Journal of Plant Physiology and Molecular Biology 2006, 32 (4): 497-503
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技术与方法 Techniques and Methods
Proteomic Technique Used in Studying Ovule Development of Chinese Pine (Pinus tabulaeformis Carr.) BAO Ren-Yan1, ZHENG Cai-Xia1*, ZHU Li-Hua2 1
College of Biological Sciences and Technology, Beijing Forestry University, Beijing 100083, China; 2Department of Crop Science, Swedish University
of Agricultural Sciences, Box 44, SE230 53 Alnarp, Sweden
Abstract: Chinese pine (Pinus tabulaeformis Carr.) is one of the most important afforestation and ornamental species in China. Ovule abortion is a serious problem influencing the output of seeds and reproduction of Chinese pine. So it is of much significance to study the mechanisms of ovule development and abortion of Chinese pine. By combining two-dimensional gel electrophoresis (2-DE) and mass spectrometry analysis, the patterns of gene expression in a specific tissue at a specific stage can be displayed and characterized. This study acquired an appropriate protein extraction method from ovules of Chinese pine and optimized the conditions for protein identification by mass spectrometry. Key words: Chinese pine (Pinus tabulaeformis Carr.); ovule; proteomic; mass spectrometry; peptide mass fingerprinting
Chinese pine (Pinus tabulaeformis Carr.) is a particular tree species in China and one of the most important afforestation species in North China (Wu et al. 2000). It is distributed in 14 provinces in north, northeast, northwest, and southwest China. Chinese pine is good for soil and water conservation and depollution of environment. It is also a popular ornamental tree in China. But abortion of ovules affects the output of seed of Chinese pine seriously in many seed orchards. This causes the seed quantity of Chinese pine cannot meet the need of large-scale afforestation. In addition, the propagation of Chinese pine is very difficult through vegetative reproduction. So far, only the graft method can be used in production, and most nursery stock is the clone produced by grafting in seed orchard of Chinese pine. So it is of much significance to study the mechanisms of ovule
development and abortion of Chinese pine for enhancing the quantity and quality of seeds (Wang and Shen 1989; Zhang et al. 1997). Proteomic technology can display and characterize the pattern of gene expression in specific tissue at a specific stage. Proteomic analysis includes protein separation and identification. For protein separation, two dimensional gel electrophoresis (2-DE) using large gel (24 cm×20 cm) can provide enough space to separate several thousand protein spots and complete in a few hours (Klose and Kobalz 1995). For protein identification, mass spectrometry (MS) is the most common method used. Since the primary structure of each protein is unique, the mass of peptides also varies after the protein is hydrolyzed by protease that specifically hydrolyzes peptide bonds at a specific site. The peptides mass map of protein, also known as peptide mass fingerprinting (PMF) (Wise et al. 1997), is an effective tool for identifying proteins since each protein has a single PMF. The method combining 2-DE with PMF has been employed on proteomic studies in some plant species such as Arabidopsis and rice (Millar et al. 2001; Imin et al. 2001). However, up to now this combined technique has been little tested in woody plants, especially gymnosperms. In this study, we compared two protein extraction methods and optimized the conditions for protein identification, established the proteomic method appropriate for the study of ovule development in Chinese pine. Received 2005-12-26, Accepted 2006-05-22. This work was supported by the National Natural Science Foundation of China (No. 30371144). * Corresponding author (E-mail:
[email protected]; Tel: 86-10-
62337717).
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The establishment of this combined method on pine would be very useful for further proteomic studies in this genus.
1 1.1
Materials and Methods
Plant materials The ovules of Chinese pine were taken out from two-year-old cones immediately after collecting, and then stored at –70ºC. 1.2 Extraction of proteins Method 1: The trichloroacetic acid (TCA) / acetone method of Imin et al. (2001) was used with some modifications. Tissues were finely ground in liquid nitrogen in a mortar with a pestle, and then 20 mg tissue powder was suspended in 1 mL cold extraction solution (10% TCA in acetone plus 0.07% β-mercaptoethanol). After kept for 1 h at –20ºC, the samples were centrifuged at 15 000×g for 15 min at 4ºC. The pellets were washed twice with prechilled acetone containing 0.07% β-mercaptoethanol, and kept at –20ºC for 30 min before centrifugation at 15 000×g for 15 min at 4ºC, and then were dried under vacuum for10 min at 4ºC. Method 2: Proteins were extracted from tissue powder with sucrose solution [5% sucrose, 5% βmercaptoethanol and 4% sodium dodecyl sulfate (SDS), pH 3.8] as described by Ekramoddoullah (1993). 1 mL sucrose solution was added to 50 mg of lyophilized, ground tissue and vortexed for 15 min. This mixture was centrifuged at 17 310×g for 15 min. The supernatant was collected, heated over boiling water for 3 min, cooled to room temperature and then purified by the addition of 1.2 mL of ice-cold acetone per 150 µL of extract. The mixture was kept at –20ºC for 1 h and the precipitated proteins were collected by centrifugation at 17 310×g for 10 min. The precipitate was washed twice with prechilled acetone containing 0.07% β-mercaptoethanol, allowed to stand for 30 min at –20ºC, centrifuged at 15 000×g for 15 min at 4ºC, and then dried under vacuum for 10 min. 1.3 Sample preparation The dry protein pellets were suspended in urea lysis buffer containing urea 9.5 mol/L, 2% NP-40, 5% βmercaptoethanol, 1.6% carrier ampholytes pH 3.5–10 (Pharmacia Biotech, Sweden) and incubated at 30ºC for
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30 min and then centrifuged at 15 000×g for 15 min at room temperature. The protein concentration of the supernatant was determined by Bradford method (Wang and Fa n 2000). The supernata nt was loaded for isoelectrofocusing. 1.4 Electrophoresis Both isoelectrofocusing (IEF) and SDS-polyacrylamide gel electrophoresis (PAGE) were carried out in Bio-Rad Mini-PROTEAN II 2-D system. The IEF gels were made in a 7.5-cm long glass capillary tube with an inner diameter of 1.0 mm. Polyacrylamide column gels were prepared according to the ma nufacturer’s instructions. IEF was performed at 200 V for 30 min, at 500 V for 60 min, and then at 750 V for 3 h. After IEF, the column gel was equilibrated in the SDS sample equilibration buffer (Tris-HCl 0.0625 mol/L, pH 6.8; 2.3% SDS; 5% β-mercaptoethanol; 10% glycerol) for 10 min. The column gel was then carefully slid onto a slab gel. The separation gel of 12% T was used to perform the second-dimensional electrophoresis. The slab gels were run at 5 mA/gel for 15 min, then at 15 mA/gel for 2.5 h. The gel was stained with 0.25% Coomassie brilliant blue (CBB) solution overnight and then destained in the solution containing 7.5% acetic acid and 5% ethanol. 1.5 MS analysis Protein spots selected were excised from the gel. Each gel piece was cut into 1 mm2 pieces and washed thrice with Milli-Q H2O in an Eppendorf tube. The gel pieces were destained with NH4HCO3 25 mmol/L in 50% acetonitrile (ACN) and dried for 15 min under vacuum. A solution of dithiotreitol (DTT) 10 mmol/L in NH 4HCO 3 25 mmol/L was added in an amount just enough to cover the gel pieces, and incubated together for 1 h at 56ºC. The DTT solution was then replaced by the same volume of solution of iodoacetamide 55 mmol/L in NH4HCO3 25 mmol/L, and incubated for 45 min at room temperature in the dark with occasional vortex. Afterwards, the gel pieces were washed with NH4HCO3 25 mmol/L solution for 10 min by vortexing, then the solution was removed and gel pieces were dried under vacuum. The gel pieces were rehydrated in 5–10 µL (according to the size of protein spot) NH4HCO3 25 mmol/L (pH
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Bao Ren-Yan et al.: Proteomic Technique in Chinese Pine
8.0, containing 0.025 g/L trypsin) and incubated at 37ºC for 12–16 h. The gel pieces were extracted with 30 µL 5% trifluoroacetic acid (TFA) / 50% acetonitrile mixture for 30 min by vortexing, and this process was repeated twice. The extracts were collected in a new tube and concentrated by reducing the final volume to 10 µL under vacuum. The peptides mixture was analyzed by MALDITOF-MS and the PMF was generated. The PMF was used to search database with Mascot which can be found at the following URL: http://www.matrixscience.com/cgi/ search_form.pl?FORMVER=2&SEARCH=PMF, to identify protein.
2
Results and Discussion
2.1 Comparisons of two protein extraction methods We compared two protein extraction methods, TCA/ acetone method (method 1) and sucrose method (method 2), and found that the protein concentration and purity with the sucrose method were higher than those with the TCA/acetone method. These proteins were separated by 2-DE and the results are in Fig.1. Gel A was 2-DE separation pattern of proteins extracted by the sucrose method, while gel B was 2-DE separation pattern of proteins extracted by the TCA/acetone method. The protein spots displayed on gel A were more than gel B, and the disturbance of vertical and horizontal streaks was little and
499
had higher reproducibility. This suggested that the sucrose method was successful in extracting protein from pine. This method ca n improve information and reproducibility. The resolution of 2-DE separation pattern of proteins extracted by the TCA/acetone method was low. Compared with the TCA/acetone method, the sample supernatant needed to be heated over boiling water for 3 min in the sucrose method, besides extract solution is different. The results implied that heating the sample supernatant could remove secondary compounds such as phenol and polysaccharide that affect protein extraction seriously. 2.2 Condition optimization of mass spectrometry analysis Three protein spots with different sizes were selected for MS identification and the PMF results of proteins are presented in Fig.2. The accuracy of mass spectrometry in the range of 5–100 µmol is now possible, and all visible spots stained with CBB can be measured in theory. However, in practice, the veracity of results can not be ensured unless the protein spot in gel is large enough. The three protein spots with different sizes were analyzed by mass spectrometry under accordant experiment conditions. The PMF result of protein spot 3 was too poor to identify it through searching database since only two peaks were detected (Fig.2). We considered that the reason was its small quantity. So the protein concentration in the samples
Fig.1 2-DE separation patterns of proteins from ovules of Chinese pine with CBB stained A: Proteins extracted by the sucrose method, three protein spots with different sizes were selected for MS identification; B: Proteins extracted by TCA/acetone method.
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Fig.2 Peptide mass fingerprinting map of protein spots from 2-DE gel Peaks marked with “ * ” mean that they are matched with the database searching.
loaded for 2-D electrophoresis should be high enough to secure quantification of target protein spots by MS analysis. Proteins and enzyme were in gel and solution respectively, so it is important for enzymatic lysis to make enzyme and proteins be in sufficiently good contact. Dye (CBB) and other complements (Tris, SDS etc.) in gel greatly interfere with mass spectrometry analysis. CBB is an anion dye, and combines with the alkaline groups of proteins. The PMF map was not detectable when CBB was present in the samples. To remove the dye, we compared three solutions, 25% ethanol / 7% acetic acid solution, NH4HCO 3 100 mmol/L pH 7.8 solution and
NH4HCO3 25 mmol/L / 50% ACN solution, and found that the NH4HCO3 / ACN solution was best. ACN can remove the blue dye and NH4HCO3 can maintain the alkaline condition, which is beneficial for the following enzymatic reaction. The excised gel containing protein spot should be chopped into small pieces, since the small pieces facilitates the removal of SDS and CBB, and increase the area of contact with the enzyme solution. In addition, the gel should be fully dehydrated before the addition of the enzyme solution, otherwise the enzyme can not infiltrate into the gel and get contact with the proteins. Trypsin and Glu C proteases are usually used for
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Bao Ren-Yan et al.: Proteomic Technique in Chinese Pine
PMF, but Glu C is very expensive. We used the Sequencing Grade Modified Trypsin purchased from Promega Company. The trypsin was modified by reductive methylation, making it resistant to proteolytic digestion. It should be avoided to add more solution than the amount that can be absorbed by the gel pieces, otherwise trypsin autolysis will occur. The enzyme to substrate ratio employed for in-gel digestion should be greater than for in-solution digestion due to the hindered access of enzyme to the protein substrate in the gel. A relatively low salt concentration of 25 mmol/L was used to reduce
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the possibility of salt interference with ionization in the mass spectrometer. 2.3 Identification of proteins by searching database Different programs for database searching on the internet allow the identification of proteins according to pI, molecular weight, PMF data and other parameters. The PMF of protein spots was matched with the Mascot search program. Searching results of protein spot 1 are presented in Table 1. 17 protein points whose scores were significant were found. All of them are ATP synthase beta chains, and the difference is that they come from differ-
Table 1 The results of database searching with PMF of spot 1 Entry name Q5N7P8_ORYSA
Mass (kD) 45.265
Score
Expect
94
8.7e-05
Queries
Description
matched 7
Putative ATP synthase beta subunit (Oryza sativa japonica cultivar-group)
Q5N7P9_ORYSA
45.937
93
9.6e-05
7
Putative ATP synthase beta subunit (Oryza sativa
O24345_SORBI
49.219
91
0.00015
7
F1-ATP synthase, beta subunit (Fragment) (Sorghum
A24355
59.933
86
0.00048
7
H+-transporting two-sector ATPase (EC 3.6.3.14)
S20504
60.335
86
0.00048
7
H+-transporting two-sector ATPase (EC 3.6.3.14)
japonica cultivar-group) bicolor, Sorghum vulgare) beta-1 chain, mitochondrial (curled-leaved tobacco) beta chain, mitochondrial (para rubber tree) Q6I636_ORYSA
59.012
86
0.00048
7
Putative ATP synthase beta chain (Oryza sativa japonica cultivar-group)
S11491
59.181
86
0.00048
7
H+-transporting two-sector ATPase (EC 3.6.3.14)
S47350
59.326
86
0.00048
7
H+-transporting two-sector ATPase (EC 3.6.3.14)
Q8H135_ARATH
48.284
75
0.0062
6
beta chain, mitochondrial (maize) beta chain, mitochondrial (wheat) Hypothetical protein At5g08670 (Fragment) (Arabidopsis thaliana mouse-ear cress) O24346_SORBI
49.239
71
0.015
6
F1-ATP synthase, beta subunit (Fragment) (Sorghum
CAC81058
63.560
70
0.019
6
ATH271468 NID (Arabidopsis thaliana)
Q9C5A9_ARATH
59.993
70
0.019
6
Putative H +-transporting ATP synthase beta chain
bicolor, Sorghum vulgare)
(Mitochondrial) (At5g08680) (Arabidopsis thaliana) O82722_NICSY
59.638
70
0.02
6
Mitochondrial ATPase beta subunit (Nicotiana sylvestris wood tobacco)
Q9SAQ0_NICSY
59.597
70
0.02
6
ATPase beta subunit (Nicotiana sylvestris wood tobacco)
Q541W2_ARATH
59.847
70
0.021
6
Putative H +-transporting ATP synthase beta chain
Q541W7_ARATH
59.805
70
0.021
6
Putative H +-transporting ATP synthase beta chain
S21988
59.327
73
0.0094
6
H+-transporting two-sector ATPase (EC 3.6.3.14)
(Mitochondrial) (Arabidopsis thaliana) (Mitochondrial) (Arabidopsis thaliana) beta chain, mitochondrial (carrot)
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ent species or tissues. We can assume that protein spot 1 is an H+-transporting ATPase beta chain of Chinese pine considered that the molecular weight of the protein spot is 60 kD. The protein spot 2 cannot be identified through database searching. This may be due to the insufficient information on PMF data for plants, especially for woody species. Or it may be a new protein which has not been identified. So we need to determine its amino acid sequence and synthesize DNA probe to express, separate and identify it. Successful identification of proteins using PMF relies on the development of mass spectrometry technology, and the peptide mass obtained by MS apparatus is more accurate, the searching result is more reliable. Its sensitivity can reach fmol grade and its accuracy can reach 0.01% when using the modernized MALDI-TOF-MS to assay peptide mixtures. The technology promotes the proteomic studies (Roepstorff et al. 1997; Walsh et al. 1998), and makes PMF become a preferred method to identify proteins on a large scale, in despite of the fact that not all protein spots on a 2-D gel can be identified with the PMF method, especially for the species the genome of which is yet unknown. In this paper we have done a research on the extraction method, 2-D electrophoresis and mass spectrometry assay about ovule proteins of Chinese pine. This will be in favor of the study on ovule development mechanism and provide the theoretical basis to increase seed output of Chinese pine. References Ekramoddoullah AKM (1993). Analysis of needle proteins and N-
terminal amino acid sequence of two photosystem II proteins of western white pine (Pinus monticola D. Don). Tree Physiol 12: 101-106 Imin N, Kerim T, Weinman JJ, Rolfe BG (2001). Characterisation of rice anther proteins expressed at the young microspore stage. Proteomics 1: 1149-1161 Klose J, Kobalz U (1995). Two-dimensional electrophoresis of proteins: an updated protocol and implications for a functional analysis of the genome. Electrophoresis 16: 10341059 Millar AH, Sweetlove LJ, Giegé P, Leaver CJ (2001). Analysis of the Arabidopsis mitochondrial proteome. Plant Physiol 127: 1711-1727 Roepstorff P (1997). Mass spectrometry in protein studies from genome to function. Curr Opin Biotech 8: 6-13 Walsh BJ, Molloy MP, Williams KL (1998). The Australian proteome analysis facility (APAF): assembling large scale proteomics through integration and automation. Electrophoresis 19: 1883-1890 Wang JZ, Fan M (2000). Protein Technology Manual. Beijing: Science Press 42-47 (in Chinese) Wang XR, Shen XH (1989). Studies on increasing seed production in seed orchards of Pinus tabulaeformis Carr.: an analysis of seed losses caused by aborted ovules and empty seeds. J Beijing For Univ 11(7): 60-65 (in Chinese) Wise MJ, Littlejohn TG, Humphery-Smith I (1997). Peptide mass fingerprinting and the ideal covering set for protein characterization. Electrophoresis 18: 1399-1409 Wu H, Cui DF, Hu ZH (2000). Initiation and development of the ovulate strobilus in Pinus tabulaeformis Carr. Acta Bot Sin 42(4): 353-357 (in Chinese) Zhang HX, Li FL, Shen XH, Li GF, Yang XH (1997). Formation of male and female gametophytes and development of embryo in Pinus tabulaeformis Carr. J Beijing For Univ 19(3): 1-7 (in Chinese)
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油松胚珠发育研究中的蛋白质组技术 包仁艳 1，郑彩霞 1*，朱丽华 2 1
北京林业大学生物科学与技术学院，北京 10 0 0 83 ；2 瑞典农业科学大学作物科学系，阿尔那普 SE2 3 0 5 3 ，4 4 号邮箱，瑞典
摘要：油松是中国特有的树种，具有重要的造林和
白点用肽质量指纹图谱的方法进行鉴定，得到了满
绿化价值。但是油松胚珠败育是油松种子生产和繁
意的结果，优化了蛋白质质谱鉴定的条件。
殖面临的一个严重的问题，因此研究油松胚珠的发 育机理和败育原因具有重要的理论和实践意义。双
关键词：油松；胚珠；蛋白质组；质谱；肽质量指纹谱
向电泳和质谱技术结合研究基因表达的蛋白质组技术
中图分类号：Q 94 5
正越来越广泛地应用到植物研究中，我们将该技术 应用到油松胚珠发育的研究中，比较了两种蛋白质 提取的方法，发现蔗糖提取方法比传统的 TCA 方法
国家自然科学基金(No. 30371144)资助。
更适合于油松蛋白质的制备。将电泳分离得到的蛋
* 通讯作者(E-mail:
[email protected]; Tel: 010-62337717)。
会 议 消 息 由亚洲大洋洲光生物学学会发起， 中国生物物理学会光生物学专业委员会负责承办的第三届亚洲 大洋洲光生物学大会（3rd AOSP）定于 2006 年 11 月 17~20 日在北京举行。会议将讨论和交流包括 光化学、光物理、光技术、光感受、节律生物学、光合作用、生物与化学发光、光医学、环境 光生物学和紫外辐射效应在内的光生物学领域的所有重要进展，会议还将为与会的光生物学、光医学 各个领域的物理学家、化学家、生物学家和医生提供相互交流的极好机会。 会议时间
2006 年 11 月 17 日报到，18~20 日会议。
会议地点 北京西郊宾馆(三星级) 有关大会报告及 15 个分会邀请报告的内容请见：http://www.AOSP2006.org.cn 会议工作语言 英语 截止日期 (1) 论文摘要：2006 年 9 月 15 日 (2) 会前注册：2006 年 9 月 15 日 会议联系人 (1) 摘要：魏舜仪，100101，北京朝阳区大屯路 15 号中国生物物理学会（电话: +86-10-64889894， 传真: +86-10-64889892，E-mail:
[email protected]）； (2) 注册：王 悦，100101，北京朝阳区大屯路 15 号中国生物物理学会（电话: +86-10-64889894， 传真: +86-10-64889892，E-mail:
[email protected]）。
