1. Introduction

Amphibian metamorphosis is a complex process, in which various organs and tissues undergo dramatically remodeling to transform from larva to juvenile(Atkinson et al., 1998; Brown and Cai, 2007; Du et al.,2017). Although complex, this development process is completely initiated and orchestrated by only one hormone, thyroid hormone (TH) (Buchholz et al., 2006).The TH signaling pathway in amphibians has been well studied: when TH is absent, the unliganded TR/9-cis-retinoic acid receptor (RXR) heterodimers recruit corepressors to repress the transcription of downstream target genes; once liganded with TH, the TR/RXR heterodimers undergo conformational change and thus allow to recruit coactivators to activate the same group of downstream genes (Morvan-Dubois et al., 2008; Grimaldi et al., 2013; Zhao et al., 2016). Therefore, TRs play important roles in the metamorphosis by acting as liganddependent transcriptional factors.

TRs, members of a large superfamily of nuclear receptors (NR), possess a similar domain structure as that found in the other NRs: an N-terminal A/B domain with binding sites for transcriptional coregulators, a central DNA-binding domain C (DBD) containing two“zinc fingers” for target gene recognition, a D domain(hinge region) containing the nuclear localization signal, and a C-terminal ligand-binding domain E/F (LBD) where thyroid hormone binds and activates the receptor (Chen et al., 2014). There are two closely related families of TRs called TRα and TRβ in vertebrate(Yaoita et al., 1990; Chen et al., 2014). TRα and TRβ are differentially expressed in various tissues of different species (Kawahara et al., 1991). Particularly, the TRα mRNA increases throughout the premetamorphosis stage of tadpole development, and falls after the climax of metamorphosis to a lower level in frogs (Yaoita and Brown, 1990). The TRβ mRNA is barely detectable during premetamorphosis. In synchrony with the onset of endogenous TH synthesis by the thyroid gland, the level of TRβ mRNA rises in parallel with endogenous TH, reaching a peak at the climax of metamorphosis and drops after metamorphosis (Choi et al., 2015). Although TH signaling pathway has been well studied, functional of TRα and TRβ during metamorphosis have not been clearly characterized. TRα-deficient tadpoles developed faster with smaller body size than their wild-type siblings suggesting that TRα played important roles in controlling the timing of Xenopus tropicalis metamorphosis (Choi et al., 2015; Wen and Shi, 2015). Furthermore, disrupted TRα had different effect on the development of larval and juveniles and the metamorphosis of different organs (Choi et al., 2017, Wen et al., 2017). Different from TRα-knockout tadpoles, significantly delayed tail regression, the reduction in olfactory nerve length and head narrowing by gill absorption were detected in TRβknockout tadpoles (Nakajima et al., 2017). The different relative abundance levels of TRα and TRβ transcripts induced by T3 where the general pattern was TRα≥ TRβ in R. catesbeiana, while TRα ≤ TRβ in Xenopus laevis(Veldhoen et al., 2014). TRβ was highly expressed during metamorphosis in M. fissipes and X.laevis, but TRα showed especially low expression in M. fissipes, implying that TRβ is essential for initiating metamorphosis, at least in M. fissipes (Zhao et al., 2016).

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Microhyla fissipes is a small-sized anuran from the family Microhylidae suborder Neobatrachia (Figure 1). Due to the special expression pattern of TRs in M.fissipes mentioned above, it is important to clone TRs and understand the molecular mechanism of them in regulating metamorphosis. Furthermore, because of its characteristics (including wide distribution, fast development, development in vitro, strong survivability,biphasic life cycle, small body size, diploid and transparent tadpoles) and being induced to metamorphose by exogenous TH, M. fissipes may be an ideal model to evaluate the possible effects of environmental compounds on the thyroid system (Liu et al., 2016).

第一，赛事标志、特许经营与竞赛项目的冠名。1)竞赛项目、火炬传递等活动的冠名与经营；2)特殊标志的分类使用；3)赛事合作者、赞助商的称号授予；4)银行、保险、商品、商场等特殊冠名；5)特许经营与代理经营。第二，赛事广告与媒体资源。1)电视转播、采访、官网、商网、新闻发布会广告等；2)场馆内外广告；3)户外广告，包括征用的机场、车站、广场、道路等区域的广告；4)印刷品广告，包括票证、竞赛指南、秩序册等广告；5)特殊赛事广告，包括热气球、动力伞等赛事广告。第三，开(闭)幕式、门票及纪念品的经营开发。第四，与赛事有关的商业性体育比赛、文艺演出、义赛等。第五，体育彩票与社会捐赠。

Figure 1 Photograph of Microhyla fissipes.

Therefore, it is necessary to characterize TRs and evaluate responsiveness to TRs agonist (such as 3,3',5-Triiodo-L-thyronine, T3) in M. fissipes.In this study, we have isolated, characterized, and phylogenetically analyzed TRα and TRβ gene in M. fissipes, examined their expression pattern after T3 treatment and explored the utility of M. fissipes as a model species for assaying TH signaling disrupting effects.

2. Materials and Methods

2.1. Animals sampling and experimental treatments Mature female and male M. fissipes were collected from Shuangliu, Chengdu, China (30.5825o N, 103.8438o E) in June, 2016. The male and female were injected luteinizing hormone-releasing hormone a (LHRHa) with 0.3 μg/g body weight resolving dosage. Fertilized eggs were obtained from one pair of frogs and incubated in the dechlorinated tap water. Five days later, tadpoles were fed with spirulina powder once daily and subjected to a 12:12 h light:dark cycle at 25 ± 0.6°C. The developmental stage of tadpole was recorded using the M. fissipes developmental table (Wang et al., 2017). Tadpoles at stage 40 (metamorphosis climax) were selected for gene cloning. Tadpoles of stage 33 (premetamorphosis, oarshaped limb bud) were selected to treat with 10 nmol/L 3,3',5-triiodo-l-thyronine sodium (T3, Sigma-Aldrich,USA) for 48 h. The chemicals were renewed after 12 h of exposure when the medium was also refreshed. Tadpoles treated for 0 h, 12 h, 24 h, 36 h, and 48 h were collected for quantitative real-time (RT) PCR (n = 3 for each time point). Tadpoles treated 0 h were set as the control group.After anesthetization by MS222, tadpole sample was frozen immediately in liquid nitrogen, and then stored at–80°C for RNA extraction.

3.1. Molecular characterization of TRs in M. fissipes The full-length cDNA sequences of TRα and TRβ were obtained by RNA-seq and 3'-RACE strategies. The full-length of TRα cDNA was 1 706 bp in length and contained an open reading frame (ORF) of 1 257 bp, which encoded a peptide of 418 amino acids (Figure 2a). The TRβ cDNA was 1 422 bp with an ORF of 1 122 bp, which encoded a peptide of 373 amino acids (Figure 2b). And the 3'untranslated region of TRα and TRβ were 449 bp and 300 bp, respectively. Two sequences were submitted to the GenBank (GenBank accession numbers: MG596879 and MG596880). The homologies of nucleotide sequences and deduced amino acid sequences between TRα and TRβ in M. fissipes were 61% and 72%, respectively. The calculated molecular weight of TRα polypeptide was 47.7 kDa, and the theoretical isoelectric point (pI) was 7.08,while the calculated molecular weight of TRβ polypeptide was 42.4 kDa with pI 6.76.

在废水pH值为4、H2O2加入量为10 mL/L的条件下，研究石墨烯加入量对制浆中段废水CODCr去除率和出水UV254值的影响，结果如图3所示。

A phylogenetic tree constructed by the ML method from a multiple alignment of nucleotide sequences of M.fissipes TRα and TRβ and a wide range of counterparts in various species including invertebrate, reptiles,birds, mammals and other amphibian (Figure 4). The phylogenetic tree showed that TRα and TRβ grouped into two highly consistent and separate clades. In both TRα and TRβ clades, TRs from M. fissipes have similar positions in the phylogenetic tree. Furthermore, phylogenetic tree constructed based on TRs cDNA sequences was consistent well with the taxonomic positions of these organisms.

2.3. RNA isolation and gene expression analysis For TRs mRNA expression analysis after exposure of T3,we conducted qRT-PCR. Total RNA was extracted from tadpoles with TranZol reagent and first strand cDNA was synthesized from the same amount of RNA (1 μg) for each sample via TranScript All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (TransGen, Beijing, China)with oligo (dT) primer. The expression of TRα and TRβ mRNA was analyzed by qRT-PCR with the primer TRα and TRβ (Table 1). Rpl37 gene was used as a reference gene to normalize mRNA expression of TRs. PCR was performed in a reaction volume of 20 μl containing 10 μl of TransStart® Tip Green qPCR SuperMix (2×), 0.4 μl of Passive Reference Dye (50×), 0.4 μl each of forward and reverse primer (10 μmol/L), 8.2 μl of ddH2O, and 1 μl of cDNA. Amplification was carried out in 7300plus(ABI, CA, USA), including 5 min at 95°C and 45 cycles of 5 s at 95°C and 31 s at 60°C, followed by a melting curve analysis. Each sample was run in triplicates. Each reaction was verified to contain a single product of the correct size by agarose gel electrophoresis. Quantitative data were shown as mean ± SD (n = 3). The fold change of TRs expression after T3 treatment was determined by 2−ΔΔCt (cycle threshold, Ct). Data were then subjected to one-way analysis of variance (ANOVA) with SPSS Statistics 13.0 (SPSS Inc., Chicago, IL, USA). P ＜ 0.05 was regarded as statistically significant.

3.3. Responsiveness of M. fissipesTRs expression to exogenous T3 In the presence of exogenous T3,the morphology of M. fissipes tadpoles has changed dramatically, and the characteristic of morphology change trend was similar to that during the natural metamorphosis(data not shown). To determine whether exogenous T3 could potentially regulate the expression pattern of TRα and TRβ, we exposed the premetamorphic tadpoles to T3 and analyzed by qPCR. These results showed that M.fissipes TRα and TRβ mRNA expression were significantly increased by T3 at 12 h and 24 h, respectively. And then TRα and TRβ expression decreased to the lower level at 48 h than 0 h (Figure 5). Furthermore, the same gene expression pattern of TRβ has been detected between exogenous T3 induced and natural metamorphosis (Zhao et al., 2016). Therefore, different responsiveness of TRα and TRβ to T3 indicated different functions of them in the metamorphosis.

Multiple cDNA sequences of TRα and TRβ from 17 species represented vertebrate (Mammalia, Aves, Reptilia,Amphibia, Pisces) and invertebrate were used in the sequence alignment by Clustal X. A phylogenetic tree was constructed by using the maximum likelihood (ML)method with the MEGA 6 (Tamura et al., 2013), and the reliability of the tree was assessed by the bootstrap method with 1,000 replications. The gene accession numbers are: Alligator mississippiensisTRα NM_001287278.1,Branchiostoma lanceolatumTRα EF672345.1,Crassostrea gigas TRα KP271450.1, Gallus gallus TRα NM_205313.1, Homo sapiens TRα AB307686.1,Oryzias latipesTRα AB114860.1, Rattus norvegicusTRα M18028.1, P. nigromaculatus TRα KC139354.1,R. catesbeiana TRα L06064.1, R. chensinensis TRα KJ579109.1, R. rugose TRα AB683466.1, Schistosoma mansoni TRα AY395038.1, S. mansoni TRβ AY395039.1,X. laevis TRαA M35343.1, X. laevis TRαB AB669465.1,X. tropicalis TRα NM_001045796.1, Nanorana parkeri TRα XM_018569566.1, Oryzias latipesTRα NM_001104705.1, Danio rerio TRα NM_131396.1,R. rugosa TRβ AB683467.1, R. chensinensis TRβ KJ579110.1, R. catesbeiana TRβ L27344.1, P.nigromaculatus TRβ KC139355.1, N. parkeri TRβ XM_018570223.1, X. laevis TRβA NM_001096713.1,X. laevis TRβB NM_001087781.1, X. tropicalis TRβ AB244214.1, G. gallus TRβ NM_205447.2, A.mississippiensis TRβ NM_001287311.2, H. sapiens TRβ M26747.1, R. norvegicus TRβ J03933.1, O. latipes TRβ AB114861.1, D. rerioTRβ NM_131340.1.

边界条件的设置中V形结构与主梁交汇处及主梁立柱连接均采用刚性连接模拟；主梁顶部中心节点和主梁立柱用刚性连接模拟；主梁立柱底部节点和支座顶部节点用刚性连接模拟；永久支座依据支座规格对应设置竖向、横桥向、纵桥向(SDx、SDy、SDz)刚度进行弹性支撑模拟，依据设计图选定的支座类型计算的支座反力即可得出弹性支撑的竖向刚度，然后依据该支座的横纵向刚度与竖向刚度比例关系(10%)来进行横、纵桥向刚度设置；支座底部用一般支承固结，即约束该点的六个方向的自由度(三个方向平动及三个方向的转动)。

3. Results

设计意图：通过铺设梯度，两个活动都巧妙地化解了本课的难点，用形象地模型建构清晰地梳理了物质循环和能量流动的规律，帮助学生实现了概念的螺旋式上升。至此，学生能够更好地解答“一山不容二虎”的奥秘。

Table 1 Primers used in this study. F and R denote forward and reverse primer, respectively.

Funticon Primers Sequence (5' to 3') Size (bp) Tm (°C)Cloning TRαGSP3-1 CCATCGCAAACACAACATTCCCCA 621 50 TRαGSP3-2 TGACTTGCGTATGATCGGAGCCTG 567 60 TRβGSP3-1 GCTGATGAAAGTCACCGACCTCCG 424 50 TRβGSP3-2 CGCCAGCAGGTTCTTGCACATG 386 60 3' CDS primer A AAGCAGTGGTATCAACGCAGACTAC(T)30 VN(N = A, C, G, or T; V = A, G, or C)UPM CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT CTAATACGACTCACTATAGGGC RT-PCR TRα F: GCAGCCGTGCGCTATGAT 57 60 R: TGCCATCTCACCGCTTAGTG TRβ F: TGGCCAAAACTGCTGATGAA 56 60 R: GCGTGGCAGGCTCCAA Rpl37 F: CCAAAAAGCGCAACAACCA 59 60 R: TTGCGAATCTGACGGACTTG

A multiple alignment of TRs deduced amino acid sequences was performed in amphibians (Figure 3).The deduced amino acids of both TRα and TRβ were composed of the N-terminal hypervariable region (A/B domain), a conserved DNA-binding domain (DBD domain), a hinge region (D domain), and a ligand-binding domain (LBD domain). TRα and TRβ in M. fissipes had high similarity in the DBD, D and LBD domains, whereas there was a deletion of 42 amino acids in TRβ compared with TRα in the A/B domain (Figure 3). These deletion between TRα and TRβ in M. fissipes corresponded to the differences found in X. laevis, X. tropicalis, R.chensinensis and P. nigromaculatus. Although there were several different sites in the A/B domain among TRs, the N-terminal signature sequence (NTSS) was well conserved. The conserved cysteine residues that comprise the two zinc fingers and the regulatory elements P-box as well as the T-box and A-box in the DBD were conserved. Furthermore, the consensus motif I (spanning helix 3–6) and motif II (from helix 8 to helix 10) and the putative AF2 activation domain core (helix 12) were also identified. Therefore, this highly-conserved feature is likely to indicate pivotal significance of TRs in thyroid signaling pathway.

3.2. Evolutionary relationships of TRs BLASTP and BLASTN in NCBI indicated that TRα and TRβ shared different levels of homology with other species. The amino acid sequences of M. fissipesTRα had highest homolgy with R. rugosa, N. parkeri, R. chensinensis(98%, 10 different sites out of 418 amino acids),while M. fissipesTRβ had highest homolgy with R.catesbeiana (98%, 8 different sites out of 373 amino acids). Nucleotide sequences of M. fissipesTRα and TRβ shared high homology with N. parkeri (92% and 93%), P. nigromaculatus (92% and 92%), R. rugosa(93% and 92%), X. tropicalis (88% and 87%), X. laevis A (88% and 80%), X. laevisB (88% and 78%) and had a lower homology with G. gallus (65% and 74%), A.mississippiensis (63% and 63%), H. sapiens (58% and 47%), D. rerio (57% and 49%), B. lanceolatum (47% and 49%), respectively.

The care and treatment of animals in this study were performed according to the Guideline for the Care and Use of Laboratory Animals in China. The animal experiments were approved by the Experimental Animal Use Ethics Committee of the Chengdu Institute of Biology (Permit Number: 2016036).

2.2. Cloning and molecular characterization of TRs Total RNA was extracted using TransZol (Transgen Biotech, Beijing, China), following the manufacturer’s instructions. Total RNA concentration was calculated using Nanodrop ND-1000 (Nanodrop, DW, USA).Partial cDNA sequence of TRs (TRα: comp77374_c0 ;TRβ: comp124487_c3) were obtained from M. fissipes transcriptome (Zhao et al., 2016). According to the partial cDNA sequence, two specific primers for each gene,GSP3-1 and GSP3-2 (Table 1) were designed to amplify the 3' terminal regions by nested PCR. The 3' DNA ends were obtained using the SMART RACE cDNA Amplification Kit (Clontech, CA, USA) in accordance with the manufacturer’s instructions. Products of rapid amplification of cDNA ends (RACE) were cloned into pMD 18-T vector (TaKaRa, Japan) and sequenced using an automated DNA sequencer ABI3730 (Thermo Fisher Scientific, CA, USA) by Sangon Biotech Co. Ltd.(Shanghai, China).

Figure 2 cDNA and deduced amino acid sequences of Microhyla fissipes thyroid hormone receptor gene. (a) TRα; (b) TRβ. The start codon(ATG) and the stop codon (TAG or TGA) are in bold.

Figure 3 Alignments of TR amino acid sequences from Microhyla fissipes with other amphibian species. Asterisk (*) indicated conserved amino acids and hyphens (−) represented spaces inserted to maximize similarity. Colon (:) indicates conservation between groups of strongly similar properties, and period (.) indicates conservation between groups of weakly similar properties. The black vertical lines indicate the borders of four domains: the N-terminal hypervariable region (A/B domain), DNA-binding domain (DBD domain), hinge region (D domain)and ligand binding domain (LBD domain). Triangle indicated the conserved cysteine residues that comprise the two zinc finger with red box of the DBD. The conserved N-terminal signature sequence (NTSS), DR-box, P-box, T-box, A-box and Helices (H3-H12) are figured out.Motif I and Motif II are boxed by blue while activation domain (AF2-AD) is purple double underlined.

The amino acid sequence was deduced from the coding region via DNAStar (version 6.13). The cDNA sequence and the deduced amino acid sequence were analyzed using BLASTN and BLASTP, respectively. Deduced amino acid sequences of amphibian were aligned for analysis of putative conserved functional residues by Clustal X. The relevant amino acid sequences were obtained from the NCBI GenBank database: Rugosa rugoseTRα BAM15695.1, Pelophylax nigromaculatus TRα AGT55994.1, Rana chensinensisTRα AIA98429.1,X. laevisTRαA NP_001081595.1, X. laevisTRαB BAL70322.1, X. tropicalis TRα NP_001039261.1,X. tropicalis TRβ NP_001039270.1, X. laevis TRβA NP_001090182.1, X. laevisTRβB NP_001081250.1, P.nigromaculatusTRβ AGT55995.1, R. chensinensisTRβ AIA98430.1.

Figure 4 Phylogenetic tree of nucleotide sequences of TRs genes of different species using the the maximum likelihood method. Numbers at the branches represent the bootstrap support values. Microhyla fissipesTRα and TRβ are highlighted in the box, respectively.

4. Discussion

This study was designed to determine TRα and TRβ sequences and analyze their expression patterns after T3 exposure to gain further insights as to how these genes may function in metamorphosis. Therefore, TRα and TRβ genes of M. fissipes were cloned by RNA-seq and RACE. Phylogenetical analysis showed M. fissipesTRα and TRβ gene had high homology with the corresponding genes of other amphibians at both the nucleotide and the amino acid level, respectively, confirming their identities. The expression patterns after T3 treatment indicated the important roles of TRα and TRβ during the metamorphosis.

Figure 5 Relative expression levels of TRs genes after exposure to 10 nmol/L T3. Data were expressed as the mean fold difference(mean ± SD, n = 3) from the control group. Significant differences between treatment groups and the controls were indicated by* (P ＜ 0.05).

Both TRα and TRβ amino acid structure identified in M. fissipes possessed the typically functional domains of the NR superfamily. All of them had a conserved DBD domain with two zinc fingers, which determined the binding specificity of nuclear receptors (Natalia and Thorsten, 2004), and a LBD domain containing the typical 12 helices (Marchand et al., 2001). The P-box determining DNA binding specificity interacted with the specific response element AGGTCA, and the T-box and A-box regions contributed to dimerization and DNA binding stabilization, respectively (Manchado et al., 2009). The conserved NTSS (GYIPS(Y/H) L(D/N)KDE(P/L)) which was the TR specific motif was also detected in the C-terminus of the variable A/B domain of M. fissipes TRs (Wu et al., 2007). The deletion of 42 amino acids in the A/B domain of TRβ indicated its different function from TRα. The conserved AF2-AD motif (LFLEVF) played an important role in recruiting a coactivator (Nagy et al., 1999; Nelson and Habibi, 2009).The conservation of structure and functional roles of the above mentioned sites in TRs would be consolidated by their high identity throughout the evolution of vertebrates.

TRα and TRβ have been demonstrated to have high homology across vertebrates (Oka et al., 2013). In this study, the amino acid and nucleotide sequence homologies of TRα and TRβ between M. fissipes and other amphibians were over 90%, but lower homology with invertebrates. In the phylogenetic tree, vertebrate TRα and TRβ were located in two clearly separated clades, in accordance with the fact that TRα and TRβ may be the products of an ancient gene duplication event during evolution (Chen et al., 2014). Furthermore, the homologies of TRα and TRβ to the corresponding genes from other species accorded with their evolutionary relationship. Only one TRα and TRβ gene were identified in amphibian except in X. laevis which is tetraploid. The homologies of nucleotide sequences and deduced amino acid sequences between TRα and TRβ in M. fissipes were 61% and 72%, respectively. The homology of TRα to TRβ in M. fissipes was lower than R. nigromaculata (72%and 86%) and X. laevis (74% and 85%). Low sequence identification of TRα and TRβ may indicate their different regulated function in M. fissipes metamorphosis, which is also implied by their different expression pattern during natural metamorphosis (Zhao et al., 2016).

Metamorphosis is a critical developmental stage mediated by TH in amphibian (Wang et al., 2008).The function of TRs as transducers of TH responses has converted NRs in targets to clarify the molecular mechanisms that govern metamorphosis (Manchado et al., 2009). To understand the potential importance of these two receptors in M. fissipes metamorphosis and function of exogenous ligand for the receptor systems, we examined their mRNA expression levels by qPCR after T3 exposure. T3 not only induced M. fissipes premetamorphic tadpoles metamorphosis at the morphology and histology level (data not shown), but also induced TRα and TRβ expression, which have also been reported in X. laevis,X. troplis and R. catesbeiana (Shi, 1999; Wang et al.,2008). During the natural metamorphosis, TRβ expression increased dramatically and correlated with the endogenous THs, while TRα expression slightly increased; and all of them decreased at the end of metamorphosis (Shi,1999; Zhao et al., 2016). In this study, TRα expression reached peak at 12 h and then decreased from 12 h to 48 h. While dramatically up-regulated TRβ expression was observed after exposure of T3 within 24 h, and it was down-regulated from 24 h and with the lowest expression observed at 48 h of T3 treatment. These results suggested that TRβ expression pattern after T3 treatment is the same as that during natural metamorphosis, and the expression of TRβ in tadpoles treated with T3 for 24h also resembled its expression in the tadpoles at the climax of metamorphosis (Zhao et al., 2016), which correlated with the morphological and histological results. Therefore,we can use T3 to simulate metamorphosis for further research on metamorphosis which will be high-efficiency and time saving. Moreover, M. fissipes could also serve as the model to assay environmental compounds on TH signaling disruption, while expression of TRs(in particular TRβ) has been also used as a molecular biomarker for assaying TH signaling disrupting actions in X. laevis and R. nigromaculata because of their response to TH (Opitz et al., 2006; Veldhoen et al., 2006; Lou et al., 2014). Due to the specific expression level of TRs during M. fissipes natural metamorphosis and dramatically up-regulated TRβ mRNA expression after T3 treatment,we will use genome editing tools such as CRISPR/Cas9 to illustrate the mechanism of TRα and TRβ in M. fissipes.In conclusion, TRα and TRβ from M. fissipes were cloned and characterized for the first time. Functional site and phylogenetic analysis indicated the conserved function of TRs from invertebrate to vertebrates. Premetamorphic tadpoles treated with T3 for 24 h resembled the climax of metamorphosis tadpoles during natural metamorphosis,and TRβ mRNA expression analysis could serve as a sensitive molecular testing approach to study effects of environmental compounds on the thyroid system in M. fissipes.

“轻用户端，重云端”是目前IT架构的发展趋势[5]，云端采用Apache做web服务器，服务端程序设计采用三层架构，即控制层、业务逻辑层和数据访问层，控制层负责接收用户端发送的数据请求，并调用业务逻辑层；业务逻辑层对控制层传来的请求进行二次处理，调用数据访问层，并将调用结果返回到控制层；数据访问层封装了对数据库的增删查改方法。用户端与云端服务器通信遵循HTTPS协议，数据交互采用轻量级JSON数据格式，对来往数据采用MD5加密，确保信息安全。云端架构如图2所示。

Acknowledgements This study was funded by the Important Research Project of Chinese Academy of Sciences (KJZG-EW-L13), 2015 Western Light Talent Culture Project of the Chinese Academy of Sciences(Y6C3021) and the Basic Application Project of Sichuan Province (2017JY0339). We thank Lanying ZHAO for the data analysis, and we also thank Liezhen FU and Shouhong WANG for the manuscript revision.

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