African Journal of Biotechnology
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White egg 2 is one of white egg mutants in silkworm, whose molecular mechanism remains unknown so far. In order to obtain an overall view on gene expression profiles at early embryo development stages, the white egg 2 near-isogenic line was constructed and the whole-genome of silkworm microarray system containing 21375 predicted genes from the silkworm whole genome sequence was employed to investigate gene expression profiles at 0, 24 and 48 h post oviposition between white egg 2 mutant and normal black egg strain. At 24 h post oviposition, 49 genes exhibited at least 2.0 fold differences at expression level, including 24 up-regulated genes and 25 down-regulated genes while at 48 h post oviposition, 52 genes, including 23 up-regulated genes and 29 down-regulated genes were expressed differentially over 2.0 change fold. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that nine differentially expression genes were involved in nine significant (p<0.05) pathways at 24 h post oviposition and 24 significant pathways at 48 h post oviposition, respectively. These pathways were related to amino acid metabolism, sugar metabolism, and series of major physiological metabolism. Our results hopefully shed light on the further study of molecular mechanism of white egg 2 mutant.
Key words: Bombyx mori, white egg 2 mutant, microarray, embryo, differentially expressed gene.
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White egg mutants in silkworm, Bombyx mori, exhibit several genotypes defined as white egg 1 (w-1), white egg 2 (w-2) and white egg 3 (w-3), caused by different deficient pigment metabolism in vivo (Lu, 1991). The mutant white egg 1 (w-1) is characterized by its white eyes and the production of white eggs as a result of its loss of the ninth and tenth exons of kynurenine 3-monooxygenase (KMO, EC1.14.13.9) gene (Quan et al., 2002). The mutant white egg 3 (w-3) has white eyes and eggs with translucent larval skin, resulting from a single-base deletion in exon 2 and a premature stop codon at the 5’ end of exon 3 (Komoto et al., 2009) while the white egg 2 (w-2) has the same phenotypes as white egg 1 and white egg 3 mutants with white egg color, but its mechanism is more complicated than white egg 1 and white egg 3 mutants based on recent report (Tatematsu et al., 2011) which suggest that the silkworm w-2 locus existed multi-allelic mutations. As of other insects, the color of the eggs of silkworm mainly depends on the color of the serosa, which is a membrane consisting of flattened polygonal cells, located between the yolk and the shell. The ommochrome pigment production is often accumulated in those specific pigment granules of serosa cells in eggs to produce the color of eggs, a process that involves several enzymes and relevant pathways. The first detected enzymes were kynurenine formamidase (Glassman, 1956) and kynureninase (Inagami, 1958) in insect homogenates, which are involved in the kynurenine pathway, and mainly contribute to the ommochrome pigment produc-tion. Subsequently, other enzymes such as tryptophan oxygenase (Egelhaaf, 1963a; Baglioni, 1959), kynurenine-3-hydroxylase (Mayer et al., 1968), and kynurenine transaminase (Leibenguth, 1967; Pinamonti et al., 1970) were detected and con-firmed to play essential roles in ommochrome biosynthesis, with any enzyme gene mutated during the evolutionary development in silkworm resulting in blocking or affecting the process of ommochrome bio-synthesis, which finally leads to egg color mutants. At present, DNA microarray technology is a cost-efficient and high-throughput method for investigating the differentially expressed genes or the gene different expression levels in different samples on the whole-genome scale, such as human (Son et al., 2005; Liu et al., 2009), rat (Walker et al., 2004), fruit fly (Arbeitman et al., 2002), rice (Ma et al., 2005), worm (Jiang et al., 2001), and yeast (DeRisi et al., 1997). It has been employed to successfully investigate gene expression profiles in multiple tissues of the domesticated silkworm using whole-genome oligonucleotide microarray (Xia Q et al., 2007). This microarray is a very efficient tool to investigate the response of the host infected by its pathogen on gene expression level (Luo et al., 2010; Wu et al., 2011). Based on cDNA microarray technology, investigators had recently analyzed gene expression patterns in eggs of silkworm at different stages during embryonic development from 2445 unique ESTs (Hong et al., 2006).In this study, we applied the whole-genome of silkworm microarray to comprehensively screen the differentially expressed genes of white egg 2 mutant compared to normal silkworm strain at early embryo development stages and tried to understand the formation mechanism involved in pathways or the differentially expressed genes undergoing white egg 2 mutation. Hopefully, this will pave the way for clear understanding of the mechanism of the mutant white egg 2 in molecular level and the use of white egg 2 mutant as biomarker in sericulture further research.
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The white egg 2 near-isogenic line construction
The white egg 2 near-isogenic line defined as Jingsong A white was constructed with the black egg sex-limited variety Suluanban as white egg 2 gene donor and the normal black egg variety Jingsong A as back-cross parent. The theoretical homozygous rate between the white egg 2 near-isogenic line and normal black egg back-cross strain was 99% tested by general genetics method after eight backcrosses. All the varieties used in this study were maintained and preserved by close inbreeding in laboratories in Sericultural Research Institute, Chinese Academy of Agricultural Sciences. The silkworm larvae were reared at standard temperature and humidity condition with a photoperiod of 12 h of light and 12 h of dark.
RNA isolation
Egg samples of white egg 2 mutant, Jingsong A white, and normal black egg strain, Jingsong A, were collected at 0, 24 and 48 h time point, respectively after eggs were laid. Total RNA was extracted from eggs using NucleoSpin® RNA II kit (MACHEREY-NAGEL, Germany), according to the manufacturer's protocol. The RNA samples were further purified using DNase (TaKaRa, Japan) to remove potential genomic DNA contamination. Purified RNA was then quantified using a NanoDrop spectrophotometer (NanoDrop Technologies, DE, USA). The ratio 28 s/ 18 s equal to 1.8 to 2.0 shows the high quality of purified total RNA without degradation. For each experimental condition, three independent samples were collected for microarray analysis.
The 23k silkworm genome array and microarray hybridization
The 23k silkworm genome array used in this study was constructed by Southwest University and CapitalBio Corporation containing 21375 predicted genes from the silkworm whole genome sequence (Xia et al., 2007). 5 µg of RNA for each sample proceeded to the fluorescent dye-labeled cDNA using the mRNA amplification procedure earlier described (Patterson et al., 2006; Guo et al., 2005), then the labeled cDNAs was dissolved in 100 μl of hybridization solution containing 3 × standard saline citrate (SSC), 0.2% sodium dodecyl sulfate (SDS), 5 × Denhardt’s solution and 25% formamide, followed by denaturing at 95°C for 3 min before hybridization. The mixed hybridization buffer was loaded onto a microarray slide, and covered with a LifeSlipTM coverslip (Erie Company, Portsmouth, NH, USA). The hybridizations were performed in a hybridization chamber (BioMixerTM, CapitalBio Corp.). After hybridization, slides were washed with washing solution I (0.2% SDS, 2 × SSC) and II (2 × SSC) respectively at 42°C for 5 min. Finally, the arrays were scanned using the Affymetrix GeneChip® Scanner 3000 7G (Affymetrix, Santa Clara, USA).
Detection of differentially expressed genes
After microarray hybridization, sample intensities were quantified using the LuxScan 3.0 image analysis software (CapitalBio). Significant analysis of microarray (SAM) (multiclass, 3.0) was applied to infer the differentially expressed genes between white egg 2 mutant and normal black egg strain. We set fold change >2 or <0.5 and p value <0.05 as cutoff values for differentially expressed genes up-regulated or down-regulated (Tusher et al., 2001).
Gene ontology and pathway analysis of the gene expression
Gene ontology (GO) categories and pathway enrichment analysis were carried out by CapitalBio® MAS software. P value used in a pathway and GO analysis was calculated by a hyper geometric distribution probability formula. P value reflects the importance of GO or the pathway in the experimental results. In the actual analysis, after selection employing the threshold of P value, we acquired the significant pathway and the GO false positive rate, called false discovery rate (FDR). It is more scientific to assign a Q value to each P value to reflect the selection of this P value as the threshold value of the FDR. Pathway and GO in the analysis of statistical results can also be integrated to consider P- and Q-value, according to the specific circumstances of experimental design set differential thresholds for analysis.
RT-PCR based validation
The total RNA for microarray hybridization was also used for RT- PCR validation. The concentration of RNA of each sample was adjusted with DEPC H2O to a final concentration of 500 ng/μL. A total of 800 ng RNA was reverse transcripted in a 20 μl reaction system using the Prime ScriptTM RT Reagent Kit (TaKaRa). Quantitative real time PCR was performed using 1 μl of 1:10 diluted first-strand cDNA in a 25 μl reaction volume according to the manufacturer’s instructions of the SYBR Premix ExTaqTM (TaKaRa). The specific primers of 15 genes and the endogenous control gene Bm Actin B are listed in Supplementary Table 1. The final concentration of the primers was 300 nM. PCR reactions were run in triplicates on an Opticon Lightcycler (BioRad) using thermal cycling parameters at 95°C for 10 s followed by 40 cycles of 95°C for 5 s, 54 to 58°C for 30 s, and 72°C for 7 s. Following amplification, melting curves were constructed. Data were analyzed and normalized to Bm Actin B transcript level by the Opticon Monitor Analysis software (MJ Research). A relative quantitative method (łCt) was used to evaluate relative expression differences.
Supplementary Table 1. List of primers of the 15 differentially expressed genes at 24 h post oviposition for RT-PCR validation.
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The white egg 2 near-isogenic line construction
Near-isogenic lines have been applied to study the (Figure 1 A and B). The gene expression profiles of each group exhibited significant differences when normal black egg strain was compared to white egg 2 mutant at 0, 24 and 48 h time points post oviposition. In normal black egg strain, the expression level of 825 genes increased sharply from 0 to 24 h, then remained steady from 24 to 48 h. Yet in white egg 2 mutant, only 449 genes exhibited the similar expression tendency. Especially, 104 genes of relationship between specific biological properties and related genes in many plants, such as rice, tomato, wheat, barley, soybean etc (Young and Tanksley, 1989). In our study, before microarray hybridization, the white egg 2 near-isogenic strain (harbored white egg 2 gene, w-2) was constructed with the black egg sex-limited variety Suluanban as white egg 2 gene donor, and the normal black egg variety Jingsong A as recurrent parent. Firstly, the hybrid (F1) was raised between the donor parent and the recurrent parent and subsequently the recurrent parent repeatedly backcrossed. At each backcrossed generation, the progeny was selected for the target gene (w-2) of the donor parent prior to each backcrossing. After eight backcrosses, the theoretical homozygous rate between the white egg 2 near-isogenic line and the normal black egg back-cross strain was more than 99%; tested by general genetics method.
Gene expression profiles at the early embryonic development stages
The microarray used in this study was constructed using 21375 predicted genes from the silkworm whole genome sequence (Xia et al., 2007). We attempted to determine the difference of the genes expression profiles at 0, 24 and 48 h time point after egg were laid between white egg 2 mutant and normal black egg strain. We selected a cutoff fold change >2.0 for the up-regulated genes at each of time point, and chose fold change <0.5 for the down-regulated genes (Tusher et al., 2001). Out of the 21375 predicted genes in the microarray, 2055 genes in
Differentially expressed genes at 24 or 48 h post oviposition between white egg 2 mutant and normal black egg strain
At 24 h post oviposition between normal black egg strain and white egg 2 mutant, the analyses identified a total of 157 genes that were statistically different. Out of them, 80 (50.96%) genes were up-regulated, and 77 (49.04%) genes were down-regulated (Supplementary Table 2). At 48 h time point, 178 genes were expressed differentially between normal black egg strain and white egg 2 mutant. 98 (55.06%) genes were up-regulated and 80 (44.94%) genes were down-regulated respectively (Supplementary Table 3). Among all differentially expressed genes identified by microarray in this study, 40 genes (25.48%) at 24 h time point and 48 genes (26.97%) at 48 h time point were unknown due to the absence of genetic information at present, respectively. Each data in this study was collected from three repeat samples. The gene expression patterns from three repeat samples at 24 or 48 h each time point between normal black egg strain and white egg 2 mutant were analyzed by Cluster analysis v3.0 software for hierarchical cluster analysis. Figure 2A and B show as a bar diagram the number of genes that were up-regulated or down-regulated at 24 and 48 h time point, respectively. The gene expression patterns of each three repeat samples were quite similar in normal black egg strain or white egg 2 mutant. However, the pattern of gene expression between normal black egg strain and white egg 2 mutant was quite different. On the other hand, there was a good reproducibility betweensamples, and the genes were successfully distinguished between white egg 2 mutant and normal black egg strain.
Supplementary Table 2. List of differentially expressed genes at 24 h post oviposition meared by microarray between white egg 2 mutant and normal black egg strain.
Table 1. Gene categories of differentially expressed genes at 24 h post oviposition.
Analysis of differentially expressed gene ontology
To investigate the global differences at gene level, the gene ontology (GO) hierarchy analysis was carried out on the differentially expressed genes. The genes were categorized according to CapitalBio Molecule Annotation System (MAS) software (MAS 3.0) (http://bioinfo. capitalbio.com/mas3). The MAS software was developed to integrate all differential gene expression datasets, and defines those GO categories that share a similar functional role from the differentially expressed genes according to a gene enrichment principle. Three GO terms (biological process, cell component, and molecular function) were evaluated in our study. P value <0.05 was considered statistically significant. At 24 h post oviposition, 102 differentially expressed genes had annotations according to MAS analysis (Table 1), and 37 genes were classified into four subgroups of molecular function, described as catalytic activity (19, 24.17%), binding (13, 16.54%), and transcription regulator activity (4, 5.09%). 56 genes were classified into eight subgroups of biological process, described as physiological process (16, 13.45%), cellular process (15, 12.61%), metabolism (15, 12.61%), biological regulation (3, 2.52%), regulation of biological process (3, 2.52%), negative regulation of biological process (1, 0.84%), andbiological adhesion (1, 0.84%). Nine genes were classified into cellular component (6.62%) (Figure 3A). Moreover, at 48 h post oviposition, 136 differentially expressed genes had annotations and were classified into five subgroups of molecular function, seven sub-groups of biological process, and ten genes were cellular component (Table 2). Five subgroups were described as catalytic activity (22, 20.95%), binding (17, 16.19%), transporter activity (5, 4.76%), molecular transducer activity (2, 1.90%) and enzyme regulator activity (2, 1.90%). Seven subgroups of biological process were included: cellular process (22, 13.78%), transducer activity, enzyme regulator activity, localization, and establishment of localization, were only detected in the ones at 48 h post oviposition.
Supplementary Table 3. List of differentially expressed genes at 48 h post oviposition meared by microarray between white egg 2 mutant and normal black egg strain.
Table 2. Gene categories of differentially expressed genes at 48 h post oviposition.
Table 3. Significant KEGG pathways at 24 h post oviposition.
Analysis of differentially expressed gene pathway
The differentially expressed genes at 24 and 48 h post oviposition were analyzed by CapitalBio MAS software for a pathway-based analysis to identify known pathways such as those in the KEGG (http://www.genome.jp/kegg), Biocarta (http://www.biocarta.com), and GenMAPP. A total of nine differentially expressed genes at 24 h post oviposition based on the KEGG database analysis were involved in nine pathways with a P-value cutoff of less than 0.05, including glycosaminoglycan degradation, phenylalanine, tyrosine and tryptophan biosynthesis, pentose and glucuronate interconversions, homologous recombination, drug metabolism-other enzymes, por-phyrin and chlorophyll metabolism, starch and sucrose metabolism, and aminoacyl-tRNA biosynthesis, except metabolism pathway with the p value 0.092528 (Table 3). Compared with differentially expressed genes at 24 h post oviposition, more than 26 differentially expressed genes were involved in 24 significant pathways with a P-value less than 0.05 at 48 h post oviposition. These pathways include metabolism, synthesis and degradation of ketone bodies, benzoate degradation via CoA ligation, pantothenate and CoA biosynthesis, drug metabolism-other enzymes, TGF-beta signaling pathway, galactose metabolism, inositol phosphate metabolism, starch and sucrose metabolism, fructose and mannose metabolism, amino-sugars metabolism, beta-alanine metabolism, propanoate metabolism, fatty acid metabolism, lysine degradation, phosphatidylinositol signaling system, tryptophan metabolism, butanoate metabolism, valine, leucine and isoleucine degradation, VEGF signaling pathway, pyrimidine metabolism, pyruvate metabolism, glycolysis/gluconeogenesis, and Wnt signaling pathway (Table 4).
Table 4. Significant KEGG pathways at 48 h post oviposition.
Quantitative reverse transcription-polymerase chain reaction (RT-PCR) validation of differentially expressed genes
We confirmed the gene expression level collected from microarray based on quantitative RT-PCR; 15 genes were selected randomly as test genes, while Bm Actin B was selected as the control to perform quantitative RT-PCR to validate the gene expression level. The results indicate that the change tendency of the expression levels of the 15 genes (normalized to Bm Actin B) had similar patterns with the changes measured by the microarray analysis at 24 h time point. The coefficient of determination was 0.7575 between the two sets of data (Figure 4).
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The white egg mutants have important biological properties in silkworm with potential application and eco-nomic value in sericulture, such as sex-limited application in sericulture, and screening marker in transgenic silkworm study (Klemenz et al., 1987; Loukeris et al., 1995; Zwiebel et al., 1995). White egg 2 mutant characterized by its white eggs and white eyes during its life cycle, is more convenient and easily used as biomarker for transgenesis screening and gene functional study. In this study, to investigate the differentially expressed genes between white egg 2 mutant and normal black egg strain, near-isogenic line of white egg 2 was constructed with the black egg sex-limited variety Suluanban as white egg 2 gene (w-2) donor, and the normal black egg variety Jingsong A as recurrent parent. Eight generations proceeded by the selection procedure of the white egg as selecting phenotype. After eight generations, the re-current parent recovery is assumed to be about 99.95%, retaining all normal black egg traits, while the donor parent genome is reduced to less than 0.05%, eliminating all the undesirable traits except the white egg 2 related genes (Ashwath et al., 2010). Theoretically, the near- isogenic line of white egg 2 mutant construction paves a high-efficiency way to investigate the differentially expressed genes between white egg 2 mutant and normal black egg strain joint with microarray analysis. The morphogenesis of the silkworm egg, which has three distinct phases: spheric, ellipsoidal, and plattened-ellipsoid, is an important procedure for its reproduction by strict transmission of genetic information and energy stores to the next generation (Tazima, 1964). As known, the color of the eggs of silkworms depends on three factors: yolk color, shell color, and the color of the serosa. The yolk and shell color of eggs are derived from the silkworm’s blood; the pigments passing from the haemolymph of the mother’s body into the eggs. However, serosa color is produced by granules which are formed in the serosa cells themselves (Tirelli, 1946). Normally, the color observed apparently concerns mainly the serosa pigment, than that of the yolk or/and shell. It is a fact that the serosa cell pigmentation may therefore be considered as the result of an enzymatic process involved in genes related to pigmentation. Furthermore, when eggs are newly laid, all silkworm eggs appear yellow, taking their color only from the yolk and shell, since the serosa pigment has not yet developed. As the serosa develops, granules of melanic pigment appear in its cells, yellowish at first, following pink, red, dark red and eventually dark brown. Notably, this dark brown serosa pigment, as modified by the pigment of the translucent shell, is what gives the normal egg its gray color. The time course of this procedure is basically around 24 h post oviposition (Kikkawa, 1941), so our hypothetical genes related white egg 2 phenotype may have been expressed before pigmentogenesis was complete in the serosa at 24 h post oviposition. In this study, we found that 157 genes were expressed differentially, including 80 up-regulated genes and 77 down-regulated genes at 24 h post oviposition, and 178 genes including 98 up-regulated genes and 80 down-regulated genes at 48 h post oviposition. The GO functional categories for these genes exhibited significant differences at both time points. It was noted that a gene encoding high affinity nuclear juvenile hormone binding protein was expressed over notable 33-fold change at 24 h post oviposition, inferring that this gene was involved in juvenile hormone signal trans-duction in the morphogenesis of the silkworm egg. Juvenile hormone exerts pleiotropic functions during insect life cycles and it primes the ecdysteroid response of developing follicles (Hartfelder, 2000). The fat body of pre-diapausing, early diapausing and mid-diapausing larvae was found to release a high affinity juvenile hormone binding protein in the southwestern corn borer, Diatraea grandiosella (Dillwith et al., 1985). We know that the diapause hormone, which is secreted from the suboesophageal ganglion is mainly responsible for the induction of diapause eggs, acts to control the metabolism of 3-hydroxykynurenine and carbohydrate in silkworm pupae. This hormone accelerates the 3-hydro-xykynurenine and glycogen accumulation in pupae ovaries of silkworm (Yamashita et al., 1966). In silkworm, the interaction of diapause hormone and juvenile hormone regulate the diapause event, although we hope to sequentially trace the upstream or downstream genes of this high affinity nuclear juvenile hormone binding protein in further study. Tryptophan metabolites are the source of pigment produced by granules accumulated in serosa of the eggs (Bernt Linzen, 1974). Bonse (1969) reported that one mutant of Drosophila was unable to accumulate tryptophan in the Malpighian tubules leading to its white color. In our study, the transcript level of gene charged for phenylalanine, tyrosine and tryptophan biosynthesis detected 1.27-fold change. Down-regulation shows that the low tryptophan content at early embryo development stage probably was assumed to be insufficient, so that pigments in serosa led to the color deficiency phenotype of the eggs observed as the white egg. At 48 h post oviposition, one gene encoding reverse transcriptase involved in tryptophan metabolism was 3.2173 fold up-regulated. We therefore inferred that the source of pigment production was from tryptophan metabolites at very early stage after oviposition. The results of KEGG analysis indicate that the pathways involved in differentially expressed genes were quite different at 24 h post oviposition and at 48 h post oviposition; only two common pathways; starch and sucrose metabolism and pyrimidine metabolism existed at both time points. The genes involved in starch and sucrose metabolism were both up-regulated at two time points. We conjectured that the genes involved in starch and sucrose metabolism were over expressed to fill the needs of the embryo development at non-diapause phase or merely survived at diapause phase. Embryogenesis is an extremely energy-intensive activity, requiring the rapid mobilization of energy source at the early embryo development stages. On the contrary, the gene involved in pyrimidine metabolism at 24 h post oviposition was up-regulated, while the one at 48 h was down-regulated. Pyrimidine nucleotides play a critical role in cellular metabolism serving as activated precursors of RNA and DNA (Evans and Guy, 2004). A gene involved in pyrimidine metabolism was up-regulated leading to more synthesized nucleic acids to meet the need of cell proliferation from newly laid to 24 h. However, at 48 h post oviposition, the eggs used in this experiment were in diapause stage and as the process of cellular morpho-genesis was almost completed, the gene expression level dropped down correspondingly. In order to demonstrate the accuracy of the microarray analysis results, we confirmed the differential expression of several randomly selected genes via RT-PCR validation, as is shown in Figure 4. The coefficient of determination was 0.7575 and the result of microarray analysis was reasonably consistent with those of the RT-PCR analysis. The microarray analysis is based on the hybridization technology; actually there is no strict linear relationship between signal strength and transcript abundance for different genes (Luo et al., 2005). In some cases, the cross-hybridization among the homologous sequences may cause the variance. Therefore, we conclude that our results are reliable, and can be used for further research of the white egg 2 mutant. In summary, we report herein genes differentially expressed in white egg 2 mutant at 24 and 48 h after oviposition compared to normal black egg strain, as detected by DNA microarray analysis. The aim was to shed more light on any cues of genetic information of white egg 2 mutant. Recent study (Tatematsu et al., 2011) showed that in silkorm w-2 there exist several multi-allelic mutations encoding the ortholog of Drosophila scarlet, which is responsible for the formation of a white/scarlet heterodimer and involved in the transport of ommo-chrome precursors. In our laboratory, we also found other new allelic mutation (data not shown). Hence, results in this study provide new clues for the exploration of the molecular mechanism of white egg 2 mutant. Further-more, in subsequent studies, close attention will be focused on those genes with unknown function due to the limitation of the silkworm database.
Acknowledgements
This project was supported by the National Natural Science Foundation of China (Grant no. 30871829). We thank Prof. Xia Qingyou (Southwest University) for the thoughtful advice and Prof. Miao Yungen (Zhejiang University) for the critical reading of the manuscript. We also thank Prof. Shen Xingjia and Prof. Xu Anying (Sericultural Research Institute, CAAS) for the materials supported.
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