Proteomic and Comparative Genomic Analysis of Two Brassica napus

Article pubs.acs.org/jpr

Proteomic and Comparative Genomic Analysis of Two Brassica napus Lines Differing in Oil Content Lu Gan,†,⊥,∥ Chun-yu Zhang,‡,∥ Xiao-dong Wang,† Hao Wang,§ Yan Long,‡ Yong-tai Yin,† Dian-Rong Li,§ Jian-Hua Tian,§ Zai-yun Li,‡ Zhi-wei Lin,† Long-Jiang Yu,† and Mao-Teng Li*,† †

Institute of Resource Biology and Biotechnology, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China ‡ National Key Laboratory of Crop Improvement, Huazhong Agricultural University, No.1, Shizishan Street, Wuhan 430070, China § Hybrid Rapeseed Research Center of Shaanxi Province, Dali 715105, China S Supporting Information *

ABSTRACT: Ultrastructural observations, combined with proteomic and comparative genomic analyses, were applied to interpret the differences in protein composition and oil-body characteristics of mature seed of two Brassica napus lines with high and low oil contents of 55.19% and 36.49%, respectively. The results showed that oil bodies were arranged much closer in the high than in the low oil content line, and differences in cell size and thickness of cell walls were also observed. There were 119 and 32 differentially expressed proteins (DEPs) of total and oil-body proteins identified. The 119 DEPs of total protein were mainly involved in the oil-related, dehydration-related, storage and defense/disease, and some of these may be related to oil formation. The DEPs involved with dehydration-related were both detected in total and oil-body proteins for high and low oil lines and may be correlated with the number and size of oil bodies in the different lines. Some genes that corresponded to DEPs were confirmed by quantitative trait loci (QTL) mapping analysis for oil content. The results revealed that some candidate genes deduced from DEPs were located in the confidence intervals of QTL for oil content. Finally, the function of one gene that coded storage protein was verified by using a collection of Arabidopsis lines that can conditionally express the full length cDNA from developing seeds of B. napus. KEYWORDS: Brassica napus, oil content, differentially expressed proteins, QTL mapping, comparative genomics, transgenic analysis

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INTRODUCTION Rapeseed is one of the most important oilseed crops in the world. Oil can account for up to 46% of dry weight of B. napus seed,1 which is also an important source of unsaturated fatty acids and proteins for human and animal nutrition as well as for nonedible use such as biodiesel production.2 Rapeseed production has experienced a rapid increase in China in recent years, and the seed yield has increased from 427.5 to 1775.10 kg/hm2; however, the oil content of commercial varieties has increased only slightly.3 Increasing the oil content is increasingly important and has become a primary goal for B. napus breeding.3,4 Some varieties with relatively higher oil content were obtained in recent years in China, such as Qin you 33 (47.80%), Zhong Shuang 11 (49.5%), and Qin Za 4 (50.01%). Oil content is a characteristic with generally high heritability, and some quantitative trait loci (QTL) for oil content with additive effects and environmentally sensitive have been identified.5,6 Microarray analysis has shown that the expression of genes related to sugar metabolism differed when B. napus was planted in different locations.7 TAG is present in oil bodies (OBs) as small, discrete, spherical intracellular organelles, © 2013 American Chemical Society

which are each surrounded by a monolayer of highly unusual oleosins.8 Oleosins are relatively hydrophobic proteins with molecular weight in the range of 15−26 kDa9 and are localized exclusively on the surface of OBs. Some other minor proteins have also been observed in seed OBs of many species.9 Protein content is generally negatively correlated with oil content in seeds, and the size of OBs is also related to oil content.10 Consequently, the morphologic analysis,11 formation mechanisms of OBs, and biosynthesis of olesins have been much studied in recent years.8,10,12 Lipids or oil storage in the form of TAG are deposited in the embryo or endosperm during seed development.13 All of these studies helped to determine the protein composition of OBs in B. napus. Recently, a significant difference for the ratio of OB organelle was observed based on the ultrastructure of B. napus with super high and low oil content14 Meanwhile, the negative relationship was also found between OBs hydrodynamic diameter and the content of oleosins.15 However, differences in protein composition or Special Issue: Agricultural and Environmental Proteomics Received: June 17, 2013 Published: September 6, 2013 4965

dx.doi.org/10.1021/pr4005635 | J. Proteome Res. 2013, 12, 4965−4978

Journal of Proteome Research

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abundance of OBs between B. napus lines differing in oil content at the proteomic and comparative genomic levels have not been reported. Proteomic technology is a useful tool for understanding complex metabolisms and cell functioning16 and allows easier analysis of the intricacies of physiology in the postgenomics era.17 To date, research on some physiological traits at the proteomic level in B. napus has been reported. Meza-Basso et al.18 observed the protein synthesis change in rapeseed seedlings during low temperature. Pi et al.19 and Mihr et al.20 observed proteomic differences in different CMS lines. Albertin et al.21 compared the proteomic differences among leaf, stem, and root in synthetic B. napus. Sheoran et al.22 researched the proteome of mature and in vitro germinating pollen of canola using DIGE technology. Proteomic analysis has also been widely used to determine the metabolism of seed formation and the regulatory networks for oil production23 in such species as soybean,24 rapeseed,25 sunflower,26 peanut,27 and castor.28 The proteomic analysis of different near-isogenic lines of sunflower differing in oil content showed that oil content in seeds was closely related to carbohydrate metabolism and protein synthesis;26 the seed-filling dynamic proteome in other oilseed corps, such as soybean,24 B. napus,25 and castor,28 has also been recently researched. However, research at the proteomic level comparing B. napus of relatively high and low oil contents has not been reported. Brassica and Arabidopsis share common ancestors and diverged ∼20 MYA,29 and early comparative studies conducted using genetic linkage maps revealed extensive collinearity between their genomes.30 The availability of genomic resources, such as the high-density comparative map of B. napus and Arabidopsis31 and the genome sequence of B. rapa (www. brassica-rapa.org), allows the identification of important candidate genes, which can be located in the QTL regions of Brassica by in silico mapping. For example, Long et al.32 identified several candidate genes underlying QTL of flowering time by comparative analysis of Arabidopsis and B. napus via in silico mapping analysis; their following experiments showed that one of the potential candidate genes, FLC, was the gene that controlled one QTL controlling flowering time.33 The present paper reports significant proteomic differences between two B. napus lines with relatively higher and lower oil contents. Many genes that encoded the proteins were confirmed by QTL mapping and in silico mapping analysis of Brassica and Arabidopsis.

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replicates. The NMR analyzer was standardized by use of known oil content B. napus seed with clean and high quality. The oil content of each sample was calculated according to Robertson and Morrison.35 The glucosinolate profiles in seeds were analyzed as described by Agerbirk et al.36 with minor modifications. The components of glucosinolate in seeds were determined using UPLC (Waters ACQUITY). Transmission Electron Microscopy and Confocal Laser Scanning Microscope Analysis of Mature Embryos

For ultrastructural observation, embryos were isolated from mature seeds and fixed immediately in 2.5% glutaraldehyde. The samples were postfixed with 1% osmium tetroxide solution after phosphate buffer rinsing, then washed with ultrapure water, and dehydrated through a graded series of acetone (20, 50, 70, 90% and 3 × 100% v/v). After infiltration through a graded acetone/Epon/Spurr’s epoxy resin series, samples were embedded in 100% (w/v) Spurr’s epoxy and polymerized at 60 °C for 24 h. Ultrathin sections were prepared using a Diatome diamond knife on a UC6 Ultratome (Leica, Germany) onto copper grids and stained with uranyl acetate and lead citrate. Images were viewed and collected under an H-7650 transmission electron microscope (TEM) (Hitachi, Japan). For confocal laser scanning microscope (CLSM) analysis of mature embryos in HO and LO lines, the sample preparation and observation method were according to Siloto et al.,37 and the images were collected with LSM 700 (Zeiss, Germany). Total Protein and Oil-Body Protein Extraction

The method for total protein extraction from mature seeds was according to Gan et al. 38 with minor modifications. Approximately 0.1 g of seeds was homogenized to fine powder in liquid nitrogen and then transferred to a 1.5 mL Eppendorf tube. Then, 1.5 mL of precooled 10% TCA/0.07% dithiothreitol (DTT) in acetone was added and incubated at −20 °C overnight, followed by centrifugation for 30 min at 20 000 g at 4 °C. The pellets were then washed three times with 1.5 mL of precooled acetone containing 0.07% DTT at −20 °C for 1 h and then centrifuged as described above. The sample pellets were then solubilized in the lysis buffer containing 7 mM urea, 2 mM thiourea, 4% CHAPS, 50 mM DTT, 0.5% Triton X-100, 1% protease inhibitor cocktail, and 2% IPG buffer. The solution was incubated at 25 °C for 1 h with gentle mixing and then centrifuged at 12 000 g for 20 min, with the supernatants collected into fresh tubes and stored at −80 °C in aliquots. The protein quantification was determined by using the 2D-QuantKit with BSA as standard. The OB isolation and protein extraction from OBs were according to the method of Katavic et al.10 0.5 mL of petroleum ether was added to 0.5 mL of isolated OBs (with 2 M NaCl washing purification) and then vortexed. The sample was first centrifuged at 13 000g for 5 min; then, the upper petroleum ether layer was removed; this procedure should performed more than two times. The petroleum ether fractions were pooled and dried by using nitrogen gas. The interfacial layer and bottom aqueous phase were sparged with nitrogen gas to remove the remaining petroleum ether. We added 0.75 mL of chloroform/methanol (2:1 v/v) to the interfacial layer and aqueous phase and then vortexed. The lower chloroform phases containing PLs were washed three times with 1 mL of methanol/water (1:1 v/v) and dried under nitrogen gas. The protein-rich interfacial layer was resuspended in 0.25 mL of water, 0.75 mL of chloroform/methanol (2:1 v/v) was added and then vortexed and centrifuged at 13 000 g for 5 min, and

MATERIALS AND METHODS

Experimental Materials

Two winter-type B. napus lines, 08QT11 with higher oil content of 55.72 ± 0.75% (HO line) and 08QT43 with lower oil content of 36.77 ± 0.39% (LO line), are derived from doubled haploid (DH) lines, which derived from hybridization between KnC8 and N53. The materials were grown in the field of Rapeseed Hybrid Center of Shaanxi Province in 2009. Mature seeds of these two lines were collected for experimental use. Oil Content, Fatty Acid, and Glucosinolate Composition Analysis

Bulked seed samples were analyzed for their fatty acid composition by gas liquid chromatography according to Rücker and Röbbelen.34 Seed oil content was determined by using nuclear magnetic resonance (NMR) and at least five biological 4966



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in 0.1% trifluoroacetic acid were mixed and loaded onto a MTP 384 massive target T and analyzed with ABI 4800 MALDITOF-TOF plus mass spectrometer (Applied Biosystems, Foster City, CA) controlled by GPS Explore software. The parameters were as follows: reflex model, 160 ns delay extraction time, 60− 65% grid voltage, and 20 kV accelerating voltage. Laser shots at 200 per spectrum were used to acquire the spectra with mass range of 800−4000 Da. The filtered precursor ions with a userdefined threshold (S/N ratio >50) were selected for the MS/ MS scan. Data were acquired in a positive MS reflector using a CalMix5 standard to calibrate the instrument (ABI4800 Calibration Mixture). Both the MS and MS/MS data were integrated and processed by using the GPS Explorer V3.6 software (Applied Biosystems) with default parameters On the basis of combined MS and MS/MS spectra, proteins were successfully identified based on 95% or higher confidence interval of their scores in the MASCOT V2.3 search engine (Matrix Science, London, U.K.): 0.25 Da for fragment ion tolerance. PMFs were searched in the NCBInr protein database with the use of the MASCOT search engine (http://www. matrixscience.com). Search parameters were set as: taxonomy, Viridiplantae (Green Plants; released in December 2010; 849 474 sequences); enzyme, trypsin; max missed cleavages, 1; fixed modifications, carbamidomethyl (C); variable modifications, oxidation (M); fragment mass tolerance, ± 0.2 Da; and mass accuracy, 50 ppm. Protein hits were validated if the identification was with at least four top-ranking peptides with p < 0.05. In the case of peptides matching multiple members of a protein family, the presented protein was selected based on the highest score and the highest member of the matching peptides. Subcellular localization of differentially expressed proteins (DEPs) were predicted using programs WoLF PSORT prediction (http://wolfpsort.org/), ESLpred (http://www. imtech.res.in/raghava/eslpred/), and Subloc (http://www. bioinfo.tsinghua.edu.cn/SubLoc/eu_predict.htm) according to Salavati et al.41

the procedure was repeated two more times. After washing, oilbody protein pellet was resuspended in 0.5 mL of water, sonicated 5 min, and precipitated in 4 volumes of cold 100% acetone for 16 h at −20 °C. The final protein pellets were dried in air and resuspended in the lysis buffer containing 7 mM urea, 2 mM thiourea, 4% CHAPS, 50 mM DTT, 0.5% Triton X-100, 1% protease inhibitor cocktail, and 2% IPG buffer at pH 3−10. Then, the supernatants were collected and quantified as previously described. 2-DE

For total protein analysis, IPG strips with linear pH gradient 4− 7 and 24 cm length were rehydrated with 450 μL of rehydration solution (7 M urea, 2 M thiourea, 4% CHAPS, 65 mM DTT, 2% IPG buffer, pH 4−7, 0.001% bromophenol blue) containing 1 mg of protein for 14 h at 20 °C in an IPG Box. IEF was carried out in the Ettan IPGphor isoelectric focusing system according to Gan et al.38 After IEF, the strips were equilibrated for 15 min each in an equilibration buffer [6 M urea, 0.375 M Tris−HCl (pH 8.8), 20% glycerol, and 2% DTT] containing 2% SDS and 2.5% iodacetamide, respectively. The second dimension separation of proteins was performed on 12.5% SDS-PAGE gel using an Ettan DALT six system. SDS-PAGE was run at a constant power of 1 W/gel for 45 min and switched to 5 W/gel until the bromophenol blue frontier reached the bottom of the gel. The proteins on gels were visualized with colloidal Coomassie Brilliant blue G-250 according to the Blue Silver method.39 For OB protein, the loading sample was 500 mg with linear, pH 3−10, 24 cm IPG strips, and the pH of IPG buffer changed to 3−10;other operations were as previously described. Data Analysis and Protein Identification by MALDI-TOF-MS-MS

The stained gels were scanned using an Ettan III scanner with a resolution of 300 μm pixel−1. The spot detection and gel matching were performed using PDquest version 8.01 software. All spots were normalized to the total density in the gel image and expressed in parts per million. The spots that changed in abundance more than two-fold and the least significant difference performed >95% (p < 0.05) were selected for protein identification. Protein digestion was performed according to Shu et al.40 with slight modification. Spots of interest were manually excised from gels and washed twice with Milli-Q water. Gel spots were incubated at 37 °C for 30 min, and the gel became lipochromous and transparent by the addition of 50 μL of destaining solution containing 50% acetonitrile and 25 mM NH4HCO3. The sample was dehydrated with 50 μL of acetonitrile and dried in a Speed-Vac after washing twice with Milli-Q water. The gel pieces were rehydrated in 5 μL of trypsin (0.02 μg/μL, Promega) on ice until the gel became lipochromous and transparent again, and 20 μL of buffer containing 10% acetonitrile and 25 mM NH4HCO3 was added and incubated at 37 °C overnight. After digestion, the supernatant was transferred to new tubes, and 50 μL of extract liquor containing 67% acetonitrile and 5% trifluoroacetic acid was added to the gel and incubated at 37 °C for 30 min. Then, it was ultrasonically treated for 15 min, and the remaining peptides were collected. The peptides were dried in a SpeedVac; finally, 10 μL of 1% trifluoroacetic acid was added to dissolve the extracted peptides for sample plating. Uniform volumes (0.4 μL) of peptide solution and 10 mM αcyano-4-hydroxycinnamic acid saturated with 50% acetonitrile

Hierarchical Cluster Analysis

Hierarchical clustering was performed using the procedures with freely available software PermutMatrix to analyze the protein expression profiles of different quantitative protein spots.42 For protein expression profiling, induction of the fold change ratios (R) of the protein abundance (HO/LO) was used, and the distance was measured based on the logtransformed values of fold change ratios of the protein abundance. The hierarchical cluster analysis was conducted using the unweighted pair group method with UPGMA using the procedures of software PermutMatrix. QTL Detection for Oil and Protein Content and Map Alignment between DEPs in B. napus with Arabidopsis, B. rapa, and B. oleracea

A DH segregating population of B. napus with 202 lines derived from hybridization between a European winter cultivar Tapidor and a Chinese semiwinter cultivar Ningyou 7 was named TNDH.43 This TNDH population was used for QTL analysis of oil and protein contents. The TNDH population and its parents were grown in three natural environments in the year 2009−2010 (Huanggang and Qichun county in Hubei Province and Dali county in Shaanxi Province). The mature seeds of every line were used for oil and protein content analyses. Oil and protein contents were determined by NMR and NIRS, respectively. 4967



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Figure 1. TEM and CLSM analysis of mature embryos in HO and LO lines. (A,B) TEM analysis of mature embryo in HO and LO lines, respectively. (C,D) CLSM analysis of mature embryo in HO and LO lines. White arrows and white arrow heads in panels A and B indicate the protein storage vacuole (PSV) and oil body (OB). The red arrows in panels A and B point to the cell wall; white arrows in panels C and D indicate the cells in mature embryo of HO and LO lines. (A,B), bar = 2 μm; (C,D) bar = 20 μm.

anchors.47 A gene is considered to be associated with a QTL if it is within the confidence interval.

The reference linkage map used in this study was according to Jiang et al.44 The QTL mapping was analyzed using the composite interval method (CIM) with WinQTL cartographer 2.5 software.45 CIM was used to scan the genetic map and estimate the likelihood of a QTL and its corresponding effect at every 2 cM. The LOD value of 2.5 was used for identifying statistically significant QTL of oil and protein contents in each environment. The DEPs corresponding to the genes of Arabidopsis were searched using the database of the Arabidopsis Information Resource (TAIR, http://www.arabidopsis.org/). The name of the DEPs was used as a query name to search the corresponding genes in Arabidopsis; the GenBank accession containing the term for the gene’s name resulting in different loci matches with different distinct gene models was considered as a corresponding gene. Comparative analysis between Arabidopsis chromosomes and TN linkage groups was based on the method of Long et al.46 The comparative mapping of homologous genes in B. napus (AACC, 2n = 38) between Arabidopsis with B. rapa (AA, 2n = 20) and B. oleracea (CC, 2n = 18) was done using BLASTN analysis. To identify physical positions in the A and C genomes, 361 molecular markers with sequencing information were used as query sequences to BLAST with the gene sequences in databases http://brassicadb.org/brad/index.php and http:// www.ocri-genomics.org/bolbase/. The map alignment between B. napus and B. rapa and between B. napus and B. oleracea was aligned and connected using these homologous genes as

Functional Analysis of Candidate Genes in Arabidopsis

To reveal the contribution of those DEPs in seeds for oil content, we employed in this study Arabidopsis lines, which are enable to conditionally express full-length cDNA from developing seeds of B. napus based on full-length cDNA over-eXpressing gene-hunting system (FOX hunting system).48 For conditional expression of B. napus cDNA in Arabidopsis, Choristoneura f umiferana ecdysone receptors (EcR)-based inducible system was applied.49 As indicated, expression of transgene can be perfectly induced through this system if the Arabidopsis host plant is exposed to water containing a 10 000fold diluted commercial ecdysone agonist (Intrepid-2F, Dow AgroSciences) by watering or spaying. In total, about 6000 independent Arabidopsis transgenic lines of T1 generation were generated, and currently 300 of them were randomly selected for amplifying the transgene by using PCR and further for DNA sequencing (unpublished data). Thereafter, those Arabidopsis plants of T2 generation containing transgenes that matched well with genes coding the DEPs were used for oil content analysis. For each type of transgenic line, seeds of T2 generation were grown side-by-side into two groups. One group was watered during the whole life cycle with 10 000-fold diluted inducer, intrepid-2F, under the 35S promoter, while the other group of plants was as control and treated only with 4968



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Figure 2. Representative 2-DE images of HO and LO proteins. (A,B) Total protein of LO and HO lines, respectively. (C,D) Oil-body protein of LO and HO lines, respectively.

fatty acid profiles of TAG were observed. The glucosinolate contents in mature seeds of HO and LO lines were 60.37 ± 0.73 and 86.69 ± 1.39 μmol/g, respectively; further analysis showed that contents of some components varied, such as desulfoproitrin, desulfoglucobrassicanapin, desulfoglucobrassicin, and desulfogluconasturtiin. Thus the main differences between HO and LO lines were the contents of oil, protein, and glucosinolate.

water. Subsequently, mature seeds with or without treatments were used for measuring oil content by GC-MS analysis.

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RESULTS

Correlations between Oil and Protein Contents, and Qualitative Analysis of HO and LO Lines

NMR analysis showed that the oil content of 08QT11 and 08QT43 was quite different, being 55.72 ± 0.75% and 36.77 ± 0.39%, respectively. Total and OB proteins were isolated from mature seed of HO and LO lines. Total protein content in LO was 0.172 ± 0.011 g/g, which was higher than that of HO with 0.113 ± 0.015 g/g; correspondingly, OB protein contents were 0.0116 ± 0.001 and 0.025 ± 0.002 g/g. Thus the oil content was negatively correlated with the oil content in mature seeds in B. napus. The fatty acid and glucosinolate compositions were also characterized in mature seeds of HO and LO lines (Figures S1 and S2 in the Supporting Information). This suggested that the fatty acid compositions of HO and LO lines were similar with no significant difference, and no dramatic differences in overall

TEM and CLSM Analysis of Mature Embryos of HO and LO Brassica Lines

TEM analysis was used to examine the ultrastructural differences in embryo cells between HO and LO lines. The average number of protein storage vacuoles (PSVs) was 9.4 ± 1.14 in the LO line, which was much greater than that of the HO line (3.8 ± 0.84) (Figure 1A,B). The OBs were arranged much closer in the HO than in the LO line and comprised much more than 50% of the total cell area in the HO line. Further observations showed that the HO line had much smaller cells (18.3 ± 1.67 μm) (Figure 1A). Compared with the LO line, with individual cells of an average size of 30.9 ± 2.85 4969



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Figure 3. Expression pattern of some DEPs in the different developmental stages between HO and LO lines. (A) Enlarged area of 2-DE gel of HSPassociated protein like (3784) and protein disulfide isomerase (1620) between HO and LO lines. (B) Change pattern of some DEPs between HO and LO lines. WAF represents the weeks after pollination.

μm (Figure 1B), this was confirmed by CLSM observations (Figure 1C,D). Most of the OB size in the LO line had a broad range of 0.09−3.52 μm (about 1.19 ± 0.47); the same phenotype of OB had a broad size range of 0.07−3.07 μm in the HO line (about 0.48 ± 0.08 on average), but there was a relatively high ratio of very large OBs in the LO line (Figure 1B). The cell walls in LO and HO lines had large differences in thickness with 1.5 ± 0.17 and 0.49 ± 0.06 μm, respectively (Figure 1A,B). These results indicated that OB size and arrangement, PSV number, and thickness of cell wall might be correlated with oil content in these two B. napus lines.

disease proteins (15.97%), oil-related proteins (15.13%), dehydration-related proteins (10.08%), protein biosynthesis proteins (8.4%), metabolism of amino acids proteins (4.2%), secondary metabolism proteins (2.52%), transporter proteins (1.68%), and other proteins of unclear classification (16.81%) (Table S1 in the Supporting Information). For OB protein, 50 DEPs including 18 spots with two-fold quantitative change and 32 with qualitative change were observed (Figure 2C,D), and 32 of them were successfully identified. These DEPs of OB protein could be categorized into six groups: storage function proteins (34.38%), oil-related proteins (15.63%), dehydration-related proteins (15.63%), defense/disease proteins (15.63%), secondary metabolism proteins (6.25%), and some proteins of unclear classification (12.50%) (Table S2 in the Supporting Information). Statistical analysis of log2 ratio of high/low oil showed that most quantitative change DEPs of OB were up-regulated in the HO line (Table S2 in the Supporting Information). The expression rules of some DEPs observed in mature seeds of HO and LO lines were traced through the different seed developmental stages (pollination after 2, 3, 4, 5, 6, and 7 weeks, represented as 2WAF−7WAF, respectively). HSPassociated proteins like putative protein disulfide isomerase, PFK2, peroxiredoxin type 2, and protein disulfide isomerase were chosen as examples. The 2-DE analysis in all stages showed that the expression pattern of HSP-associated proteins like peroxiredoxin type 2, putative protein disulfide isomerase, and protein disulfide isomerase in 7WAF between HO and LO lines was the same, with the exception of mature seeds (Figure 3). Further research revealed the expression of HSP-associated proteins like peroxiredoxin type 2 and protein disulfide isomerase showed the same pattern in all developmental stages; however, putative protein disulfide isomerase showed the opposite pattern at 7WAF to the other five stages (Figure 3). There was no obvious expression pattern for PFK2 in the

Establishment of 2-DE Reference Maps and Expression Profiles of Total and OB Protein of Mature Seed of HO and LO Lines

2-DE with biological triplicates was conducted for total and OB protein from mature seeds to detect the DEPs between HO and LO lines. Total and OB proteins of 2-DE reference maps were obtained using IPG strips with pH 4−7 and 12% SDSPAGE (Figure 2). Protein spots were detected and quantified independently from each 2-DE image using the software of PDquest version 8.01. Approximately 1000 and 300 protein spots were detected for total and OB proteins, respectively. For further understanding of the proteomic differences of total and OB protein between HO and LO lines, the DEPs that changed in abundance more than two-fold and that exceeded the least significant difference (p < 0.05) were selected for MALDITOF-MS-MS analysis (Figure 2). A total of 143 DEPs, including 96 with quantitative change of more than two-fold and 47 qualitative change spots, were found between HO and LO lines using t-test analysis (Figure 2A,B), and 119 of these DEPs were successfully identified. When the 119 DEPs were statistically analyzed as a log2 ratio of high/low oil, 43 proteins were up-regulated in the HO and 44 in the LO line. These DEPs in HO and LO lines could be categorized into nine groups: storage functions proteins (22.69%), defense/ 4970



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Figure 4. Protein expression profiles of DEPs in total proteins from mature seeds of HO and LO lines. Left and right represent the hierarchical clustering of the quantitative change proteins and protein functional classification of five most abundant clusters, respectively.

proteins with high expression in the LO line, with defense/ disease proteins and storage functions dominant. Cluster C, also highly expressed in the LO line, represented 16 proteins with dehydration-related and oil-related proteins as the dominant classification. Cluster E, the largest cluster, accounted for 30 proteins that all showed high expression in the HO line; this cluster contained all of the nine functionally classified. Proteins involved in storage functions, oil-related, and unclear classification were the majority in Cluster E. Cluster G was composed of eight proteins, all highly expressed in the HO line, with proteins of storage functions the largest group.

development of seeds (Figure 3). On the basis of the previously mentioned results, DEPs detected on mature seed could not completely reflect a difference that is observed throughout seed development. If we want to make clear the mechanism of high oil formation, then the DEP expression pattern in different developmental seeds should be taken into consideration. Hierarchical Clustering of DEPs

To facilitate the biological interpretation of DEPs of total protein of HO and LO lines, hierarchical clustering analysis of the quantitative DEPs was conducted using PermutMatrix software. Seven clusters were recognized, which included five major clusters and another two minor clusters (Figure 4). This demonstrated that the DEPs between HO and LO lines showed significant changes in their expression patterns. Further research revealed that Cluster A included seven proteins, all highly expressed in the LO line. Cluster B accounted for 18

QTL Mapping and Candidate Gene Confirmation Based on Comparative Analysis on the Genes That Corresponding to DEPs

The phenotypic and genetic correlations between the two traits were evaluated and the corresponding genes of these DEPs 4971



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Figure 5. QTLs for oil content and protein content on A4 chromosome and comparative analysis between A. thaliana, B. napus, and B. rapa on the genes that corresponding to DEPs. (A) QTL scanning results from three environments with TN DH population. Curves of different colors represent QTLs scanned from different environments, and the right vertical bar shows the positions of QTLs. (B) Genome blocks, which were defined by comparative mapping of B. napus and A. thaliana and using the locus boundaries of the blocks reported by Schranz et al.47 (C) A4 linkage map of TN DH population. Left characters represent the molecular markers that existed on A4 chromosome, right characters represent the genes of N block that located in the QTLs confidence interval, and the red characters represent the candidate genes that correspond to DEPs. (D) A4 linkage map of B. rapa. Right characters represent the genes that are homologous to the genes of B. napus and red characters represent the genes in B. rapa homologous to the genes for DEPs. (E) Different linkage group of A. thaliana..

for oil content with the negative additive effect of −0.667, which showed that the QTL could decrease the oil content Besides, the QTL qOCQC-4 for oil content with a positive additive effect of 0.7392 was colocalized with the QTL qPCQC-9 for protein content with a negative additive effect of −0.3784 in the confidence interval region 2, and the negative-effect QTL qPCQC-10 for protein content was closely positioned with the QTL qOCQC-5 for oil content with positive additive effect in the confidence interval region 3. (Figure S3A,B in the Supporting Information). These findings indicated that the allele increasing protein content was in coupling phase with the allele decreasing oil content and vice versa, explaining the negative correlation between oil and total protein contents. The DEPs that changed in abundance by more than two-fold for total and OB proteins in HO and LO lines were considered together, and 154 identified DEPs were found to correspond to 562 homologous genes in Arabidopsis, which corresponded to 378 key genes in protein-related metabolism, and those genes were used as candidate genes. The corresponding relationship between the whole candidate genes and QTLs for oil content is shown in Table S4 in the Supporting Information. The QTLs for different traits clustered in the same region and sharing the same molecular markers were considered to be one QTL. We selected the QTLs that existed in chromosomes A4 and C3 and that controlled oil and protein contents at the same time as an example: qOCQC-1 and qOCDA-1, qOCDA-2 and qPCDA-3,

were used for alignment with the QTL intervals for oil and total protein contents to verify whether the DEPs between HO and LO lines were related to oil and total protein content. Significantly negative correlations between oil content and protein content were obtained after evaluating the two phenotype traits; the correlation coefficient was −0.855 (data was not on shown), which was also verified by QTL analysis of the two traits using TNDH population. A total of 37 QTLs were detected based on the data obtained in the year 2009 for plantings in three different environments, of which 17 QTLs were for seed oil content and four of them showed a close linkage with QTL for protein contents (Table S3 in the Supporting Information). QTLs that were mapped in the same chromosome that controlled protein and oil content were observed: for example, the QTLs qOCHG-1 and qOCHG-2 with positive additive effects of 2.1057 and 1.3549 for oil content, while the QTLs qPCHG-1 and qPCHG-2 with negative additive effects of −0.7104 and −0.7957 for protein content, respectively (Figure S3A,B in the Supporting Information). The QTL that controlled oil and protein contents mapped in the same confidence interval region with opposite additive effect were also found in chromosome 13: for example, QTLs qPCQC-6, qPCDA-8, and qOCQC-3 in the confidence interval region of 1, in which qPCQC-6 and qPCDA-8 were the QTLs for protein content with the positive additive effect of 0.4688 and 0.4104, indicating that these QTLs could increase protein content, while qOCQC-3 was the QTL 4972



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Figure 6. Phylogenetic and functional analysis of candidate gene of BngNAP1 gene for napin seed-storage protein. (A) Phylogenetic tree of BngNAP1 gene for napin seed-storage protein obtained by ClustlW and Mega 4.0. (B) Comparative of oil content in the seeds and the content of different fatty acid compositions in the oil between control and inducible transgenic materials.

these candidate genes were recognized as the major candidate genes. (Figure 5). Further comparative analysis between the A genome of the TNDH population (B. napus) and the A genome of B. rapa showed four homologous genes that corresponded to the above-mentioned candidate genes in B. rapa (Figure 5). For genes on chromosome C3, four of 17 candidate genes belonging to C block were mapped on the QTL qC3-2 confidence interval: BGLU1 (β-glucosidase related protein), BGLU34 (encodes a myrosinase), AT1G50120 (unknown), and AT1G50590 (RmlC-like cupins superfamily protein), and the five candidate genes should be paid more attention because they are assigned to the major QTL. No block matched to qC3-1, but U block was just matched to the QTL qPCQC-8 confidence interval, and 36 genes were aligned (Figure S3 in the Supporting Information). Comparing the C genome of the TNDH population (B. napus) and the C genome of B. oleracea showed 18 homologous genes of B. oleracea (Figure S4 in the Supporting Information). 117 candidate genes that corresponded to DEPs showed a good corresponding relationship with QTL confidence intervals for oil content, indicating that these DEPs might be involved in oil or protein formation.

on chromosome A4 were considered as two QTLs (named qA4-1 and qA4-2, respectively). qOCQC-4 and qPCQC-9, qOCQC-5 and qPCQC-10, on chromosome C3 were considered as another two QTLs (named qC3-1 and qC3-2, respectively). Because the QTLs qPCDA-3, qOCQC-5, and qPCQC-10 could explain about or more than 10% of phenotypic variance, the QTLs qA4-2 and qC3-2 were considered as the major QTLs, and the candidate genes that were underlying the two QTL were recognized as the major candidate genes. In chromosomes A4 and C3, 24 candidate genes were underlying the N block, 36 genes were underlying the U block, and 17 genes were underlying the C block. These 77 genes were used as candidate genes for in silico mapping in chromosomes A4 and C3 of the TN linkage map (Figure 5 and Figure S4 in the Supporting Information). Oil and total protein QTLs in the TNDH population were compared with candidate genes that corresponded to DEPs; 6 of the 24 genes underlying the N block of chromosome A4 were mapped on the QTL qA4-1 confidence interval: AT3G56350 (iron/manganese superoxide dismutase family protein), AT3G57620 (glyoxal oxidase-related protein), PGL34 (plastoglobulin 34kD), AT3G58450 (adenine nucleotide α hydrolases-like superfamily protein), BGLU27 (β glucosidase 27), and BGLU30 (encodes a protein similar to β-glucosidase and is a member of glycoside hydrolase family 1). Five genes were mapped on the major QTL qA4-2 (qOCDA-2) confidence interval: AT3G52470 (late embryogenesis abundant hydroxyproline-rich glycoprotein family), ATELP (encodes the vacuolar sorting receptor-1/epidermal growth factor receptorlike protein1), MDAR1 (encodes a peroxisomal monodehydroascorbate reductase), FBA8 (aldolase superfamily protein), and AT3G53040 (late embryogenesis abundant protein), and

Function Analysis of Candidate Genes in Arabidopsis

154 DEPs were observed in the present study, and some could be aligned to QTL confidence intervals for oil content. The genes that coded for storage proteins were selected as examples for function analysis by screening Arabidopsis transgenic lines, which are enabled to conditionally express full-length cDNA from developing seeds of B. napus DNA of 300 independent T1 Arabidopsis transgenic plants, and were amplified by using the specific primer designed from the recombinant loci of T-DNA, 4973



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then sequencing the amplified cDNA of B. napus. All of the sequences were used for homology analysis using BLAST analysis (http://blast.ncbi.nlm.nih.gov/Blast.cgi; www. arabidopsis.org), and one sequence that has 99% identity to the B. napus BngNAP1 gene for napin seed storage protein (homologous to AT2S3, SESA3/seed storage albumin 3 in Arabidopsis) was observed (Figure 6A). Subsequently, this Arabidopsis transgenic line that can conditionally express napin was then planted to produce seeds for oil content analysis. (One group was watered with 10 000-fold diluted inducer intrepid-2F during the whole life cycle, and the other group was treated only with water as controls.) The oil content of the induced transgenic group for B. napus BngNAP1 gene for napin seed-storage oil content decreased from 30.47 ± 0.76 to 25.67 ± 0.55% (Figure 6B); further analysis showed that the fatty acid composition changed very little (Figure 6B). It shows that increasing the seed storage protein in seeds could induce decreasing the oil content and not affect the content of fatty acids. This results indicated that proteomics was a powerful method for determining the useful genes.

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content in oilseed crops has also been reported; 77 DEPs were identified in two near-isogenic sunflower varieties differing in oil content,26 revealing that oil content was tightly linked to carbohydrate metabolism and protein synthesis in a complex manner. Devouge et al.50 revealed that the protein involved in defense systems and carbohydrate metabolism were the main DEPs in four near-isogenic B. napus varieties differing in erucic acid and glucosinolate contents. In the present study, 119 DEPs between HO and LO lines were classified into nine groups, the majority of which were involved in storage functions, defense/ disease, and oil-related, which was in agreement with some of the previously mentioned cases. There were 32 DEPs identified from OBs between HO and LO lines. Katavic et al.10 revealed that the OB proteins mainly included myrosinase, myrosinasebinding protein, 12S storage protein, heat stock protein, cruciferin, and β glucosidase. The present study showed that oil-related, dehydration-related, and storage function proteins were the main DEPs between HO and LO lines. Storage function proteins represented 34.38% of the total. The detection of storage protein was not unexpected due to the separation method of OBs because it is always difficult to purify any organelle to homogeneity.

DISCUSSION

OB Observation in HO and LO Lines and QTL Analysis for Oil Content and Protein in B. napus

DEPS Involved in the Oil-Related Synthesis

For genes involved in the oil-related pathways, 18 DEPs were observed, including genes involved in the Calvin cycle, glycolysis, TCA cycle and some other carbohydrate metabolic pathways. DEPs involved in the oil-related were also observed in OB proteins and were all more highly expressed in the HO line. Ribulose 1,5-bisphosphate carboxylase (RuBP, spot 2123) is common in plants and is involved in Calvin’s reductive pentose phosphate cycle and catalyzes the initial step of carbon fixation;56 it is the key enzyme of carbon dioxide fixation in photosynthesis and catalyzes either the carboxylation or the oxygenation of RuBP and oxygenized the fragmentation of the substrate into 3-phosphoglyceric acid and 2-phosphoglycolic acid.57 Hajduch et al.25 detected 11 Rubisco large subunits that were highly expressed throughout seed filling, and recent study also revealed that oil content was closely correlated with BnRBCS1A expression levels and Rubisco activities in the silique wall.58 The results of Schwender et al. also found that Rubisco acted without the Calvin cycle and improved the efficiency of carbon use during the formation of oil in developing embryos of B. napus.59 Nevertheless, a similar study in castor showed that Rubisco was 11 times less prominent in castor compared with rapeseed.28 The present study also revealed that ribulose 1,5-bisphosphate carboxylase was highly expressed in the HO line, indicating that Rubisco activity expressed throughout seed filling of the HO line might enhance the carbon fixation in photosynthesis and increase the oil content. Comparative analysis of the genes corresponding to DEPs with QTLs for oil content showed that some candidate genes were underlying the QTL confidence intervals for oil content, and BGLU27, BGLU30, FBA8, BGLU1, and BGLU34 were involved in the carbohydrate metabolic process. This again confirmed that the oil content was closely correlated with these DEPs. For protein related to glycolysis, three DEPs (7309, 7402, and 9401), highly expressed in the HO line, were identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an enzyme involved in conversion of glyceraldehyde 3-phosphate in the sixth step of glycolysis by coupling with the reduction of NAD+ to NADH.60 Houston et al.28 observed differential

Seed oil content is a very complex quantitative trait, and improving oil content is a major breeding goal for oilseed crops. However, the molecular mechanism for oil content is not clear because it is controlled by multigenes and is also sensitive to environmental influence. A negative correlation between the protein content and lipids in seeds has often been highlighted50 and was verified in the present study. The relationship between OB structure and oil content has also been investigated in B. napus10 and indicated a positive relationship between unusually large OBs and low oil content. The larger OB was also observed in LO line in the present studies, it indicated that the appearance of larger OB in low oil materials of B. napus was a universal phenomena. However, few large OBs (>2.5 μm) were also observed in many HO lines (data not shown). Oil and protein content in the seeds has been reported to be closely linked through QTL analysis in B. napus. Gül et al. identified four QTLs for oil content being closely linked to QTL for protein content.51 Zhao also observed a strong genetic relationship between oil and protein content with six QTL, and nine epistatic locus pairs had pleiotropic effects on both traits.52 The similar phenomenon was also found in other plants, for example, in soybean. Shoemaker et al. reported that QTL for seed protein and oil content showed correspondence across homeologous regions.53 In B. juncea, Mahmood et al. identified 6 and 5 QTLs for oil and protein content, respectively, and three of these QTLs with opposite effects were a tight linkage.54 In the present study, significantly negative correlations with correlation coefficient of −0.855 between oil content and protein content were obtained, and four QTLs for oil content colocalized with protein content but with opposite effects were also detected. Differentially Expressed Enzymes Involved in total and OB Proteins

Proteomic research on developing embryos,55 seed filling,25 and mature seed50 in B. napus has been reported, but these have not focused on oil content. Research has shown that more common protein spots exist when the genotypes of materials are closely related.50 Research on the oil accumulation process and oil 4974



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glucosinolate system has been suggested to act as a defense system against general herbivorous pests, for it hydrolyzes glucosinolate into various compounds, such as isothiocyanates, thiocyanates, nitriles, or epithionitriles.68

expression of GAPDH in the seed-filling process of castor and considered that it might be related to an increased need for reducing equivalents or glycolytic intermediates. Fructosebisphosphate aldolase (FBA) (spot 8412) was highly expressed in the LO line and participates in catalyzing the reversible aldol cleavage or condensation of fructose-1,6-bisphosphate to dihydroxyacetone-phosphate and glyceraldehyde 3-phosphate.61 A relatively large number of cytosolic and plastidial FBA spots were also detected during seed filling in B. napus;25 30% higher expression of this enzyme was detected during seed filling of castor, but higher expression was not detected in soybean.28 Phosphofructokinase (PFK) (spot 6501) phosphorylates fructose to 6-phosphate in glycolysis, and PFK is located in the cytosol of the cell and regulated by Fru-2, 6 bisphosphate in plants.25 For TCA and other carbohydrate metabolism pathway, succinyl co-A synthetase (spot 4524) facilitates the reaction to form a high-energy phosphate bond and directly forms ATP in plants. Pyruvate orthophosphate dikinase (PPDK) (spot 3805) regenerates the primary CO2 acceptor PEP in the mesophyll cell chloroplasts involved in the C4 pathway and is also present in C3 plants mostly in the photosynthetic mesophyll cells.62 Aldose reductase-like protein (AR) (spot 8403) is an NADPHdependent oxidoreductase that catalyzes the conversion of glucose to sorbitol.63 β glucosidase is known to be involved in polysaccharide catabolism;64 seven DEPs of β glucosidase were detected in four near-isogenic B. napus varieties differing in erucic acid and glucosinalate contents,50 which was also observed in protein composition of OBs of B. napus.12 Three DEPs of β glucosidase (spots 9519, 8619, and 8629) were detected in the present study. Previous transcriptional study on four developing oilseeds including B. napus showed that some ESTs of carbon metabolism such as plastidial and cytosolic glycolysis were present,65 and similar results of comparative transcriptome and metabolite analysis in oil palm also detected that the high oil content in oil palm was associated with much higher transcript levels of key enzymes of plastidial carbon metabolism including PFK, pyruvate kinase, and pyruvate dehydrogenase, which were also detected in our research.66 Collectively, these proteins involved in glycolysis, the TCA cycle, and other glycometabolism pathways appeared to be enhanced in the LO lines and may lead to more carbon equivalents consumed or flowing to other metabolic pathway, thus reducing the corresponding materials participating in fatty acid synthesis and accumulation.

DEPs Involved in Secondary Metabolism, Storage Protein, Protein Biosynthesis, Metabolism of Amino Acids, and Dehydration-Related

Proteins detected and involved in secondary metabolism suggested that in the HO line the secondary metabolism process was at least fractionally induced, because S-adenosyl-Lhomocystein hydrolase (spot 5510) increased by more than two-fold in the HO line. For genes involved in dehydrationrelated, late embryogenesis abundant (LEA) protein was widely existed in high plant, which was rapidly accumulated during seed desiccation in late stage of seed development.69 LEA proteins have been associated with desiccation tolerance70 and also play roles in storage protein of seeds and in whole-plant stress resistance to drought, salt, and cold.71 During the embryo maturation period, cell division and cell growth proteins are highly expressed to satisfy these two fundamental cell functions to finish the postembryonic development of plant seeds. In the present study, seven LEA DEPs (spots 3605, 1314, 0309, 6018, 1304, 0310, and 0314) were detected in the total proteins between HO and LO lines, of which six were highly expressed in the LO line. These results showed that cell growth was more rapid in the LO line than the HO line during the embryo maturation period, consistent with TEM analysis showing much larger average cell size for the LO compared with the HO line. Further research revealed that five LEA DEPs observed in OB proteins were highly or only expressed in the HO line, indicating that OBs divided more quickly in the HO than in the LO line. The greater number of OBs in cells of the HO line seems to confirm this. Comparative analysis also showed that AT3G52470 and AT3G53040 that corresponded to the LEA proteins also occurred in the QTL confidence interval. For desiccation-induced 1VOC-like protein, a seed-specific gene in desiccation-sensitive plants that is activated by water loss, adult plants constitutively expressing Xhdsi-1VOC showed higher growth rates, less photo-oxidative damage, and lipid peroxidation in drought treatment.72 During the seed maturing process of B. napus, high temperature is frequently encountered in China, and water loss would be rapid. Thus higher expression of desiccation-induced 1VOC-like protein in the HO line might protect embryo development from harm due to drought. In contrast, it is not surprising that protein biosynthesis and metabolism of amino acids appear to be enhanced in the LO line. The oil and protein traits were negatively correlated, as also found in near-isogenic sunflower varieties differing in seed oil traits, in which amino acid synthesis was up-regulated in the low oil variety while some storage proteins were up-regulated in the high oil variety,26 showing similar results to the present research. DEPs belonging to cupin family proteins were also observed between the HO and LO lines, which were also highly expressed in seeds and siliques of Arabidopsis (http://www.ncbi.nlm.nih.gov/ UniGene/ESTProfileViewer.cgi?uglist=At.21535). This was also verified by transgenic analysis, indicating that these DEPs might have a relationship with oil content. Nevertheless, no enzymes involved in de novo fatty acid syntheses were detected between mature seeds of the B. napus lines with different oil contents. Mature seed is the final expression pattern of proteins and fatty acids and does not

DEPs Involved in Defense/Disease Pathway

DEPs involved in the defense/disease pathway were also observed in HO and LO lines, such as protein disulfide isomerase (spots 1618, 1627, 1621, and 1616), putative dehydroascorbate reductase (spot 4212), lactoylglutathione lyase family protein (spot 3021), type 2 peroxiredoxin (spot 1116), and universal stress protein (USP) family protein (spot 8225) that was highly expressed in the HO line. Myrosinase (spot 6304), myrosinase-binding protein (spots 4906, 4905, 5910, and 2103), superoxide dismutase family proteins (spots 7224, 9205, 6019, and 7109), and other antistress proteins or EST (spots 2925, 2309, 3621, and 6206) were highly expressed in the LO line. Myrosinase and myrosinase-binding protein comprised the glucosinolate degrading enzyme occurring in the family of Brassicaceae67 and were always detected in OBs of mature seeds,12,13 which might be related to the higher glucosinate content of the LO line. The myrosinase/ 4975



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years ago; NADP, nicotinamide adenine dinucleotide phosphate; NCBInr, national center for biotechnology information nonredundant; NIRS, near-infrared spectroscopy technology; NMR, nuclear magnetic resonance; OBs, oil bodies; PEP, phosphoenolpyruvate; PFK, phosphofructokinase; PMF, peptide mass fingerprinting; PPDK, pyruvate orthophosphate dikinase; PSVs, protein storage vacuoles; QTL, quantitative trait loci; RuBP, ribulose-1,5-bisphosphate; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; TAG, triacylglyceride; TCA, trichloroaceticacid; TCA cycle, tricarboxylic acid cycle; TEM, transmission electron microscopy; UPGMA, unweighted pair group method with arithmetic mean; UPLC, ultra performance liquid chromatography; USP, universal stress protein; VOC, vicinal oxygen chelate; WAF, week after flowering

totally reflect all metabolism and biosynthesis during the whole fatty acid de novo synthesis and accumulation. Further investigation of protein different expression pattern between HO and LO B. napus lines during the seed filling development stage is needed in the future.

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ASSOCIATED CONTENT

S Supporting Information *

Fatty acid compositions of mature seeds in HO and LO lines. Glucosinolate component of mature seeds in HO and LO lines. Location of QTLs for oil content and total protein content on the partial linkage map for TN-DH population. The QTLs for oil content and protein content on C3 chromosome and comparative analysis between A. thaliana, B. napus, and B. rapa on the genes that corresponding to DEPs. The differentially expressed proteins for total protein in HO and LO B. napus lines. The differentially expressed proteins for oil body protein in HO and LO B. napus lines. Putative QTL for protein content and oil content in the TNDH population. The candidate gene in QTL confidence interval. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +86 (27) 87792432. Fax: +86 (27) 87792265. Present Address ⊥

National Key Facility for Crop Gene Resources and Genetic Improvement, Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing 100081, China Author Contributions ∥

REFERENCES

Lu Gan and Chun-yu Zhang contributed equally to this paper.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. Liebo Shu from Shanghai Boyuan Biotechnology Co., Ltd. for assistance of protein MS analysis. This work was supported by the National Natural Science Foundation of China (31171582 and 31071453), the National 863 High Technology Program (2011AA10A104), the Natural Science Funds for Distinguished Young Scholars of Hubei Province of China (2010CDA097), the New Century Talents Support Program by the Ministry of Education of China (NCET110172), and the Key Natural Science Foundation of Shannxi Province (2012JZ3001).

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ABBREVIATIONS 2-DE, two-dimensional gel electrophoresis; AR, aldose reductase; ATP, adenosine-5-triphosphate; CIM, composite interval method; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate; CLSM, confocal laser scanning microscope; CMS, cytoplasmic male sterility; DEPs, differentially expressed proteins; DH, doubled haploid; DIGE, difference gel electrophoresis; DTT, dithiothreitol; EST, expressed sequence tag; FBA, fructose-bisphosphate aldolase; GAPDH, glyceraldhyde-3-phosphate dehydrogenase; GC-MS, gas chromatography−mass spectrometry; HSP, heat shock proteins; IEF, isoelectric focusing; IPG, immobilized pH gradient; LEA, late embryogenesis abundant; MYA, million 4976



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Proteomic and Comparative Genomic Analysis of Two Brassica napus

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