Molecular characterization of thyroid hormone receptor beta from Schistosoma japonicum and assessment of its potential as a vaccine candidate antigen against schistosomiasis in BALB/c mice
© Qiu et al.; licensee BioMed Central Ltd. 2012
Received: 12 April 2012
Accepted: 6 August 2012
Published: 13 August 2012
Thyroid hormones (TH) modulate growth, development and differentiation and metabolic processes by interacting with thyroid hormone receptors (THRs). The purpose of this study was to identify a novel thyroid hormone receptor beta encoding gene of Schistosoma japonicum (SjTHRβ) and to investigate its potential as a vaccine candidate antigen against schistosomiasis in BALB/c mice.
The full-length cDNA sequence of SjTHRβ, its gene organization, and its transcript levels were characterized, and the phylogenetic relationship between THR, RAR and RXR from other organisms were analysis, the ability of this protein binding to a conserved DNA core motif, and its potential as a vaccine candidate antigen against schistosomiasis in BALB/c mice were evaluated.
The SjTHRβ cDNA was cloned, verified by 5’ and 3’ Rapid Amplification of cDNA Ends and shown to be polyadenylated at the 3’end, suggesting the transcript is full-length. SjTHRβ is homologous to THRs from other species and has a predicted conservative DNA binding domain and ligand binding domain that normally characterizes these receptors. A comparative quantitative PCR analysis showed that SjTHRβ was the highest expressed in 21d worms and the lowest in 7 d and 13 d schistosomula. The cDNA corresponding to DNA binding domain (SjTHRβ-DBD) and ligand binding domain (SjTHRβ-LBD) were cloned and subsequently expressed in E coli. The expressed proteins were used to immunize mice and generate specific serum against recombinant SjTHRβ (rSjTHRβ). Western blotting revealed that anti-rSjTHRβ-LBD serum recognized two protein bands in extracts from 21 d worm with molecular sizes of approximately 95 kDa and 72 kDa. Electrophoretic mobility shift assay (EMSA) analysis showed that rSjTHRβ-DBD could bind to a conserved DNA core motif. Immunization of BALB/c mice with rSjTHRβ-LBD could induce partial protective efficacy(27.52% worm reduction and 29.50% liver eggs reduction)against schistosome infection. Enzyme-linked immunosorbent assay showed that mice vaccinated with recombinant SjTHRβ-LBD (rSjTHRβ-LBD) generated increased levels of specific IgG, IgG1 and IgG2a antibody. Bio-plex analysis demonstrated that rSjTHRβ-LBD induced considerably higher levels of T helper 1 cytokines (IL-2, IL-12 and TNF-α) than T helper 2 cytokines (IL-10, IL-4), suggesting that rSjTHRβ-LBD vaccination could stimulate mixed Th1/Th2 types with Th1 dominant immune responses.
Our study presented here identified SjTHRβ as a new schistosome THR that might play an important role in host-parasite interaction and be a vaccine candidate for schistosomiasis.
Thyroid hormones (TH) modulate growth, development and differentiation and metabolic processes by interacting with THRs [1, 2]. THR which belongs to nuclear receptor (NR) superfamily 1 possesses a typical nuclear receptor modular motif, including a poorly conserved N-terminal A/B domain, a highly conservative DNA binding domain (DBD), a hinge region and a moderately conservative C-terminal ligand binding domain . In the traditional model of THR action, THR regulates gene expression through its binding to the promoter region of the target gene called specific thyroid hormone- response elements (THRE) by the DBD . A number of artificial and natural THREs are a direct, inverted or everted repeat or palindrome of the DNA sequence AGGTCA. THR can bind to the THRE as a monomer, a homodimer or as a heterodimer with RXR and positively or negatively control the gene transcription and expression .
Schistosomiasis caused by parasitic blood flukes is a major public health problem worldwide. Approximately 200 million people are infected with pathogenic schistosomes, of which 20 million have advanced to life-threatening disease [5, 6].
Schistosomes live in the vascular system of the host, where they feed on blood components and respond to molecular signals from the host to develop normally .
Earlier researches have revealed that schistosomes can take advantage of signals from each other and from the host to facilitate their development, however, little is known about the molecular characterization of these signals [8–10]. Previous studies suggest that the existence of THR in S. japonicum and the parasite may use an endogenous TH/THR signalling pathway for growth and development . Herein, a novel mammalian orthologue of thyroid hormone receptor beta of NR superfamily 1 from S. japonicum (SjTHRβ) was identified and characterized. Study of schistosome NR will help us to understand how they regulate signaling pathways in the schistosome itself and add to our knowledge of the molecular relationship between the schistosome and its hosts .
Parasites and animals
The life cycle of S. japonicum (Chinese mainland strain, Anhui isolate) was maintained in New Zealand rabbits and Oncomelania hupensis in Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences. Specific pathogen free (SPF) male BALB/c mice, 6 weeks old, were purchased from Shanghai laboratory animal center, Chinese academy of sciences (Shanghai). 7d, 13d, 21d, 28d, 35d and 42d Schistosomes were collected by perfusion of New Zealand rabbits infected percutaneously with 1000 to 6000 cercariae of S. japonicum respectively, shed from Oncomelania hupensis snails as described . Animal care and all procedures involving animals were conducted according to the principles of the Shanghai Veterinary Research Institute, CAAS.
Cloning of S. japonicum thyroid hormone receptor
The total RNAs were extracted from 21d worms using Trizol reagent (Invitrogen), following the manufacturer’s instructions. DNA remaining in the RNA solution was digested with DNase I (TaKaRa) and then purified by an RNeasy mini kit (Qiagen, Germany). The concentration and purity of the RNAs were evaluated by spectrophotometry (Eppendorf, German). The cDNAs were synthesized using the PrimeScriptTM RT reagent Kit (TaKaRa, China), according to the user manual. According to putative thyroid hormone receptor(predicted Sjc_0027230) sequence deposited in http://lifecenter.sgst.cn/schistosoma/cn/schdownload several pairs of primer were designed. Two correct PCR products corresponding to partial fragment of putative thyroid hormone receptor were amplified individually by primer1 (Forward primer: 5′-AACAAACTACGAACGCAAAG-3′ and reverse primer: 5′-TTAAAGCTACCGCACGAA-3′) and primer2 (Forward primer: 5′-ATTATCGTGCTATGACTTG-3′ and reverse primer: 5′-GTCTTTGCGTTCGTAGTT-3′). In order to obtain complete mRNA sequence, two pairs of RACE primers were designed based on amplified PCR sequence. After total RNAs were extracted, reverse transcription (RT) and PCR were performed according to the user’s manual of the SMARTTM RACE cDNA Amplification Kit (Clontech). The transcription initiation site and the 3′-end of SjTHRβ were amplified by 5′-RACE and 3′-RACE, respectively. The 3′-RACE was performed with the following primers: forward gene-specific primer, 5′-TCACTTGGAATCGTCGAACCTA-3′; forward gene-specific nested primer, 5′-GCCCAAATGATTCGTGCGGTAG-3′. The 5′-RACE was performed with the following primers: reverse gene-specific primer, 5′-CCATCCCACCGCTTATGCACCGATCAAA-3′; reverse gene-specific nested primer, 5′-GCTTATCAGAAACAGAGCAGCGACCTTG-3′; PCR products obtained from RACE were ligated with pMD19-T vector (TaKaRa) and were then transformed into DH5α competent cells (Invitrogen). The positive clones were detected and sequenced. DNAStar software was employed to assemble the sequences of 3′-RACE, 5′-RACE, and mRNA into a complete cDNA sequence.
Real-time PCR analysis
The mRNA expression of SjTHRβ was evaluated in 6 different developmental stages including 7, 13, 21, 28, 35 and 42 d worms of S. japonicum. The cDNA products were obtained by the method described above. The primers specific to SjTHRβ (forward: 5′-AGTTCCTGAATTTGAGCTGT-3′ and reverse: 5′-TTTCTTGTTGATGAGACGGC-3′) can generate an amplicon of 218 bp. S. japonicum NADH-ubiquinone reductase was used as a housekeeping gene for this study . Primers specific for NADH-ubiquinone reductase gene were 5′-CGAGGACCTAACAGCAGAGG-3′ (sense) and 5′-TCCGAACGAACT TTGAATCC-3′ (antisense), and the PCR product size of the internal control is 174 bp. The PCR amplification was carried out using the reverse-transcribed cDNA as template with the SYBR Premix Ex TaqTM (TaKaRa, Japan) using the Mastercycler ep realplex4 (Eppendorf, Germany) real-time PCR detection System. The cycling protocol was as follows: 95°C for 10 s and 40 cycles of 95°C for 15 s, 55°C for 15 s, 72°C for 15 s. The value of fluorescence was detected at the end of each extension step. Specificity of the PCR products was determined by melting curve analysis and agarose gel electrophoresis. Negative control reactions, which contain all of the reagents except cDNA template, were included to make sure that the reaction system was not contaminated. Each reaction was performed in triplicate the entire experiment was carried out at least three times.
The web-based query system (http://lifecenter.sgst.cn/blast/cn/) was used to BLAST search the cDNA sequence. The exon/intron boundaries were determined.
Phylogenetic analysis of SjTHRβ
Fifteen nuclear receptor protein sequences from different species were evaluated in the phylogenetic analysis. The phylogenetic tree was constructed from deduced amino acid sequences of SjTHRβ. After alignment with CLUSTAL program in Mega4, an evolutionary tree was drawn by the neighbor-joining method.
Generation of anti rSjTHRβ-DBD and anti rSjTHRβ-LBD specific antibodies
DNA sequence encoding DNA binding domain (amino acid 119–250) of SjTHRβ was amplified by PCR using the forward primer 5′-GCGGAATTC CCTCCTAAAGTTAAAAAAAGAG-3′ and the reverse primer 5′-GCGCTCGAG AGTAGTCAGTGAATCTGTAAC-3′. The DBD fragment was then subcloned into the pET28a (+) expression vector between the restriction recognition sites of Eco R I (5′end underline) and Xho I (3′end underline). The recombinant plasmid expressed SjTHRβ-DBD in E. coli BL21 (DE3). LBD sequence corresponding to the predicted LBD (amino acid 556–817) of SjTHRβ was amplified by PCR using the forward primer 5′-GCGGGATCC GATATAGACAAAGATCCTG-3′ and the reverse primer 5′-GCGCTCGAG ATCAGTCTGCTGGATCAT-3′. The LBD fragment was subcloned into the pET32a (+) expression vector between the restriction recognition sites of Bam H I (5′end underline) and Xho I (3′end underline). The recombinant plasmid expressed SjTHRβ-LBD fusion protein in E. coli BL21 (DE3). These fusion proteins were purified by affinity chromatography on Ni2+-NTA resin. After dialysis against PBS, the purified fusion proteins of SjTHRβ-DBD and SjTHRβ-LBD were used to immunize BALB/c mice. The mice were injected subcutaneously four times at two week intervals with the purified protein in Montanide ISA 206 adjuvant (20 μg/100μL/mouse; Seppic). Antiserums were collected two weeks after the last injection.
For Western blot, 21d worm antigen was prepared from worms of S. japonicum and unrelated protein was extracted from E. coli BL21 (DE3) as described (You et al. 2010). The extracts of 21d worm and unrelated protein were separated by SDS/PAGE (12% gel) and then transferred onto nitrocellulose membrane (Whatman, Germany). The membranes were blocked with 5% non fat milk in PBS (blocking buffer) containing 0.05% Tween20 for 1 h. After washing, the membrane was incubated in a 1:100 dilution of anti SjTHRβ-LBD mouse serum overnight at 4°C. Subsequently, horseradish peroxidase conjugated goat anti-mouse secondary IgG (Sigma, USA) was used to bind first antibody. Three washes with 10mins each were performed after each step. Finally, detection was performed with the 3, 3′5, 5′-Tetramethyl Benzidine dihydrochloride (TMB, Sigma, USA) according to the instruction of manufacturer. Image was obtained by using ImageQuant 300 Capture Imaging System (GE Healthcare, USA).
EMSA was performed using a LightShift chemiluminescent EMSA kit (Thermo scientific, pierce, USA) with biotin-labeled oligonucleotide (Invitrogen, China), corresponding to sequence of Half-site (5′-GTACCGTAAGGTCA CTCGCG-3′) and DR0 (5′-CCGTAAGGTCAAGGTCA CTCG-3′). The binding reactions were incubated on ice for 30 mins in 20 μL reaction mixture containing 2 μL 10 × binding buffer, 10 μL ultrapure water, 1 μL 50% glycerol, 1μL 100 mM MgCl2, 1 μg ploy(dI: dC), 1 μL 1%NP-40, 2 μL dialyzed rSjTHRβ-DBD and 2 μL biotin-probe. After binding reaction, samples were loaded onto 6.5% polyacrylamide gel in 0.5 × Tris-borate- EDTA buffer and electrophoresed at 100 V at 4°C for 1 h. Biotin-labeled, double-stranded DNA was transferred to positively charged nylon membrane (Thermo scientific, pierce, USA). After cross-linking the transferred DNA to membrane by UV-light, biotin-labeled DNA was integrated with streptavidin-horseradish peroxidase conjugate. Finally, the result was detected using enhanced chemiluminescence and the images were taken by Image Quant LAS 4000MINI (GE, USA). For the non-specific competition experiment, the sequence (5′-CTAGTATACGTACTACTCTGA-3′) was designed and annealed to its complementary oligonucleotide and then added into the binding reaction mixture. Half-site and DR0 without biotin-label were synthesized and added into the binding reaction in the specific competition experiment. For the supershift experiment, 2 μg dialyzed rSjTHRβ-DBD was incubated with 1μL anti rSjTHRβ-DBD serum for 10 mins before incubation with the biotin-labeled probe.
Evaluation of immune protection against S. japonicum challenge induced by the r SjTHRβ-LBD
Thirty-six mice were randomly allotted into 3 groups of 12 mice per group and injected subcutaneously (SC) three times at 2 week intervals with 206 adjuvant (Seppic, France) in PBS (100 μl/mouse), rSjTHRβ-LBD in 206 adjuvant (20 μg/100 μl/mouse), and PBS only (100 μl/mouse), respectively. Two weeks after the last injection, all mice were challenged with 40 ± 2 cercariae. The worm and egg counting reduction rates were used to evaluate the efficacy of immunization. Adult worms perfused from BALB/c mice were obtained and counted. The worm reduction rates were calculated using the formula: percentage reduction in worm burden = (mean worm burden of control group − mean worm burden of vaccinated group)/mean worm burden of control group × 100%. Livers of each infected mouse were weighed and homogenized in 5 ml of PBS, and then 5 ml of 10% NaOH was added and mixed. The mixture was incubated at 56°C for 45 min and then mixed sufficiently. 10 μl mixture was used to count the number of eggs and it was performed in triplicate. The data was transformed to eggs per gram (EPG). Egg counting reduction rate was calculated by the formula (mean EPG of control group – mean EPG of vaccinated group)/mean EPG of control group × 100%.
Detection of SjTHRβ-LBD specific antibodies
Mouse sera was collected by retro-orbital bleeding before the first vaccination, 10 days post each vaccination and before the mouse perfusion. ELISA assay was performed to detect specific IgG antibodies against rSjTHRβ-LBD. rSjTHRβ-LBD at a working concentration of 10 μg/ml (pH 9.6) was coated on 96-well microtiter plates (Costar, USA) overnight at 4°C. The wells were blocked with 5% skimmed milk in PBS containing 0.05% Tween20 (PBST) for 1 h at 37°C, thereafter washed 3 times by PBST. All test sera were diluted in 1:100 in PBST, added onto plates and incubated for 1 hr. The goat anti-mouse IgG conjugated horseradish peroxidase (Sigma, USA) in 1:2500 dilutions was added to bind the primary antibody. Thorough washes were taken after each antibody incubation step. Substrate of 100 μl soluble 3,3′5, 5′-tetramethyl benzidine dihydrochloride (TMB, Sigma, USA) was added to each well. The plates were incubated at 37°C in the dark for 10 min. The reaction was terminated by 2 M sulfuric acid (50 μl/well). The absorbance was read at 450 nm on microplate reader (BioTek, USA). For IgG1 and IgG2a detection, we used goat anti-mouse IgG1 and IgG2a conjugated horseradish peroxidase (Sigma, USA) in 1:2000 dilutions as secondary antibodies, and the other steps were the same with those described in IgG detection.
Bio-Plex mouse cytokine assay for simultaneous quantization of interleukin (IL)-2, IL-4, IL-10, IL-12, tumor necrosis factor (TNF)-α, interferon (IFN)-γ was employed according to the recommended procedure. In brief, the premixed standards generated a stock concentration of 32,000 pg/mL for each cytokine. The standard stock was serially diluted in PBS to generate 9 points for the standard curve. The assay was performed in a 96-well filtration plate supplied with the assay kit. Premixed beads (50 μL) coated with target capture antibodies were transferred to each well of the filter plate and washed twice with Bio-Plex wash buffer. Premixed standards or anti rSjTHRβ-LBD serum (50 μL) were added to each well containing washed beads. The plate was shaken for 30 s with 1,100 rmp and then incubated at room temperature for 30 min with lower shaking speed. After incubation and 3 washes, premixed detection antibodies (50 μL) were added to each well. The incubation was terminated after shaking for 10 min at room temperature. After 3 washes, the beads were resuspended in 125 μL of Bio-Plex cytokine assay buffer. Beads were read on the Bio-Plex suspension array system, and the data was analyzed using Bio-Plex ManagerTM software with 5PL curve fitting.
Results in this work were acquired from at least three replicates on independent experiments with identical protocols. All values are expressed as means ± SD. Statistical analyses were performed by Student's t test, and P < 0.05 was defined as statistical significance.
Sequence analysis of S. japonicum thyroid hormone receptor
Gene organization analysis
The cDNA sequence of SjTHRβ was queried by a BLASTN search against the published S. japonicum genomic supercontigs in order to elucidate the SjTHRβ gene organization. Five shotgun sequences (SJC_C070306, SJC_C070307, SJC_C016210.
SJC_C016211 and SJC_C016212), which covered the 5′-UTR, the entire ORF, and the 3′-UTR, were obtained. It was found that the SjTHRβ gene spanned more than 32 kb and consisted of eight exons (bp sizes of 153, 168, 413, 217, 1028, 359,131 and 678) and seven introns (Figure 2B). Comparison with the genomic supercontigs revealed that the A/B domain and 3′-UTR are encoded by one exon. 5′-UTR, DNA binding domain and Hinge domain of SjTHRβ protein are composed by two exons. The ligand binding domain (LBD) is encoded by three exons. The sizes of each intron were shown in Figure 2B.
Phylogenetic analysis of SjTHRβ
mRNA expression level of SjTHRβ in the development of schistosomula into adult worms obtained from rabbit
rSjTHRβ-DBD protein binds to Half-site and DR0 in vitro
Protective immune efficacy in mice induced by rSjTHRβ-LBD
Comparison of protective efficacy against S. japonic um challenge in mice induced by SjTHRβ-LBD
Average worm burden
worm reduction rate(%)
Liver egg reduction rate(%)
25.25 ± 4.23
77,210.85 ± 15,437.34
26.13 ± 7.99
73,623.43 ± 12,063.51
18.3 ± 4.58
54,432.46 ± 5,341.33
Specific antibody detection
S. japonicum has a complex life cycle including free-living, aquatic stages and parasitic stages in the intermediate host snail or in the definitive mammalian host and is mainly responsible for intestinal and hepatosplenic schistosomiasis in China, the Philippines, and Indonesia . The genomic information of S. japonicum suggests the existence of an integral hypothalamic–pituitary– thyroid axis in this species . TH is synthesized in the thyroid gland under the control of thyroid stimulating hormone (TSH) secreted by the pituitary. TSH secretion is controlled by thyrotropin-releasing hormone (TRH), which is secreted from the hypothalamus . In addition, TRH, TSH and TH perform their functions by TRH, TSH and TH receptors . Among THRs, two principal THR types (THRα and THRβ) with specific physiological functions have been identified from various species, including S. mansoni. According to putative sequences of THR deposited in http://lifecenter.sgst.cn/schistosoma/cn/schdownload, several pairs of primer were designed to amplify SjTHRs. Based on these primers; we firstly identified a sequence encoding SjTHRβ. However, none of the primers resulted in SjTHRα sequence. The RT-PCR confirmed that SjTHRβ is constitutively expressed with higher levels from 21d and 35d worms, suggesting that SjTHRβ may have a more significant role in the developmental stages of 21d and 35d worms. The SjTHRβ contained a highly conserved DBD domain and a moderately conserved LBD domain. Our EMSA show that SjTHRβ- DBD can bind to a conserved DNA core motif AGGTCA, indicating that the function of THR is conserved during evolution. Freebern and his colleagues have proved the binding ability between SmRXR and cis-elements of the P14 gene, which is an eggshell precursor, providing evidence for a possible role in regulation of female-specific gene expression . THR DNA binding is involved in both positive and negative gene regulation . Further identification of the THRE in specific target gene in S. japonicum will help to understand the regulation mechanism of THR.
Even though praziquantel is highly effective in curing schistosomiasis, it still has its limitations that cannot prevent high frequency of re-infection and repair injury . Thus, the discovery of an effective vaccine remains the most potentially powerful means of control for this disease. Previous studies have described the cross-talk of host-parasite relationships between endocrine and immune mediators . The influence of TH on the immune system is well characterized and could mediate its action on infection. Saule et al. showed that the variation of host TH and interleukin-7 could favor the development of S. mansoni. Considering that S. japonicum might have an integral hypothalamic–pituitary–thyroid axis in its genome, we speculate that THRβ of S. japonicum could bind to ligand (TH) of both itself and its host. TH might act through THR, a member of the nuclear receptor superfamily I that intervenes in the expression of numerous genes, on both host and parasite. So, we assumed that SjTHR might have a role in immune regulation during the S. japonicum infection process. Here, we cloned and over-expressed SjTHRβ-LBD to evaluate its protective efficacy against schistosoma. In this experiment, rSjTHRβ-LBD could induce partial levels of protection against S. japonicum challenge. The mice vaccinated with rSjTHRβ-LBD elicited high levels of specific IgG as well as its subtypes IgG1 and IgG2a antibody via ELISA. IgG2a might predominantly activate T helper 1 cells, while IgG1 antibody mainly differentiated Th0 precursor in the Th2 direction in mice . Taken together, antibody profiles of vaccinated mice revealed the protective response to be a mixed Th1/Th2 types. Cytokines acting on lymphocytes play an important regulatory and signaling role in the development of an immune response. It is well known that IL-12, together with TNF-α, IL-2 and IFN-γ have long been classified as a type 1 immune cytokines [24, 25]. The presence of IL-2, IFN-γ, and IL-12 activates the Th0 precursor cells to become Th1 inflammatory T cells. IL-12 gene polarizes the immune responses towards Th-1 cell development and stimulates the strongest CTL activity [26, 27]. In addition, TNF-α is a pro-inflammatory cytokine involved in the cytokine cascade and leukocyte recruitment. On the other hand, IL-4 as well as IL-10 induces a Th0 precursor becoming an armed Th2 helper cell . IL-4 is a prominent Th2 cytokine that plays an important part in stimulating humoral immune responses . Given that the cytokines IL-2, IL-12 and TNF-α in the SjTHRβ-LBD group were significantly enhanced, whereas, the levels of serum IL-4 and IL-10 in the rSjTHRβ-LBD group and 206 adjuvant group were not obviously different, we suggested that rSjTHRβ-LBD can induce considerably higher levels of Th-1 response enhancing cytokines (IL-2, IL-12 and TNF-α) than Th-2 response enhancing cytokines (IL-10, IL-4). rSjTHRβ- LBD vaccination could stimulate a mixed Th1/Th2 type with Th1 dominant in humoral and T cell responses.
Herein, we identified SjTHRβ as the first member of NR superfamily in S. japonicum. Multiple sequence alignment and constructed evolutionary tree showed that SjTHRβ belongs to nuclear superfamily I. EMSA analysis showed that SjTHRβ-DBD could bind to a conserved DNA core motif, suggesting SjTHRβ might play an important role in gene regulation in S. japonicum. In addition, immunization of BALB/c mice with rSjTHRβ-LBD vaccination could induce partial protective efficacy against schistosome infection, indicating SjTHRβ might be a vaccine candidate for schistosomiasis. A full understanding of the role of SjTHRβ in the biology of S. japonicum will add to our knowledge of nuclear receptor gene regulation in schistosomes. Ongoing studies to identify more nuclear receptors involved in host-parasite interactions will aid in mechanistic studies to determine useful drug targets to modulate parasite or host biochemical pathways  and treat schistosomiasis.
We are deeply indebted to Yaojun Shi and Hao Li for helping us to infect rabbits in this work. We are also grateful to Zhixin Zhao for providing laboratory facilities. This work was supported by the National Natural Science Foundation of China (no. 31172315) and Special Fund for Agro-Scientific Research in the Public Interest (no. 200903036).
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