Article pubs.acs.org/jpr
Quantitative Proteomic and Transcriptomic Analyses of Molecular Mechanisms Associated with Low Silk Production in Silkworm Bombyx mori Shao-hua Wang,† Zheng-ying You,† Lu-peng Ye,† Jiaqian Che,† Qiujie Qian,† Yohei Nanjo,‡ Setsuko Komatsu,*,‡ and Bo-xiong Zhong*,† †
College of Animal Sciences, Zhejiang University, 866 Yuhangtang Road, Hangzhou 310058, P.R. China National Institute of Crop Science, NARO, Kannondai 2-1-18, Tsukuba 305-8518, Japan
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S Supporting Information *
ABSTRACT: To investigate the molecular mechanisms underlying the low fibroin production of the ZB silkworm strain, we used both SDS-PAGE-based and gel-freebased proteomic techniques and transcriptomic sequencing technique. Combining the data from two different proteomic techniques was preferable in the characterization of the differences between the ZB silkworm strain and the original Lan10 silkworm strain. The correlation analysis showed that the individual protein and transcript were not corresponded well, however, the differentially changed proteins and transcripts showed similar regulated direction in function at the pathway level. In the ZB strain, numerous ribosomal proteins and transcripts were down-regulated, along with the transcripts of translational related elongation factors and genes of important components of fibroin. The proteasome pathway was significantly enhanced in the ZB strain, indicating that protein degradation began on the third day of fifth instar when fibroin would have been produced in the Lan10 strain normally and plentifully. From proteome and transcriptome levels of the ZB strain, the energy-metabolismrelated pathways, oxidative phosphorylation, glycolysis/gluconeogenesis, and citrate cycle were enhanced, suggesting that the energy metabolism was vigorous in the ZB strain, while the silk production was low. This may due to the inefficient energy employment in fibroin synthesis in the ZB strain. These results suggest that the reason for the decreasing of the silk production might be related to the decreased ability of fibroin synthesis, the degradation of proteins, and the inefficiency of the energy exploiting. KEYWORDS: Bombyx mori, complex trait, silk production, proteomics, transcriptomics
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INTRODUCTION 1−3
worm is an ideal model for studying molecular mechanisms underlying complex traits. The silk production varies from different varieties of silkworm. The major components of fibroin include the heavy fibroin chain, the light fibroin chain, and P25/fibrohexamerin. There are two strains of silkworm with extremely low fibroin production that is caused by gene mutation. One is a naked-type called “Nd”; the mutated gene is located at 0.0 site in chromosome 25, and the heavy fibroin chain derived from the Nd allele is structurally abnormal, which results in the naked pupa.10 The other one is a strain of sericin cocoon called “Nd-s”; the mutated gene is located at 19.2 site in chromosome 14, and the light fibroin chain gene of Nd-s allele cannot synthesize the normal light fibroin chain that produced the sericin cocoon.11 During the bioreactor experiments of our lab, T7 RNA polymerase gene, controlled by the fibroin light chain promoter, was introduced into silkworm embryo and expressed specifically in the posterior silk gland. A mutated individual with distinguishable low silk production was
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Multiple human diseases including hypertension, diabetes, and adiposity5 are complex traits. Various economic characteristics of crops and animals, such as the yield of the rice,6 soybean oil concentration,7 and the lactation traits of cattle,8 are also complex traits. Complex traits are controlled by multiple genes and closely associated with the environment. Determining the genetic mechanisms is considerably complicated, as phenotypic variation arises from interactions between multiple and environmentally sensitive alleles.9 The silkworm (Bombyx mori), a Lepidoptera insect, is an important economic insect providing fibroin for human consumption and has been domesticated for over 5000 years for silk production. The production of fibroin is a typical complex trait. The life span of a silkworm is ∼40 days. Similar to controlled plant growth in a greenhouse and controlled mice or rat strains, silkworms survive by eating mulberries and can be reared indoors, thus enabling the design of various controlled experiments that will reduce the experimental errors caused by environmental impacts dramatically. Consequently, the silk© 2014 American Chemical Society
Received: August 14, 2013 Published: January 2, 2014 735
dx.doi.org/10.1021/pr4008333 | J. Proteome Res. 2014, 13, 735−751
Journal of Proteome Research
Article
found, and the character of it is different from Nd and Nd-s lines. It was named ZB, which had a significantly shorter posterior silk gland and a more slender cocoon compared with that of the original strain, Lan10. This transgenic strain is useful for investigation of the molecular mechanisms of low silk production. Proteomic and transcriptomic techniques are often used to investigate the molecular mechanisms involved in complex traits.12−14 The investigation of the molecular mechanics in low silk production enables researchers to design bioactive agents to elevate silk yield and thus facilitates screening and cultivation of high fibroin-yielding silkworm varieties. In the present study, to investigate the molecular mechanisms of low silk production, which means the low fibroin production, we analyzed ZB transgenic strain using SDS-PAGE- and gel-free-based proteomic techniques and transcriptomic sequencing technique. Furthermore, subsequent bioinformatics analysis based on data from proteomic and transcriptomic research was performed.
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mM 50% NH4HCO3 for 10 min, dehydrated with 100% ACN, and then air-dried. The samples were digested with 20 μL of trypsin (12.5 μg/mL in 50 mM NH4HCO3) for 30 min on ice and incubated overnight at 37 °C. The resulting peptides were extracted twice with 30 μL of 50% ACN/2% formic acid, dried, vacuum centrifuged, and reconstituted to 10 μL with 2% formic acid. The resulting peptide solutions were desalted with SPE CTip (Nikkyo Technos, Tokyo, Japan) and subjected to nanoliquid chromatography−mass spectrometry (nanoLC− MS/MS). Gel-Free Digestion
According to the methods of Komatsu et al.,17 100 μg of protein was subjected to chloroform/methanol precipitation to remove contaminants and detergent. The precipitated proteins were resuspended with 50 mM NH4HCO3 and were reduced with 30 mM DTT and alkylated with 50 mM iodoacetamide. Proteins were digested with trypsin at a 1:100 enzyme/protein concentration at 37 °C for 16 h. The resulting peptide solutions were desalted with SPE C-Tip (Nikkyo Technos) and subjected to nanoLC-MS/MS.
MATERIALS AND METHODS
Transgenic Silkworm and Organ Isolation
Data Acquisition by Nanoliquid-Chromatography−Tandem MS
The transgenic ZB strain was obtained by introducing a piggyBac vector that contained an EGFP reporter gene and T7 RNA polymerase under the control of actin-3 and fibroin light-chain promoter, respectively, into silkworm embryos of the Lan10 strain and screening the EGFP positive individuals. The traits of the transgenic ZB strain were hereditary stable. In the transgenic silkworm, the cocoon weight was significantly decreased compared with that of original strain Lan10. The posterior silk gland was dissected at the developmental stage on the third day of the fifth instar and frozen in liquid nitrogen for later investigation. The weights of the posterior silk gland and cocoon were measured in 30 individual ZB silkworms.
The nanoLC−MS/MS procedure was performed as previously described.18 The tryptic peptides were analyzed using a nanospray LTQ Orbitrap MS (Thermo Fisher Scientific, San Jose, CA) operated in a data-dependent acquisition mode through Xcalibur software (version 2.0.7, Thermo Fisher Scientific). Peptides in 0.1% formic acid were loaded onto a C18 PepMap trap column (300 μm ID × 5 mm, Dionex, Germering, Gemany) and eluted with linear acetonitrile gradient (15−40% for 85 min for 1D-gel proteomic; 15−40% for 115 min for gel-free proteomic) in 0.1% formic acid at a flow rate of 200 nL/min using an Ultimate 3000 nanoLC system (Dionex).17 The eluted peptides from the trap column were separated and sprayed on a C18 Tip column (75 μm 1D × 120 mm, nano HPLC capillary column, NTTC-360/75-3)(Nikkyo Technos). The spray voltage was 1.8 kV. Full scan mass spectra were obtained in the Orbitrap over a mass coverage of 400−15,000 m/ z at a resolution of 30 000. To obtain high mass accuracy, a lock mass function was used.17 During the acquisition of mass spectra, the top six most intense precursor ions were subjected to collision-induced fragmentation in the linear ion trap with normalized collision energy of 35%. In the fragmentation, the dynamic exclusion was set at 90 s to prevent repetitive selection of peptides.18
Protein Extraction
Protein extraction was performed as previously described.15 In brief, samples of posterior silk gland from 10 silkworms were mechanically homogenized in 10 μL of lysis buffer containing of 2.5% SDS, 10% glycerin, 5% 2-mercaptoethanol, and 62.5 mM Tris-HCl (pH 6.8) per 1 mg of sample using a motor-driven plastic pestle. The homogenization was conducted on ice. The homogenate remained at room temperature for 10 min and was then subjected to continued drill shock treatment in an ice-bath four times, each time for 30 s with an interval of 30 s. The supernatant was centrifuged twice at 20 000g at 25 °C for 10 min. The supernatant was stored at −20 °C. The samples were boiled for 2 min and centrifuged at 20 000g for 10 min, and the proteins were separated by SDS-PAGE.
Protein Identification
SDS-PAGE and In-Gel Digestion
Identification of proteins was performed by MASCOT search engine (version 2.3.0.2, Matrix Science, London, U.K.). DTA files were first generated from acquired raw data and then converted to MASCOT generic files by BioWorks software (version 3.3.1, Thermo Fisher Scientific). Parameters used in MASCOT searches have been previously described by Komatsu et al.17 Trypsin was used to cleave the peptides. One missed cleavage was allowed in the database search. Peptide and fragment ion mass tolerances were 5 ppm and 0.5 Da, respectively. MASCOT results were filtered with MASCOT percolator for accuracy and sensitivity, thus improving peptide identification.19 For all searches, the false discovery rate of peptide identification was
