multiple benefits, including easy cultivation, low cost and high production potential, thus allowing production from laboratory to industrial scale [START_REF] Rosano | Recombinant protein expression in Escherichia coli: advances and challenges[END_REF]. E. coli as a factory was used for production of recombinant GnRH in some studies as well as the current study [START_REF] Gupta | Engineering, cloning, and expression of genes encoding the multimeric luteinizing-hormone-releasing hormone linked to T cell determinants in Escherichia coli, Protei expression and purification[END_REF][START_REF] Xu | Cloning, expression, and purification of a highly immunogenic recombinant gonadotropinreleasing hormone (GnRH) chimeric peptide[END_REF].

Generally, proteins with molecular weight below 60 kDa are successfully expressed in soluble forms of E. coli [START_REF] Soufi | Recombinant Proteins in Escherichia coli: Optimizing Production Strategies, American-Eurasian[END_REF]. Weber et al. [START_REF] Weber | Evidence that gonadotropinreleasing hormone (GnRH) functions as a prolactin-releasing factor in a teleost fish (Oreochromis mossambicus) and primary structures for three native GnRH molecules[END_REF] isolated three forms of GnRH from native tissue of pituitary gland of tilapia . Extraction from the pituitary gland is accompanied by some problems including the transmission of the disease from the source to the hormone receiver, the process of extraction is time-consuming and the amount of production is low, requiring more fish to euthanize [START_REF] Aizen | Biologically active recombinant carp LH as a spawning-inducing agent for carp[END_REF]. Therefore, the technology of recombinant production or peptide synthesis is more beneficial than extraction of pituitary tissue. In previous study reported the chemical synthetic of GnRH (GnRHa) designed to withstand enzymatic degradation in the blood stream had a higher resistance than normal GnRH against enzymatic degradation and had a half-life of 23 minutes [START_REF] Gothilf | Clearance of different forms of GnRH from the circulation of the gilthead seabream, Sparus aurata, in relation to their degradation and bioactivities[END_REF]. Further studies to estimate the half-life of this recombinant GnRH in the plasma of sturgeon and the kinetics of GnRH enzymatic degradation is under way.

In this study, the expressed peptide was soluble in cytoplasm and there was not any detection in insoluble precipitation (data not shown). Purification process was performed in native condition and there was no need of refolding process of the purified peptide. Xu et al. [START_REF] Xu | Cloning, expression, and purification of a highly immunogenic recombinant gonadotropinreleasing hormone (GnRH) chimeric peptide[END_REF] reported that chimeric peptide (recombinant GnRH) was expressed in an inclusion body and the target peptide purified in denaturing condition [START_REF] Xu | Cloning, expression, and purification of a highly immunogenic recombinant gonadotropinreleasing hormone (GnRH) chimeric peptide[END_REF]. Two fusion proteins, GnRH-P53 and GnRHΙΙΙ-p53 were expressed intracellularly and dissolved in 8M urea and the purification process was performed in denaturing condition with Ni-NAT affinity chromatography [START_REF] Jia | The Novel Fusion Proteins, GnRH-p53 and GnRHIII-p53,Expression and Their Anti-Tumor Effect[END_REF].

The results of the in vivo study showed that DHP level increased 17 h and 24 h after injection by recombinant GnRH/GAP but no change was observed in DHP level in the control fish. All females injected with recombinant GnRH/GAP spawned, while none of the control fish spawned.

The ovulation-induction effect of GnRH was observed in common carp (Cyprinus carpio) [START_REF] Drori | Spawning induction in common carp (Cyprivtus carpio) using pituitary extract or GnRH superactive analogue combined with metoclopramide: analysis of hormone profile, progress of oocyte maturation and dependence on temperature[END_REF].

The ability of mGnRHa to induce ovulation goldfish is similar to several species [START_REF] Podhorec | The effects of GnRHa with and without dopamine antagonist on reproductive hormone levels and ovum viability in tench Tinca tinca[END_REF]. The potential of DHP to induce final maturation was demonstrated in several fish species [START_REF] Canario | Structure activity relationships of C21 steroids in an in vitro oocyte maturation bioassay in rainbow-trout, Salmo-gairdneri[END_REF](52)(53)(54). In this study, recombinant GnRH/GAP stimulated a surge of blood DHP occurring 17 h after injection subsequently decline at 24 h. Podhorec [START_REF] Podhorec | The effects of GnRHa with and without dopamine antagonist on reproductive hormone levels and ovum viability in tench Tinca tinca[END_REF] reported that GnRH treatments stimulated a surge of DHP detected at 12 h post-injection in tench (Tinca tinca). Pinillos et al (55) reported a peak of DHP occurring 6 h later with the subsequent decline seen at 24 h in tench. The effectiveness of GnRH/GAP injection to promote DHP elevation and spawning is the demonstration of the biological activity of the recombinant peptide.

Conclusion

The recombinant GnRH/GAP was successfully produced in E.coli expression system in soluble form and then purified and a significant concentration of the peptide obtained. The specificity was checked at both the DNA and protein levels using PCR, SDS-PAGE and western blot respectively.

Mass analysis confirmed that the sequence of the recombinant peptide was exactly the same as the designed sequence. Due to the negative effects of multiple hormone injections in fish, it is now necessary to develop a hormonal therapy that has a higher half-life and that does not require multiple injections to induce sexual maturation in fish.

Fig. 1 .

 1 Fig. 1. Multiple alignment of GnRHs from fish. Signal peptide, amino acids 1-24; Decapeptide,

Fig 2 .Fig. 3 .

 23 Fig 2. The amino acid sequences of GnRH designed. Decapeptide, the grey color (10 aa),

Fig 4 .Fig. 5 .Fig. 6 .

 456 Fig 4. 3D-structure model constructed according to Swiss-Pdb-Viewer in Swiss-Model. The N-

Fig. 7 .

 7 Fig. 7. SDS-PAGE gel electrophoresis of the recombinant peptide. Line1, Protein molecular

Fig 8 .Fig. 11 .

 811 Fig 8. Western blot analysis of rGnRH/GAP after purification. Line1, The band of 8 kDa of

  After 2 washing, cycles in BA and BA/acetonitrile, 1:1, 15 min each, gel bands were dried in a speed-vac for 1 hour, and they were digested with 100 ng trypsin in 20 ul BA overnight. Peptides were extracted twice consecutively in 25 ul acetonitrile, 80%, TFA 1% for 10 min under

	sonication in a water bath sonicator. Extracted peptides were pooled, dried in a speed-vac and
	resuspended in 10 ul 2% acetonitrile, 0.1% TFA.
	HPLC was performed using Ultimate 3000 equipment (Thermo). Peptides (6.4 ul) were injected
	in the micro liter pickup mode onto a desalting column and loaded onto a 15 cm Acclaim pep

map RSLC C18 column (Thermo) and eluted over a 30 min run by a 2-45% acetonitrile linear gradient and 170 fractions were collected onto a 1536 TF anchorchip MALDI plate via a Proteineer FcII fractionator (Bruker) and mixed with α-Cyano-4-hydroxycinnamic acid (HCCA)

Table 1

 1 All females goldfish injected with recombinant GnRH/GAP spawned which is promising to use this recombinant peptide to induce spawning in other teleost fish species of interest for aquaculture. 52. F.W. Goetz. Hormonal-control of oocyte final maturation and ovulation in fishes, Fish Pinillos., A.I. Guijarro., M.J. Delgado., P.C. Hubbard., A.V.M. Canario., A.P. Scott.Production, release and olfactory detection of sex steroids by the tench (Tinca tinca L.), Primer pairs used in the amplification of rGnRH.

	Physiol. 9 (1983) 117-170.
	53. K. Yamauchi., H. Kagawa., M. Ban., N. Kasahara., Y. Nagahama.. Changes in plasma
	estradiol-17-beta and 17-alpha,20-beta-dihydroxy-4-pregnen-3-one levels during final
	oocyte maturation of the masu salmon Oncorhynchus masou, Bull. Jpn. Soc. Sci. Fish.
	2137.
	54. M.L. Fish Physiol Biochem. 26 (2002) 197-210
	Gene		Primer Sequence (5' -3')	Size (bp)
	GnRH	Forward CCATGGGAACATTGGAGCCATAGC	186
		Revers	AAGCTTCTTCTTCCTCTGCATCTG
	.

Acknowledgements

The authors are gratefully thanks for providing the facility and assistance of the Cell and Molecular and Fisheries Labs, Sari Agricultural Sciences and Natural Resources University and

Mass spectrometry was performed at the proteomics core facility of UMS 2008, UL-CNRS-INSERM, IBSLor, http://umsibslor.univ-lorraine.fr"